DNA-CYTOS-METHYLTRANSFERASE (S)

此应用程序的长期目标是获得详细的 对细胞的结构、作用机制和调节的理解 人DNA甲基转移酶(S)(E.C.2.1.1.37)。有确凿的证据 这表明由这一点催化的DNA合成后修饰 酶在细胞分化和体细胞遗传中起作用。脱氧核糖核酸 甲基化也可能参与异位蛋白的产生，如肿瘤 启动和肿瘤进展。 我们建议制备和研究均相的毫克量 人类胎盘中的酶。为研究这种酶而设计的实验 结构包括亚单位化学计量比和分子的测定 重量和氨基酸序列的部分测定。纯净的 酶将用于构建和优化体外DNA甲基化 系统。该系统和纯化酶的抗血清将用于 识别可能促进或抑制DNA甲基化的因素的实验 在细胞增殖过程中，以及可能导致 在某些转化的细胞系中观察到DNA的低甲基化。 氨基酸序列信息将用于合成 用作杂交探针的寡核苷酸，以帮助分离 甲基转移酶的cdna克隆。如此分离的cDNA克隆(S) 将用于确定基因组组织和转录 甲基转移酶基因的调控(S)。

The long-term objectives of this application are to obtain a detailed understanding of the structure, mechanisms of action, and regulation of the human DNA methyltransferase(s) (E.C.2.1.1.37). Available evidence strongly suggests that the postsynthetic modification of DNA catalyzed by this enzyme has a role in cellular differentiation and somatic inheritance. DNA methylation may also be involved in ectopic protein production, tumor initiation, and tumor progression. We propose to prepare and study milligram quantitites of the homogeneous enzyme from human placenta. Experiments designed to study the enzyme structure include the determination of sub-unit stoichiometry and molecular weight, and a partial determination of amino acid sequence. The purified enzyme will be used to construct and optimize an in vitro DNA methylation system. This system and antisera to the purified enzyme will be used in experiments to identify factors that may promote or inhibit DNA methylation during cell proliferation, and factors that may be responsible for the hypomethylation of DNA observed in certain transformed cell lines. The amino acid sequence information will be used to synthesize oligonucleotides for use as hybridization probes to aid in the isolation of a cDNA clone for the methyltransferase. The cDNA clone(s) thus isolated will be used to determine the genomic organization and the transcriptional regulation of the methyltransferase gene(s).