Haas S, Thatikunta P, Steplewski A, Johnson E M, Khalili K, Amini S
Jefferson Institute of Molecular Medicine, Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Cell Biol. 1995 Sep;130(5):1171-9. doi: 10.1083/jcb.130.5.1171.
The MB1 regulatory sequence of the myelin basic protein (MBP) gene spanning between nucleotides -14 to -50 with respect to the transcription start site is critical for cell type-specific transcription of the MBP gene, which encodes the major protein component of myelin sheath in cells derived from the central nervous system (CNS). This regulatory sequence has the ability to interact with a developmentally controlled DNA-binding protein from mouse brain that stimulates transcription of MBP promoter in an in vitro system (Haas, S., J. Gordon, and K. Khalili. 1993. Mol. Cell. Biol. 13:3103-3112). Here, we report the purification of a 39-kD protein from mouse brain tissue at the peak of myelination and MBP production that binds to the MB1 regulatory motif. Following partial amino acid sequence analysis, we have identified a complementary DNA encoding a 39-kD DNA-binding protein called pur alpha. Expression of pur alpha cDNA in the prokaryotic and eukaryotic cells resulted in the synthesis of a protein with characteristics similar to the purified brain-derived 39-kD protein in band shift competition assays. Cotransfection of the recombinant pur alpha expressor plasmid with MBP promoter construct indicated that Pur alpha stimulates transcription of the MBP promoter in oligodendrocytic cells, and that the nucleotide sequence required for binding of the 39-kD Pur alpha to DNA within the MB1 region is crucial for this activity. Moreover, transient expression of Pur alpha caused elevation in the level of endogenous MBP RNA in oligodendrocytic cells. Thus, Pur alpha, a sequence-specific DNA-binding protein upon binding to MB1 regulatory region may play a significant role in determining the cell type-specific expression of MBP in brain.
髓鞘碱性蛋白（MBP）基因的MB1调控序列，相对于转录起始位点，跨度为核苷酸-14至-50，对于MBP基因的细胞类型特异性转录至关重要，该基因编码中枢神经系统（CNS）来源细胞中髓鞘的主要蛋白质成分。这个调控序列能够与来自小鼠大脑的一种受发育调控的DNA结合蛋白相互作用，该蛋白在体外系统中刺激MBP启动子的转录（哈斯，S.，J.戈登，和K.哈利利。1993.《分子与细胞生物学》13:3103 - 3112）。在这里，我们报告了从小鼠脑组织中纯化出一种39-kD的蛋白质，它在髓鞘形成和MBP产生的高峰期与MB1调控基序结合。经过部分氨基酸序列分析，我们鉴定出一个编码39-kD DNA结合蛋白的互补DNA，称为嘌呤α。嘌呤α cDNA在原核和真核细胞中的表达导致合成了一种在凝胶迁移竞争试验中具有与纯化的脑源性39-kD蛋白相似特征的蛋白质。将重组嘌呤α表达质粒与MBP启动子构建体共转染表明，嘌呤α在少突胶质细胞中刺激MBP启动子的转录，并且39-kD嘌呤α与MB1区域内DNA结合所需的核苷酸序列对于该活性至关重要。此外，嘌呤α的瞬时表达导致少突胶质细胞内源性MBP RNA水平升高。因此，嘌呤α作为一种序列特异性DNA结合蛋白，在结合到MB1调控区域后，可能在决定大脑中MBP的细胞类型特异性表达中发挥重要作用。
