CHARACTERIZATION OF GENES IMPORTANT IN LIMB DEVELOPMENT

这个项目的长期目标是了解这些机制 参与细胞内不同类型细胞的分化 发育中的肢体。肢体分子生物学研究进展 发展强调了对生物多样性结构和调控的研究 在肢体发育过程中表达的表型基因，特别是那些 软骨(蛋白多糖核心蛋白和II型胶原)和肌肉 (肌动蛋白和肌球蛋白)，而负责调节或 促进单个细胞的分化是未知的。 一种结合单抗技术和技术的综合方法 重组DNA技术将被用于识别和表征基因 以及在肢体早期细胞多样化中重要的基因产物 发展。与独有的mRNA相对应的互补DNA文库 将构建早期肢体中的特定细胞群。单人 用于构建这些文库的链(第一链)将是 通过缺乏杂交来选择与来自一种密切相关的过剩mRNA的 细胞数量。由此产生的克隆的cDNA将通过 与不同细胞群体的RNA杂交和原位杂交 与组织杂交。与特定基因相对应的克隆 兴趣将在大肠杆菌中表达，由此产生的多肽将 用于产生更多蛋白质和组织的抗体 人物刻画。一种补充方法将是使用选定的 从其他实验室获得的抗体显示出有趣的模式 在肢体发育过程中定位以分离相应的基因 通过筛选表达文库或通过免疫沉淀 多聚体和随后的cdna构建。 然后将使用抗体和DNA探针来识别和 鉴定以前未鉴定的基因和基因产物，它们是 在调节肢体早期发育方面很重要。

The long term objectives of this project are to understand the mechanisms involved in the differentiation of different cell types within the developing limb. Previous studies of the molecular biology of limb development have emphasized the study of the structure and regulation of phenotypic genes expressed during limb development particularly those of cartilage (proteoglycan core protein and type ii collagen) and muscle (actin and myosin), whereas the genes responsible for regulating or promoting differentiation of individual cells are unknown. An integrated approach combining monoclonal antibody technology and recombinant DNA technology will be used to identify and characterize genes and gene products important in the early cell diversification during limb development. Complementary DNA libraries corresponding to mRNAs unique to specific cell populations in the early limb will be constructed. Single stranded cDNA (first strand) for construction of these libraries will be selected by lack of hybridization to excess mRNA from a closely related cell population. The resulting cloned cDNAs will be analyzed by hybridization to RNA from various cell populations and by in situ hybridization to tissue. Clones corresponding to genes of particular interest will be expressed in E. coli and the resulting polypeptides will be used to generate antibodies for further protein and tissue characterization. A complementary approach will be to use selected antibodies obtained from other laboratories which show interesting patterns of localization during limb development to isolate the corresponding genes either by screening expression libraries or by immunoprecipitation of polysomes and subsequent cDNA construction. Both antibodies and DNA probes will then be used to identify and characterize previously unidentified genes angd gene products which are important in regulating early limb development.