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Assay for movement of Lepidopteran transposon IFP2 in insect cells using a baculovirus genome as a target DNA.

Mutagenesis of baculoviruses by host mobile elements occurs spontaneously and frequently during propagation of the viruses in Lepidopteran cell cultures. Most of the transposons identified as insertions in baculovirus genomes are relatively small Class II elements that exhibit a remarkable specificity for TTAA target sites. We have developed a transposition assay to analyze the movement of these TTAA-specific Lepidopteran transposons using the baculovirus genome as a target and a lacZ gene under control of the polyhedrin gene promoter as a selective marker for the transposon. This assay provides the first demonstration that a Lepidopteran transposon is capable of transposing while carrying a marker gene in insect cells. The data generated from this assay provide strong evidence that IFP2 encodes a protein that facilitates its own movement. This element may be used in a manner analogous to the P-element to mobilize genes in at least some Lepidopteran insect cells. Transposon tagging within the baculovirus genome identified several known genes and two previously undescribed open reading frames as nonessential to in vitro replication of the virus.

Animals↗

Interstrain transfer of the large pathogenicity island (PAPI-1) of Pseudomonas aeruginosa.

The large Pseudomonas aeruginosa pathogenicity island PAPI-1 of strain PA14 is a cluster of 108 genes that encode a number of virulence features. We demonstrate that, in a subpopulation of cells, PAPI-1 can exist in an extrachromosomal circular form after precise excision from its integration site within the 3' terminus of the tRNA(Lys) gene. Circular PAPI-1 can reintegrate into either of the two tRNA(Lys) genes, including the one that was used for integration of small pathogenicity island PAPI-2 in strain PA14. The excision requires PAPI-1-encoded integrase, a member of the tyrosine recombinase family. PAPI-1 Soj contains the conserved domains of proteins that are related to chromosome and plasmid partition. soj plays a role in maintaining PAPI-1 and mutations in soj result in the loss of PAPI-1 from P. aeruginosa. We further demonstrate that, during coculture, the PAPI-1-containing strains are able to transfer it into P. aeruginosa recipient strains that do not harbor this island naturally. After transfer, PAPI-1 integrates into either of the two tRNA(Lys) genes. PAPI-1 encompasses many features of mobile elements, including mobilization and maintenance modules. Together with the virulence determinants, PAPI-1 plays an important role in the evolution of P. aeruginosa, by expanding its natural habitat from soil and water to animal and human infections.

Attachment Sites, Microbiological↗

Exogenous isolation of mobilizing plasmids from polluted soils and sludges.

Exogenous plasmid isolation was used to assess the presence of mobilizing plasmids in several soils and activated sludges. Triparental matings were performed with Escherichia coli (a member of the gamma subgroup of the Proteobacteria) as the donor of an IncQ plasmid (pMOL155, containing the heavy metal resistance genes czc: Co, Zn, and Cd), Alcaligenes eutrophus (a member of the beta subgroup of the Proteobacteria) as the recipient, and indigenous microorganisms from soil and sludge samples as helper strains. We developed an assay to assess the plasmid mobilization potential of a soil ecosystem on the basis of the number of transconjugants obtained after exogenous isolations. After inoculation into soil of several concentrations of a helper strain (E. coli CM120 harboring IncP [IncP1] mobilizing plasmid RP4), the log numbers of transconjugants obtained from exogenous isolations with different soil samples were a linear function of the log numbers of helper strain CM120(RP4) present in the soils. Four soils were analyzed for the presence of mobilizing elements, and mobilizing plasmids were isolated from two of these soils. Several sludge samples from different wastewater treatment plants yielded much higher numbers of transconjugants than the soil samples, indicating that higher numbers of mobilizing strains were present. The mobilizing plasmids isolated from Gent-O sludge and one plasmid isolated from Eislingen soil hybridized to the repP probe, whereas the plasmids isolated from Essen soil did not hybridize to a large number of rep probes (repFIC, repHI1, repH12, repL/M, repN, repP, repT, repU, repW, repX). This indicates that in Essen soil, broad-host-range mobilizing plasmids belonging to other incompatibility groups may be present.

