DNA METHYLATION AND GENE EXPRESSION IN MAMMALIAN CELLS

将进行实验以分析DNA甲基化在以下方面的作用： 培养的哺乳动物细胞中基因表达的调节。 的 实验将利用生物化学技术，体细胞技术， 遗传学和重组DNA，以确定改变 甲基化模式对基因表达的影响 这些研究将在 在2种不同的系统中：（1）转化的小鼠L细胞，其含有 单纯疱疹病毒DNA片段，包括 胸苷激酶（TK），和（2）中国仓鼠V79细胞，其含有 失活的苯丙氨酸羟化酶（PH）基因。 我们已经得到了 有证据表明，病毒TK基因的表达和 仓鼠PH基因可以被5-氮杂胞苷诱导， DNA低甲基化通过取代DNA中的胞嘧啶。 我们还 显示病毒TK基因的表达与 及其在一系列转化株系中的甲基化状态。 通过 分析用以下物质处理细胞的生物学和分子学效应： 诱导剂的hypothylation，我们将试图确定以何种方式和通过 DNA甲基化影响基因表达的机制。 使用 可用的病毒TK基因的分子探针，这些研究将 包括测定两种细胞中的特定甲基化模式， 结构和调控序列及其关系的分析 特定的甲基化模式和基因表达之间的联系 我们还将 试图克隆仓鼠PH基因， DNA甲基化的实验也可以在该系统中进行。 这两种生物系统提出了不同类型的问题， 作为一种系统（TK）涉及外源病毒基因的表达 整合到细胞基因组中，而另一个系统（PH）涉及 一种正常的细胞基因，其表达受到严格控制， 分化 这些研究的结果应该提供信息， 细胞和外源基因的调控机制， 哺乳动物细胞，无论是在正常发育和疾病状态。 在 此外，PH系统的研究应朝着详细的 人类苯丙酮尿症的分子分析。

Experiments will be carried out to analyze the role of DNA methylation in the regulation of gene expression in mammalian cells in culture. The experiments will utilize the techniques of biochemistry, somatic cell genetics, and recombinant DNA to determine the effects of altering methylation patterns on gene expression. The studies will be carried out in 2 different systems: (1) transformed mouse L cells containing a fragment of Herpes Simplex virus DNA that includes the viral gene for thymidine kinase (TK), and (2) Chinese hamster V79 cells which contain an inactive phenylalanine hydroxylase (PH) gene. We have already obtained evidence indicating that the expression of both the viral TK gene and the hamster PH gene can be induced by 5-azacytidine, a compound which causes DNA hypomethylation by substituting for cytosine in DNA. We have also shown an inverse correlation between the expression of the viral TK gene and its state of methylation in a series of transformed lines. Through the analysis of the biological and molecular effects of treating cells with inducers of hypothylation, we will attempt to determine in what ways and by which mechanisms does DNA methylation effect gene expression. Using available molecular probes for the viral TK gene, these studies will include the determination of specific methylation patterns in both structural and regulatory sequences and the analysis of the relationship between specific methylation patterns and gene expression. We will also attempt to clone the hamster PH gene, so that detailed molecular experiments on DNA methylation can be performed in that system as well. The two biological systems to be studied pose different types of questions, as one system (TK) involves the expression of an exogenous viral gene incorporated into the cellular genome, while the other system (PH) involves a normal cellular gene whose expression is rigidly controlled in differentiation. The results of these studies should provide information on the mechanisms by which cellular and exogenous genes are regulated in mammalian cells, both in normal development and in disease states. In addition, the studies of the PH system should progress towards a detailed molecular analyses of the human disease phenylketonuria.