IS4 is found between eleven or twelve base pair duplications

Summary Three mutations caused by the integration of IS4 in galT in both possible orientations were shown by DNA sequence analysis to be integrated between a duplication of eleven base pairs of gene galT.IS4 has been cloned from its single position on the E. coli K12 chromosome. Here, 12 base pairs are duplicated adjacent to IS4. This sequence is unrelated to the duplicated sequence in galT.     

IS4 at its chromosomal site in E. coli K12

Summary IS4 DNA has been isolated in pure form. Hybridization of this DNA against restricted DNA of several E.coli K12 strains by Southern's blotting technique has shown that, in most strains, only one copy of IS4 is present. Though the restriction fragments around this site differ in size, IS4 can be shown always to be located at the same site. In one strain, one additional copy has been found in a new location. In this strain, IS4 in its original location has been retained.     

Isolation of the mini insertions IS6 and IS7 of E. coli

Summary The mini IS elements IS6 and IS7 have been detected in constitutive gal ⁺ revertants of galOP-308::IS2 (I), in which the expression of the gal operon is turned off by IS2 in orientation I. Both, IS6 and IS7, are integrated into IS2 proximal to the gal structural genes. IS6 is 115 base pairs long and causes 50% constitutive expression of the gal genes. IS7 is only 65 base pairs long and the gal operon is expressed 20% constitutively compared to the gal ⁺ wild type operon. Both IS6 and IS7 are excised frequently, in the absence of selective pressure. These findings are discussed with respect to the evolution of gene expression.     

Close vicinity of IS1 integration sites in the leader sequence of the gal operon of E. coli.

Summary Four insertions of IS1 in the leader sequence of the gal operon of E. coli have been analysed.Two of them occur at the same position, but in opposite orientations. The other two are inserted one nucleotide to one side and four nucleotides to the other side, respectively.In each case, nine base pairs of the leader sequence of the gal operon are duplicated directly, and are found flanking the termini of IS1 at its junction with the gal operon. These repeated sequences differ from each other as expected from the different insertion sites.     

IS<Emphasis Type="Italic">Ppy1</Emphasis>, a novel mobile element of the ancient <Emphasis Type="Italic">Psychrobacter maritimus</Emphasis> permafrost strain: Translocations in <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 cells and formation of composite transposons

It was shown that IS element ISPpy1 isolated earlier in the permafrost strain Psychrobacter maritimus MR29-12 has a high level of functional activity in cells of the heterologous host Escherichia coli K-12. ISPpy1 can be translocated in E. coli cells by itself and mobilize adjacent genes and can also form composite transposons flanked by two copies of this element. Apart from translocations between different plasmids, the composite ISPpy1-containing transposon Tn5080a is capable of translocation from the plasmid into the E. coli chromosome with high frequency and from the chromosome into the plasmid. Among products of Tn5080a transposition into plasmid R388, simple insertions were predominantly formed together with cointegrates. Upon mobilization of adjacent genes with the use of one ISPpy1 copy, only cointegrates arise.     

Absence of insertions among spontaneous mutants of Salmonella typhimurium

Summary While insertion sequences (IS) in Escherichia coli transpose frequently to generate spontaneous insertion mutants, such mutations are rare in Salmonella typhimurium: the only documented insertion mutation is a hisD mutation caused by the Salmonella-specific IS element IS200. To obtain more examples of IS200 insertion mutations and to seek additional types of IS elements in Salmonella, we selected and characterized 422 independent, spontaneous His^– mutants and some 2100 additional mutants that are not necessarily independent. None of the mutants showed the absolute polar effect characteristic of insertion mutations or the reversion properties characteristic of insertions (low spontaneous reversion frequency and no reversion induction by chemical mutagens). A few mutants, showing a high spontaneous reversion frequency, were screened physically. No insertion mutations were found. Thus insertion mutations appear to be rare in S. typhimurium, in strong contrast to E. coli and despite the possession in Salmonella of at least one type of insertion element (IS200). These results suggest that in Salmonella transposition of the endogenous elements has been controlled. The transposition ability of the elements may have been reduced or favored target sites removed from the host genome.     

Nature of deletions formed in response to IS2 in a revertant of the gal3 insertion of E. coli.

