Krebs M P, Reznikoff W S
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Gene. 1988 Mar 31;63(2):277-85. doi: 10.1016/0378-1119(88)90531-8.
We constructed a derivative of Tn5, Tn5 ORFlac, that is capable of creating lacZ translational fusions upon transposition. Lac- strains carrying this construct formed red papillae when plated on MacConkey-lactose media. Lac+ cells isolated from independent papillae expressed distinct beta-galactosidase fusion proteins, suggesting that the Lac+ phenotype resulted from transposition. In support of this, analysis of plasmids carrying Tn5 ORFlac prepared from these cells indicated that the Lac+ phenotypes arose as a result of intermolecular rearrangements. Furthermore, a derivative of Tn5 ORFlac that contains an ochre mutation in the transposase gene formed papillae only in a supB strain. Tn5 ORFlac is useful for obtaining mutants that affect Tn5 transposition and for creating lacZ fusions. We used the papillation phenotype to isolate a spontaneous revertant of IS50L that promotes transposition at a 3.6-fold higher rate than IS50R. The mutation altered the amino acid sequence of both transposase and inhibitor.
我们构建了Tn5的一个衍生物Tn5 ORFlac，它能够在转座时产生lacZ翻译融合体。携带此构建体的Lac-菌株在麦康凯乳糖培养基上平板培养时形成红色乳头状菌落。从独立乳头状菌落中分离出的Lac+细胞表达不同的β-半乳糖苷酶融合蛋白，这表明Lac+表型是由转座产生的。支持这一点的是，对从这些细胞制备的携带Tn5 ORFlac的质粒进行分析表明，Lac+表型是分子间重排的结果。此外，在转座酶基因中含有一个赭石突变的Tn5 ORFlac衍生物仅在supB菌株中形成乳头状菌落。Tn5 ORFlac可用于获得影响Tn5转座的突变体以及创建lacZ融合体。我们利用乳头状菌落表型分离出IS50L的一个自发回复突变体，其促进转座的速率比IS50R高3.6倍。该突变改变了转座酶和抑制剂的氨基酸序列。
