Sohocki M M, Sullivan L S, Harrison W R, Sodergren E J, Elder F F, Weinstock G, Tanase S, Daiger S P
Human Genetics Center, School of Public Health, University of Texas Health Science Center, Houston 77225, USA.
Genomics. 1997 Mar 1;40(2):247-52. doi: 10.1006/geno.1996.4604.
Two frequent protein variants of glutamate pyruvate transminase (GPT) (E.C.2.6.1.2) have been used as genetic markers in humans for more than two decades, although chromosomal mapping of the GPT locus in the 1980s produced conflicting results. To resolve this conflict and develop useful DNA markers for this gene, we isolated and characterized cDNA and genomic clones of GPT. We have definitively mapped human GPT to the terminus of 8q using several methods. First, two cosmids shown to contain the GPT sequence were derived from a chromosome 8-specific library. Second, by fluorescence in situ hybridization, we mapped the cosmid containing the human GPT gene to chromosome band 8q24.3. Third, we mapped the rat gpt cDNA to the syntenic region of rat chromosome 7. Finally, PCR primers specific to human GPT amplify sequences contained within a "half-YAC" from the long arm of chromosome 8, that is, a YAC containing the 8q telomere. The human GPT genomic sequence spans 2,7 kb and consists of 11 exons, ranging in size from 79 to 243 bp. The exonic sequence encodes a protein of 495 amino acids that is nearly identical to the previously reported protein sequence of human GPT-1. The two polymorphic GPT isozymes are the results of a nucleotide substitution in codon 14, coding for a histidine in GPT-1 and an asparagine in GPT-2, which causes a gain or loss of an NlaIII restriction site. In addition, a cosmid containing the GPT sequence also contains a previously unmapped, polymorphic microsatellite sequence, D8S421. The cloned GPT gene and associated polymorphisms will be useful for linkage and physical mapping of disease loci that map to the terminus of 8q, including atypical vitelliform macular dystrophy (VMD1) and epidermolysis bullosa simplex, type Ogna (EBS1). In addition, this will be a useful system for characterizing the telomeric region of 8q. Finally, determination of the molecular basis of the GPT isozyme variants will permit PCR-based detection of this world-wide polymorphism.
二十多年来，谷氨酸丙酮酸转氨酶（GPT）（E.C.2.6.1.2）的两种常见蛋白质变体一直被用作人类的遗传标记，尽管20世纪80年代GPT基因座的染色体定位产生了相互矛盾的结果。为了解决这一冲突并开发该基因的有用DNA标记，我们分离并鉴定了GPT的cDNA和基因组克隆。我们使用多种方法将人类GPT明确地定位到8q的末端。首先，两个显示含有GPT序列的黏粒来自8号染色体特异性文库。其次，通过荧光原位杂交，我们将含有人类GPT基因的黏粒定位到染色体带8q24.3。第三，我们将大鼠gpt cDNA定位到大鼠7号染色体的同线区域。最后，人类GPT特异的PCR引物扩增了来自8号染色体长臂的一个“半YAC”内包含的序列，即一个含有8q端粒的YAC。人类GPT基因组序列跨度为2.7kb，由11个外显子组成，大小从79到243bp不等。外显子序列编码一个495个氨基酸的蛋白质，与先前报道的人类GPT-1蛋白质序列几乎相同。两种多态性GPT同工酶是由于密码子14中的核苷酸取代导致的，该密码子在GPT-1中编码组氨酸，在GPT-2中编码天冬酰胺，这导致了一个NlaIII限制性酶切位点的获得或缺失。此外，一个含有GPT序列的黏粒还包含一个先前未定位的多态性微卫星序列D8S421。克隆的GPT基因及相关多态性将有助于对定位到8q末端的疾病基因座进行连锁和物理定位，包括非典型卵黄样黄斑营养不良（VMD1）和奥尼亚型单纯性大疱性表皮松解症（EBS1）。此外，这将是一个用于表征8q端粒区域的有用系统。最后，确定GPT同工酶变体的分子基础将允许基于PCR检测这种全球范围内的多态性。
