IFN-Y RESISTANT MUTANTS OF MAMMALIAN CELLS

人宫颈癌ME-180细胞系的一系列突变体 已经分离出对干扰素-γ的细胞毒性作用具有抵抗力的药物。 这种突变体被发现在吲哚胺2，3-双加氧酶中存在缺陷 (IDO)诱导，色氨酸分解代谢途径的第一种酶。 第二轮突变体也被分离出来，完全缺乏IDO 并在其他干扰素诱导的反应中也受到影响，如 作为人类白细胞抗原和寡聚A合成酶的诱导。IDO之间的关系 色氨酸的诱导、运输和代谢将在 以确定这一途径的生理意义。使用 我们将研究IDO突变体是否是点突变 突变或由使用凝胶的调节序列中的突变引起的- 发育迟缓分析。北方斑点和南方分析将与 调查这类突变是否是由缺失导致的 或信号通路的改变。受体结合研究使用 还将进行I125干扰素治疗。 IDO基因组DNA中干扰素反应序列的结构将是 调查过了。干扰素-γ对该基因的调节将是 使用突变和野生型细胞进行了研究。最后是一个遗传分析 将对这些突变体进行定位，以确定影响他们的基因(S) 以确定这些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.