Ercolani L, Florence B, Denaro M, Alexander M
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114.
J Biol Chem. 1988 Oct 25;263(30):15335-41.
Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.2.12) (GAPDH) mRNA levels, protein, and enzymatic activity increase in 3T3-F442A adipocytes after exposure to physiological concentrations of insulin (Alexander, M., Curtis, G., Avruch, J., and Goodman, H. (1985) J. Biol. Chem. 260, 11978-11985). In order to understand the mechanism of this regulation, we have isolated and sequenced 5.4 kilobase pairs of a 12-kilobase pair human genomic clone encoding a functional GAPDH gene. The gene consists of 9 exons and 8 introns with eukaryotic signals necessary for the transcription and translation of GAPDH mRNA. The exon sequence confirms previously published cDNA sequences for human GAPDH in muscle, liver, and erythrocytes. The organization of the human and the unique chicken GAPDH genes is strikingly similar. Although chicken exons VIII-XI have been fused into human exon 8, introns which separate exons encoding the NAD binding, catalytic, and helical domains of the GAPDH protein have been retained. Stable transfection of rodent cells with the intact human GAPDH gene resulted in the expression of a correctly initiated human GAPDH mRNA and an enzymatically active human GAPDH polypeptide. Thus, the gene contains a functional promoter and intact coding sequences. Although many processed GAPDH pseudogenes and GAPDH-like sequences are present in the human genome, Southern blot analysis of human genomic DNA using a probe derived from the 3'-untranslated region of the GAPDH gene detected only two genes, a 10-copy processed pseudogene and a single copy of the isolated gene. In contrast, a probe derived from an intron segment of the isolated gene detected only a single copy of the GAPDH gene. Collectively, these findings strongly suggest that the human genome encodes a single functional GAPDH gene.
在暴露于生理浓度的胰岛素后，3T3 - F442A脂肪细胞中的甘油醛 - 3 - 磷酸脱氢酶（EC 1.2.2.12）（GAPDH）的mRNA水平、蛋白质及酶活性均增加（亚历山大，M.，柯蒂斯，G.，阿夫鲁奇，J.，古德曼，H.（1985年）《生物化学杂志》260，11978 - 11985）。为了理解这种调节机制，我们分离并测序了一个12千碱基对的人类基因组克隆中5.4千碱基对的片段，该克隆编码一个功能性GAPDH基因。该基因由9个外显子和8个内含子组成，带有GAPDH mRNA转录和翻译所需的真核信号。外显子序列证实了先前发表的人类肌肉、肝脏和红细胞中GAPDH的cDNA序列。人类和独特的鸡GAPDH基因的组织惊人地相似。虽然鸡的外显子VIII - XI已融合到人类外显子8中，但分隔编码GAPDH蛋白的NAD结合、催化和螺旋结构域的外显子的内含子得以保留。用完整的人类GAPDH基因稳定转染啮齿动物细胞，导致正确起始的人类GAPDH mRNA和具有酶活性的人类GAPDH多肽的表达。因此，该基因包含一个功能性启动子和完整的编码序列。虽然人类基因组中存在许多加工过的GAPDH假基因和GAPDH样序列，但使用源自GAPDH基因3' - 非翻译区的探针进行人类基因组DNA的Southern印迹分析仅检测到两个基因，一个10拷贝的加工假基因和一个分离基因的单拷贝。相反，源自分离基因内含子片段的探针仅检测到GAPDH基因的单拷贝。总体而言，这些发现强烈表明人类基因组编码单个功能性GAPDH基因。
