Mielke D L, Russel M
Rockefeller University, New York, NY 10021.
Gene. 1992 Sep 1;118(1):93-5. doi: 10.1016/0378-1119(92)90253-l.
The TnphoA transposon constructed by Manoil and Beckwith [Proc. Natl. Acad. Sci. USA 82 (1985) 8129-8133] has been modified to permit easy isolation of single-stranded (ss) DNA of target plasmids. The intergenic region (IG) of filamentous phage f1, which consists of the phage origin of replication and packaging signal, was inserted into a nonessential region of TnphoA. This modified transposon should be useful for the analysis of genes cloned in plasmids that lack a filamentous phage IG. Transposition of TnphoA-IG into a plasmid carries the IG with it; subsequently, after infection with a filamentous helper phage, ss plasmid DNA suitable for sequence analysis and useful for oligodeoxyribonucleotide-mediated mutagenesis of TnphoA-generated fusions can be isolated. The utility of TnphoA-IG was confirmed by analysis of 'blue hops' into the bla (encoding beta-lactamase) and pspE (encoding phage shock protein) genes whose products are secreted into the Escherichia coli periplasm.
马诺伊尔和贝克威思构建的TnphoA转座子[美国国家科学院院刊82 (1985) 8129 - 8133]已被改造，以便能够轻松分离靶质粒的单链(ss) DNA。丝状噬菌体f1的基因间隔区(IG)，它由噬菌体复制起点和包装信号组成，被插入到TnphoA的一个非必需区域。这种改造后的转座子对于分析克隆在缺乏丝状噬菌体IG的质粒中的基因应该是有用的。TnphoA - IG转座到质粒中时会携带IG；随后，在用丝状辅助噬菌体感染后，可以分离出适合进行序列分析且可用于对TnphoA产生的融合体进行寡脱氧核糖核苷酸介导诱变的ss质粒DNA。通过分析“蓝色跳跃”进入bla（编码β - 内酰胺酶）和pspE（编码噬菌体休克蛋白）基因（其产物分泌到大肠杆菌周质中），证实了TnphoA - IG的实用性。
