REGULATION OF COLLAGEN EXPRESSION IN CHONDROCYTES

拟议的研究旨在确定DNA序列，无论是在 以及I型和II型鸡胶原蛋白基因， 参与这些基因的正或负调控， 分化成纤维细胞和软骨细胞。 I型和II型胶原蛋白基因的甲基化状态将是 使用改良的凝胶转移程序重新研究， 检测非常小的碎片。 染色质结构的变化，如 DNA酶I超敏位点，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和侧翼基因区域 核，以确定是否发生变化，如果它们发生， 表情 为了鉴定参与调节成纤维细胞的序列， 软骨细胞将用含有来自 2000至小于100 bp的pro的5'侧翼基因序列 α 1（I）、pro α 2（I）和pro α 1（II）胶原基因位于5'端至 其表达可容易地在蛋白质和RNA水平上测试的基因， 如SV 40大T和小T抗原。 类似瞬时表达 将使用胶原模拟基因进行实验， 侧翼区和前三个或更多个外显子连接到部分的 最后一个内含子、3 ′最大外显子和3 ′侧翼基因区域，以确定是否 基因组区域和/或3 ′侧翼区域参与调控。 一旦鉴定了调控序列，就可以用它们来 隔离参与调节的交易因素。 显然，失败 为了正确地调节I型的精确定时的顺序表达， 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.