Ruvkun G B, Ausubel F M
Nature. 1981 Jan 1;289(5793):85-8. doi: 10.1038/289085a0.
The genetic analysis of genes from prokaryotic species for which experimental genetic systems have not yet been developed is often limited by the difficulty of producing mutations in those genes. We report here a general technique applicable to Gram-negative prokaryotes for site-directed mutagenesis of cloned DNA fragments which we have applied to the study of the symbiotic nitrogen fixation genes of Rhizobium meliloti. In particular, we mutagenized cloned R. meliloti restriction fragments in Escherichia coli with transposon Tn5 and then replaced the wild-type parental DNA sequences with the mutant DNA sequences in the R. meliloti genome. Using this method we show that an R. meliloti DNA restriction fragment, cloned previously on the basis of homology to Klebsiella pneumoniae nif genes, contains gene(s) essential for symbiotic nitrogen fixation. In addition, we use this method to construct a physical genetic map of a subset of the R. meliloti nif genes.
对于尚未开发出实验性遗传系统的原核生物物种的基因进行遗传分析，常常受到在这些基因中产生突变的困难的限制。我们在此报告一种适用于革兰氏阴性原核生物的通用技术，用于对克隆的DNA片段进行定点诱变，我们已将该技术应用于苜蓿根瘤菌共生固氮基因的研究。具体而言，我们用转座子Tn5在大肠杆菌中诱变克隆的苜蓿根瘤菌限制性片段，然后用突变的DNA序列替换苜蓿根瘤菌基因组中的野生型亲本DNA序列。使用这种方法，我们表明，先前基于与肺炎克雷伯氏菌nif基因的同源性而克隆的苜蓿根瘤菌DNA限制性片段包含共生固氮所必需的基因。此外，我们使用这种方法构建了苜蓿根瘤菌nif基因子集的物理遗传图谱。
