TRANSCRIPTIONAL REGULATION OF TYPE I COLLAGEN GENES BY C

I型胶原，由两个(L)和一个 α2(L)多肽，是在皮肤、骨骼、 还有肌腱。I型胶原蛋白的合成发生了许多变化 病态的疾病。为了了解致病的分子机制 几种顺式作用元件对I型胶原基因表达的调控 和它们的同源DNA结合蛋白已经在 α(L)和α2(L)胶原基因启动子。一次关键的交易 因子是一种CCAAT结合因子(CBF)，可在体外激活转录本 α(L)和α(L)胶原启动子以及其他 推动者。 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，它们都是DNA结合所必需的。CDNA3 已分离出CBF-A、CBF-B和CBF-C多肽的克隆。这些 三个重组的CBF亚基，由它们的cDNA表达，一起形成 CBF-DNA复合体和所有三个CBF亚基都存在于DNA中- 蛋白质复合体。CBF-A和CBF-B的一部分具有高度的氨基 猪瘟病毒HAP3和HAP2亚基片段的酸序列同源性 酵母多聚体转录因子。在拟议的研究中，重组 CBF亚单位将被用来分析不同步骤的关联 形成CBF蛋白的三个亚基。重组的CBF 将用于确定CBF结合的共识DNA序列 聚合酶链式反应介导的随机选点方法。CBF的联系方式 DNA中的分子将通过各种DNA-蛋白质相互作用分析来揭示 以确定CBF是否与DNA的小沟或大沟相互作用。 将使用光交联法将CBF亚基交联到 DNA，并鉴定CBF亚基中的特异性DNA接触。一个 CBF依赖的启动子将被构建来确定 CBF对启动子的转录激活依赖于 CBF结合位点相对于CBF的距离和方向 塔塔箱。抑制DNA的CBF亚基的显性负性突变 野生型CBF的结合将在成纤维细胞中表达 通过四环素反应启动子的严格控制来灭活 内源性CBF蛋白。在本研究中，CBF在转录中的作用 I型胶原基因的激活与成纤维细胞的生长 将会被确定。

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.