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Mobile inverted-repeat elements of the Tourist family are associated with the genes of many cereal grasses.

Tourist was originally described as a 128-bp insertion mutation in the maize wx-B2 allele. Subsequent analysis revealed that Tourist elements are in the introns or flanking sequences of 11 maize genes and a single barley gene. In this study we report that Tourist elements are frequently associated with the wild-type genes of two other grasses, rice and sorghum. Six of 35 rice and 5 of 8 sorghum complete gene sequences reported to date contain Tourist elements. Furthermore, 11 additional maize genes have been found to contain Tourist elements, bringing the current total of elements associated with maize genes to 23. Sequence comparison of Tourist elements has led to the identification of four subfamilies, designated A-D. Evidence is presented for the recent mobility of elements in three of these subfamilies and in three of the four grass species. These data suggest that Tourist elements are highly repetitive in the genomes of some and perhaps all members of the grasses.

Alcohol Dehydrogenase↗

Mutations in the Lactococcus lactis Ll.LtrB group II intron that retain mobility in vivo.

BACKGROUND: Group II introns are mobile genetic elements that form conserved secondary and tertiary structures. In order to determine which of the conserved structural elements are required for mobility, a series of domain and sub-domain deletions were made in the Lactococcus lactis group II intron (Ll.LtrB) and tested for mobility in a genetic assay. Point mutations in domains V and VI were also tested. RESULTS: The largest deletion that could be made without severely compromising mobility was 158 nucleotides in DIVb(1-2). This mutant had a mobility frequency comparable to the wild-type Ll.LtrB intron (DeltaORF construct). Hence, all subsequent mutations were done in this mutant background. Deletion of DIIb reduced mobility to approximately 18% of wild-type, while another deletion in domain II (nts 404-459) was mobile to a minor extent. Only two deletions in DI and none in DIII were tolerated. Some mobility was also observed for a DIVa deletion mutant. Of the three point mutants at position G3 in DV, only G3A retained mobility. In DVI, deletion of the branch-point nucleotide abolished mobility, but the presence of any nucleotide at the branch-point position restored mobility to some extent. CONCLUSIONS: The smallest intron capable of efficient retrohoming was 725 nucleotides, comprising the DIVb(1-2) and DII(ii)a,b deletions. The tertiary elements found to be nonessential for mobility were alpha, kappa and eta. In DV, only the G3A mutant was mobile. A branch-point residue is required for intron mobility.

Journal Article↗

Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia.

By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after gamma-ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 bp insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 bp terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5' and 3' ends of dTnp1 together with a perfect palindrome located after the 5' inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.

Amino Acid Sequence↗

Widespread occurrence of phage-encoded exotoxin genes in terrestrial and aquatic environments in Southern California.

Many human diseases are caused by pathogens that produce exotoxins. The genes that encode these exotoxins are frequently encoded by mobile DNA elements such as plasmids or phage. Mobile DNA elements can move exotoxin genes among microbial hosts, converting avirulent bacteria into pathogens. Phage and bacteria from water, soil, and sediment environments represent a potential reservoir of phage- and plasmid-encoded exotoxin genes. The genes encoding exotoxins that are the causes of cholera, diphtheria, enterohemorrhagic diarrhea, and Staphylococcus aureus food poisoning were found in soil, sediment, and water samples by standard PCR assays from locations where the human diseases are uncommon or nonexistent. On average, at least one of the target exotoxin genes was detected in approximately 15% of the more than 300 environmental samples tested. The results of standard PCR assays were confirmed by quantitative PCR (QPCR) and Southern dot blot analyses. Agreement between the results of the standard PCR and QPCR ranged from 63% to 84%; and the agreement between standard PCR and Southern dot blots ranged from 50% to 66%. Both the cholera and shiga exotoxin genes were also found in the free phage DNA fraction. The results indicate that phage-encoded exotoxin genes are widespread and mobile in terrestrial and aquatic environments.

Bacterial Infections↗

Two copies of a DNA element, 'Wendy', in the chloroplast chromosome of Chlamydomonas reinhardtii between rearranged gene clusters.

