STRUCTURE AND REGULATION OF LIVER ACUTE PHASE GENES

这一提议旨在检验一个假设，即 在大鼠α 2-巨球蛋白（A2 M）和家兔血清浓度中， C-反应蛋白（CRP），发生在急性炎症，是由于一个 相应的基因转录增加。 的 目的是描述一个系统，研究炎症基因调控 在培养的肝细胞，并确定顺式作用的控制序列 这些基因。 该项目包括五个阶段：1）筹备小组 大鼠和兔肝cDNA克隆用作核酸 杂交探针 该组包括大鼠A2 M和补体组分 C3和C5，结构相关，但不是主要急性期 proteins. 2)A2 M和CRP的分离和部分表征 作为用于转化的控制区DNA来源的基因 实验 大鼠A2 M基因的序列分析。 3)滴定 在实验性炎症期间相应的肝脏RNA浓度。 尝试区分转录和转录后 控制机制，通过测量转录率在肝细胞核， 刺激和非刺激的动物。 征聘新工作人员的设想 通过肝切片的原位杂交进行转录。 四、 短期和长期培养条件的开发， 兔肝细胞可表达急性期mRNA。 将努力 用于确定用血清因子处理培养物的条件 从受刺激的动物，以实现增加的转录 急性期基因 对于长期培养， 将使用用温度敏感的 (tsA)DNA肿瘤病毒SV 40的突变体。 肝脏特异性表达 通过改变培养温度， 从33摄氏度到40摄氏度 5)顺式作用对照的鉴定 参与炎症反应的A2 M和CRP基因的序列 调控 构建携带对照序列的测试质粒 急性期基因和可检测标记。 这些DNA的包装 腺病毒5颗粒中。 感染肝细胞培养物， 用炎性刺激物诱导和增加转录的测定 源自急性期基因的控制序列。 通过重组DNA操作剖析控制序列 （缺失、接头插入、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).