CHARACTERIZATION OF GENES IMPORTANT IN LIMB DEVELOPMENT

本项目的长期目标是了解 参与不同细胞类型的分化， 发展肢体。 肢体的分子生物学研究进展 发展强调的结构和监管的研究 在肢体发育过程中表达的表型基因， 软骨（蛋白聚糖核心蛋白和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。 一种结合单克隆抗体技术和 重组DNA技术将被用于鉴定和表征基因 和基因产物的重要性，在早期细胞的多样化， 发展 互补DNA文库对应于 将构建早期肢体中的特定细胞群。 单个 用于构建这些文库的单链cDNA（第一链）将被 选择缺乏杂交过量的mRNA从密切相关的 细胞群 所得克隆的cDNA将通过 与来自各种细胞群体的RNA杂交， 与组织杂交。 对应于特定基因的克隆 将在E中表达兴趣。大肠杆菌中，并且所得多肽将 用于产生抗体， 特征化 一种补充方法是使用选定的 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 多核糖体和随后的cDNA构建。 然后，抗体和DNA探针将用于识别和 表征先前未鉴定的基因和基因产物， 在调节早期肢体发育中起重要作用。

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.