Journal Article↗

Characterization of IS1110, a highly mobile genetic element from Mycobacterium avium.

A highly mobile insertion sequence designated IS1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20') were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited in M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.

Amino Acid Sequence↗

Elements of mobility as predictors of survival in elderly patients with dementia: findings from the Canadian Study of Health and Aging.

In order to identify elements of mobility that predict survival in elderly people with dementia, we conducted a two-year follow-up of a cohort of dementia subjects from the population-based Canadian Study of Health and Aging. There were 749 prevalent cases of Alzheimer's disease (AD) and 208 prevalent cases of vascular dementia. Elements of mobility that predicted death during the two-year follow-up period included difficulty in dressing (OR = 2.08, 95% CI 1.41 3.07), difficulty in getting about (OR = 1.69, 95% CI 1.18 2.40), history of falls (OR = 1.43, 95% CI 1.05 1.94), abnormal gait (OR = 1.61, 95% CI 1.08 2.40) and abnormal motor strength (OR = 1.51, 95% CI 1.07 2.15). Sociodemographic factors such as older age and male sex were also significant predictors of decreased survival. These associations are potentially useful to clinicians and health professionals by providing prognostic information to supply to families and suggesting areas in which interventions to improve survival might be focused

Activities of Daily Living↗

[Multiple induction of transpositions of Mobile Genetic Element B104 in Drosophila with severe heat shock].

Induction of transpositions by heavy heat shock (HHS) was demonstrated for the copia-like mobile genetic element B104. The probability of transposition induction lambda HHS was equal to 8.1 x 10(-2) per site per spermatozoon per spermatozoon per generation, which is an order of magnitude higher than the upper limit of spontaneous transposition probability estimate, lambda k < 1.7 x 10(-3). "Hot" and "cold" transposition sites were found. Many multisite heat shock haplotypes were shown to be nonrandom combinations of independent single-site transpositions. Multiples of numerous heat shock haplotypes exceeded expected values, which suggest-s that they were selected either in male germline cells or at the level of gametic fertility. These data confirm our earlier results obtained for Dm412. Heat shock transposition induction can be considered as a general property of different copia-like mobile genetic elements in Drosophila.

Animals↗

Mutations and rearrangements in the genome of Sulfolobus solfataricus P2.

The genome of Sulfolobus solfataricus P2 carries a larger number of transposable elements than any other sequenced genome from an archaeon or bacterium and, as a consequence, may be particularly susceptible to rearrangement and change. In order to gain more insight into the natures and frequencies of different types of mutation and possible rearrangements that can occur in the genome, the pyrEF locus was examined for mutations that were isolated after selection with 5-fluoroorotic acid. About two-thirds of the 130 mutations resulted from insertions of mobile elements, including insertion sequence (IS) elements and a single nonautonomous mobile element, SM2. For each of these, the element was identified and shown to be present at its original genomic position, consistent with a progressive increase in the copy numbers of the mobile elements. In addition, several base pair substitutions, as well as small deletions, insertions, and a duplication, were observed, and about one-fifth of the mutations occurred elsewhere in the genome, possibly in an orotate transporter gene. One mutant exhibited a 5-kb genomic rearrangement at the pyrEF locus involving a two-step IS element-dependent reaction, and its boundaries were defined using a specially developed "in vitro library" strategy. Moreover, while searching for the donor mobile elements, evidence was found for two major changes that had occurred in the genome of strain P2, one constituting a single deletion of about 4% of the total genome (124 kb), while the other involved the inversion of a 25-kb region. Both were bordered by IS elements and were inferred to have arisen through recombination events. The results underline the caution required in working experimentally with an organism such as S. solfataricus with a continually changing genome.

