IFN-Y RESISTANT MUTANTS OF MAMMALIAN CELLS

人宫颈癌ME-180细胞系的一系列突变体 已经分离出抗干扰素-γ的细胞毒性作用的细胞。 已发现这类突变体在吲哚胺2，3-双加氧酶中有缺陷 (IDO)诱导，色氨酸分解代谢途径的第一个酶。 第二轮突变体也已分离出完全缺乏IDO 诱导和影响也在其他干扰素诱导的反应，如 如HLA和oligo A合成酶诱导。 IDO之间的关系 诱导、色氨酸转运和代谢将在 以确定这一途径的生理意义。 使用 克隆的cDNA，我们将研究IDO突变体是否是点 突变或由调节序列中的突变产生，使用凝胶电泳， 阻滞测定。 北方印迹和南方印迹分析将使用 “全局”突变体，以研究此类突变体是否由缺失引起 或信号通路的改变。 受体结合研究，使用 I125干扰素也将完成。 IDO基因组DNA中的干扰素应答序列的结构将被分析。 研究了 干扰素γ对该基因的调控将是 使用突变体和野生型细胞进行研究。 最后是基因分析 将进行突变体的定位基因（S）影响到他们的 相应的染色体，并确定突变是否是等位基因。

A series of mutants of the ME-180 (human cervical carcinoma) cell line resistant to the cytotoxic effects of interferon-gamma have been isolated. Such mutants have been found to be defective in indoleamine 2,3-dioxygenase (IDO) induction, the first enzyme of the tryptophan catabolic pathway. Second round mutants have also been isolated lacking completely IDO induction and affected also in other interferon-inducible responses, such as HLA and oligo A synthetase induction. The relationship between IDO induction, tryptophan transport and metabolism will be investigated in order to ascertain the physiological significance of this pathway. Using a cloned cDNA we shall investigate whether the IDO-mutants are point mutations or result from mutation in regulatory sequences using gel- retardation assays. Northern blot and Southern analyses will be done with "global" mutants to investigate whether such mutants result from deletions or alterations in the signalling pathway. Receptor binding studies using I125 interferon will also be done. The structure of interferon responsive sequences in IDO genomic DNA will be investigated. The regulation of this gene by interferon-gamma will be investigated using mutant and wild-type cells. Finally a genetic analysis of the mutants will be undertaken to locate the gene(s) affected to their respective chromosomes, and to ascertain whether the mutations are allelic.