Le T T, Nguyen T M, Love D R, Helliwell T R, Davies K E, Morris G E
Research Division, North East Wales Institute, Deeside, Clwyd, United Kingdom.
Am J Hum Genet. 1993 Jul;53(1):131-9.
The first three exons of the human muscle dystrophin gene were expressed as a beta-galactosidase fusion protein. This protein was then used to prepare two monoclonal antibodies (mAbs) which react with native dystrophin on frozen muscle sections and with denatured dystrophin on western blots but which do not cross-react with the dystrophin-related protein, utrophin. Both mAbs recognized dystrophin in muscular dystrophy (MD) patients with deletions of exon 3, and further mapping with 11 overlapping synthetic peptides showed that they both recognize an epitope encoded by the muscle-specific exon 1. Neither mAb recognizes the brain dystrophin isoform, confirming the prediction from mRNA data that this has a different N-terminus. One Becker MD patient with a frameshift deletion of exons 3-7 is shown to produce dystrophin which reacts with the N-terminal mAbs, as well as with mAbs which bind on the C-terminal side of the deletion. The data suggest that transcription begins at the normal muscle dystrophin promoter and that the normal reading frame is restored after the deletion. A number of mechanisms have been proposed for restoration of the reading frame after deletion of exons 3-7, but those which predict dystrophin with an abnormal N-terminus do not appear to be major mechanisms in this patient.
人类肌肉营养不良蛋白基因的前三个外显子被表达为β-半乳糖苷酶融合蛋白。然后用该蛋白制备了两种单克隆抗体(mAb),它们能与冰冻肌肉切片上的天然营养不良蛋白以及蛋白质印迹上的变性营养不良蛋白发生反应,但不与肌养蛋白(dystrophin相关蛋白)发生交叉反应。两种单克隆抗体在3号外显子缺失的肌肉营养不良(MD)患者中均识别出营养不良蛋白,并且用11种重叠合成肽进行的进一步定位表明,它们均识别由肌肉特异性外显子1编码的一个表位。两种单克隆抗体均不识别脑型营养不良蛋白异构体,这证实了来自mRNA数据的预测,即其具有不同的N端。一名患有3 - 7号外显子移码缺失的贝克型MD患者被证明产生的营养不良蛋白能与N端单克隆抗体发生反应,也能与在缺失的C端一侧结合的单克隆抗体发生反应。数据表明转录起始于正常的肌肉营养不良蛋白启动子,并且在缺失后正常的阅读框得以恢复。已经提出了多种机制来解释3 - 7号外显子缺失后阅读框的恢复,但那些预测具有异常N端的营养不良蛋白的机制在该患者中似乎不是主要机制。