GENETICS AND BIOCHEMISTRY OF VIRAL LEUKEMOGENESIS

这项拟议研究的总体目标是确定 Moloney鼠白血病病毒转录增强子调节 病毒在淋巴和髓系造血细胞中的表达，以及 有助于T细胞特异性转化。离体 对Moloney增强子的表征已经确定了一组复杂的 核因子结合部位。我们最近的基因分析使我们能够 为了确定其中两个站点，直播站点和邻近的核心元素为 Moloney胸腺疾病特异性的重要决定因素 病毒。我们现在计划利用分子遗传学和生物化学来进一步 描述与直播/核心区相关的核蛋白 莫洛尼增强剂的。我们还计划确定莫洛尼增强剂 调节髓系细胞中病毒基因表达的序列。 我们的具体目标是： 1.鉴定与直播/核心相结合的核蛋白 莫洛尼增强剂中的元素。 微亲和DNA沉淀法将用于鉴定和 区分无处不在的、组织特异性的和TPA诱导的核 Moloney增强器的直播/核心区的绑定因子。 2.获得并鉴定编码T细胞特异性c DNA的克隆 核心结合蛋白。 将对从T细胞制备的cDNA文库进行核心结合筛选 通过2.1-2.3分离克隆中列出的方法之一获得的因子将 对DNA结合的特异性进行表征、测序和分析 组织特异性表达。 3.鉴定介导转录的增强子序列 髓系细胞中的Moloney增强剂。 一组Moloney增强子突变体，每个都包含一个点突变 将分析增强子上的核因子结合位点之一 在髓系白血病细胞系中的转录活性 通过瞬时CAT检测，细胞分化进入进行性阶段。

The overall goal of the proposed research is to determine how the transcriptional enhancer of the Moloney murine leukemia virus regulates the expression of the virus in lymphoid and myeloid hematopoietic cells, and contributes to the T cell specificity of transformation. In vitro characterization of the Moloney enhancer has identified a complex array of nuclear factor binding sites. Our recent genetic analysis has enabled us to identify two of these sites, the LVb site and adjacent core element as important determinants of the thymic disease specificity of the Moloney virus. We now plan to use molecular genetics and biochemistry to further characterize the nuclear proteins that associate with the LVb/core region of the Moloney enhancer. We also plan to identify Moloney enhancer sequences that regulate viral gene expression in myeloid cells. Our specific aims are: 1. To identify and characterize nuclear proteins that bind to the LVb/core element in the Moloney enhancer. Microaffinity DNA precipitation assays will be used to identify and distinguish between ubiquitous, tissue specific, and TPA inducible nuclear factors that bind to the LVb/core region of the Moloney enhancer. 2. To obtain and characterize T cell specific cDNA clones that encode the core-binding protein. cDNA libraries prepared from T cells will be screened for core binding factors by one of the methods listed in 2.1 - 2.3 isolated cDNA clones will be characterized for DNA binding specificity, sequenced, and analyzed for tissue-specific expression. 3. To identify enhancer sequences that mediate transcription from the Moloney enhancer in myeloid cells. A panel of Moloney enhancer mutants, each containing a point mutation in one of the nuclear factor binding sites on the enhancer, will be analyzed for transcriptional activity in myeloid leukemia cell lines representing progressive stages of differentiation, by transient CAT assays.