REGULATION OF CD4 GENE EXPRESSION

描述：(改编自申请人摘要)本申请书为 旨在了解基因的转录调控 编码CD4。Cd4细胞表面分子是一种必需的 负责识别外周血淋巴细胞的受体成分 抗原，并介导T细胞激活的细胞内信号。它的 细胞表面的表达在细胞周期中以阶段特异性的方式被调节 T淋巴细胞的个体发育，并与CD8一起被用来定义 发育中的胸腺细胞的谱系关系。此外，它还可用作 结合人类免疫缺陷病毒的主要受体 (HIV)至CD4+T淋巴细胞和单核细胞。CD_4细胞表面分子 因此，在正常的生理免疫反应中是最重要的， 在HIV感染的情况下，它的异常相互作用可能是 对后天获得性肺炎的免疫缺陷部分负有责任 免疫缺陷综合征(艾滋病)。对法律规范的理解 CD4分子的表达将是关于T细胞的信息 淋巴细胞发育，并可能在设计治疗方法时有用 免疫缺陷疾病。一个75千碱基的染色体片段，包含 Cd4基因已被定位为编码外显子和转录起始。 网站，这项建议是针对了解结构和 顺式作用DNA序列和反式作用因子的活性 确定CD4基因表达的细胞类型和发育调控。 转录起始点上游的不同DNA片段将 连接到报告基因(荧光素酶)，转录活性将 通过测量荧光素酶活性来测量在转基因的CD4+T细胞中的表达。 将进一步分析显示活动的每一种不同结构 通过引物延伸，以确保转录被正确启动。 所有的转基因都将在CD4+/-T细胞和B细胞中复制。 似乎对阳性或阴性对照很重要的基因片段 将通过接头扫描突变分析来检查转录， 这些基因片段将被进一步分析，以确定是否存在 可能会特别结合的核因素。第二组实验 将针对定位更远的顺式作用区域，这些区域充当 增强剂和核基质联合区域(MARS)。大片区域 Cd4基因及其周围的序列将连接到无增强子 启动子的构建，并通过转基因进行转录检测 增强功能。实际的增强子元件将在转基因生物中进行测试 以确定所分离的序列是否足以指导 适当的组织和发育控制。CD4的差异性调节 和CD8在胸腺分化过程中的表达将通过 重组这两个基因的调控和结构元件。

DESCRIPTION: (Adapted from Applicant's Abstract) This application is directed at understanding the transcriptional regulation of the gene encoding CD4. The CD4 cell surface molecule serves as an essential receptor component responsible for the T lymphocyte recognition of foreign antigens, and mediates an intracellular signal for T-cell activation. Its cell surface expression is regulated in a stage-specific manner during the ontogeny of T lymphocytes, and, along with CD8, it is used to define the lineage relationships of developing thymocytes. In addition, it serves as the primary receptor for the binding of the human immunodeficiency virus (HIV) to CD4+ T lymphocytes and monocytes. The CD4 cell surface molecule is, thus, most important in the normal physiological immune response, and its abnormal interactions in the presence of HIV infections may be responsible, in part, for the immunodeficiency portion of acquired immunodeficiency syndrome (AIDS). An understanding of the regulation of expression of the CD4 molecule will be informative with respect to the T lymphocytes development, and may be useful in designing therapies for immunodeficiency diseases. A 75 kilobase chromosomal segment containing the CD4 gene has been mapped for coding exons and a transcriptional start site, and this proposal is directed at understanding the structure and activity of the cis-acting DNA sequences and trans-acting factors that determine the cell-type and developmental control of CD4 gene expression. Various segments of DNA upstream of the transcriptional start site will be ligated to a reporter gene (luciferase), and transcriptional activity will be measured in transfected CD4+ T-cells by measuring luciferase activity. Each of the difference constructs showing activity will be further analyzed by primer extension to insure that transcription is correctly initiated. All transfections will be reproduced in CD4+/- T-cells and in B-cells. Gene segments that appear to be important for positive or negative control of transcription will be examined by linker-scanning mutational analysis, and these gene segments will be further analyzed for the presence of nuclear factors that may specifically bind. A second set of experiments will be directed at locating more distant cis-acting regions that serve as enhancers and nuclear matrix association regions (MARS). Large regions of the CD4 gene and its surrounding sequence will be ligated into enhancerless promoter constructs, and assayed by transfection for transcription enhancement. The actual enhancer elements will be tested in transgenic mice to determine whether the isolated sequences are sufficient to direct appropriate tissue and development control. Differential regulation of CD4 and CD8 expression during thymic differentiation will be analyzed by recombining the regulatory and structural elements of the two genes.