ADENOVIRUS GENOME EXPRESSION: PHYSICAL MAPPING STUDIES

这项研究旨在阐明腺病毒DNA在 序列在基因表达、DNA折叠和 进化多样化 最近描述的自动调节， 腺病毒E1 A基因为研究提供了一个模型系统。 E1a 启动子序列或衍生变体已经连接到探针基因 例如氯霉素乙酰转移酶，以方便地测量 在培养的转染人类细胞中的基因表达。 的影响 在E1 A-探针的表达上连接野生型或突变型E1 A基因 很容易确定。 有证据表明，负性自我抑制和两个 E1 A自动调节中的正反馈模式。 这些试验将 用于定位启动子响应E1 A的顺式靶点， E1 A肽内的结构域。 一种极性DNA的顺式元件 腺苷酸化位于E1 A的转录控制区内， 基因，并将进一步表征。 腺病毒E1 A是参与免疫应答的两种腺病毒基因之一。 腺病毒诱导细胞致癌转化的过程。 的 腺病毒的致瘤性也与E1 A基因有关， 其方式随人腺病毒的特定亚群而变化 血清型 这增强了对监管性质的兴趣， 腺病毒E1 A基因的进化。 人腺病毒的B亚群，中度致癌，由以下组成： 两个密切相关但在流行病学和病原学上不同的家族 或子亚组。 B1病毒可引起严重的流行性呼吸道疾病 而B2病毒通常从无症状 免疫抑制患者。 E1 A的DNA序列分析 和纤维基因将进行代表性血清型的每一个 子亚群，以表征其同源性和进化限制 调节基因和结构基因。 这可能会提供进一步的见解， 基因内的功能域及其在进化中的命运。 将来自B和C亚组腺病毒的序列克隆到细菌中， 表达载体，并使用来自已被 用特定的腺病毒感染或接种。 免疫阳性克隆 将通过杂交和DNA序列映射到相应的病毒 基因组以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制定一项 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.