Involvement of transposable elements in the formation of hybrid phages between bacteriophage P1 and the R plasmid NR1

Homologous recombination between IS1 elements present on both replicons, P1 and NR1, resulted in P1-NR1 cointegrates and P1-RTF and P1-r-det phages. Cointegration between P1 and NR1-B, and NR1 derivative with multiple DNA rearrangements including insertion of the transposable element γδ, was also mediated by reciprocal recombination in IS1 sequences. However, all 4 hybrids studied carried deletions promoted by γδ residing on NR1-B. Further IS1-mediated deletions on the hybrid genomes resulted in plaque-forming P1Cm phages.     

Comparison of left-end DNA sequences of bacteriophages Mu and D108

The nucleotide sequences of the left ends of bacteriophage Mu DNA and that of its close relative D108 have been determined. The first 100 bp of phages Mu and D108 are substantially the same except for an octanucleotide change from bp 53 to 61 and other small interspersed base-pair changes from bp 61 to 200. The first five host nucleotides preceding the host-phage junction are generally, but not always, G + C-rich and these five nucleotides display no obvious consensus sequence. Both phages Mu and D108 share striking similarity in their end DNA sequences to the end sequences of the newly described Escherichia coli movable genetic element IS30.     

An insertion of Escherichia coli transposable element IS1K into the site immediately before tetracycline-resistance determinant of Bacillus subtilis chromosomal DNA fragment in cloning in E. coli

In cloning in Escherichia coli C600 of a 4.5-kbp HindIII DNA fragment with the tetracycline-resistance determinant (tetBS908) from Bacillus subtilis GSY908 chromosome using a plasmid vector, a 5.2-kbp HindIII DNA fragment was also isolated at a ratio of 2 to 89. The two independently obtained 5.2-kbp fragments were an insertion derivative of the 4.5-kbp fragment and carried E. coli transposable element ISlK, which was inserted at the same site immediately before tetBS908 in the same direction. For the ISlK insertions, the 8-bp sequence CAAATTTT was used as a target, this having no similarity to any published sequences.     

Structures of oversized genomes of phage P1 derivatives carrying lac genes of Escherichia coli K12

Physical analyses of two newly isolated oversized P1lac phage genomes showed that they are partly diploid in P1 genes and that they carry a 60–70-kb segment of host DNA. The transposable element γδ is present at one of the junctions between host and P1 DNA, and IS1 is at the other junction. These elements must thus have been actively involved in the formation of these P1lac prophages. The genome of a third oversized P1lac has a segment with dispensable P1 genes deleted. The absence of any known recombinogenic element at one of its junctions between P1 and host DNA suggests non-homologous recombination to have been involved in its formation. Non-homologous recombination might have also taken place in one of the final steps of the formation of the former P1lac genomes.     

Analysis of Tag1-like elements in Arabidopsis thaliana and their distribution in other plants.

Analysis of genomic DNA of Arabidopsis Columbia (Col.) ecotype using a transposon Tag1-specific primer showed the presence of Tag1 homologues which was confirmed by Southern hybridization with a Tag1 probe. Further analysis showed that the homologue, 0.75 kb in length, had inverted repeats at both ends, 8-bp duplicated sequences at the site at which it is located and about 80% homology with Tag1, and was randomly distributed in the Arabidopsis genome. Based on these results, we concluded that these elements are non-autonomous variants of Tag1 and we termed this element sTag1. Using the polymerase chain reaction fragment hybridization technique, we found the distribution of such homologues in other plant species.     

Transformation of Bacillus subtilis and Escherichia coli by a hybrid plasmid pCD1.

The gene thyP3 from Bacillus subtilis bacteriophage phi 3T was cloned in the plasmid pMB9. The resulting chimeric plasmid, pCD1, is effective in transforming both Escherichia coli and Bacillus subtilis to thymine prototrophy. The activity of the thyP3 gene product, thymidylate synthetase, was assayed and found to be 9 times greater in a transformed strain of Escherichia coli than in a phi 3T lysogen of Bacillus subtilis. The physical location of restriction sites has been determined for two related plasmids pCD1 and pCD2. Hybridization studies clearly indicate that the plasmid gene responsible for Thy+ transformation is the gene from the bacteriophage phi 3T. The lack of restriction in this transformation process is consistent with our previous studies using bacterial DNA in heterospecific exchanges indicating that the nucleotide sequence surrounding the gene is the dominant factor in determining interspecific transformation.     

