ADENOVIRUS GENOME EXPRESSION: PHYSICAL MAPPING STUDIES

这项拟议的研究旨在阐明腺病毒dna的作用。 基因表达调控、DNA包埋和基因调控中的序列 进化多样化。最近描述的自动调节由 腺病毒E1a基因为研究提供了一个模型系统。E1a 启动子序列，或衍生变种，已经连接到探测基因 例如氯霉素乙酰转移酶，以方便地测量程度 在培养的转基因人类细胞中的基因表达。的影响 共转染野生型和突变型E1a基因对E1a-探针表达的影响 是很容易确定的。有证据表明消极的自我压抑和两个 E1a自动调节中的正反馈模式。这些化验结果将是 用于定位启动子对E1a和功能性反应的顺式靶点 E1a多肽中的结构域(S)。指定极性DNA的顺式元件 包膜位于E1a的转录控制区内 基因，这将得到进一步的表征。 腺病毒E1a是两个腺病毒基因之一，参与 腺病毒诱导细胞致癌转化的过程。这个 腺病毒的致瘤性也与E1a基因有关，在 随人腺病毒特定亚群的不同而变化的方式 血清型。这提高了人们对监管属性的兴趣，并 腺病毒E1a基因的进化。 人腺病毒B亚群，中等致癌性，由以下组成 两个关系密切但在流行病学和病原学上截然不同的家族 或者说子组。B1型病毒可引起严重的呼吸道疫情 疾病，而B2型病毒通常从无症状的情况下恢复 处于免疫抑制状态的患者。E1a基因的DNA序列分析 并将对每种具有代表性的血清型执行纤维基因 描述它们的系统发育和进化制约因素的亚群 关于调控和结构基因。这可能会为我们提供关于 基因内的功能区及其通过进化的命运。 来自B和C亚群腺病毒的序列将被克隆到细菌中 表达载体，并用来自已被 用特定的腺病毒感染或接种的。免疫阳性克隆 将通过杂交和DNA序列映射到各自的病毒 基因组来鉴定编码抗原决定簇的DNA序列。 这一背景将有助于进一步努力开发一种 实用的腺病毒疫苗载体。

The proposed research is designed to elucidate the roles of adenovirus DNA sequences in the regulation of gene expression, DNA encapsidation and evolutionary diversification. Recently described autoregulation by the adenovirus E1A gene provides a model system for the investigation. E1A promoter sequences, or derivative variants, have been joined to probe genes such as chloramphenicol acetyltransferase to conveniently measure extents of gene expression in transfected human cells in culture. The effects of cotransfecting wild type or mutant E1A genes on expression of the E1A-probe is readily determined. Evidence indicates negative autorepression and two modes of positive feedback in E1A autoregulation. These assays will be used to localize cis-targets for promoter response to E1A and functional domains within the E1A peptide(s). A cis-element specifying polar DNA encapsidation lies within the transcriptional control region of the E1A gene and this will be further characterized. The adenovirus E1A is one of the two adenovirus genes involved in the process of adenovirus induced oncogenic transformation of cells. The tumorigenicity of adenoviruses is also related to the E1A gene, in a fashion which varies with the particular subgroup of human adenovirus serotypes. This enhances interest in the regulatory properties and evolution of the adenovirus E1A gene. The B subgroup of human adenoviruses, moderately oncogenic, is comprised of two closely related but epidemiologically and pathogenically distinct clans or sub-subgroups. The B1 viruses can cause serious epidemic respiratory disease while the B2 viruses are generally recovered from asymptomatic patients subject to immunosuppression. DNA sequence analysis of the E1A and fiber genes will be performed for representative serotypes of each sub-subgroup to characterize their phylogeny and evolutionary constraints on regulatory and structural genes. This may provide further insights on the functional domains within the genes and their fate through evolution. Sequences from B and C subgroup adenoviruses will be cloned into bacterial expression vectors and screened using sera from individuals having been infected or vaccinated with specific adenoviruses. Immunopositive clones will be mapped by hybridization and DNA sequence to the respective viral genomes to identify DNA sequences which encode antigenic determinants. This background will be useful in the further efforts to develop a practical adenovirus vaccine vector.