REGULATION OF COLLAGEN EXPRESSION IN CHONDROCYTES

拟议的研究旨在识别DNA序列，两者都在 围绕着I型和II型鸡胶原基因，这两个基因是 参与对这些基因的正向或负向调控 分化的成纤维细胞和软骨细胞。 I型和II型胶原基因的甲基化状态将是 使用改进的凝胶转移程序重新研究，这将允许 探测到非常小的碎片。染色质结构的变化，被视为 DNase I超敏部位，将在I型和II型中寻找 成纤维细胞和软骨细胞中的胶原基因及其侧翼基因区域 确定变化，如果它们发生，是否与 表情。鉴定与成纤维细胞调控相关的序列 软骨细胞将被瞬时转化为含有来自 2000~小于100个碱基的PRO基因5‘侧翼基因序列 α1(I)、前α2(I)和前α1(II)胶原基因位于5‘- 在蛋白质和RNA水平上可以很容易地测试其表达的基因， 如SV40大T抗原和小T抗原。类似的瞬时表达 实验将使用胶原蛋白模拟物进行，其中5‘ 侧翼区域和前三个或更多外显子连接到部分 最后一个内含子，3‘最大外显子和3’侧翼基因区 基因组区域和/或3‘侧翼区域参与调控。 一旦确定了调控序列，使用它们可能是可行的 隔离涉及监管的交易因素。显然，这一失败 适当调控I型精确定时顺序表达 II型胶原基因会导致肢体发育异常。

The proposed research is directed at identifying DNA sequences, both within and surrounding the type I and type II chicken collagen genes, which are involved in either positive or negative regulation of these genes in differentiated fibroblasts and chondrocytes. The state of methylation of both type I and type II collagen genes will be reinvestigated using a modified gel transfer procedure which will permit detection of very small fragments. Changes in chromatin structure, seen as DNAse I hypersensitive sites, will be looked for in type I and type II collagen genes and flanking gene regions in both fibroblast and chondrocyte nuclei to determine whether changes, if they occur, are associated with expression. To identify sequences involved in regulation fibroblasts and chondrocytes will be transiently transformed with plasmids containing from 2000 to less than 100 bp of 5' flanking gene sequences of the pro Alpha1(I), pro Alpha2(I) and pro Alpha1(II) collagen genes placed 5' to genes whose expression can be readily tested at the protein and RNA level, such as SV40 large T and small t antigens. Analogous transient expression experiments will be carried out using collagen mimigenes in which the 5' flanking region and first three or more exons are ligated to part of the last intron, 3' most exon and 3' flanking gene region to determine if genomic regions and/or 3' flanking regions are involved in regulation. Once regulatory sequences are identified, it may be feasible to use them to isolate transacting factors involved in regulation. Obviously the failure to properly regulate the precisely timed sequential expression of type I and type II collagen genes would result in abnormal limb development.