STRUCTURE AND REGULATION OF LIVER ACUTE PHASE GENES

这一提议旨在检验这样一种假设，即 大鼠和兔血清中α2-巨球蛋白(A2M)浓度 C-反应蛋白(CRP)，发生在急性炎症中，是由于 相应基因转录的相应增加。这个 目的是建立一个研究炎症基因调控的系统 并鉴定其顺式作用调控序列。 这些基因。该项目包括五个阶段：1)准备一个小组 大鼠和兔肝cdna克隆用作核酸 杂交探针。该面板包括RAT A2M和补体成分 C3和C5，它们在结构上是相关的，但不是重大急性期 蛋白质。2)A2M和CRP的分离和部分鉴定 基因作为控制区DNA的来源用于转化 实验。大鼠A2M基因的序列分析。3)滴定 在实验性炎症过程中相应的肝脏RNA浓度。 试图区分转录和转录后转录 通过测量肝细胞核中的转录速率来控制机制 受刺激和未受刺激的动物。招聘新员工的可视化 肝切片原位杂交转录细胞。4) 大鼠短期和长期培养条件的研究进展 兔肝细胞可表达急性期mRNAs。一项努力将是 用血清因子确定培养物的处理条件 从受刺激的动物身上，为了实现转录的增加 急性期基因。长期培养已建立的大鼠肝细胞 将使用转化为对温度敏感的细胞系 DNA肿瘤病毒SV40的(TSA)突变体。肝脏特异性表达 通过改变培养温度，可以在这些细胞中诱导出基因 从33摄氏度到40摄氏度5)顺式作用控制的识别 参与炎症反应的A2M和CRP基因序列 监管。携带控制序列的检测载体的构建 一个急性期基因和一个可分析的标记。这些DNA的包装 序列化为腺病毒5颗粒。肝细胞培养的感染， 炎性刺激诱导和转录增加的检测 起源于急性期基因的控制序列。 重组DNA操作对控制序列的剖析 (删除、链接器插入、片段交换)。

This proposal is designed to test the hypothesis that the dramatic increase in serum concentrations of rat alpha2-macroglobulin (A2M) and rabbit C-reactive protein (CRP), which occurs in acute inflammation, is due to a corresponding increase in transcription from the respective genes. The goal is to characterize a system to study the inflammatory gene regulation in cultured liver cells and to identify the cis-acting control sequences of these genes. The project comprises five stages: 1) Preparation of a panel of rat and rabbit liver cDNA clones to be used as nucleic acid hybridization probes. The panel includes rat A2M and complement components C3 and C5, which are structurally related but are not major acute phase proteins. 2) Isolation and partial characterization of the A2M and CRP genes as source of control region DNA for use in transformation experiments. Sequence analysis of the rat A2M gene. 3) Titration of the corresponding liver RNA concentrations during experimental inflammations. Attempt to discriminate between transcriptional and post-transcriptional control mechanisms by measuring rates of transcription in liver nuclei from stimulated and non-stimulated animals. Visualization of recruitment of new cells for transcription by in-situ hybridization of liver sections. 4) Development of short-term and long-term culture conditions under which rat and rabbit hepatocytes can express acute phase mRNAs. An effort will be made to define conditions of treatment of the cultures with serum factors from stimulated animals, in order to achieve increased transcription of acute phase genes. For long-term cultures an established rat hepatocyte cell line will be used which is transformed with a temperature sensitive (tsA) mutant of the DNA-tumor-virus SV40. Expression of liver-specific genes can be induced in these cells by a shift in the culture temperature from 33 degrees C to 40 degrees C. 5) Identification of cis-acting control sequences of the A2M and CRP genes involved in the inflammatory regulation. Construction of test plasmids carrying control sequences from an acute phase gene and an assayable marker. Packaging of these DNA sequences into adenovirus 5 particles. Infection of liver cell cultures, induction with inflammatory stimuli and assay for increased transcription originating from the control sequences of the acute phase genes. Dissection of the control sequences by recombinant DNA manipulations (deletions, linker insertions, fragment exchange).