Archaeal Proteins↗

A broad-host-range plasmid for isolating mobile genetic elements in gram-negative bacteria.

Plasmid pGBG1 was constructed to isolate mobile genetic elements in a wide variety of gram-negative bacteria. The mutation target, carried on a broad-host-range vector, allows positive selection for tetracycline resistance. In tests using several gram-negative bacteria we could detect transposition events of either insertion sequences or transposons. A new insertion sequence (IS) element was identified in Ralstonia eutropha.

Agrobacterium tumefaciens↗

ACLAME: a CLAssification of Mobile genetic Elements.

The ACLAME database (http://aclame.ulb.ac.be) is a collection and classification of prokaryotic mobile genetic elements (MGEs) from various sources, comprising all known phage genomes, plasmids and transposons. In addition to providing information on the full genomes and genetic entities, it aims to build a comprehensive classification of the functional modules of MGEs at the protein, gene and higher levels. This first version contains a comprehensive classification of 5069 proteins from 119 DNA bacteriophages into over 400 functional families. This classification was produced automatically using TRIBE-MCL, a graph-theory-based Markov clustering algorithm that uses sequence measures as input, and then manually curated. Manual curation was aided by consulting annotations available in public databases retrieved through additional sequence similarity searches using Psi-Blast and Hidden Markov Models. The database is publicly accessible and open to expert volunteers willing to participate in its curation. Its web interface allows browsing as well as querying the classification. The main objectives are to collect and organize in a rational way the complexity inherent to MGEs, to extend and improve the inadequate annotation currently associated with MGEs and to screen known genomes for the validation and discovery of new MGEs.

Bacteriophages↗

Trypanosoma brucei s.l.: characterisation of stocks from Central Africa by PCR analysis of mobile genetic elements.

To better understand the epidemiology of sleeping sickness in the Central African sub-region, notably the heterogeneity of Human African Trypanosomiasis (HAT) foci, the mobile genetic element PCR (MGE-PCR) technique was used to genotype Trypanosoma brucei s.l. (T. brucei s.l.) isolates from this sub-region. Using a single primer REV B, which detects positional variation of the mobile genetic element RIME, via amplification of flanking regions, MGE-PCR revealed a micro genetic variability between Trypanosoma brucei gambiense (T. b. gambiense) isolates from Central Africa. The technique also revealed the presence of several T. b. gambiense genotypes and allowed the identification of minor and major ubiquitous genotypes in HAT foci. The presence of several T. b. gambiense genotypes in HAT foci may explain the persistence and the resurgence phenomena of the disease and also the epidemic and the endemic status of some Central African sleeping sickness foci. The MGE-PCR technique represents a simple, rapid, and specific method to differentiate Central African T. brucei s.l. isolates.

Africa, Central↗

Target specificity of insertion element IS30.

The Escherichia coli resident mobile element IS30 has pronounced target specificity. Upon transposition, the element frequently inserts exactly into the same position of a preferred target sequence. Insertion sites in phages, plasmids and in the genome of E. coli are characterized by an exceptionally long palindromic consensus sequence that provides strong specificity for IS30 insertions, despite a relatively high level of degeneracy. This 24-bp-long region alone determines the attractiveness of the target DNA and the exact position of IS30 insertion. The divergence of a target site from the consensus and the occurrence of 'non-permitted' bases in certain positions influence the target activity. Differences in attractiveness are emphasized if two targets are present in the same replicon, as was demonstrated by quantitative analysis. In a system of competitive targets, the oligonucleotide sequence representing the consensus of genomic IS30 insertion sites proved to be the most efficient target. Having compared the known insertion sites, we suppose that IS30-like target specificity, which may represent an alternative strategy in target selection among mobile elements, is characteristic of the insertion sequences IS3, IS6 and IS21, too.

Binding Sites↗

Ion mobility based on column leaching of South African gold tailings dam with chemometric evaluation.

