REGULATION AND ENZYMOLOGY OF DNA METHYLASE IN CANCER

最近的实验表明，酶促DNA甲基化参与了 高等真核生物基因表达的控制。 如今 调节机制和酶的特异性，DNA 甲基转移酶（DMase）使DNA甲基化的作用尚不清楚。 研究 显示出组织特异性基因组甲基化模式的畸变 在致癌过程中，肿瘤低甲基化，最近致癌 干扰DMase催化的甲基转移反应。 以来 癌症的特征是基因表达异常， DNA复制，致癌物结合引起的基因组甲基化变化可能 与致癌起始和/或恶性演变有因果关系。 建议的研究重点是DMase的调控和酶学， 正常肝脏和肝癌发生过程中;因素的阐明 用于忠实地传递特定序列的甲基化， 致癌物可能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 产生抗主要DM酶种类的单克隆抗体。 这些将用于简化纯化方案以及检测 活性和非活性DM酶在致癌过程中，并测试 可能存在多种酶。 这些抗体也将 用于在体外翻译测定中鉴定DMase多肽， 菌落裂解物，以便可以产生、克隆和检测DMase cDNA， 表达（pUC）cDNA文库或非表达载体（pBR）中。 获得这些探针的目的是最终分离DMase 基因 因为我们对甲基化作用的理解受到了 无法保留分子克隆的甲基化模式， 我们在表达载体中克隆全长DMase的目标是 能够在细菌宿主中进行基因工程表达， 目的. 这样，测试DNA的功能将更容易 甲基化，并确定它是否是一个 癌症发展中的一个必要因素。

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.