MOLECULAR BASIS OF NORMAL AND MUTANT LIMB DEVELOPMENT

研究的总体目标是了解发展中的 发生在鸟类胚胎肢体中的过程。 在这个系统中，我们 建议检查软骨细胞的合成活性， 正常胚胎软骨细胞外基质的建立 以及由于基因损伤导致软骨异常的胚胎。 我们将研究在分化过程中的转录活性 软骨细胞，在软骨细胞中，其分化在 原分化的状态，并在那些导致经历 “去分化”。 我们还将研究染色质的变化 结构伴随着转录活性的变化。 这些 将使用四个含有编码区的cDNA探针进行测量 对于软骨特异性蛋白质（II型胶原，54kDa基质蛋白， 软骨蛋白聚糖核心蛋白和连接蛋白）和两个cDNA探针 含有非软骨蛋白（I型α 1和 Alpha2）。 利用蛋白聚糖核心蛋白cDNA探针，我们将确定 软骨突变体中核心蛋白缺陷的遗传基础， 侏儒症 我们将研究结构和功能之间的关系， 组成连接稳定的蛋白聚糖聚集体的组分 通过产生单克隆抗体， 蛋白聚糖核心蛋白与连接蛋白相互作用， 透明质酸和那些在与核心蛋白相互作用连接蛋白上的 和透明质酸。 单克隆抗体也将用于 鉴定由转化有以下物质的细菌合成的多肽 表达含有cDNA插入片段的质粒，所述插入片段编码不同的 核心蛋白和连接蛋白的区域。 以这种方式， 蛋白质的结构域将被识别，它们的物理极限将 通过绘制表达cDNA插入片段的范围来确定。

The overall goal of the research is to understand the developmental processes which take place in the avian embryonic limb. In this system we propose to examine the synthetic activity of the chondrocytes during the establishment of the extracellular matrix of cartilage by normal embryos and by embryos whose cartilage is abnormal owing to a genetic lesion. We will investigate the transcriptional activity in differentiating chondrocytes, in chondrocytes whose differentiation is arrested in the protodifferentiated state, and in those that are caused to undergo "de-differentiation". We will also investigate changes in chromatin structure which accompany changes in transcriptional activity. These measurements will be done using four cDNA probes containing coding regions for cartilage-specific proteins (Type II collagen, 54 k Da matrix protein, cartilage proteoglycan core protein, and link protein) and two cDNA probes containing coding regions for non-cartilaginous proteins (Type I Alpha1 and Alpha2). With the proteoglycan core protein cDNA probes we will determine the genetic basis for the core protein defect in the cartilage mutant, nanomelia. We will study the relationship between structure and function of the components which make up the link stabilized proteoglycan aggregates of cartilage by generating monoclonal antibodies which can identify the sites on proteoglycan core protein which interact with link protein and hyaluronic acid, and those on link protein which interact with core protein and hyaluronic acid. The monoclonal antibodies will also be used to identify polypeptides synthesized by bacteria which were transformed with expressing plasmids containing cDNA inserts which code for different regions of core protein and link protein. In this manner the functional domains of the proteins will be identified and their physical limits will be determined by mapping the extent of the expressing cDNA inserts.