MYOSIN EXPRESSION AND FUNCTION IN A NON-MUSCLE SYSTEM

对肌球蛋白在非肌肉组织中的作用知之甚少 系统。这项提议的目的是阐明生理作用。 肌球蛋白及其在非肌肉系统中的调节。我们建议进行 这些研究是在酿酒酵母上进行的。酵母代表着一种优势 进行这些研究的系统，因为它可以很容易地培养 和操纵，传统的和分子遗传的方法学 已经开发出了操纵和其他结构中的基因突变 蛋白质基因随处可见。 在本研究中，生化、传统遗传学和分子遗传学 将使用各种方法来识别和描述功能区域 酵母肌球蛋白。肌球蛋白在细胞内组装中的作用 肌动蛋白-肌球蛋白纤维及其在细胞中的功能 将对过程进行调查。除了我们的结构研究，我们还 将分析肌球蛋白在整个细胞周期中的调节。培训 重点是研究生和本科生。 其具体目的是：1)检验肌球蛋白是一种 酵母细胞骨架的基本成分，通过免疫化学 方法研究肌球蛋白和肌球蛋白中肌动球蛋白组件的定位。 肌动蛋白突变体；2)检验肌球蛋白在 酵母细胞周期，通过定量分析肌球蛋白的mRNA和蛋白 在同步培养中；3)测试通过以下方式调节的假设 酵母肌球蛋白羧基末端的磷酸化是重要的 [~(32)P]P_1标记的肌球蛋白的SDS-PAGE分析 体内、体外诱变、细胞转化及功能分析 活体内的突变体；4)培训学生分子生物学技术 怀着攻读研究生学位的期望。

Relatively little is known about the function of myosin in non-muscle systems. The goal of this proposal is to elucidate the physiological role of myosin and its regulation in a non-muscle system. We propose to conduct these studies in the yeast S. cerevisiae. Yeast represents an advantageous system in which to conduct these studies because it can be easily cultured and manipulated, the methodologies for traditional and molecular genetic manipulations have been developed and genetic mutants in other structural protein genes are readily available. In this study, biochemical, traditional genetic and molecular genetic approaches will be used to identify and characterize the functional regions of yeast myosin. The role of myosin in the assembly of intracellular actin-myosin fibers and the function of such assemblies in cellular processes will be investigated. In addition to our structural studies, we will analyze the regulation of myosin throughout the cell cycle. Training of students at graduate and undergraduate level is stressed. The specific aims are: 1) to test the hypothesis that myosin is an essential component of the yeast cytoskeleton, by using immunochemical methods to study the localization of actomyosin assemblies in myosin and actin mutants; 2) to test the hypothesis that myosin is regulated during the yeast cell cycle, by quantitatively analyzing myosin mRNA and protein in synchronized cultures; 3) to test the hypothesis that regulation by phosphorylation at the carboxy terminus of yeast myosin is important for myosin function, by SDS-PAGE analysis of myosin labelled with [32P]P1 in vivo, in vitro mutagenesis, cell transformation, and functional analysis of mutants in vivo and; 4) to train students in molecular biology techniques with the expectation that graduate degrees will be pursued.