STRUCTURAL ORGANIZATION OF MYELIN PROTEIN GENES

在本申请中，我们提出测试两个新的假设 关于MBP基因 这些假设是基于数据 从两类不寻常的髓磷脂的鉴定中得出 碱性蛋白（MBP）相关的转录本分离自筛选的 小鼠脑和人胎儿脊髓cDNA文库。 的 第一个假设是MBP基因的结构目前 不完整的，并含有至少一个以上的外显子上游什么 目前认为是外显子1和另外的启动子。 第二个假设是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变体以外的蛋白质的mRNA 特征到目前为止。 任何额外外显子的位置 序列将通过常规程序确定， 使用MBP cDNA对基因组克隆进行定位和测序， 含有对应于附加外显子区域的序列。 在这个新的上游外显子的转录起始位点将是 通过S-1核酸酶和引物延伸分析测定。 将进行实验以确定是否启动 转录在这个起始位点和一个以前确定的在 MBP基因在发育过程中或在不同的区域变化， CNS。 还将进行实验以确定是否所有 当转录开始时，产生MBP变体形式。 新的转录位点 全长cDNA对应于 分离第二类MBP基因相关转录物， 测序和鉴定。 的结构关系 这第二类转录本和MBP基因之间的关系将是 测定 基因编码的内含子/外显子排列 这些MBP相关的cDNA将被阐明， 确定该基因的所有外显子是否由基因组编码， 完全包含在MBP基因内的片段，或者如果一些 外显子位于MBP基因之外。 为了确定这些记录 在脑以外的组织中或在其他细胞类型中表达 与少突胶质细胞相比， 将进行各种组织和培养的细胞。 的 大脑中转录本的发育外观 将正常小鼠和髓鞘形成障碍突变体与 MBP mRNA的表达。 原位杂交研究 将被执行，以确定蜂窝和区域 成绩单的本地化。

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.