MOLECULAR DETERMINANTS OF CYTOSKELETAL ORGANIZATION

细胞骨架结构是一个主要的和特征的特征 真核细胞的特性和功能。这个项目的目标是 研究协调细胞骨架组织的基因的活性 利用重组DNA载体体内培养细胞和两栖类胚胎 包括反义质粒。 反义基因抑制是一种有价值的分子策略 脊椎动物中不可接受的表型复制基因突变 经典的遗传分析。DNA介导的作用机制研究 “翻转”的基因反义抑制还将继续。反义鸡肉 胸苷激酶内含子和反义内含子-外显子连接质粒将 通过与野生型基因共显微注射到 培养的细胞核和非洲爪哇卵母细胞生发泡。基因产品 活性和靶mRNA转录物的代谢将仔细 被监视着。将探索基因替换的相关策略。 含有多种细胞骨架相关基因的反义和正义质粒 蛋白质基因(原肌球蛋白、产量-肌动蛋白和200kd 微管相关蛋白)将被构建并导入 培养的细胞和非洲爪哇胚胎。由此导致的细胞缺陷 将对体系结构进行研究，以评估每个基因对 对细胞形态、病毒转化和胚胎 形态发生。 非洲爪哇cDNA库的插入，富含胚胎特异序列，将 将其亚克隆到反义表达载体中，然后导入 非洲爪哇胚胎识别指导胚胎模式的新基因 队形。目的是利用反义基因抑制作为一种 伪遗传工具，用于识别以前未表征的因素， 除了使用这些分子方法外，还可以调节细胞组织 用于分析克隆基因在活细胞中的功能。

Cytoskeletal architecture is a predominant and characteristic feature of eukaryotic cell identity and function. The goal of this project is to study the activity of genes that coordinate cytoskeletal organization in cultured cells and amphibian embryos in vivo using recombinant DNA vectors including anti-sense plasmids. Anti-sense gene inhibition is a valuable molecular strategy for phenocopying gene mutations in vertebrate organisms that are not amenable to classical genetic analysis. Studies on the mechanism of DNA mediated "flipped" gene anti-sense inhibition will continue. Anti-sense chicken thymidine kinase intron and anti-sense intron-exon junction plasmids will be constructed and tested by comicroinjection with the wild type gene into cultured cell nuclei and Xenopus oocyte germinal vesicles. Gene product activity and target mRNA transcript metabolism will be carefully monitored. Related strategies for gene replacement will be explored. Anti-sense and sense plasmids containing several cytoskeleton-associated protein genes (tropomyosins, yield-actinin, and a 200 kd microtubule-associated protein) will be constructed and introduced into cultured cells and Xenopus embryos. The resultant deficiencies in cellular architecture will be studied to assess the contribution that each gene makes to cellular morphology, viral transformation, and embryonic morphogenesis. Xenopus cDNA library inserts, enriched in embryo-specific sequences, will be subcloned into anti-sense expression vectors and then introduced into Xenopus embryos to identify novel genes that direct embryonic pattern formation. The objective is to use anti-sense gene inhibition as a pseudo-genetic tool to identify previously uncharacterized factors that regulated cell organization in addition to using these molecular approaches for the analysis of cloned gene function in living cells.