Small bacteriophage Mu transposable elements containing the lac operon structural genes were constructed to facilitate the isolation and use of Mu insertions and lac gene fusions. These mini-Mu elements have selectable genes for either ampicillin or kanamycin resistance and can be used to form both transcriptional and translational lac gene fusions. Some of the mini-Mu-lac elements constructed are deleted for the Mu A and B transposition genes and form stable insertions that cannot undergo transposition unless complemented for these functions. A procedure was developed for selecting mini-Mu insertions specifically into plasmids, including commonly used high-copy-number cloning vectors such as pBR322. Mu itisertions in pBR322 were found to be distributed around the plasmid, but insertions in certain regions occurred more frequently than in others.

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 ...

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The inducible tetracycline resistance determinant isolated from Proteus mirabilis cloned into the plasmid pACYC177 was mutagenized by insertion of a mini-Mu-lac phage in order to define the regions in the cloned sequences encoding the structural and regulatory proteins. Three different types of mutants were obtained: one lost the resistance phenotype and became Lac + ; another expressed the resistance at lower levels and constitutively; the third was still dependent on induction but showed a lower minimal inhibitory concentration. The mutant phenotypes and the locations of the insertions indicate that the determinant is composed of a repressor gene and a structural gene which are not transcribed divergently as are other known tetracycline determinants isolated from Gram-negative bacteria.

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