Studies on Transposition Mechanisms and Specificity of IS4

The major hot spot of transposon TnS insertion in plasmid pBR322 (hot spot I) is in the promoter for the tetracycline resistance gene (tet). We made a series of pBR322 derivatives with mutations in and around this promoter and assayed their effects on insertion of TnS into hot spot I. Those mutations which reduced transcription from the tet promoter also reduced the frequency of insertion into hot spot I. Transcription and translation of tet are thought to cause the formation of paired domains of negative and positive supercoiling in pBR322. An amber codon in tet, 345 base pairs from hot spot I, decreases the negative supercoiling of the DNA segment containing hot spot I because it terminates translation of tet prematurely. We report here that this amber mutation also reduces insertion into hot spot I. These results suggest that the ability of Tn5 to insert into its major hot spot in pBR322 depends directly on negative supercoiling of the target DNA.The bacterial transposon TnS inserts into many sites within a single gene but shows a preference for certain sites. Five hot spots of TnS insertion were identified in the tet gene of plasmid pBR322 (2, 16). Of 150 independent transpositions to tet, 40 were in a single site, hot spot I, and 15 were in a second site, hot spot II. The only obvious sequence features common to TnS insertion hot spots are GC base pairs at each end of the 9 base pairs (bp) duplicated by TnS insertion. Mutation of these GC pairs to AT caused a sharp reduction of insertion into hot spot I (15). However, there are hundreds of GC pairs spaced 9 bp apart which are not hot spots, and therefore other features must also help guide Tn5 to its preferred insertion sites.TnS insertion hot spots do not match any obvious consensus sequence, either in the internal base pairs, which are equivalent to the 6-bp consensus sequence for TnJO (9), or in the flanking sequences, such as those for IS4 (12); nor are there known protein binding sites adjacent to the hot spots equivalent to integration host factor binding sites found adjacent to an IS] hot spot (8). Possibly, TnS prefers to insert into transcriptionally active DNA or into DNA with a particular topology.The 9 bp duplicated by TnS insertion at hot spot I includes the -10 region of the tet promoter (Ptet) and the transcription start site for the divergent antitet promoter (Pantitet) (for a review, see reference 1) (Fig. 1). To determine whether the activities of these promoters affect TnS insertion into hot spot I, a series of deletion and insertion mutations in and near the promoters were made and the effects of these mutations on TnS insertion into hot spot I were assessed. The results presented here suggest that transcription or coupled transcription and translation from the tet promoter is important in hot spot I selection.

mentioning

confidence: 99%

Mutations that affect Tn5 insertion into pBR322: importance of local DNA supercoiling

Lodge

Berg

1990

“…Insertional hot spots have been found for most transposable elements, with consensus sequences often present at the location of the insertions (2,8,10,15,17,19).…”

mentioning

confidence: 99%

Specificity of mini-Mu bacteriophage insertions in a small plasmid

Castilho

Casadaban

1991

Target site selection for bacteriophage Mu transposition was studied in pools of over 107 independent mini-Mu insertions in pUC9, selected by transduction of the plasmid. Insertions in both orientations were clustered in three regions and, within these, at preferred sites.Bacteriophage Mu insertions within defined genetic intervals have indicated that insertions occur preferentially at certain locations (3,4,6,12,13). Insertional hot spots have been found for most transposable elements, with consensus sequences often present at the location of the insertions (2,8,10,15,17,19).The insertion specificity of Mu at the nucleotide level was investigated with pools of insertions of the mini-Mu Mu dII4041 (4) in pUC9 (16, 18), selected as described in Fig. 1. This procedure yields approximately 5 x 104 independent transductants per ml of lysate, 95% of which contain pUC9 plasmids with mini-Mu insertions. These should represent, on a random basis, the saturation of pUC9 with insertions at all possible positions, in both orientations. In order to reduce the statistical fluctuations in the insertion distribution, the experimental procedures were scaled up to obtain pools of over 107 transductants.BamHI digestion of plasmid DNA extracted from pools of the Kmr transductants should result in two sets of fragments: one, ranging from 7.5 to 10.2 kb, containing the left end of Mu dII4041 plus a variable length of plasmid sequences; and another set, from 117 bp to 2.8 kb, containing the right terminus of Mu dII4041 and the remaining plasmid sequences. If insertions were random, these would each form a smear on a gel within their size ranges, except for interruptions from the lack of insertions in the essential replication region of the plasmid. Figure 2 shows the pattern of the small BamHI fragments on an acrylamide gel, implying that insertions are clustered in discrete regions. This pattern is reproducible in different lysates and with different lysogens, except for one set of fragments (C) present only in pools 4 and 5. Pooled insertion plasmid DNA cut with EcoRI, which has only one recognition site in the pUC9 plasmid and none in the mini-Mu, generated only one fragment of 10.2 kb, indicating that the majority of the plasmids in the pool had only one insertion of Mu dII4041 without detectable rearrangements (data not shown).The orientation of the insertions originating this pattern of fragments was determined (Fig. 3) labeled with _y32p. The EcoRI-digested DNA was then digested with BamHI. Both samples were electrophoresed on a 4% acrylamide gel. The size standard was pBR322 plasmid DNA digested with Hinfl and end labeled with y_32p. A similar experiment was performed to have the insertions in the (+) orientation only labeled, digesting the pool with Sall, instead of EcoRI, which confirmed these results (data not shown). By labeling the plasmid pools at the BamHI ends, fragments F and G, corresponding to insertions adjacent to the plasmid BamHI site, could also be visualized. Three preferred regions for Mu insertions in pUC9 ...