New column leaching experiments were designed and used as an alternative rapid screening approach to element mobility assessment. In these experiments, field-moist material was treated with an extracting solution to assess the effects of acidification on element mobility in mine tailings. The main advantage of this version of column leaching experiments with partitioned segments is that they give quick information on current element mobility in conditions closely simulating field conditions to compare with common unrepresentative air-dried, sieved samples used for column leaching experiments. Layers from the tailings dump material were sampled and packed into columns. The design of columns allows extracting leachates from each layer. The extracting solutions used were natural (pH 6.8) and acidified (pH 4.2) rainwater. Metals and anions were determined in the leachates. The concentrations of metals (Ca, Mg, Fe, Mn, Al, Cr, Ni, Co, Zn, and Cu) in sample leachates were determined using ICP OES. The most important anions (NO3-, Cl-, and SO4(2)-) were determined using the closed system izotacophoresis ITP analyser. The chemical analytical data from tailings leaching and physico-chemical data from field measurements (including pH, conductivity, redox potential, temperature) were used for chemometric evaluation of element mobility. Principal factor analysis (PFA) was used to evaluate ions mobility from different layers of tailings dump arising from varied pH and redox conditions. It was found that the results from the partitioned column leaching illustrate much better complex processes of metals mobility from tailings dump than the total column. The chemometric data analysis (PFA) proofed the differences in the various layers leachability that are arising from physico-chemical processes due to chemical composition of tailings dump deposit.

Anions↗

The role of mobile genetic elements in bacterial adaptation to xenobiotic organic compounds.

Retrospective studies clearly indicate that mobile genetic elements (MGEs) play a major role in the in situ spread and even de novo construction of catabolic pathways in bacteria, allowing bacterial communities to rapidly adapt to new xenobiotics. The construction of novel pathways seems to occur by an assembly process that involves horizontal gene transfer: different appropriate genes or gene modules that encode different parts of the novel pathway are recruited from phylogenetically related or distant hosts into one single host. Direct evidence for the importance of catabolic MGEs in bacterial adaptation to xenobiotics stems from observed correlations between catabolic gene transfer and accelerated biodegradation in several habitats and from studies that monitor catabolic MGEs in polluted sites.

Adaptation, Physiological↗

High prevalence of multidrug resistance and random distribution of mobile genetic elements among uropathogenic Escherichia coli (UPEC) of the four major phylogenetic groups.

One hundred and ten UTI Escherichia coli strains, from Ljubljana, Slovenia, were analyzed for antibiotic resistances, mobile DNA elements, serotype, and phylogenetic origin. A high prevalence of drug resistance and multidrug resistance was found. Twenty-six percent of the isolates harbored a class 1 integron, while a majority of the strains (56%) harbored rep sequences characteristic of F-like plasmids. int as well as rep sequences were found to be distributed in a random manner among strains of the four major phylogenetic groups indicating that all groups have a similar tendency to acquire and maintain mobile genetic elements frequently associated with resistance determinants.

Conjugation, Genetic↗

Identification in methicillin-susceptible Staphylococcus hominis of an active primordial mobile genetic element for the staphylococcal cassette chromosome mec of methicillin-resistant Staphylococcus aureus.