Summary The gal3 mutation of E. coli, which arose by the insertion of IS2 in the OP region of the gal operon, reverts spontaneously by excision of the IS2 to produce inducible revertants or by mutational alterations of IS2 to produce constitutive revertants. However, gal3() strains bearing chlD-pgl deletions produce constitutive revertants alone. We proposed that deletions formed in the presence of IS2 terminate specifically at its right end, so that revertants arising by excision of IS2 fuse the gal genes to other promoters. Therefore, the revertants are exclusively constitutive.The above hypothesis was tested by electron microscopy of IS2-specific deletions. Spontaneous chlD-pgl deletions were isolated from gal ^c331 (a revertant of gal3 which retains IS2) and transferred to gal genomes. Electron microscopy of DNA heteroduplexes from these phages confirmed that all of the deletions examined have one end-point fixed at the right end of IS2, whereas their other end-points are variable. In each case, the complete IS2 element was apparently retained. This specificity was also detectable in a revertant (gal ^c200) which retains only the right 1/5 portion of the IS2. The frequencies of these deletions were generally increased in constitutive revertants of gal3. Since a galO ^cmutant did not show a similar increase, it seems that this effect depends upon a base sequence provided by IS2. Moreover, the presence of prophage contributes to the specificity and, in some instances, the frequency of IS2-specific deletions.A mechanism for the formation of the IS2-specific deletions has been proposed. A base sequence located at, or near, the right end of IS2 is recognized and nicked by a specific endonuclease. The nick is enlarged by unidirectional, exonucleolytic degradation to produce deletions extending outwards from the insertion. In constitutive revertants, the nicking site may be exposed to endonucleolytic attack more frequently.     

Replicon fusions promoted by insertion sequences on Pseudomonas cepacia plasmid pTGL6

Summary Plasmid pMR5 (pRP1ts) failed to replicate in Pseudomonas cepacia at 47° C. Selection at this temperature for maintenance of tetracycline resistance associated with this plasmid allowed isolation of cointegrate plasmids formed by fusion of pMR5 with pTGL6, a 170 kb plasmid harbored by P. cepacia 249. In the cointegrate plasmids pTGL100, pTGL101, and pTGL102, different regions of pTGL6 were involved in fusion with the same tra-2-containing region of pMR5. Formation of all three plasmids was promoted by insertion sequences on pTGL6, which were also represented in the chromosome.Two different copies of a 1.3 kb element, IS401, were involved in formation of pTGL100 and pTGL101. Another insertion sequence, IS402 (1 kb), promoted the fusion which formed pTGL102. Southern hybridization experiments indicated that each of the cointegrate plasmids contained an additional copy of the fusion mediating element. Plasmid pTGL100 was observed to resolve into two independent replicons: pTGL6 and pTGL105 (pMR5::IS401), a novel derivative of pMR5 containing a copy of IS401.The third cointegrate plasmid, pTGL102, evolved in two steps: fusion of pTGL6 and pMR5 mediated by IS402, and transposition of IS411 (1.9 kb) to a region of pMR5 distinct from that involved in the fusion. Plasmid pTGL6 contained one copy of IS402 and IS411 while pTGL102 contained two copies of each of these elements.     

Isolation, characterization and transposition of an (IS2)2 intermediate

We have isolated and characterized a dimer derivative of the extensively studiedEscherichia coli insertion sequence IS2. The dimer structure — called (IS2)₂ — consists of two IS2 elements arranged as a direct repeat, separated by 1 bp. The junction between the (IS2)₂ dimer and target sequences is located at various positions in independent isolates; however, one position was preferred. The transposition of (IS2)₂ into a target plasmid resulted in cointegrate-type structures. The transposition frequency of the (IS2)₂ dimer itself was significantly higher than that of the isogenic monomer IS2 insertion. The poor stability and high activity of (IS2)₂ indicates that this is an active transposition intermediate. The mode of transposition of (IS2)₂ is analogous to the joined dimer model described in the case of (IS21)₂ and (IS30)₂.     

Formation of the tandem repeat (IS30)2 and its role in IS30-mediated transpositional DNA rearrangements

Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS30 elements. The resulting covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp. This (IS30)₂ structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA ^– bacteria, replicated copies of the introduced (IS30)₂ structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS30 transposition, various transposition products of (IS30)₂ are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS30 transposase gene and of both proximal ends of the two IS30 elements concerned not only for the formation of (IS30)₂, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS30)₂ intermediates play a key role in IS30 transposition pathways in which the formation of (IS30)₂ may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS30)₂. Evolutionary implications of these findings are discussed.     

