Endo H, Hasegawa K, Narisawa K, Tada K, Kagawa Y, Ohta S
Department of Biochemistry, Jichi Medical School, Tochigi-ken, Japan.
Am J Hum Genet. 1989 Mar;44(3):358-64.
A patient with lactic acidosis showed a lowered pyruvate dehydrogenase E1 activity and fatigued on slight exercise. The cDNA encoding the pyruvate dehydrogenase E1 alpha-subunit from his lymphocytes, transformed by infection of Epstein-Barr virus, was cloned and sequenced. The nucleotide sequence determination revealed that the gene had a deletion of four nucleotides at the second codon upstream from the termination codon. This deletion would lead to a reading-frame shift and make a new termination codon at the 33d codon downstream from the "normal" termination codon. An S1 nuclease-protection experiment confirmed the presence of mRNA with its deletion in the patient. Amplification, by the polymerase chain reaction method, of the genomic-DNA region from his peripheral blood cells showed that the deletion was localized in an exon and that it was not caused by an abnormal splicing at the intron/exon junction. This is the first report on cloning a defective gene of the pyruvate dehydrogenase complex.
一名乳酸酸中毒患者丙酮酸脱氢酶E1活性降低，轻微运动后即感到疲劳。通过感染爱泼斯坦-巴尔病毒对其淋巴细胞中编码丙酮酸脱氢酶E1α亚基的cDNA进行转化，然后进行克隆和测序。核苷酸序列测定显示，该基因在终止密码子上游第二个密码子处有四个核苷酸缺失。这种缺失会导致读框移位，并在“正常”终止密码子下游第33个密码子处产生一个新的终止密码子。S1核酸酶保护实验证实该患者存在带有缺失的mRNA。通过聚合酶链反应方法对其外周血细胞基因组DNA区域进行扩增，结果表明该缺失位于一个外显子中，并非由内含子/外显子交界处的异常剪接所致。这是关于克隆丙酮酸脱氢酶复合体缺陷基因的首次报道。
