STRUCTURAL ORGANIZATION OF MYELIN PROTEIN GENES

在这个应用程序中，我们建议测试两个新的假设 关于MBP基因。这些假设是以数据为基础的 来源于对两类异常髓鞘的鉴定 碱性蛋白(MBP)相关转录本的筛选 小鼠脑和人胎脊髓cDNA库。这个 第一个假设是MBP基因的结构目前是 不完整，并且包含至少一个以上的外显子 目前被认为是外显子1和一个额外的启动子。 第二种假设是MBP基因的一部分(包括 至少一个外显子和一个内含子的部分)转录成 编码MBP变异体以外的蛋白质的mRNAs 到目前为止所特有的。任何额外外显子的位置 序列将通过以下常规程序确定 使用MBP cDNA对基因组克隆进行定位和测序 包含与附加外显子区域对应的序列。 这个新的上游外显子的转录起始位置将是 经S-1核酸酶和引物延伸分析确定。 将进行实验以确定是否启动 这个起始点的转录和之前在 MBP基因在发育过程中或在不同的区域有差异 中枢神经系统。还将进行实验，以确定是否所有 当转录开始时，会产生MBP的变异形式 新的转录站点。全长cDNA对应于 第二类MBP基因相关转录本将被分离， 进行了排序，并进行了表征。结构关系 在这第二类转录本和MBP基因之间 下定决心。编码基因的内含子/外显子排列 这些与MBP相关的cDNA将被阐明，并将被 确定该基因的所有外显子是否都由基因组编码 完全包含在MBP基因中的片段，或者如果一些 外显子位于MBP基因之外。以确定这些文字记录是否 在脑以外的组织或在其他类型的细胞中表达 而不是少突胶质细胞，Northern印迹分析 将进行各种组织和培养细胞的培养。这个 小鼠脑内转录产物的发育形态 正常小鼠和髓鞘障碍突变体将与 MBP mRNAs的表达。原位杂交研究 将被执行以确定细胞和区域 记录的本地化。

In this application, we propose to test two new hypotheses concerning the MBP gene. These hypotheses are based upon data derived from the identification of two classes of unusual myelin basic protein (MBP)-related transcripts isolated from screens of mouse brain and human fetal spinal cord cDNA libraries. The first hypothesis is that the structure of the MBP gene is presently incomplete and contains at least one more exon upstream of what is currently believed to be exon 1 and an additional promoter. The second hypothesis is that portions of the MBP gene (including parts of at least one exon and one intron) are transcribed into mRNAs encoding proteins other than the MBP variants characterized thus far. The location of any additional exon sequences will be determined by conventional procedures of mapping and sequencing genomic clones using an MBP cDNA that contains sequence corresponding to the additional exon region. Sites of transcription initiation at this new upstream exon will be determined by S-1 nuclease and primer extension analysis. Experiments will be performed to determine if initiation of transcription at this start site and the one previously identified in the MBP gene varies during development or in different regions of the CNS. Experiments will also be performed to determine if all MBP variant forms are produced when transcription begins at this new transcription site. Full length cDNAs corresponding to the second class of MBP-gene related transcripts will be isolated, sequenced, and characterized. The structural relationship between this second class of transcripts and the MBP gene will be determined. The intron/exon arrangement of the gene coding for these MBP-related cDNAs will be elucidated and it will be determined if all the exons of this gene are encoded by genomic segments completely included within the MBP gene or if some exons lie outside the MBP gene. To determine if these transcripts are expressed in tissues other than brain or in cell types other than oligodendrocytes, Northern blot analyses of mRNA from a variety of tissues and cultured cells will be performed. The developmental appearance of the transcripts in the brains of normal mice and dysmyelinating mutants will be compared with the expression of the MBP mRNAs. In situ hybridization studies will be performed to determine the cellular and regional localization of the transcripts.