GENETIC ELEMENTS REGULATIONG MYELIN GENE EXPRESSION

我们实验室最近的研究表明，32kb的髓鞘碱性 蛋白质(MBP)转录单位包含在重叠的 转录单位，称为Golli-MBP基因，约为105 KB在鼠标中。Golli-MBP转录单位由11个 部分或全部最后七个外显子形成MBP 转录单位。MBP转录单位的表达开始于 Golli-MBP基因外显子5b上游的启动子，并表达 Golli-MBP转录单位从外显子1开始，产生许多 组织、细胞和发育调节的剪接产品至少有三种 其中包含在MBP转录中发现的一个或多个外显子 单位。这项提案的总体目标是确定 Golli-MBP的发育和组织特异性调控元件 转录单位在小鼠中使用体外细胞和 转基因小鼠。我们将分析这些元素在 根据水平调控MBP转录的表达 转录水平以及组织、细胞和发育阶段的表达 这种基因的。首先，我们将分析Golli-MBP上游地区 与组织、细胞和发育有关的元素的外显子1 这个转录单位的调节。我们还将研究 找到一个或多个这些监管要素的可能性 在基因的其他地方，特别是第一内含子。我们会做好准备 推测的调控区域加上一个报告基因的构建和测试 通过在有表达和不表达的细胞系中进行转染分析 Golli-MBP基因。然后我们将测试最有希望的构造 转基因动物提供的证据表明假定的调控区域 都在活体内运作。在其他实验中，我们将检查 Golli-MBP启动子和其他调控元件对WE的影响 发现MBP转录的表达水平和性质 单位。颤抖小鼠基因组中的缺失至少影响到两个 除Mbps外，Golli-MBP转录单元的产品。在……里面 关于抖动删除的多效性的观点 少突胶质细胞功能我们将引入Golli-MBP微型基因到 发抖的老鼠，仔细检查所有已知的影响 与寒战突变相关(超越MBP表达和 “颤抖”表型)。这些研究可以帮助我们定义 Golli-MBP基因产物表达的后果。他们 也能帮助我们确定产品本身是否对 MBP转录单位在少突胶质细胞中的表达 发展。

Recent studies in our laboratory have shown that the 32 kb myelin basic protein (MBP) transcription unit is contained within an overlapping transcription unit, termed the Golli-mbp gene, which is approximately 105 kb in the mouse. The Golli-mbp transcription unit consists of eleven exons of which parts or all of the last seven exons form the MBP transcription unit. Expression of the MBP transcription unit begins at a promoter upstream of exon 5b of the Golli-mbp gene, and expression of the Golli-mbp transcription unit begins at exon 1, producing a number of tissue, cell and developmentally regulated splice products at least three of which contain one or more exons found within the MBP transcription unit. The overall objective of this proposal is to determine the developmental and tissue-specific regulatory element of the Golli-mbp transcription unit in the mouse using in vitro transfection of cells and transgenic mice. We will analyze the role of these elements in modulating the expression of the MBP transcription with respect to levels of transcription as well as the tissue, cell and developmental expression of the gene. Initially, we will analyze the region upstream of Golli-mbp exon 1 for elements involved in the tissue, cell and developmental regulation of this transcription unit. We w ill also examine the possibility that one or more of these regulatory elements are found elsewhere in the gene, particularly the first intron. We will prepare constructs of putative regulatory regions plus a reporter gene and test them by transfection analysis in cell lines that do and do not express the Golli-mbp gene. We will then test the most promising constructs in transgenic animals to provide proof that the putative regulatory regions are operative in vivo. In other experiments we will examine the influence of the Golli-mbp promoter and any other regulatory elements we find on the levels and nature of expression of the MBP transcription unit. The deletion in the shiverer mouse genome affects at least two products of the Golli-mbp transcription unit in addition to MBPs. In view of the pleiotropic nature of the shiverer deletion on oligodendrocyte function we will introduce a Golli-mbp minigene into shiverer mice and carefully examine all the effects known to be associated with the shiverer mutation (beyond MBP expression and the "shivering" phenotype). These studies could help us define the consequences of the expression of the Golli-mbp gene products. They would also help us determine if the produces themselves had an effect on the expression of the MBP transcription unit during oligodendrocyte development.