Walker D C, Christodoulou J, Craig H J, Simard L R, Ploder L, Howell P L, McInnes R R
Department of Genetics, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
J Biol Chem. 1997 Mar 7;272(10):6777-83. doi: 10.1074/jbc.272.10.6777.
To determine the molecular and biochemical basis of intragenic complementation observed at the human argininosuccinate lyase (ASL) locus, we identified the ASL alleles in ASL-deficient cell strains with two unique complementation phenotypes: (i) frequent complementers, strains that participated in the majority of complementation events, and (ii) high activity complementers, strains in which complementation was associated with a relatively high level of restoration of ASL activity. Four mutations (Q286R, D87G, A398D, and a deletion of exon 13) were identified in the four strains examined. One of the two frequent complementers was homozygous, and the other heterozygous, for the Q286R allele. Similarly, one of the two high activity complementers was homozygous, and the other heterozygous, for the D87G allele. When the Q286R and D87G mutations were introduced by site-directed mutagenesis into wild-type ASL cDNA, each conferred loss of ASL activity in COS cell transfection assays. To test directly the hypothesis that intragenic complementation occurs at the ASL locus, one of the major complementation events observed previously, between strains carrying the Q286R and D87G alleles, was reconstructed in COS cell transfection assays. A partial restoration of ASL activity, comparable with the increase seen in the fibroblast complementation analysis, was observed on joint cotransfection of these two alleles. The results provide molecular confirmation of the major features of the ASL mutant complementation map, identify the Q286R and D87D alleles as the frequent and high activity complementing alleles, respectively, and provide direct proof of intragenic complementation at the ASL locus.
为了确定在人精氨琥珀酸裂解酶（ASL）基因座观察到的基因内互补的分子和生化基础，我们在具有两种独特互补表型的ASL缺陷细胞株中鉴定了ASL等位基因：（i）频繁互补株，即参与大多数互补事件的细胞株；（ii）高活性互补株，即互补与ASL活性相对较高水平的恢复相关的细胞株。在所检测的四个细胞株中鉴定出四个突变（Q286R、D87G、A398D和外显子13缺失）。在两个频繁互补株中，一个对Q286R等位基因为纯合子，另一个为杂合子。同样，在两个高活性互补株中，一个对D87G等位基因为纯合子，另一个为杂合子。当通过定点诱变将Q286R和D87G突变引入野生型ASL cDNA时，在COS细胞转染试验中，每个突变都导致ASL活性丧失。为了直接检验ASL基因座发生基因内互补的假说，在COS细胞转染试验中重建了先前观察到的携带Q286R和D87G等位基因的细胞株之间的一个主要互补事件。在这两个等位基因共同转染时，观察到ASL活性部分恢复，与成纤维细胞互补分析中看到的增加相当。这些结果为ASL突变互补图谱的主要特征提供了分子证实，分别将Q286R和D87D等位基因鉴定为频繁互补和高活性互补等位基因，并为ASL基因座的基因内互补提供了直接证据。
