Stone E M, Rothblum K N, Alevy M C, Kuo T M, Schwartz R J
Proc Natl Acad Sci U S A. 1985 Mar;82(6):1628-32. doi: 10.1073/pnas.82.6.1628.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an evolutionarily conserved glycolytic enzyme, is constitutively expressed in most cell types yet is induced to high levels during the development of fast twitch muscle fibers. To analyze the organization and regulation of the chicken GAPDH gene, we first constructed a nearly full-length GAPDH cDNA clone (pGAD-28). pGAD-28 was used in the current study to screen a genomic library, and several overlapping clones were selected. The GAPDH coding region was detected within a 4.65-kilobase Xho I/EcoRI genomic fragment that was completely sequenced by using the M13 cloning vector system. A small portion of pGAD-28 was used as a primer to extend a 33-nucleotide sequence from the 5' end of GAPDH mRNA. The canonical promoter "TATA" region was found 22 base pairs from the 5' end of the mRNA. The 5' end of the GAPDH gene is extraordinarily G+C-rich (80%) and contains two inverted sequences with a 9-base-pair homology found at -58 (G-G-G-G-C-G-G-G-C) and -93 (G-C-C-C-G-C-C-C-C) nucleotides from the transcription start site. Sequencing also revealed the location of 11 introns within the transcribed portion of the GAPDH gene. The placement of at least 3 of the introns corresponds to the boundaries of protein domains within prokaryotic and eukaryotic GAPDHs that were previously detected by x-ray crystallography. This concordance suggests that introns may have participated in the construction of the earliest GAPDH gene.
甘油醛-3-磷酸脱氢酶（GAPDH）是一种进化上保守的糖酵解酶，在大多数细胞类型中组成性表达，但在快肌纤维发育过程中会被诱导至高水平表达。为了分析鸡GAPDH基因的组织和调控，我们首先构建了一个几乎全长的GAPDH cDNA克隆（pGAD-28）。在本研究中，pGAD-28被用于筛选基因组文库，并挑选出了几个重叠克隆。通过使用M13克隆载体系统对一个4.65千碱基的Xho I/EcoRI基因组片段进行完全测序，检测到了GAPDH编码区。pGAD-28的一小部分被用作引物，从GAPDH mRNA的5'端延伸出一段33个核苷酸的序列。在mRNA的5'端22个碱基对处发现了典型的启动子“TATA”区域。GAPDH基因的5'端富含G+C（80%），并且包含两个反向序列，在距转录起始位点-58（G-G-G-G-C-G-G-G-C）和-93（G-C-C-C-G-C-C-C-C）核苷酸处发现有9个碱基对的同源性。测序还揭示了GAPDH基因转录部分内11个内含子的位置。至少3个内含子的位置与先前通过X射线晶体学检测到的原核和真核GAPDH中蛋白质结构域的边界相对应。这种一致性表明内含子可能参与了最早的GAPDH基因的构建。
