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的非常相似，由7个外显子组成。PLP基因的转录从多个位点开始。在转录区域的上游有一个独特的序列重复了四次。Jimpy小鼠，一个X连锁的隐性髓鞘障碍突变体，已经被证明含有异常的PLP-mRNA。为了了解突变的分子基础，我们使用针对小鼠PLP基因每个外显子的探针，通过RNase作图程序分析了PLP-nRNA的结构。我们发现plp基因的第五个外显子在Jimpy中没有被利用。我们分析了Jimpy PLP-mRNA，发现该转录起始于与正常小鼠相同的位置。对jimpy plp基因5‘侧翼区的克隆和测序表明，jimpy plp基因的启动子区域没有突变。将野生型和幼小鼠PLP基因的上游片段亚克隆到pIP111载体中，用该载体可以检测插入片段在细胞内的启动子活性。由于在C6细胞中检测到PLP-mRNA，并且在血清剥夺后PLP-mRNA的表达显著增加，因此将其导入大鼠C6胶质瘤细胞。在C6细胞中，PLP启动子的活性是NIH3T3细胞的3倍，而在血清剥夺后，其活性增加了6倍。因此，该片段含有组织限制性表达plp基因的顺式作用元件(S)。然而，野生型和Jimpy型plp基因的启动子活性没有差异，因此，很可能是在jimpy plp基因第五外显子的剪接受体处存在“A”到“G”的转换，导致了第五外显子的跳过，并直接影响了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.