Lerner Claude G, Kakavas Stephan J, Wagner Christian, Chang Richard T, Merta Philip J, Ruan Xiaoan, Metzger Randy E, Beutel Bruce A
Global Pharmaceutical Research Division, Abbott Laboratories, R4CC, AP10-1, 100 Abbott Park Road, Abbott Park, Illinois 60064, USA.
Antimicrob Agents Chemother. 2005 Jul;49(7):2767-77. doi: 10.1128/AAC.49.7.2767-2777.2005.
Restriction enzyme modulation of transformation efficiencies (REMOTE) is a method that makes use of genome restriction maps and experimentally observed differences in transformation efficiencies of genomic DNA restriction digests to discover the location of mutations in genomes. The frequency with which digested genomic DNA from a resistant strain transforms a susceptible strain to resistance is primarily determined by the size of the fragment containing the resistance mutation and the distance of the mutation to the end of the fragment. The positions of restriction enzyme cleavage sites immediately flanking the resistance mutation define these parameters. The mapping procedure involves a process of elimination in which digests that transform with high frequency indicate that the restriction enzyme cleavage sites are relatively far away from the mutation, while digests that transform with low frequency indicate that the sites are close to the mutation. The transformation data are compared computationally to the genome restriction map to identify the regions that best fit the data. Transformations with PCR amplicons encompassing candidate regions identify the resistance locus and enable identification of the mutation. REMOTE was developed using Haemophilus influenzae strains with mutations in gyrA, gyrB, and rpsE that confer resistance to ciprofloxacin, novobiocin, and spectinomycin, respectively. We applied REMOTE to identify mutations that confer resistance to two novel antibacterial compounds. The resistance mutations were found in genes that can decrease the intracellular concentration of compounds: acrB, which encodes a subunit of the AcrAB-TolC efflux pump; and fadL, which encodes a long-chain fatty acid transporter.
限制酶介导的转化效率调节（REMOTE）是一种利用基因组限制图谱以及基因组DNA限制酶切片段转化效率的实验观察差异来发现基因组中突变位置的方法。来自抗性菌株的酶切基因组DNA将敏感菌株转化为抗性菌株的频率主要由包含抗性突变的片段大小以及突变到片段末端的距离决定。紧邻抗性突变的限制酶切割位点的位置定义了这些参数。定位过程涉及一个排除过程，其中高频转化的酶切片段表明限制酶切割位点离突变相对较远，而低频转化的酶切片段表明这些位点靠近突变。将转化数据与基因组限制图谱进行计算比较，以识别最符合数据的区域。用包含候选区域的PCR扩增子进行转化可确定抗性位点并鉴定突变。REMOTE是利用分别对环丙沙星、新生霉素和壮观霉素具有抗性的gyrA、gyrB和rpsE发生突变的流感嗜血杆菌菌株开发的。我们应用REMOTE来鉴定对两种新型抗菌化合物具有抗性的突变。抗性突变存在于可降低化合物细胞内浓度的基因中：acrB，其编码AcrAB - TolC外排泵的一个亚基；以及fadL，其编码一种长链脂肪酸转运蛋白。
