Functional restoration of genetically inherited neuronal disorder - Molecular biological approach using heriditary mutant mice -

将小鼠蛋白脂蛋白（PLP）基因克隆到噬菌体Charon 4A中。小鼠PLP基因外显子的结构和核苷酸序列与人类PLP基因的外显子非常相似，由7个外显子组成。plp基因的转录从多个位点开始。在转录区上游，有一个独特的序列连续重复4次。已经证明，这种x连锁的隐性髓鞘异常突变小鼠含有异常的PLP-mRNA。为了了解突变的分子基础，我们使用小鼠plp基因的每个外显子特异性探针，通过rnase定位程序分析了plp - nrna的结构。我们发现PLP基因的第5外显子没有被利用。我们分析了突变的PLP-mRNA，发现转录起始位点与正常小鼠相同。对jimpy PLP基因5′侧区域的克隆和测序结果显示，该基因的启动子区域未发生突变。将野生型小鼠和瘸子小鼠的PLP基因上游区域亚克隆到pIP111载体中，通过该载体可以测量插入片段在细胞内的启动子活性。将质粒引入大鼠C6胶质瘤细胞，因为在C6细胞中检测到PLP-mRNA，并且在血清剥夺后其表达显著增加。C6细胞的plp启动子活性比NIH3T3细胞高3倍，血清剥夺后plp启动子活性增加6倍。因此，该片段包含顺式作用元件，赋予PLP基因的组织限制性表达。然而，野生型和跳跃型PLP基因的启动子活性没有差异。因此，可能是在PLP基因第5外显子剪接受体位点存在“A”到“G”的转换，导致第5外显子跳变，直接影响mRNA水平。

The mouse proteolipid protein (PLP) gene was cloned into the phage Charon 4A. The organization and the nucleotide sequence of the exons of the mouse PLP gene were quite similar to those of their human counterparts, consisting of seven exons. The transcription of the plp gene started from multiple sites. There was a unique sequence tandemly repeated four times, upstream from the transcribed region.The jimpy mouse, an X-linked recessive dysmyelinating mutant, has been shown to contain abnormal PLP-mRNA. In order to understand the molecular basis of the mutation, we analyzed the structure of PLP-nRNA by an RNase-mapping procedure, using the probes specific to each exon of the mouse plp gene. We found that the fifth exon of the PLP gene is not utilized in the jimpy. We analyzed the jimpy PLP-mRNA and showed that the transcription initiated from the same sites as those in normal mice. Cloning and sequencing of the 5'-flanking region of the jimpy PLP gene revealed that there were no mutations in the promoter region of the jimpy PLP gene. The upstream region of the mouse PLP gene from wild-type and jimpy mice was subcloned into a pIP111 vector, with which the promoter activity of the inserted fragments within the cells can be measured. The plasmids were introduced into rat C6 glioma cells, since the PLP-mRNA was detected in C6 cells and its expression increased greatly following serum deprivation. The PLP-promoter activity was three times higher in C6 cells than in NIH3T3 cells and it increased six times upon serum deprivation. Thus this fragment contained cis-acting element(s) conferring tissue-restricted expression of the PLP gene. However, there was no difference in the promoter activity between the wild type and the jimpy type PLP gene.Therefore, it is likely that an "A" to "G" conversion existing at the splicing acceptor site of the fifth exon of the jimpy PLP gene caused the skipping of the fifth exon and directly affected the mRNA level.