Screening for the presence of the insertion sequence IS1 in the genus Bacteroides

Abstract A specific DNA probe, containing a conserved region of the insertion sequence IS1, was hybridised to dot blots of total genomic DNA from 2 oral and 5 intestinal Bacteroides spp. Using Escherichia coli K12 as a positive control and Pseudomonas aeruginosa as a negative control, DNA homologous to the probe could not be detected in Bacteroides corporis, Bacteroides intermedius, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron or 2 strains of Bacteroides fragilis . The total DNA included plasmid DNA of 30.2, 42.7 and 42.7 MDa from B. fragilis, B. intermedius and B. corporis , respectively.
IS1 is commonly found in members of the Enterobacteriaceae, and it was concluded that the 2 groups of bacteria are not closely related.     

Identification and structural characterization of further DNA elements in the potato and pepper genomes homologous to the transposable element-like insertion Tst1

Summary The molecular cloning and nucleotide sequence of elements from potato and pepper that are related to the recently identified Tst1 element are described. Sequence analysis reveals considerable conservation of sequences internal to both the Tst1 element and two of the related elements identified here. In six potato clones analysed, the II by inverted repeat first identified in the Tst1 element is conserved. Several of the elements are flanked by an 8 by direct repeat. DNA fragments which were amplified from several pepper genomes by polymerase chain reaction (PCR) amplification using the inverted repeat as sequence primers also display considerable conservation of sequences internal to the Tst1 element. These data further support the possibility that Tst1 is a non-autonomous transposable element and that Tst1 might be the first example of a transposable element which occurs in several genera of solanaceous plants.     

The genomes of most animals have multiple members of the Tc1 family of transposable elements

A PCR assay was employed to detect sequences homologous to the transposase gene of the Tc1 family of transposable elements in a wide variety of animals. Amplification products of the appropriate size were obtained from most insects (92 of 108 examined; 85%), most other invertebrates (33 of 43; 77%), and many vertebrates (18 of 36; 50%). Sequencing a sample of cloned PCR products from eight insects, one hydra, and two frogs revealed that each had multiple distinct members of the family in their genomes. In the most extreme case, the horn fly Haematobia irritans yielded evidence of seventeen distinct types of Tc1 family elements. Most of the sequences obtained indicate that the elements are within the range of variation already known from fungi, nematodes, files, fish and frogs. Some, however, had novel length variants or divergent sequences, indicating that they represent new subfamilies of these transposons. These results indicate that this family of transposons is extremely common in animal genomes, with multiple representatives in most genomes.     

Genetic variability of the frameshift region in IS911 transposable elements from Escherichia coli clinical isolates

The IS911 bacterial transposable element has been analyzed for its mechanism of transposition and for the way it controls the expression of its genes by programmed -1 translational frameshifting. In the present study the prevalence of IS911 has been determined in the Enterobacteriaceae family and in other Gram-negative bacilli. Three variants, found in Escherichia coli clinical isolates and having mutations in the region implicated in frameshifting, were functionally characterized. All three were altered in their frameshifting and transposition abilities, suggesting that the frameshift region of IS911 may constitute a target for mutations reducing the transposition frequency of this mobile element in natural populations of E. coli.     

Identification and mapping of expressed genes, simple sequence repeats and transposable elements in centromeric regions of rice chromosomes.

