REGULATION AND ENZYMOLOGY OF DNA METHYLASE IN CANCER

最近的实验表明，酶的DNA甲基化参与了 高等真核生物中基因表达的调控。目前， DNA酶的调节和专一性的机制 甲基转移酶(DMase)对甲基化DNA的作用尚不清楚。研究表明， 在基因组甲基化的组织特异性模式中显示出异常 在致癌过程中，肿瘤的低甲基化，以及最近的致癌物 对DMase催化的转甲基化反应的干扰。自.以来 癌症的特点是基因表达异常和顽固性 DNA复制，致癌物结合导致的基因组甲基化变化可能 与致癌启动和/或恶变有因果关系。 建议的研究集中在DMase的调节和酶学方面。 正常肝脏和肝癌发生过程中的因素阐明 用于忠实地传递特定序列的甲基化，以及 致癌物在模板水平上改变这一过程的机制 通过与酶的直接相互作用。在这次更新中，一个目标将是 将生产针对主要DMase种(S)的单抗。 这些将用于简化纯化方案以及检测 在致癌过程中的活性和非活性的DMase，并检测 可能存在多种酶。这些抗体也会 可用于体外翻译实验中DMase多肽的鉴定和 菌落裂解物，以获得、克隆和检测DMase基因 表达(PUC)cDNA文库或非表达载体(PBR)。 获得这些探针的目的是最终分离DMase 吉恩。因为我们对甲基化作用的理解受到以下因素的阻碍 在分子克隆中不能保持甲基化模式，一个 我们在表达载体中克隆全长DMase的目标之一是 能够通过基因工程在细菌宿主中表达这一点 目的。这样，DNA的功能(S)就更容易检测了 由DMase控制的甲基化，并确定它是否是 在癌症的发展过程中起主要作用的成分。

Recent experiments indicate that enzymatic DNA methylation participates in the control of gene expression in higher eukaryotes. Presently, the mechanics of regulation and specificity of the enzyme, DNA methyltransferase (DMase) to methylate DNA are unclear. Studies have demonstrated aberrations in tissue-specific patterns of genomic methylation during carcinogenesis, tumor hypomethylation, and recently carcinogen interference in the transmethylation reaction catalyzed by DMase. Since cancer is charaterized by abnormalities in gene expression and persistant DNA replication, changes in genomic methylation by carcinogen binding may be causally related to carcinogenic initiation and/or malignant evolution. The proposed research focuses on regulation and enzymology of DMase in normal liver and during hepatocarcinogenesis; the elucidation of factors for faithful transmission of the methylation of specific sequences, and the mechanics by which carcinogens may alter this process at the template level of by direct interaction with the enzyme. In this renewal, one goal will be to produce monoclonal antibodies against the major DMase specie(s). These will be used to simplify the purification scheme as well as detect active and inactive DMase during carcinogenesis, and to test for the possible presence of multiple enzyme species. These antibodies will also be used to identify DMase polypeptides in in vitro translation assays and colony lysates so that DMase cDNA can be generated, clone and detected in either expression (pUC) cDNA libraries or in nonexpression vectors (pBR). The purpose of obtaining these probes is to eventually isolate the DMase gene. Because our understanding of the role of methylation is hampered by the inability to preserve the methylation pattern on molecular cloning, one of our goals in cloning full-length DMase in an expression vector is to be able to genetically engineer the expression in a bacterial host for this purpose. In this way, it will be easier to test the function(s) of DNA methylation as controlled by DMase and to determine whether it is an obligate component in the development of cancer.