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.

在过去的几年里，主要的转录调控组织相容性复合体 (MHC) II 类基因已被证明可产生来自 5' 侧翼顺式作用调控元件的相互作用 II类基因的内含子区域，以及一系列的交易可以与这些顺式作用元件结合的因子。五个主题显然参与转录调控的保守序列已在 5' 侧翼区域的启动子区域内描绘 II 类基因，这些基因被表示为 Z、X 和 Y 框，八聚体和 TATA 盒。 X 和 Y 框似乎对于高效转录。我们开发了一种基于以下方法的方法西南技术，其中用 32P- 探测 lambda gt11 文库末端标记的双链寡核苷酸对应于监管主题。我们已经分离并测序了 cDNA 编码一种称为 YB-1 的 Y 盒结合蛋白，并显示出 YB-1 的特异性 1 为 Y 框。在此应用中，我们首先提出表征 YB- 1 关于绑定和功能参数。身体条件结合将通过我们的西南测定法进行测定，并且核苷酸结合的关键将由西南DNA酶保护进行测定，和甲基化干扰测定。 γ-干扰素的诱导能力 YB-1 mRNA 和蛋白质将分别由 Northern 评估分析，以及用特异性抗体对 YB-1 进行免疫沉淀。 YB- 1 将cDNA转染至诱导型U937细胞中，测试其能力 YB-1 本身，然后与其他结合因子一起诱导 II 类表达。 YB-1基因组基因将被克隆和测序，特别注意 5' 侧翼区域可能涉及 YB-1 本身的转录调控。 cDNA编码蛋白质与其他调控基序（X 盒、Z 盒和八聚体）的结合将通过西南技术分离并测序。绑定蛋白质将被表征，基因组基因将被克隆。这些实验应阐明蛋白质与 II类基因的调控区在II类表达中发挥作用。