Non-random distribution of deletion endpoints in the gal operon of E. coli

Summary The physical distances between endpoints of deletions in the gal operon of dgal phages previously isolated and mapped by genetic methods have been measured by electron microscopy of heteroduplex DNA. Thus, the galT and galK sites previously mapped by genetic methods, may be assigned to 13 and 4 intervals, respectively, of known physical lengths. The physical data clearly show a nonrandom distribution of these deletion endpoints.On the other hand, a large number of deletions in the gal operon obtained as survivors of a _cI857 lysogenic strain at 42°C also show a non-random distribution of endpoints of deletions in the gal genes. There are at least two sites in the gal genes at which we observed a clustering of endpoints of deletions within the same regions of dgal del-phages and in the bacterial deletion mutants.     

Demonstration of IS<Emphasis Type="Italic">711</Emphasis> transposition in <Emphasis Type="Italic">Brucella ovis</Emphasis> and <Emphasis Type="Italic">Brucella pinnipedialis</Emphasis>

Background  

The Brucella genome contains an insertion sequence (IS) element called IS711 or IS6501, which is specific to the genus. The copy number of IS711 varies in the genome of the different Brucella species, ranging from 7 in B. abortus, B. melitensis and B. suis to more than 30 in B. ovis and in Brucella strains isolated from marine mammals. At present, there is no experimental evidence of transposition of IS711, but the occurrence of this element with a high copy number in some species, and the isolation of Brucella strains with "ectopic" copies of IS711 suggested that this IS could still transpose.     

Evidence for a prokaryotic insertion-sequence contamination in eukaryotic sequences registered in different databases

An insertion-sequence of prokaryotic origin was detected in a genomic clone obtained from a Phaseolus vulgaris bacterial artificial chromosome (BAC) library. This BAC clone, characterized as part of a contig constructed near a virus resistance gene, exhibited restriction fragment length polymorphism with an overlapping clone of the contig. Restriction analysis of DNA obtained from individual colonies of the stock culture indicated the presence of a mixed population of wild-type and insertional mutants. Sequence analysis of both members of the population revealed the presence of IS10R, an insertion-sequence from Escherichia coli. A BLAST search for IS10-like sequences detected unexpected homologies with a large number of eukaryotic sequences from Homo sapiens, Arabidopsis thaliana, Drosophila melanogaster and Caenorhabditis elegans. Southern analysis of a random sample of BAC clones failed to detect IS10 in the BAC DNA. However, prolonged sub-culturing of a set of 15 clones resulted in transposition into the BAC DNA. Eventually, all cultures acquired a 2.3-kb fragment that hybridized strongly with IS10. Sequence analysis revealed the presence of a preferred site for transposition in the BAC vector. These results indicate that a large number, if not all, of the BAC libraries from different organisms are contaminated with IS10R. The source of this element has been identified as the DH10B strain of E. coli used as the host for BAC libraries. Received: 5 December 2000 / Accepted: 25 April 2001     

Inter- and intramolecular transposition of Tn903.

Summary We have performed a detailed analysis of intra-and intermolecular endproducts of transposition of the compound transposon Tn903 and we show that, in our system, the transposition activity is almost entirely driven by one of the flanking insertion sequences, IS903L. The relatively inactive state of IS903R can be conferred on IS903L by changing the orientation of the internal Tn region. IS903L mediates the formation of the majority of adjacent deletions, insertion/inversions nd cointegrates, all of which are representative of replicative transposition; only a very low level of conservative transposition can be observed. Our results are discussed in relation to those showing that Tn903 uses predominantly the conservative pathway.     

Cointegrate formation by IS50 requires multiple donor molecules

Summary The insertion sequence, IS50R, promotes cointegrate formation between a lambda::IS50R phage and the chromosome of Escherichia coli strain C. We show that formation of cointegrates mediated by IS50R between the non-replicating phage genome and the bacterial chromosome requires multiple donor molecules and depends on homologous recombination functions. We conclude that the two copies of IS50 present in the cointegrate originate in two different molecules. Thus, the existence of the cointegrate structure cannot be used as evidence for replication of IS50 sequences during IS50 transposition.     