“…A DNA segment which is flanked by the same IS elements can also transpose together with the IS elements as a unit called a composite transposon (Tn). Transposition of IS and Tn elements occurs at various sites with or without specificity (2,8,11,15,22,29).IS630 is a 1,153-bp element present in the Shigella sonnei chromosome in multiple copies (19,29). A composite transposon, Tn4731, which has two inverted IS630 insertion sequences that flank the sequence of the tetracycline resistance plasmid pHS1, has been shown to transpose to the dinucleotide 5'-TA-3' in the core of at least 4-bp palindromic sequences, such as CTAG, TTAA, and ATAT, in ColEl in Escherichia coli (29).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Preferential transposition of an IS630-associated composite transposon to TA in the 5'-CTAG-3' sequence

Tenzen

Ohtsubo

1991

A composite transposon, Tn4731, associated with IS630 has been shown to transpose preferentially to 5'-TA-3' sequences that are located at two sites in a rho-dependent transcription terminator in plasmid ColEl in Escherichia coli (T. Tenzen, S. Matsutani, and E. Ohtsubo, J. Bacteriol. 172:3830-3836, 1990). Here we demonstrated that Tn4731 preferentially transposes to TA sequences at four sites in plasmid pUC118 and its derivatives: the TA sequence (hot spot I) in the intergenic region of phage M13 within the pUC sequence, the TA sequence (hot spot II) in the XbaI site in multiple cloning sites of the lacZ coding region, the TA sequence (hot spot III) in a spacer region flanked by inverted repeat sequences of a transcription terminator located downstream of the bla gene, and the TA sequence (hot spot IV) in the middle of bla. Transposition of Tn4731 to hot spot III was found not to require the inverted repeats in the terminator. Transposition of Tn4731 to hot spot II, which is located immediately downstream of the lacZ promoter, was not affected by mutations introduced into the promoter. There appear to be no particular sequences important for transposition of Tn4731 around each of the hot spots, except a palindromic sequence, 5'-CTAG-3', that contains the target sequence. Mutations introduced within the CTAG sequence at a hot spot inhibited Tn4731 from transposing to it, indicating that the CTAG sequence is responsible for the preferential transposition of Tn4731.An insertion sequence (IS) is a discrete DNA segment which can transpose from one site to another in bacterial plasmids, chromosomes, and phages (for a recent review, see reference 5). A DNA segment which is flanked by the same IS elements can also transpose together with the IS elements as a unit called a composite transposon (Tn). Transposition of IS and Tn elements occurs at various sites with or without specificity (2,8,11,15,22,29).IS630 is a 1,153-bp element present in the Shigella sonnei chromosome in multiple copies (19,29). A composite transposon, Tn4731, which has two inverted IS630 insertion sequences that flank the sequence of the tetracycline resistance plasmid pHS1, has been shown to transpose to the dinucleotide 5'-TA-3' in the core of at least 4-bp palindromic sequences, such as CTAG, TTAA, and ATAT, in ColEl in Escherichia coli (29). There are two hot spots for transposition of Tn4731 within each of the inverted repeat sequences of 13 bp in a rho-dependent transcription terminator located downstream of the cea gene in ColEl (29).To clarify the determinants for the site-specific transposition of Tn4731, we have studied transposition of Tn4731 to pUC118 and its derivatives. We report here that Tn4731 can transpose also to TA sequences at four sites in pUC118 and its derivatives. We also show that the preferential transposition is determined by the CTAG sequence containing the target site, but not by the sequences surrounding the CTAG sequence. The two transpositional hot spots previously identified in ColEl by Tenzen et al. (29) are actu...