ISOLATION OF CDNA'S FOR HLA CLASS II TRANSACTING FACTORS

HLA II 类交易因子的 CDNA 分离

基本信息

批准号：
3141629
负责人：
BENJAMIN D SCHWARTZ
金额：
$ 15.53万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-01-01 至 1993-12-31
项目状态：
已结题

项目摘要

Over the past several years, the transcriptional regulation of major histocompatibility complex (MHC) class II genes has been shown to result from the interaction of cis-acting regulatory elements in the 5' flanking and intronic regions of the class II genes, and a series of transacting factors which can bind to these cis-acting elements. Five motifs of conserved sequence apparently involved in transcriptional regulation have been delineated within the promoter region of the 5' flanking region of class II genes, and these have been denoted the Z, X, and Y boxes, the octamer and the TATA box. The X and Y boxes appear to be critical for efficient transcription. We have developed a methodology based on the Southwestern technique in which a lambda gt11 library is probed with 32P- end labeled double-stranded oligonucleotides corresponding to the regulatory motifs. We have already isolated and sequenced a cDNA encoding a Y box binding protein termed YB-1, and have shown the specificity of YB- 1 for the Y box. In this application, we first propose to characterize YB- 1 with respect to binding and functional parameters. Physical conditions of binding will be assayed by our Southwestern assay, and nucleotides critical to binding will be assayed by the Southwestern, DNAase protection, and methylation interference assays. The inducibility by gamma-interferon of YB-1 mRNA and protein will be assessed respectively by Northern analysis, and by immunoprecipitation of YB-1 with specific antibodies. YB- 1 cDNA will be transfected into inducible U937 cells to test the ability of YB-1 by itself, and later with other binding factors to induce class II expression. The YB-1 genomic gene will be cloned and sequenced, with particular attention to 5' flanking regions potentially involved in the transcriptional regulation of YB-1 itself. cDNA's encoding proteins binding to the other regulatory motifs (the X box, Z box,and octamer) will be isolated by the Southwestern technique and sequenced. The binding proteins will be characterized, and the genomic genes cloned. These experiments should elucidate the mechanism by which proteins binding to the regulatory region of class II genes play a role in class II expression.

在过去的几年中，主要的转录调节组织相容性复合物（MHC）II类基因已显示出导致从5'侧面的顺式作用调节元件的相互作用 II类基因的内含子区域以及一系列交易可以与这些顺式作用元件结合的因素。五个主题保守的序列显然参与了转录调节被描绘在5'侧面区域的启动子区域内第二类基因，这些基因已表示为z，x和y盒， Octamer和Tata盒子。 X和Y盒似乎对有效的转录。我们已经开发了一种基于西南技术，其中lambda GT11库用32p-探测最终标记的双链寡核苷酸对应于监管图案。我们已经分离并测序了cDNA编码 y盒结合蛋白称为yb-1，并显示了yb-的特异性 y盒子1。在此应用中，我们首先建议表征YB- 1相对于结合和功能参数。身体状况结合将通过我们的西南测定法和核苷酸分析对结合至关重要的西南DNAase保护将测定和甲基化干扰测定。伽马互换的诱导性 YB-1 mRNA和蛋白质的含量将分别由北部评估分析，并通过特异性抗体对YB-1进行免疫沉淀。 yb- 1个cDNA将转染到诱导型U937细胞中，以测试 YB-1本身，后来与其他结合因子诱导II类表达。 YB-1基因组基因将被克隆和测序，并用特别注意5'的侧翼区域可能涉及 YB-1本身的转录调节。 cDNA的编码蛋白质与其他调节图案（X框，Z框和八点）结合将通过西南技术隔离并进行了测序。结合蛋白质将被表征，并克隆基因组基因。这些实验应阐明蛋白质结合的机制 II类基因的调节区域在II类表达中起作用。