ISDra2 transposition in Deinococcus radiodurans is downregulated by TnpB

Transposable elements belonging to the recently identified IS200/IS605 family radically differ from classical insertion sequences in their transposition mechanism by strictly requiring single‐stranded DNA substrates. This IS family includes elements encoding only the transposase (TnpA), and others, like ISDra2 from Deinococcus radiodurans, which contain a second gene, tnpB, dispensable for transposition and of unknown function to date. Here, we show that TnpB has an inhibitory effect on the excision and insertion steps of ISDra2 transposition. This inhibitory action of TnpB was maintained when ISDra2 transposition was induced by γ‐irradiation of the host cells and required the integrity of its putative zinc finger motif. We also demonstrate the negative role of TnpB when ISDra2 transposition was monitored in a heterologous Escherichia coli host, indicating that TnpB‐mediated inhibition does not involve Deinococcus‐specific factors. TnpB therefore appears to play a regulatory role in ISDra2 transposition.     

Two kinds of insertions in bacterial genes

Summary Six insertion mutations in the gal operon of E. coli and two insertion mutations in the xycIIOP operon of bacteriophage lambda were tested for homology by annealing separated strands of lambda dgal DNA carrying the insertions, and inspection in the electron microscope.Class 1, consisting of the gal mutations OP 128, OP 141, T-N 116, OP 306, T-N 102 and the lambda mutation r14 are about 800 nucleotide pairs long, completely homologous and not circularly permuted. The first three insertions of class 1 are integrated in one direction with respect to the adjacent genes, the other three in the opposite direction. The DNA inserted in this class of mutations is called IS1.Class 2 consists of the gal insertion OP 308 and the lambda insertion r32. They are about 1400 nucleotide pairs long. The two are integrated in opposite direction with respect to the chromosome of dgal. The DNA in insertion mutations of class 2 will be called IS 2. IS1 and IS2 do not share any detectable homology.These data are supported by cross-hybridization experiments using RNA transcribed in vitro from lambda dgal or lambda DNA carrying one insertion and DNA carrying either the same or a different insertion.Similar results were obtained by Malamy, Fiandt, Szybalski and Fiandt, Szybalski, Malamy (accompanying papers).     

Transposon mutagenesis of coryneform bacteria

The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831. The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria. Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 10⁴ mutants per microgram DNA. Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants. Auxotrophs could be recovered at a frequency of approximately 0.2%. Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%). No multiple insertions were observed. Chromosomal loci of B. flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria.     

Analysis of relative reversion frequencies for IS2 insertion mutations in the regulatory region of the galOPETK operon of Escherichia coli

Two previously characterized mutations in the galOPETK operon of Escherichia coli, galOP-3 and galOPE-490, contain IS2 insertions only 1 bp apart in the gal regulatory region; yet only the former yields Gal⁺ phenotypic revertants at a detectable frequency. We have shown that the galOPE-490 allele comprises two mutations—an IS2(I) insertion at bp+(2–6) (relative to the gal mRNA start site) plus a C/G to A/T transversion at bp+59. The latter creates an ochre stop codon and lies within the internal site of the bipartite gal operator; it acts as an operator mutation in an in vivo repressor titration assay. Analysis of a newly isolated allele (galOP-490^*) which retains the IS2 of galOPE-490 but is galE⁺ reveals a reversion frequency approximately 30-fold higher than that of galOP-3. Reversion of galOPE-490 is at least 10,000-fold lower and has not been detectable even under conditions conducive to enhanced double mutations in other systems.     

Unexpectedly rapid IS1 transposition into an Arabidopsis chromatin remodeling gene

Common cloning is often associated with instability of certain classes of DNA. Here we report on IS1 transposition as possible source of such instability. During the cloning of Arabidopsis thaliana gene into commercially available vector maintained in widely used Escherichia coli host the insertion of complete IS1 element into the intron of cloned gene was found. The transposition of the IS1 element was remarkably rapid and is likely to be sequence-specific. The use of E. coli strains that lower the copy number of vector or avoiding the presence of the problematic sequence is a solution to the inadvertent transposition of IS1. The transposition of IS1 is rare but it can occur and might confound functional studies of a plant gene.