GENE ORGANIZATION AND EXPRESSION IN EUKARYOTES

研究计划继续调查结构，表达， 和原肌球蛋白在正常和转化细胞中的功能。 全长 大鼠胚胎原肌球蛋白5种异构体的cDNA克隆 将分离成纤维细胞，并测定其DNA序列，以推断 其主要结构。 这些信息将用于准备 用作免疫原的合成肽 抗体的 这种抗体将有助于确定亚细胞 每种形式的原肌球蛋白的分布，如果这种定位是 在变形时发生了变化 携带λ噬菌体的重组体 将分离大鼠原肌球蛋白的基因， 和基因的组织。 这些研究将 确定是否所有五种形式的成纤维细胞原肌球蛋白都来自于单独的 但是相关基因或者如果一些基因是通过差异表达从单个基因产生的， 消息的处理。 克隆的DNA将用于研究 原肌球蛋白表达，以确定是否有改变， 原肌球蛋白信息的转录、加工和翻译 转化细胞 为了研究改变的功能意义， 原肌球蛋白在转化成纤维细胞中的模式，原肌球蛋白将是 通过（1）显微注射纯化的mRNA或（2） 编码原肌球蛋白的克隆DNA的表达，例如，通过 金属硫蛋白启动子。 这些研究将确定是否表达 转化细胞中的原肌球蛋白将导致细胞形状的变化， 微丝的组织。 （五）

Studies are planned to continue to investigate the structure, expression, and function of tropomyosin in normal and transformed cells. Full-length cDNA clones encoding all five isoforms of tropomyosin from rat embryonic fibroblasts will be isolated and their DNA sequences determined to deduce their primary structure. This information will be used to prepare synthetic peptides to be used as immunogens for making isoform-specific antibodies. Such antibodies will be useful in determining the subcellular distribution of each form of tropomyosin and if this localization is altered upon transformation. Bacteriophage lambda recombinants which carry the genes for rat tropomyosin will be isolated and the general structure and organization of the genes determined. For example, these studies will determine if all five forms of fibroblast tropomyosin arise from separate but related genes or if some arise from a single gene via differential processing of the message. The cloned DNAs will be used to study tropomyosin expression to determine if there are alterations in transcription, processing, and translation of tropomyosin messages in transformed cells. To study the functional significance of the altered pattern of tropomyosins in transformed fibroblasts, tropomyosin will be introduced into living cells by (1) microinjection of purified mRNA or (2) the expression of cloned DNAs encoding tropomyosin, for example, via the metallothionein promoter. These studies will determine if expression of tropomyosin in transformed cells will result in changes in cell shape or organization of microfilaments. (V)