GENETIC ELEMENTS REGULATIONG MYELIN GENE EXPRESSION

我们实验室最近的研究表明，32 kb的髓鞘碱性 蛋白（MBP）转录单位包含在一个重叠的 转录单位，称为Golli-mbp基因，其约为105 KB在老鼠身上。 Golli-mbp转录单位由11个 最后7个外显子的部分或全部形成MBP的外显子 转录单位 MBP转录单位的表达开始于 Golli-mbp基因外显子5 b上游的启动子，以及 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物至少三种 其中含有一个或多个在MBP转录中发现的外显子 单位 本提案的总体目标是确定 Golli-mbp的发育和组织特异性调节元件 在小鼠中使用细胞的体外转染和 转基因小鼠 我们将分析这些元素在 调节MBP转录的表达， 转录以及组织、细胞和发育表达 的基因。 首先，我们将分析Golli-mbp上游的区域 参与组织、细胞和发育的元件的外显子1 这个转录单位的调控。 我们亦会研究 一个或多个这些调节元件被发现的可能性 在基因的其他地方，特别是第一个内含子。 我们将准备 推定的调节区加上报告基因的构建体和测试 通过在表达和不表达的细胞系中进行转染分析， Golli-mbp基因 然后，我们将测试最有前途的结构， 转基因动物提供证据， 在体内起作用。 在其他实验中，我们将检查 Golli-mbp启动子和任何其他调控元件的影响， 发现MBP转录的表达水平和性质 单位 Shiverer小鼠基因组中的缺失影响了至少两个 Golli-mbp转录单位的产物。 在 鉴于shiverer缺失的多效性， 少突胶质细胞功能，我们将引入Golli-mbp小基因， 颤抖的老鼠，仔细检查所有已知的影响， 与shiverer突变相关（除了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.