MOLECULAR BASIS OF NORMAL AND MUTANT LIMB DEVELOPMENT

这项研究的总体目标是了解儿童的发展 发生在鸟类胚胎肢体上的过程。在这个系统中，我们 建议检测关节软骨细胞的合成活性 利用正常胚胎建立软骨细胞外基质 以及由于遗传损伤导致软骨异常的胚胎。 我们将研究分化过程中的转录活性。 软骨细胞，软骨细胞的分化受阻于 原分化状态，以及在那些被导致经历 “去分化”。我们还将研究染色质的变化 伴随转录活性变化的结构。这些 将使用四个包含编码区的cdna探针进行测量 对于软骨特异性蛋白(II型胶原，54k Da基质蛋白， 软骨蛋白多糖核心蛋白和连接蛋白)和两个cdna探针。 含有非软骨蛋白的编码区(I型α1和 字母2)。利用蛋白多糖核心蛋白cDNAs探针，我们将确定 软骨突变中核心蛋白缺陷的遗传基础， 奈米莉亚。 我们将研究结构和功能之间的关系 组成连接的成分稳定的蛋白多糖聚合体 通过产生可以识别位置的单抗来识别软骨 与连接蛋白相互作用的蛋白多糖核心蛋白的研究 透明质酸，以及连接蛋白上与核心蛋白相互作用的那些 和透明质酸。这些单抗也将被用于 转化细菌合成的多肽的鉴定 含有编码不同基因的cDNAs的表达载体 核心蛋白和连接蛋白的区域。以这种方式，功能 蛋白质的结构域将被识别，它们的物理限制将被 通过定位表达的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.