The genomic sequences derived from rice centromeric regions were analyzed to facilitate the comprehensive understanding of the rice genome. A rice centromere-specific satellite sequence, RCS2/TrsD/CentO, was used to screen P1-derived artificial chromosome (PAC) and bacterial artificial chromosome (BAC) genomic libraries derived from Oryza sativa L. ssp. japonica cultivar Nipponbare. Physical maps of the centromeric regions were constructed by DNA fingerprinting methods and the aligned clones were analyzed by end sequencing. BLAST analysis revealed the composition of genes, centromeric satellites and other repetitive elements, such as RIRE7/CRR, RIRE8, Squiq, Anaconda, CACTA and miniature inverted-repeat transposable elements. Fiber-fluorescent in situ hybridization analysis also indicated the presence of distinct clusters of RCS2/TrsD/CentO satellite interspersed with other elements, instead of a long homogeneous region. Several expressed genes, sequences representative of ancestral organellar insertions, relatively long simple sequence repeats (SSRs), and sequences corresponding to 5S and 45S ribosomal RNA genes were also identified. Thirty-one gene sequences showed high-similarity to rice full-length cDNA sequences that had not been matched to the published rice genome sequence in silico. These results suggest the presence of expressed genes within and around the clusters of RCS2/TrsD/CentO satellites in unsequenced centromeric regions of the rice chromosomes.     

DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans

Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive γ irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the molecular basis of strand discrimination and transposase action. The structures also show that previously established structural rules of target site recognition that allow different specific sequences to be targeted are only partially conserved among family members. Furthermore, we have captured a fully assembled active site including the scissile phosphate bound by a divalent metal ion cofactor (Cd2(+)) that supports DNA cleavage. Finally, the observed active site rearrangements when the transposase binds a metal ion in which it is inactive provide a clear rationale for metal ion specificity.     

Overlap hybridization screening: isolation and characterization of overlapping DNA fragments surrounding the leu2 gene on yeast chromosome III.

A set of four plasmids containing overlapping segments comprising a total of about 30 kbp of cloned DNA from chromosome III of yeast (Saccharomyces cerevisiae) has been isolated and characterized by restriction endonuclease analyses and DNA:DNA hybridizations. Colony hybridization was carried out with labeled pYe(leu2)10, a plasmid carrying the yeast leu2 gene, to a bank of bacterial colonies containing recombinant plasmids constructed from the vector ColE1 and random fragments of yeast DNA. This resulted in the detection of two plasmids, pYe11G4 and pYe40C3, with DNA inserts which partially overlap the original cloned segment and contain additional DNA extending in opposite directions on the chromosome. By carrying out a second round of colony hybridization with pYe40C3, the cloned region was further extended in one direction. A region of DNA that is repeated at least ten times in the yeast genome was identified by hybridization of pYe11G4 to an EcoRI digest of total yeast DNA. The procedure described in this paper should allow the isolation of large sections of chromosomes, including non-transcribed regions, surrounding cloned genes.     

Mechanisms involved in the formation of plaque-forming derivatives from over-sized hybrid phages between bacteriophage P1 and the R plasmid NR1

Three case histories document how subsequent events of genomic rearrangements and selection interplay in the evolution of infectious bacteriophage genomes carrying acquired genes. Two of the phages studied were plaque-forming P1CmTc recombinants derived from P1Cm1 and P1Tc1, both of which are hybrids between phage P1 and the R plasmid NR1. In the formation of the P1CmTc4 genome a postulated intermediate underwent IS1-mediated deletion formation. From the same intermediate P1CmTc1 must have evolved by IS1-mediated inversion followed by homologous recombination with a parental phage DNA. The third case documents formation of the P1Cm2 genome by “illegitimate” intramolecular recombination in the genome of P1-r-det, a hybrid between P1 and NR1.     

Heteroduplex electron microscopy of phage Mu mutants containing IS1 insertions and chloramphenicol resistance transposons.

We have examined by electron microscopy the DNA heteroduplexes of six bacteriophage Mu mutants, Mu X cam, generated by the insertion of the Tn9 transposon for chloramphenicol resistance. Tn9 was found to be 2.8 +/- 0.2 kilobases (kb) in length and to consist of a cam determinant flanked by two IS1 sequences arranged in a direct order. In two of the six Mu X cam mutants, the Tn9 insertion was at a fixed location, 3.9 kb from the left, or c, end. In the other four mutants, the position of the insertion varied, even though the lysogenic cultures induced were grown from single colonies. The insertion was located at either 3.3 kb, 3.9 kb, or, less frequently, at 4.4 kb from the left end of the DNA. Furthermore, at low frequencies, the insertions were found to be in an orientation opposite to what predominated in the preparation. Thus, Tn9 in the Mu X cam mutants examined could appear to undergo rapid rearrangements during Mu growth or over a few generations of cell growth. One of the Tn9 insertion sites was apparently the same as that for a 0.8 kb insertion found in a Mu X mutant. This latter insertion was identified as an IS1 sequence. The DNA molecules from all the Mu X cam mutant phage particles were found to be missing the bacterial DNA at the S (right) end, along with a variable amount of the adjoining Mu DNA in the beta region. This observation supports the headful packaging model for Mu DNA.     

