CYTOPLASMIC MYOSIN FUNCTION IN VITRO AND IN VIVO

我们的长期目标是在分子水平上， 胞浆肌球蛋白功能的体内外机制 并确定哪些细胞运动需要肌球蛋白 力生产。 我们着重于细胞质肌球蛋白的功能 有两个原因。 首先， 有趣的运动为空间结构提供了基础 胚胎的分化和各种特征 在其他细胞中观察到的运动。 其中包括核分裂和 迁移、极细胞形成和细胞化，以及 原肠胚形成过程中复杂的细胞迁移和形状变化。 此外，这些运动可以由一系列的研究人员进行研究。 强大的方法。 我们使用了经典的蛋白质生化和 纯化和表征细胞质免疫学方法 培养的果蝇细胞中的肌球蛋白。 这种蛋白质是肌球蛋白， 五个结构和三个功能标准。 它不同于 通过肽图谱分析肌球蛋白重链同种型， 免疫化学标准。 这种生物真实的优势是 当然，它的遗传，分子生物学和 现代分子遗传操作 我们用抗体 筛选果蝇基因组DNA表达文库 载体λ gt 11，并分离了该基因的片段， 编码细胞质肌球蛋白。 它的基因产物共享表位 具有细胞质而非肌肉肌球蛋白重链， 与足够大的7.4kb消息杂交， 编码该205 kD多肽。 杂交研究， 果蝇DNA的限制性片段和编码 肌肉肌球蛋白重链证实它不是 肌球蛋白基因。 此外，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60 E，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 细胞质肌球蛋白的分析，包括进一步 体外细胞质肌球蛋白功能的表征， 这种蛋白质在胚胎和细胞中的定位， 特异性抗体，以评估肌球蛋白的功能，在体外和 体内，表征编码该肽的基因， 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 详细分析蛋白质结构/功能的系统 关系，以及细胞质的遗传操作， 通过培养细胞转化在活细胞和胚胎中的肌球蛋白 以及P元件介导的生殖系转化。 一起 这些研究形成了一个全面的调查， 胞浆肌球蛋白功能的机制及其在细胞内的作用 动作

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.