Jansen R, Kalousek F, Fenton W A, Rosenberg L E, Ledley F D
Howard Hughes Medical Institute, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Genomics. 1989 Feb;4(2):198-205. doi: 10.1016/0888-7543(89)90300-5.
The polymerase chain reaction was used to clone a full-length human methylmalonyl-CoA mutase cDNA from a human liver library by priming with sequences from the 5' end of a partial cDNA and sequences in the phage vector. The amino acid sequence predicted from the cDNA corresponds to the authentic amino acid sequences of peptide fragment from purified methylmalonyl-CoA mutase. The open reading frame of the cDNA encodes 742 amino acids (82,283 Da) comprising a 32 amino acid mitochondrial leader sequence and a mature protein of 710 amino acids (78,489 Da). The use of the polymerase chain reaction to "screen" the cDNA library represents a novel application of this technique. The full length will enable analysis of mutations underlying inherited methylmalonic acidemias caused by deficiency of the methylmalonyl-CoA mutase apoenzyme.
通过用部分互补脱氧核糖核酸（cDNA）5′端的序列和噬菌体载体中的序列进行引物延伸，利用聚合酶链反应从人肝脏文库中克隆出全长人甲基丙二酰辅酶A变位酶cDNA。从该cDNA预测的氨基酸序列与纯化的甲基丙二酰辅酶A变位酶肽片段的真实氨基酸序列相对应。该cDNA的开放阅读框编码742个氨基酸（82,283道尔顿），包括一个32个氨基酸的线粒体前导序列和一个由710个氨基酸组成的成熟蛋白（78,489道尔顿）。利用聚合酶链反应“筛选”cDNA文库代表了该技术的一种新应用。全长cDNA将有助于分析由甲基丙二酰辅酶A变位酶脱辅基酶缺乏引起的遗传性甲基丙二酸血症的潜在突变。
