TRANSCRIPTIONAL REGULATION OF TYPE I COLLAGEN GENES BY C

I型胶原，由两个（α（I）和一个（α（II））组成的异源三聚体， α 2（1）多肽是在皮肤，骨骼， 的筋1型胶原蛋白合成的变化发生在一些 的病理条件。为了了解这种疾病的分子机制， I型胶原基因表达的调控，几种顺式作用元件 和它们的同源DNA结合蛋白已经在 α 1（I）和α 2（I）胶原基因启动子。一个关键的交易 因子是一种CCAAT结合因子（CBF），可激活体外转录 α（I）和α（I）胶原蛋白促进剂以及其它 发起人。 CBF是一种多聚体蛋白，由三种不同的多肽组成， CBF-A、CBF-B和CBF-C，所有这些都是DNA结合所需的。的cDNA 已经分离出CBF-A、CBF-B和CBF-C多肽的克隆。这些 由它们的cDNA表达的三个重组CBF亚基一起形成 CBF-DNA复合物，并且所有三个CBF亚基都存在于DNA中。 蛋白质复合物CBF-A和CBF-B部分具有高度的氨基化， HAP 3和HAP 2亚基的区段的氨基酸序列同一性。 酵母多聚体转录因子在拟议的研究中，重组 CBF亚单位将用于分析不同的步骤，协会的 三个亚基形成CBF蛋白。重组CBF 将用于鉴定CBF结合的共有DNA序列， PCR介导的随机位点选择法。 CBF的联系人 DNA中的分子将通过各种DNA-蛋白质相互作用测定来揭示 以确定CBF是否与DNA的小沟或大沟相互作用。 将使用光交联方法来交联CBF亚基， 目的：探讨CBF亚基间特异性DNA接触的可能性。一 将构建CBF依赖性启动子以确定是否 CBF对启动子的转录激活依赖于 CBF结合位点相对于 塔塔盒子。抑制DNA的CBF亚基的显性负突变体 野生型CBF的结合将在成纤维细胞中表达， 四环素反应性启动子的严格控制， 内源性CBF蛋白。在这项研究中，CBF在转录中的作用， I型胶原基因和成纤维细胞生长的激活 将被确定。

Type I collagen, a heterotrimer consisting of two(alpha(l) and one alpha2(l) polypeptides, is the most abundant protein found in skin, bone, and tendons. Changes in the synthesis of type 1 collagen occur in a number of pathological conditions. To understand the molecular mechanism of the regulation of type I collagen gene expression, several cis-acting elements and their cognate DNA-binding proteins have been identified in the alphal(l) and alpha2(l) collagen gene promoters. One critical transacting factor is a CCAAT binding factor (CBF) that activates in vitro transcript ion of both the alpha(l) and alpha(l) collagen promoters as well as other promoters. CBF is a multimeric protein and consists of three different polypeptides, CBF-A, CBF-B, and CBF-C, all which are needed for DNA binding. The cDNA clones for CBF-A, CBF-B, and CBF-C polypeptides have been isolated. These three recombinant CBF subunits, expressed from their cDNAs, together form a CBF-DNA complex, and all three CBF subunits are present in the DNA- protein complex. Portions of CBF-A and CBF-B have a high degree of amino acid sequence identity to segments of the HAP3 and HAP2 subunits of a yeast multimeric transcription factor. In the proposed study, recombinant CBF subunits will be used to analyze different steps of association of the three subunits for formation of the CBF protein. The reconstituted CBF will be used to identify consensus DNA sequences for CBF binding by the PCR-mediated random site selection method. The contact of the CBF molecule in DNA will be revealed by various DNA-protein interaction assays to determine whether CBF interacts with the minor or major groove of DNA. A photo-crosslinking method will be used to crosslink the CBF subunits to DNA and to identify the specific DNA contact in the subunits of CBF. A CBF-dependent promoter will be constructed to determine whether transcriptional activation of the promoter by CBF is dependant upon distances and orientations of the CBF-binding site with respect to the TATA box. Dominant negative mutants of the CBF subunits that inhibit DNA binding of the wild-type CBF will be expressed in fibroblast cells under a tight control by a tetracycline-responsive promoter to inactivate the endogenous CBF protein. in this study, the role of CBF in transcription activation of both the type I collagen genes and in fibroblast cell growth will be determined.