INDUCTION OF A PROTHROMBINASE GENE DURING HEPATITIS

该基金建议研究生物学诱导的机制， 一种重要的炎症介质，巨噬细胞/单核细胞促凝剂（ 凝血酶原酶）。 这种分子是一种重要的介体， 小鼠肝炎病毒（MHV）引起肝损害的发病机制 感染 代表MHV诱导的促凝血基因的cDNA将被 合成克隆测序 这些cDNA克隆将用于 直接确定促凝血基因的上调是否发生在 转录水平。 翻译上调和翻译后调节的可能性 还将研究促凝血前体蛋白的活化。 将分离MHV诱导的促凝血基因的基因组克隆。 这些将是和特点，因为他们的组织由南方印迹 杂交和S1核酸酶作图以及测序。 序列 在促凝血剂编码序列的上游将被表征为 它们作为该基因的启动子元件的功能。 缺失 将进行启动子区的分析以确定启动子区中的启动子。 不同序列基序对促凝血启动子的贡献， 以及MHV感染对它们的诱导作用。 DNA足迹将 进一步定义了该基因中存在的顺式作用调控元件。 确定哪些MHV基因对诱导 启动促凝血剂。 代表个体的分子克隆 MHV基因将在巨噬细胞中表达及其对促凝血功能的影响 活动确定。 与推定的促凝血剂结合的MHV基因产物 将通过DNA结合测定来寻找调节元件。

This grant proposes to study the mechanism of induction of a biologically important inflammatory mediator, a macrophage/monocyte procoagulant (a prothrombinase). This molecule is an important mediator in the pathogenesis of hepatic lesions produced during mouse hepatitis virus (MHV) infection. cDNAs representing the MHV-induced procoagulant gene will be synthesized, cloned, and sequenced. These cDNA clones will be used to directly determine if up-regulation of the procoagulant gene occurs at the level of transcription. The possibilities of translational up-regulation and post-translational activation of a procoagulant precursor protein will also be investigated. Genomic clones of the MHV-induced procoagulant gene will be isolated. These will be and characterized as to their organization by Southern blot hybridization and S1 nuclease mapping as well as sequenced. Sequences upstream from the procoagulant coding sequences will be characterized as to their ability to function as promoter element for this gene. A deletion analysis of the promoter region will be performed to determine the contribution different sequence motifs make to the procoagulant promoter, and as to their inducibility by MHV infection. DNA footprinting will further define cis-acting regulatory elements present in this gene. Studies to determine which MHV genes are important to the induction of procoagulant will be initiated. Molecular clones representing individual MHV genes will be expressed in macrophages and their effect on procoagulant activity determined. MHV gene products which bind to putative procoagulant regulatory elements will be sought through DNA binding assays.