GENETICS AND BIOCHEMISTRY OF VIRAL LEUKEMOGENESIS

拟议研究的总体目标是确定 莫洛尼鼠白血病病毒的转录增强子调节 病毒在淋巴和骨髓造血细胞中的表达，和 有助于转化的T细胞特异性。 体外 Moloney增强子的表征已经确定了一系列复杂的 核因子结合位点。 我们最近的基因分析使我们能够 为了确定这些位点中的两个，即LVb位点和相邻的核心元件， 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， 表征与LVb/核心区域相关的核蛋白 莫洛尼增强剂 我们还计划鉴定莫洛尼增强剂 在骨髓细胞中调节病毒基因表达的序列。 我们的具体目标是： 1. 鉴定和表征与LVb/核心结合的核蛋白 莫洛尼增强剂中的元素。 微亲和DNA沉淀试验将用于鉴定和 区分普遍存在的、组织特异性的和TPA诱导的核 与莫洛尼增强子的LVb/核心区域结合的因子。 2. 为了获得和表征编码T细胞特异性cDNA克隆， 核心结合蛋白 从T细胞制备的cDNA文库将筛选核心结合 通过2.1 - 2.3中列出的方法之一分离的cDNA克隆， 表征DNA结合特异性，测序并分析 组织特异性表达。 3. 为了鉴定介导转录的增强子序列， 骨髓细胞中的莫洛尼增强子。 一组莫洛尼增强子突变体，每一个都含有一个点突变， 将分析增强子上的核因子结合位点之一 对于髓性白血病细胞系中的转录活性， 进行性分化阶段，通过瞬时CAT测定。

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.