We previously reported that the methicillin resistance gene mecA is carried by a novel type of mobile genetic element, SCCmec (staphylococcal cassette chromosome mec), in the chromosome of methicillin-resistant Staphylococcus aureus (MRSA). These elements are precisely excised from the chromosome and integrated into a specific site on the recipient chromosome by a pair of recombinase proteins encoded by the cassette chromosome recombinase genes ccrA and ccrB. In the present work, we detected homologues of the ccr genes in Staphylococcus hominis type strain GIFU12263 (equivalent to ATCC 27844), which is susceptible to methicillin. Sequence determination revealed that the ccr homologues in S. hominis were type 1 ccr genes (ccrA1 and ccrB1) that were localized on a genetic element structurally very similar to SCCmec except for the absence of the methicillin-resistance gene, mecA. This genetic element had mosaic-like patterns of homology with extant SCCmec elements, and we designated it SCC(12263) and considered it a type I staphylococcal cassette chromosome (SCC). The ccrB1 gene identified in the S. hominis strain is the first type 1 ccrB gene discovered to retain its function through the excision process as judged by two criteria: (i) SCC(12263) was spontaneously excised during cultivation of the strain and (ii) introduction of the S. hominis ccrB1 into an MRSA strain carrying a type I SCCmec whose ccrB1 gene is inactive generated SCCmec excisants at a high frequency. The existence of an SCC without a mec determinant is indicative of a staphylococcal site-specific mobile genetic element that serves as a vehicle of transfer for various genetic markers between staphylococcal species.

Amino Acid Sequence↗

Bacteria and Archaea Group II introns: additional mobile genetic elements in the environment.

Self-splicing group II introns are present in the organelles of lower eukaryotes, plants and Bacteria and have been found recently in Archaea. It is generally accepted that group II introns originated in bacteria before spreading to mitochondria and chloroplasts. These introns are thought to be related to the progenitors of spliceosomal introns. Group II introns are also mobile genetic elements. In bacteria, they appear to spread using either other mobile genetic elements or low-expression regions as target sites. Bacteria and Archaea genome sequence annotations have revealed the diversity of group II intron classes and that they are involved in vertical and horizontal inheritance.

Archaea↗

Undermethylation associated with retroelement activation and chromosome remodelling in an interspecific mammalian hybrid.

Genetic models predict that genomic rearrangement in hybrids can facilitate reproductive isolation and the formation of new species by preventing gene flow between the parent species and hybrid (sunflowers are an example). The mechanism underlying hybridization-induced chromosome remodelling is as yet unknown, although mobile element activity has been shown to be involved in DNA rearrangement in some dysgenic Drosophila hybrids. It has been proposed that DNA methylation evolved as a means of repressing the movement of mobile elements (the host defence model). If such a protective mechanism were to fail, mobile elements could be activated, and could cause major and rapid genome alterations. Here we demonstrate the occurrence of genome-wide undermethylation, retroviral element amplification and chromosome remodelling in an interspecific mammalian hybrid (Macropus eugenii x Wallabia bicolor). Atypically extended centromeres of Macropus eugenii derived autosomes in the hybrid were composed primarily of an unmethylated, amplified retroviral element not detectable in either parent species. These results, taken with the observation of deficient methylation and de novo chromosome change in other mammalian hybrids, indicate that the failure of DNA methylation and subsequent mobile-element activity in hybrids could facilitate rapid karyotypic evolution.

Animals↗

UV light and ionizing radiations cause programmed death of rat chloroleukaemia cells by inducing retropositions of a mobile DNA element (L1Rn).

The long interspersed repetitive DNA, L1 or LINE, is a class of mobile genetic elements which can amplify in the cell genome by retroposition, i.e. by a mechanism similar to that of retroviruses. We have shown before that in rat chloroleukaemia cells, maintained in suspension culture in vitro, this element is spontaneously transcriptionally activated at about half of the maximal population density. About 24 h later an explosive amplification of the L1 element is seen in DNA: about 300,000 copies are inserted into apparently random locations in the cell genome, thus creating an outburst of lethal mutations. Dead cells display morphological features typical to programmed death. The present results show that UV light and ionizing radiation induce rapid, premature activation of the L1Rn element during the fast exponential growth of chloroleukaemia cells, and that also this exogenously induced activation is followed by programmed cell death. Transcriptional activation of the L1Rn element can be very strong after the UV exposure: at least 70-fold. Severe hyperthermia, lethal to the cells, does not lead to L1Rn activation (actually a marked suppression is seen) and the mode of phenomic death is necrosis. Some biological implications of the results are discussed.

Animals↗