Makris J C, Nordmann P L, Reznikoff W S
Department of Biochemistry, University of Wisconsin-Madison 53706-1569.
Proc Natl Acad Sci U S A. 1988 Apr;85(7):2224-8. doi: 10.1073/pnas.85.7.2224.
Insertion sequence 50 (IS50) transposition utilizes a 19-base-pair "outside" end and a 19-base-pair "inside" end in inverted orientation relative to each other, whereas transposon 5 (Tn5) transposition utilizes two inverted outside ends. The frequency of transposition events that involve an inside end is regulated 1000-fold by the host dam methylase system. The end sequence requirements for transposition and its regulation by dam methylase were analyzed in Escherichia coli by generating random single base pair mutations in either an IS50 inside end or outside end placed in inverted orientation with respect to an unmutagenized outside end. The mutations were then isolated, assayed for transposition phenotype, and sequenced. Mutations were isolated at 15 of the 19 sites in the outside end. All of these mutations except those at position 4 decreased transposition. Mutations at position 4 (which is the only nonidentical base pair in a region of homology between the outside and inside ends) had no effect on transposition. Mutations were isolated at 11 of the 19 sites in the inside end. All of these mutations, including one at position 4, decreased transposition in dam- cells. Mutations at position 10 (within a dam recognition sequence) and 2 (not within a dam recognition sequence) reduced the magnitude of dam regulation. A mutation within a dam recognition sequence adjacent to the required 19 base pairs of the inside end did not reduce the magnitude of dam regulation.
插入序列50(IS50)转座利用一个19个碱基对的“外侧”末端和一个与其呈反向排列的19个碱基对的“内侧”末端,而转座子5(Tn5)转座则利用两个反向的外侧末端。涉及内侧末端的转座事件频率受宿主dam甲基化酶系统调控达1000倍。通过在与未诱变的外侧末端呈反向排列的IS50内侧末端或外侧末端产生随机单碱基对突变,在大肠杆菌中分析了转座的末端序列要求及其受dam甲基化酶的调控。然后分离这些突变,检测其转座表型并进行测序。在外侧末端的19个位点中的15个位点分离到了突变。除了第4位的突变外,所有这些突变均降低了转座效率。第4位的突变(这是外侧和内侧末端同源区域中唯一不同的碱基对)对转座没有影响。在内侧末端的19个位点中的11个位点分离到了突变。所有这些突变,包括第4位的一个突变,在dam-细胞中均降低了转座效率。第10位(在一个dam识别序列内)和第2位(不在dam识别序列内)的突变降低了dam调控的幅度。在内侧末端所需的19个碱基对相邻的一个dam识别序列内的一个突变并没有降低dam调控的幅度。