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CYTOPLASMIC MYOSIN FUNCTION IN VITRO AND IN VIVO

体外和体内细胞质肌球蛋白功能

基本信息

批准号：
3283911
负责人：
DANIEL PETER KIEHART
金额：
$ 19.53万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-09-30 至 1992-08-31
项目状态：
已结题

项目摘要

Our long range goals are to understand, on the molecular level, the mechanism of cytoplasmic myosin function in vitro and in vivo and to determine which cellular movements require myosin for force production. We focus on the function of cytoplasmic myosin in Drosophila for two reasons. First, a diverse array of interesting movements provide the structural basis for spatial differentiation of the embryo and are characteristic of various movements seen in other cells. They include nuclear divisions and migrations, pole cell formation and cellularization, and the complex cellular migrations and shape changes of gastrulation. Moreover, these movements are amenable to study by a range of powerful approaches. We used classical protein biochemical and immunological methods to purify and characterize cytoplasmic myosin from Drosophila cells in culture. This protein is myosin by five structural and three functional criteria. It is distinct from the muscle myosin heavy chain isoform by peptide mapping and immunochemical criteria. The real advantage of this organism is of course its accessibility to genetic, molecular biological and modern molecular genetic manipulation. We used antibodies to screen a library of Drosophila genomic DNA in the expression vector lambda gtll and have isolated a fragment of the gene that encodes cytoplasmic myosin. Its gene product shares epitopes with cytoplasmic but not muscle myosin heavy chain and hybridizes with a 7.4 kb message that is sufficiently large to encode this 205 kD polypeptide. Hybridization studies with restriction fragments of Drosophila DNA and clones that encode muscle myosin heavy chain confirm that it is not part of the muscle myosin gene. Moreover, preliminary mapping studies by in situ hybridization with polytene chromosomes show that this gene maps to 60E,F, distinct from the location of the muscle myosin heavy chain gene. Here we propose a multidisciplinary approach to the analysis of cytoplasmic myosin that includes further characterization of cytoplasmic myosin function in vitro, localization of this protein in embryos and cells, the use of site specific antibodies to evaluate myosin function in vitro and in vivo, characterization of the gene that encodes this peptide and its transcription unit, synthesis of cytoplasmic myosin heavy chain or its fragments in bacterial and eukaryotic expression vector systems for detailed analysis of protein structure/function relationships in vitro, and genetic manipulation of cytoplasmic myosin in living cells and embryos by cultured cell transformation and P-element mediated germ line transformation. Together these studies forge a comprehensive investigation of the mechanism of cytoplasmic myosin function and its role in cellular movements.

我们的长期目标是在分子水平上理解，细胞质肌球蛋白的体内外作用机制并确定哪些细胞运动需要肌球蛋白部队生产。我们关注的是细胞质肌球蛋白的功能。有两个原因。首先，一系列不同的有趣的运动为空间提供了结构基础胚胎的分化，并具有各种不同的特点在其他细胞中看到的运动。它们包括核部门和迁移，极细胞的形成和细胞化，以及原肠胚复杂的细胞迁移和形态变化。此外，这些运动可以通过一系列的研究来研究强大的方法。我们使用了经典的蛋白质生化和细胞质纯化和鉴定的免疫学方法果蝇细胞培养中的肌球蛋白。这种蛋白质是肌球蛋白五个结构标准和三个功能标准。它有别于肌球蛋白重链异构体的多肽图谱和免疫化学标准。这种有机体的真正优势是当然，它可获得遗传、分子生物学和现代分子遗传操作。我们用抗体来果蝇基因组DNA文库的筛选及表达载体lambda gtll，并分离出该基因的一段编码细胞质肌球蛋白。它的基因产品具有相同的表位具有细胞质而不是肌肉肌球蛋白重链与7.4kb的消息混合，该消息足够大到编码这个205kD的多肽。杂交研究与果蝇DNA的限制性内切酶片段及其编码克隆肌肉肌球蛋白重链证实它不是肌肉肌球蛋白基因。此外，印度的初步测绘研究与多线染色体的原位杂交表明，该基因映射到60E，F，与肌肉肌球蛋白的位置不同重链基因。在这里，我们提出了一个多学科的方法对细胞质肌球蛋白的分析，包括进一步细胞质肌球蛋白功能的体外鉴定，该蛋白在胚胎和细胞中的定位、SITE的使用用特异性抗体评价肌球蛋白的体外和体外功能活体，编码这种多肽的基因的特征和其转录单位、胞浆肌球蛋白重链的合成或其片段在细菌和真核表达载体中的表达蛋白质结构/功能的详细分析系统细胞质的体外关系和遗传操作培养细胞转化活细胞和胚胎中的肌球蛋白 P-元件介导的种系转化。同舟共济这些研究伪造了一项关于胞浆肌球蛋白的作用机制及其在细胞内的作用动静。