Ookawara T, Davé V, Willems P, Martin J J, de Barsy T, Matthys E, Yoshida A
Department of Biochemical Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Arch Biochem Biophys. 1996 Mar 1;327(1):35-40. doi: 10.1006/abbi.1996.0089.
The molecular abnormality of a phosphoglycerate kinase variant which was associated with severe tissue enzyme deficiency and episodes of muscle contractions and myoglobinuria was examined. Analysis of the patient's DNA showed the existence of a nucleotide transversion A/T - C/G in exon 7. No other nucleotide change was detected in the coding region of the variant gene. The mutation should produce a single amino acid substitution Glu - Ala at protein position 251 counting from the NH2-terminal acetyl serine residue. The protein abnormality caused by the amino acid substitution cannot explain the enzyme deficiency. Northern blot hybridization indicated that the PGK mRNA content of the patient's lymphoblastoid cells was only about 10% of that of normal. Nucleotide sequence analysis revealed the existence of two PGK mRNA components in the patient's cells. The major component corresponds to the normal PGK mRNA except for A - C change at nucleotide position 755 counting from adenine of the chain initiation codon. The minor component contains 5' region (52 bases) of intron 7 between exon 7 and exon 8. An inframe chain termination codon exists in the minor mRNA component, and the COOH-terminal half is expected to be deleted in the translation product. These results indicate that the low PGK activity in the patient's tissues is mainly due to retarded and aberrant pre-mRNA splicings caused by the change of the consensus 5' splice sequence AGgt to a nonconsensus sequence CGgt at the junction between exon 7 and intron 7 of the variant gene.
对一种与严重组织酶缺乏以及肌肉收缩和肌红蛋白尿发作相关的磷酸甘油酸激酶变体的分子异常进行了研究。对患者DNA的分析显示，外显子7中存在核苷酸颠换A/T - C/G。在变体基因的编码区未检测到其他核苷酸变化。该突变应在从NH2末端乙酰丝氨酸残基开始计数的蛋白质第251位产生单个氨基酸取代Glu - Ala。由氨基酸取代引起的蛋白质异常无法解释酶缺乏。Northern印迹杂交表明，患者淋巴母细胞样细胞的PGK mRNA含量仅为正常细胞的约10%。核苷酸序列分析揭示了患者细胞中存在两种PGK mRNA成分。主要成分除了从链起始密码子的腺嘌呤开始计数的核苷酸位置755处有A - C变化外，与正常PGK mRNA相对应。次要成分在第7外显子和第8外显子之间包含第7内含子的5'区域（52个碱基）。在次要mRNA成分中存在一个框内链终止密码子，预计翻译产物中COOH末端的一半会被缺失。这些结果表明，患者组织中PGK活性低主要是由于变体基因第7外显子和第7内含子交界处的共有5'剪接序列AGgt变为非共有序列CGgt，导致前体mRNA剪接延迟和异常。
