INDUCTION OF A PROTHROMBINASE GENE DURING HEPATITIS

这项拨款建议研究从生物学上诱导一种 重要的炎症介质，巨噬细胞/单核细胞促凝剂(a 凝血酶原酶)。这种分子是一种重要的介体。 小鼠肝炎病毒(MHV)引起的肝脏病变的发病机制 感染。代表MHV诱导的促凝血基因的cDNA将被 合成、克隆和测序。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整的可能性 促凝血剂前体蛋白的激活也将被研究。 将分离MHV诱导的促凝血剂基因的基因组克隆。 这些将通过Southern印迹来表征它们的组织 杂交和S1核酸酶作图以及测序。数列 促凝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能力。删除 将进行启动子区域的分析以确定 不同的序列基序对促凝剂启动子的贡献， 以及它们被MHV感染的诱导性。DNA足迹将 进一步定义该基因中存在的顺式作用调控元件。 研究确定哪些MHV基因对诱发MHV 促凝血剂将被启动。代表个体的分子克隆 MHV基因在巨噬细胞中的表达及其对促凝剂的影响 活动已确定。与可能的促凝剂结合的MHV基因产物 将通过DNA结合分析来寻找调控因素。

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.