REGULATION OF CD4 GENE EXPRESSION

说明：（改编自申请人的摘要）本申请是 旨在了解基因的转录调控 编码CD 4。 CD 4细胞表面分子作为一种重要的 受体成分负责T淋巴细胞识别外源性 抗原，并介导T细胞活化的细胞内信号。 其 细胞表面的表达是以阶段特异性的方式调节的， T淋巴细胞的个体发生，并且沿着CD 8，它被用来定义 发育中胸腺细胞的谱系关系。 此外，它还作为 结合人类免疫缺陷病毒的主要受体 (HIV)CD 4 + T淋巴细胞和单核细胞 CD 4细胞表面分子 因此，在正常生理免疫应答中是最重要的， 在HIV感染的情况下，它的异常相互作用可能是 在某种程度上，艾滋病毒/艾滋病的免疫缺陷部分， 免疫缺陷综合征（艾滋病）。 对监管的理解 CD 4分子的表达将提供关于T细胞的信息。 淋巴细胞发育，并可能有助于设计治疗 免疫缺陷疾病。 一个含有75个组氨酸酶的染色体片段， CD 4基因已被定位为编码外显子和转录起始点 网站，本建议旨在了解结构和 顺式作用DNA序列和反式作用因子的活性， 确定CD 4基因表达的细胞类型和发育控制。 转录起始位点上游的各种DNA片段将被 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在转染的CD 4 + T细胞中测量。 将进一步分析显示活性的每个差异构建体 通过引物延伸以确保转录正确启动。 所有转染将在CD 4 +/-T细胞和B细胞中重现。 对阳性或阴性对照似乎重要的基因片段 将通过连接子扫描突变分析来检查转录， 这些基因片段将被进一步分析是否存在 核因子，可以特异性结合。 第二组实验 将针对定位更远的顺式作用区域， 增强子和核基质结合区（MARS）。 的大区域 将CD 4基因及其周围序列连接到无增强子的 启动子构建体，并通过转染测定转录 增强 实际的增强子元件将在转基因小鼠中进行测试。 小鼠以确定分离的序列是否足以指导 适当的组织和发育控制。 CD 4的差异调节 胸腺分化过程中的CD 8和CD 8表达将通过 重组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.