The distribution of the transposable elementBari-1 in theDrosophila melanogaster andDrosophila simulans genomes

The distribution of the transposable elementBari-1 inD. melanogaster andD. simulans was examined by Southern blot analysis and byin situ hybridization in a large number of strains of different geographical origins and established at different times.Bari-1 copies mostly homogeneous in size and physical map are detected in all strains tested. Both inD. melanogaster and inD. simulans a relatively high level of intraspecific insertion site polymorphism is detectable, suggesting that in both speciesBari-1 is or has been actively transposing. The main difference between the two sibling species is the presence of a large tadem array of the element in a well-defined heterochromatic location of theD. melanogaster genome, whereas such a cluster is absent inD. simulans. The presence ofBari-1 elements with apparently identical physical maps in allD. melanogaster andD. simulans strains examined suggests thatBari-1 is not a recent introduction in the genome of themelanogaster complex. Structural analysis reveals unusual features that distinguish it from other inverted repeat transposons, whereas many aspects are similar to the widely distributedTc1 element ofC. elegans.     

Detection and characterisation of pr1 virulent gene deficiencies in the insect pathogenic fungus Metarhizium anisopliae

The method of suppressive subtractive hybridization was employed to map out genomic differences between the highly pathogenic Yersinia enterocolitica (Ye) biogroup 1B, serotype O:8 strain (WA-314) and the closely related apathogenic Y. enterocolitica biogroup 1A, serotype O:5 strain (NF-O). A novel IS10-like element, IS1330, uncovered by this technique was found to be uniquely present in high copy numbers among the highly pathogenic Y. enterocolitica 1B strains, while a single copy of the element was found in the low pathogenic Ye biogroup 4 serotype O:3 strain. The 1321-bp repetitive element has 19-bp imperfect inverted terminal repeats and is bracketed by a 10-bp duplication of the target sequence. The predicted transposase shares high homology with the IS10 open reading frame of the large virulence plasmid pWR501, of Shigella flexneri, with IS10 transposase of Salmonella typhi, and with IS1999 (tnpA) of Pseudomonas aeruginosa. The IS1330 tnp gene is transcribed in vitro and in vivo in HeLa cells. At least one copy of IS1330 flanks the recently described chromosomal type III secretion cluster in Y. enterocolitica WA-314, O:8, and future studies should shed light on whether this novel transposase mediates transposition events in highly pathogenic Y. enterocolitica strains, thus enhancing the genetic plasticity of this species.     

Local hopping of IS3 elements into the A+T-rich part of the high-pathogenicity island in Yersinia enterocolitica 1B, O:8

The high-pathogenicity island (Yen HPI) of Yersinia enterocolitica biogroup (BG) 1B strains is associated with mouse virulence. Three repeated sequences are clustered on the A+T-rich part of the Yen HPI downstream of the fyuA yersiniabactin receptor gene in Y. enterocolitica O:8 strains WA-314 and 8081. In addition to IS1328 and IS1400, the RS3 repeated sequence consists of a novel insertion sequence, IS1329, inserted into the remnants of IS1222. This partial IS retains both 44-bp inverted terminal repeats (ITRs) of IS1222 but has suffered deletions of different sizes in strains WA-314 and 8081. IS1329 is 1243-bp long, carries 25-bp imperfect ITRs and two consecutive orfs capable to encode 110-amino acid (aa) and 249-aa proteins, respectively. IS1329 is present only in BG 1B Y. enterocolitica strains. Similarly to IS1400, IS1329 and IS1222 belong to the IS3 group of mobile elements and seem to have preference for the 'local hopping' into the A+T-rich part of the Yen HPI. These insertion sequences may be responsible for the imprecise deletions of the Yen HPI in strain WA-314.