FACTORS AFFECTING MOUSE BETA-GLOBIN GENE EXPRESSION

为了分析诱导小鼠的序列要求 β(Maj)-珠蛋白基因，我们在小鼠体内建立了一个瞬时检测系统。 红白血病(MEL)细胞。这些细胞，已经被 Friend病毒复合体，可以在化学诱导下经历终末 在分化过程中，内源性α和β转录 珠蛋白基因大大增加。如果我们能在一个 暂时性化验，只需要4到5天，那么它应该是 可以用来快速准确地分析质粒的结构 将不同数量的DNA 5‘或3’连接到β-珠蛋白启动子以确定 归纳需要哪些区域。瞬时化验条件 已经针对未诱导和诱导的MEL细胞进行了优化，我们目前 希望确定诱导是否影响位于转染体上的基因 在这些实验中保持异构体的质粒。 我们之前已经证明，被称为增强剂的DNA序列增加了 小鼠β-珠蛋白启动子在瞬时试验中的活性。增强剂 是在转录水平上起作用的顺式作用DNA序列 增加基因表达。它们可以在两个方向中的任何一个方向上工作，都可以在3‘ 和5‘到靶基因，它们的激活水平相对较低 与位置无关的。虽然没有高度的顺序性 目前已确定的增强子之间的同源性，两类短链 观察到了“核心”区域。我们有兴趣确定是否有 增强子DNA序列的其他共同特征是否存在，如果存在，是否 他们可能提出了小鼠激活增强子的可能机制。 β-珠蛋白启动子以及其他增强子激活的启动子。 对五个增强子的分析表明，每个增强子都表现出对偶对称；我们 正在扩展这一分析，以包括其他增强剂。我们还有 表明已知的增强子突变体比野生的表现出更少的二联体对称性 键入Enhancer，表明增强器之间可能存在关联 二分体的函数和对称度。

In order to analyze the sequence requirements for induction of the mouse beta(maj)-globin gene, we have developed a transient assay system in mouse erythroleukemia (MEL) cells. These cells, which have been transformed by the Friend virus complex, can be chemically induced to undergo terminal differentiation during which transcription of endogenous alpha and beta globin genes is greatly increased. If we can mimic this effect in a transient assay, which only requires 4 to 5 days, then it should be possible to quickly and accurately analyze plasmid constructions with varying amounts of DNA 5' or 3' to the beta-globin promoter to determine what regions are required for induction. Transient assay conditions have been optimized for both uninduced and induced MEL cells and we currently wish to determine if induction affects genes located on transfected plasmids which in these experiments remain episomal. We have previously shown that DNA sequences known as enhancers increase the activity of the mouse beta-globin promoter in transient assays. Enhancers are cis-acting DNA sequences which act at the level of transcription to increase gene expression. They can function in either orientation both 3' and 5' to the target gene and their level of activation is relatively independent of position. While there is no high degree of sequence homology among the presently identified enhancers, two categories of short "core" regions have been observed. We are interested in determining if any other common features of enhancer DNA sequences exist and, if so, whether they might suggest possible mechanisms of enhancer activation of the mouse beta-globin promoter as well as other enhancer activated promoters. Analysis of five enhancers has shown that each exhibits dyad symmetry; we are extending this analysis to include additional enhancers. We have also shown that known enhancer mutants exhibit less dyad symmetry than the wild type enhancer, suggesting there may be a correlation between enhancer function and degree of dyad symmetry.