CELL CYCLE REGULATION OF MAMMALIAN GENES

这是一个研究基因调控机制的建议， 利用组蛋白和细胞周期 胸苷激酶基因作为模型系统。 这个项目的主要目标是 一项建议是对顺式作用DNA进行深入分析， 细胞周期所需的反式作用因子 调节体内基因表达。 近日有 表明哺乳动物组蛋白侧翼的序列 基因可以赋予异源融合的转录控制 基因，导致其mRNA水平的细胞周期调节。 在 根据这项提议，组蛋白基因周围的序列将被 进行体外诱变。 一系列重组体将 转染到受体哺乳动物细胞中， 细胞周期转录控制上的缺失将被 考察 该方法已成功用于定义 对调节功能至关重要的DNA结构域。 类似分析 将进行细胞胸苷激酶基因的 技术已经建立了组蛋白基因系统。 我们 重点是剖析转录调控组件的 胸苷激酶基因系统。 从我们的删除分析，我们 将决定细胞周期调控的共有序列 序列可以识别。 此外，我们还将检测细胞周期 调节元件具有增强子样性质。 为 参与细胞周期的细胞因子的表征 调控、缺失突变体构建体将被转染到 哺乳动物受体细胞，以探测这些因子是否在阳性细胞中起作用。 或消极的方式。 将进行竞赛实验 确认因素的监管性质，并确定 它们的相对结合亲和力，细胞内的浓度， 细胞周期及其对其他细胞周期调节的特异性 基因. 提取的DNA和蛋白质组分的结合试验 从分裂和G1期阻滞的细胞中进一步鉴定DNA 能够与蛋白质因子相互作用的区域， 提供用于其纯化的测定系统。 这 这项研究将有助于我们对协同基因的了解， 在哺乳动物细胞周期的控制。 因此，它可能 证明有助于了解疾病，如癌症， 是由细胞周期中基因表达的畸变引起的。

This is a proposal to study mechanisms of gene regulation during the mammalian cell cycle using the histone and the cellular thymidine kinase genes as model systems. The major goal of this proposal is to carry out an in-depth analysis of the cis-acting DNA sequence and the trans-acting factors required for the cell cycle regulated gene expression in vivo. Recently, it has been demonstrated that sequences flanking the mammalian histone genes can confer transcriptional control on heterologous fusion genes, resulting in cell cycle regulation of their mRNA levels. In this proposal, the sequences around the histone gene will be subjected to in vitro mutagenesis. A series of recombinants will be transfected into recipient mammalian cells and the effect of deletions on the cell cyclic transcriptional control will be examined. This approach has been used successfully to define domains of DNA critical for regulatory function. Similar analysis of the cellular thymidine kinase gene will be performed with techniques already established for the histone gene system. Our focus is to dissect the transcriptional regulatory component of the thymidine kinase gene system. From our deletion analysis, we will determine if a consensus sequence for cell cycle regulatory sequence can be identified. In addition, we will test if cell cycle regulatory element has enhancer-like properties. For the characterization of the cellular factors involved in the cell cycle regulation, deletion mutant constructs will be transfected into mammalian recipient cells to probe if the factors act in a positive or negative manner. Competition experiments will be performed to confirm the regulatory nature of the factors, and to determine their relative binding affinities, concentrations during the cell cycle and their specificity toward other cell cycle regulated genes. Binding assays of the DNA and protein fractions extracted from dividing and G1 arrested cells will further identify the DNA regions capable of interacting with the protein factors and provide for assay systems towards their purification. This research will contribute to our knowledge of co-ordinate gene control during the mammalian cell cycle. Consequently, it may prove useful in understanding diseases, such as cancer, that may result from aberrations of gene expression during the cell cycle.