We describe three related DNA alterations associated with transposon Tn10: precise excision of Tn10, nearly precise excision of Tn10 and precise excision of the nearly precise excision remnant. DNA sequence analysis shows that each of these alterations results in excision of all or part of the Tn10 element, and each involves specific repeat sequences at or near the ends of the element. Furthermore, all three events are structurally analogous: in each case, excision occurs between two short direct-repeat sequences, with resulting deletion of all intervening material plus one copy of the direct repeat; and in all three cases, the direct repeats involved occur at either end of an inverted repeat. Analysis of mutant Tn10 elements and characterization of bacterial host mutations suggest that all three types of excision events occur by pathways that are fundamentally distinct from the pathway(s) for Tn10-promoted transposition and other DNA rearrangements (deletions and inversions) actively promoted by the element. In addition, precise excision and nearly precise excision appear to occur by very closely related or identical pathways; and several lines of evidence suggest that the 1400 bp inverted repeats at the ends of Tn10 may play a structural role in both of these events. The third excision event appears to occur by yet another pathway.

The sequences of insertions of the translocatable tetracycline-resistance element Tn10 into the repressor (cl) gene of bacteriophage lambda have been analyzed. Each insertion contains the same discrete set of Tn10 sequences flanked by a direct repetition of a 9 bp cl-gene sequence. The flanking repititions are generated by duplication of information present only in the target DNA molecule rather than by a Campbell-type recombination event between one 9 bp sequence on the target DNA and a second one provided on the incoming element. The repetitions do not contain genetic or structural information important for translocation. A genetically constructed Tn10 insertion which lacks flanking repetitions is fully functional in translocation to a new position. Tn10 insertions cluster at preferred positions along a target DNA (Kleckner et al., 1979). Sequence analysis shows that four independently isolated cl::Tn10 insertions occur at identical positions in the cl gene. We speculate that homology between Tn10 and its target, at some distance from the site of the actual recombination event, could be relevant to the preference of Tn10 for particular insertion sites.

By heteroduplex and hybridization analysis we showed that the inverted repetition (here called IS10) at the ends of the translocatable tetracycline resistance element Tn10 is not IS3, as had previously been reported by Ptashne and Cohen (J. Bacteriol. 122:776--781, 1975). Further analysis confirmed the homology between IS3 and the alpha beta sequence of F and demonstrated that IS10 was not present in the genomes of Salmonella typhimurium LT2 or Escherichia coli K-12.

We describe an unusual DNA alteration, "nearly precise excision," which has been identified among tetracycline-sensitive deletion derivatives of lambda phages carrying the translocatable tetracycline-resistance element Tn10. DNA sequence analysis of two such derivatives demonstrates that each retains exactly 50 bp of Tn10 material. The original junctions between lambda and Tn10 sequences remain intact; however, an internal deletion has occurred within Tn10 which eliminates all but the last few base pairs at each end of the element. This deletion occurs within a short A + T-rich inverted repeat which is present near each end of Tn10. Nearly precise excisions occur at frequencies comparable to Tn10-promoted deletions, inversions and translocations, and, like these other events, are independent of phage and bacterial functions for homologous recombination (recA, recB, red). It is not yet clear, however, whether nearly precise excisions are specifically promoted by Tn10 or whether they arise during the course of normal DNA replication processes as a consequence of unusual symmetries present in the DNA sequence at the ends of Tn10.

We report here the physical structures of deletions and inversions promoted by the translocatable tetracycline-resistance element Tn10. DNA/DNA heteroduplex and restriction enzyme analyses of alterations in the genome of bacteriophage lambda suggest that both types of DNA alterations almost always originate at the internal termini of the 1400 bp terminal inverted repetitions of Tn10. Tn10-promoted deletions remove a single contiguous DNA segment beginning at one such terminus; Tn10-promoted inversions are more complex, and involve both an inversion and a specific deletion of Tn10 DNA.

Genetic analysis of 131 independent transpositions of the tetracycline-resistance element Tn10 from a single site in phage P22 into the histidine operon of Salmonella typhimurium reveals that Tn10 insertions are not randomly distributed along this chromosomal target. The insertions occur in 22 different "clusters"; insertions within each cluster are very tightly linked in recombination tests. Tn10 insertions are not evenly distributed among the identified clusters. The existence of these clusters suggests that this chromosomal target contains particular genetic signals that guide Tn10 to particular preferred positions for insertion. Insertions within each cluster occur in both orientations with roughly equal frequency.--The relationship among different insertions within each cluster has been examined. The resolution of genetic mapping places an upper limit of about 50 basepairs on the distance between different insertions within a cluster. Different insertions within a cluster usually have the same reversion frequency; however, heterogeneity in reversion frequency has been detected in at least two clusters. For most clusters, the available data are consistent with the simple possibility that all insertions within a cluster are at identical positions; however, the data do not exclude other possibilities.

Insertions of Tn10 are characterized by the presence of a direct repetition of a 9-bp target gene sequence on either side the insertion. The occurrence of these repetitions undoubtedly reflects an important feature of the way in which DNA molecules are broken and joined during translocation. Our experiments further suggest, however, that these 9-bp sequences are probably not responsible for Tn10-insertion specificity and that their presence is not required for normal Tn10 translocation elsewhere. We therefore suggest that the genetic information which controls the quality and quantity of Tn10 translocation actually resides somewhere other than these 9-bp sequences. We presume that much of this information lies within the ends of Tn10 itself and that signals on the target DNA which guide Tn10 to preferred positions must occur near, but not actually at, the eventual physical site of the integration event. Consideration of Tn10-promoted deletions and inversions reemphasizes the role of these ends in Tn10-promoted recombination events. Since Tn10-promoted events almost always consist in joining the physical end of one of the putative IS sequences of Tn10 to some other DNA segment, one comes again to the conclusion that these ends must contain important genetic information governing recombination events.

A number of independent insertions into bacteriophage lambda of the translocatable tetracycline-resistance element Tn10 have been isolated and characterized. The physical positions and relative orientations of several such insertions were determined. Two independent insertions appear to lie in the same orientation at or very near the same site in the cI gene, and two more lie in opposite orientations at or near the same position in or near the rex gene. Insertions in or near genes cI, rex, and cIII have been characterized genetically for their effects on expression of nearby genes. Tn10 appears to exert a polar effect on expression of distal genes when it is inserted within an operon, even when expression of that operon is under the influence of lambda N-function. In addition, Tn10 insertions in rex appear to influence in some way expression of an "upstream" gene, cI. Lambda derivatives carrying Tn10 give rise to spontaneously occurring, tetracycline-sensitive deletions at high frequencies. It is likely that formation of these deletions is promoted in some way by the Tn10 element. Lambda::Tn10 phages carrying a Tn10 element that has undergone several successive cycles of translocation since its first isolation and characterization have been analyzed. The results confirm that Tn10 often retains its physical and functional integrity during many cycles of translocation. Lambda derivatives carrying Tn10 have been used to generate insertions of Tn10 in the chromosome of Escherichia coli. This process is independent of recA function, and seems to be quite analogous to the translocation of Tn10 in Salmonella typhimurium as studied previously.

Individual progeny of two-factor crosses between A1am and A2am T5 phages give rise to bursts containing more than one type of plaque. The simplest explanation for these mixed bursts is that the A1 and A2 genes are located within the terminally repeated portion of the T5 genome and that the mixed bursts are made by "terminal redundancy heterozygotes". The observation of genetic heterozygosity means that the A1 and A2 genes are repeated intact. This implies that the terminal segments of T5 are genetically interchangeable.