ADENOVIRAL ONCOGENE EXPRESSION AND TRANSFORMATION

腺病毒癌基因表达和转化

• 批准号: 3178888
• 负责人: JAMES B LEWIS
• 金额: $ 11.54万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-03-01 至 1988-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3178888
• 关键词: Adenoviridae; cancer registry /resource; gene expression; genetic manipulation; genetic transcription; infant animal; mutant; neoplastic cell; nucleic acid sequence; oncogenes; oncogenic virus; plasmids; platelet derived growth factor; regulatory gene; transforming virus; viral carcinogenesis; virus genetics

Transcription units Ela and Elb of the human adenoviruses encode at least five proteins and all of the functions needed for the oncogenic transformation of primary rodent cells to cell lines that are immortalized and have altered morphology and growth properties, both in vitro and in vivo. This transformation process will be characterized for its dependence on the level at which E1 products are expressed, the effects of certain E1 products upon the expression of others, the degree to which different El products are required and at what threshold levels for each of several different characteristics of transformed cells, and the effects of E1 proteins on selected aspects of cellular structure and gene expression. These studies will help to elucidate the mechanisms involved in transforming normal to neoplastic cells, and should suggest strategies for controlling the growth of tumor cells. Chimetric DNA molecules will be constructed in which the expression of these E1 genes is controlled by regulatory sequences derived from other genes so that expression will occur at a variety of levels and in some cases can be modulated by factors in the medium. These molecules will be introduced into rodent cells by co-transfection with selectable markers, and the resulting colonies scored for levels of individual El gene products, for effects on morphology, anchorage independent growth, and saturation density, and for effects on host cell gene expression. The variation of these parameters with the level of expression of adenovirus gene will be noted. Transient overexpression of viral gene products will be induced in an effort to amplify and thereby identify effects on host cells that may be otherwise undetectable in established transformed cells. To investigate the individual effects and interactions of the five proteins, appropriate plasmids will be constructed that each express only one or a few El proteins. The effect of each of these plasmids alone, or in combination with other such plasmids, on the expression of other El genes, on the initiation and maintenance of the transformed phenotype, and on parameters of cell growth, structure, and gene expression will be investigated. Similarly, individual domains within multifunctional proteins will be identified by constructing and testing plasmids containing appropriate mutations.

人类腺病毒的转录单元ELA和ELB至少编码 五种蛋白质以及致癌性所需的所有功能 将原代啮齿动物细胞转化为永生的细胞系 并改变了体外和中的形态和生长特性 体内。 这种转换过程将以其依赖性为特征 在表达E1产品的水平上，某些E1的影响 产品表达的产品，不同的el的程度 需要产品，并且在几个中的每个阈值水平下 转化细胞的不同特征以及E1的影响 蛋白质在细胞结构和基因表达的选定方面。 这些研究将有助于阐明涉及的机制 将正常转化为肿瘤细胞，应提出 控制肿瘤细胞的生长。 将构建嵌合DNA分子，其中的表达 这些E1基因由从其他的调节序列控制 基因，因此表达将在多种级别发生，在某些层面 案例可以通过培养基中的因素调节。 这些分子将是 通过与可选标记共转染后将其引入啮齿动物细胞， 所得的菌落为单个EL基因的水平得分 产品，以对形态学，锚定独立生长和 饱和密度，以及对宿主细胞基因表达的影响。 这 这些参数随腺病毒的表达水平的变化 基因将被注意。 病毒基因产物的瞬时过表达将 被诱导以放大并从而确定对宿主的影响 在已建立的转化细胞中可能无法检测到的细胞。 研究五个的个人效果和相互作用 蛋白质，适当的质粒将仅构建，每个质粒仅表示 一个或几个EL蛋白。 仅这些质粒的效果，或 与其他此类质粒结合使用其他EL的表达 基因，关于转化表型的启动和维护，以及 关于细胞生长，结构和基因表达的参数将是 调查。 同样，多功能的单个域 蛋白质将通过构建和测试含有含有的质粒来鉴定 适当的突变。