Shull J D, Pitot H C
Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha 68105.
In Vitro Cell Dev Biol. 1989 Oct;25(10):971-6. doi: 10.1007/BF02624012.
Gyrate atrophy (GA), a degenerative disease of the human chorioretina, is associated with a deficiency of ornithine aminotransferase (OAT) activity, hyperornithinemia, and ornithinuria. We have characterized a cDNA clone for OAT (HLOAT) that was isolated from a cDNA library constructed from mRNA prepared from Hep G2 cells, a human hepatoma cell line. We have used HLOAT and a nearly full length OAT cDNA clone isolated from a rat liver library (RLOAT) to examine in cultured fibroblasts from individuals with GA and control individuals, the expression of OAT mRNA and the gross structure of the OAT gene. Northern blot analyses of total cellular RNA indicated that 3 of 3 control cell lines and 5 of 6 GA cell lines are capable of expressing an OAT related mRNA of approximately 2100 bases, the size of OAT mRNA. To date, this is the only case of GA in which a complete lack of OAT mRNA has been observed. Southern blot analyses of DNA isolated from these cell lines indicated that the gross structure of the OAT gene is usually not detectably altered in individuals with GA. However, a unique pattern of restriction fragments was observed upon digestion with Eco RI or Hind III of DNA from the GA cell line that does not express OAT mRNA. These unique Eco RI and Hind III fragments arise from the OAT structural gene and will serve as useful molecular markers that allow this particular defective OAT allele to be identified. When the cellular DNAs were digested with Hinf I and examined with a probe that corresponds to at least a portion of the active site of the enzyme, i.e., the pyridoxal phosphate binding site, identical patterns of fragments were detected in all samples. Therefore, it appears unlikely that the loss of OAT activity associated with these GA cases, 4 of which are pyridoxal phosphate responders, is the result of insertions or deletions in this region of the OAT gene. This study indicates that the lack of OAT enzyme activity associated with GA is the result of a variety of different molecular defects within the OAT gene.
回旋状萎缩（GA）是一种人类脉络膜视网膜的退行性疾病，与鸟氨酸转氨酶（OAT）活性缺乏、高鸟氨酸血症和鸟氨酸尿有关。我们鉴定了一个OAT的cDNA克隆（HLOAT），它是从由人肝癌细胞系Hep G2细胞制备的mRNA构建的cDNA文库中分离出来的。我们使用HLOAT和从大鼠肝脏文库中分离出的一个几乎全长的OAT cDNA克隆（RLOAT），来检测GA患者和对照个体的培养成纤维细胞中OAT mRNA的表达以及OAT基因的总体结构。对总细胞RNA的Northern印迹分析表明，3个对照细胞系中的3个以及6个GA细胞系中的5个能够表达一种约2100个碱基的与OAT相关的mRNA，即OAT mRNA的大小。迄今为止，这是唯一一例观察到完全缺乏OAT mRNA的GA病例。对从这些细胞系中分离的DNA进行Southern印迹分析表明，GA患者中OAT基因的总体结构通常没有可检测到的改变。然而，在用Eco RI或Hind III消化不表达OAT mRNA的GA细胞系的DNA时，观察到了一种独特的限制性片段模式。这些独特的Eco RI和Hind III片段来自OAT结构基因，将作为有用的分子标记，用于识别这个特定的有缺陷的OAT等位基因。当用Hinf I消化细胞DNA并用与该酶的至少一部分活性位点（即磷酸吡哆醛结合位点）相对应的探针进行检测时，在所有样品中检测到相同的片段模式。因此，与这些GA病例（其中4例是磷酸吡哆醛反应者）相关的OAT活性丧失似乎不太可能是OAT基因该区域插入或缺失的结果。这项研究表明，与GA相关的OAT酶活性缺乏是OAT基因内多种不同分子缺陷的结果。