We have characterized two copies of a 2.4 kb DNA element that we call 'Wendy', in the chloroplast chromosome of Chlamydomonas reinhardtii. The two copies of Wendy reside in different single-copy regions at opposite positions in the chloroplast genome. Like many mobile DNA elements, both copies of Wendy are bordered by inverted repeats and contain several additional degenerate copies of these repeat sequences in direct or inverted orientation. In addition, four basepairs are repeated in direct orientation. Two major open reading frames (ORFs) are predicted from the DNA sequence of Wendy I. These ORFs are co-transcribed from a promoter inside the element. The deduced amino acid sequence of the larger of these ORFs shares some weak similarities with sequence motifs of transposases and integrases of other mobile elements. Wendy II appears to be altered relative to Wendy I by point mutations and small deletions and insertions which destroy the ORFs. The leader sequence of the Wendy transcript is nearly identical with the leader sequence of the rbcL transcript of C. reinhardtii, but not of C. moewusii (where the complete Wendy was also undetectable). Furthermore, both copies of Wendy are bracketed by gene clusters that are separated in C. reinhardtii but are contiguous in C. moewusii where they exist in an inverted orientation compared with C. reinhardtii. Wendy was not found in any of the completely sequenced chloroplast genomes of rice, tobacco, pine, Euglena or Marchantia, nor in any other GenBank entry. Our results suggest that Wendy has invaded C. reinhardtii after divergence from other species. Subsequent Wendy-dependent illegitimate homologous or site-specific recombination events or both may have contributed to scrambling of the C. reinhardtii chloroplast genome relative to genomes of other species.

Amino Acid Sequence↗

Evidence of mobilization of hobo transposons in a P-element mutagenesis screen.

In Drosophila melanogaster, mutations in Su(var) and En(var) loci either suppress or enhance position effect variegation, respectively. Towards the cloning of these genes we have induced, recovered, and characterized a series of Su(var) mutations located on the third chromosome. These mutations were recovered from a P-element mutagenesis scheme in which the third chromosome P[ry+ delta 2-3](99B) element would provide transposase in trans to mobilize a single nonautonomous P transposon, P[pUCHSNeo](9C), located on the X chromosome. Although this cross scheme induces mobilization of the X-chromosome P transposons and its subsequent reinsertion onto the autosomes, none of the Su(var) mutations recovered could be associated with P-transposon insertions. Further investigation of these mutations by in situ hybridization showed that another mobile element that can be mobilized in hybrids, hobo, was associated with these mutations. We conclude that the mobilization and reinsertion of the hobo elements, through an inadvertent hybrid HE dysgenic cross, is most likely responsible for the Su(var) mutations recovered from this P-element mutagenesis. The association of hobo element with this Su(var) locus will facilitate its cloning and offers the possibility of hobo transposon tagging other Su(var) loci.

Animals↗

Genomic demography: a life-history analysis of transposable element evolution.

Retrotransposons are ubiquitous mobile genetic elements that have played a significant role in shaping eukaryotic genome evolution. The genome of the yeast Saccharomyces cerevisiae harbours five families of retrotransposons, Ty1-Ty5. With the publication of the S. cerevisiae genome sequence, for the first time a full genomic complement of retrotransposon sequences is available. Analysis of these sequences promises to yield insight into the nature of host--transposon coevolution. Evolutionary change in Ty elements depends on their replication and excision rates, which have been determined in the laboratory. Rates measured in the laboratory may differ from those that have operated over evolutionary time. Based on an analysis of sequence data for the Ty1, Ty2 and hybrid Ty1/2 families, we develop a novel 'genomic demography' model to estimate long-term transposition and excision rates and to estimate how long ago these elements entered the yeast genome. We find that rates of excision and transposition have averaged 7.2-8.7 x 10(-8) per generation over evolutionary time. Two separate models provide upper- and lower-bound estimates for the age of the system, suggesting that the first elements entered the genome between approximately 50 million and 250 million generations ago.

Evolution, Molecular↗

Effect of microbial activity on trace element release from sewage sludge.

The microbial role in mobilization of trace elements from land-applied wastewater sludge is not well-defined. Our study examined the leachability of trace elements (Cd, Cr, Cu, Mo, Ni, P, Pb, S, and Zn) from dewatered sludge as affected by treatments designed to alter microbial activity. Different levels of microbial activity were achieved by incubating sludge columns at 4, 16, 28, and 37 degrees C and by the addition of AgNO3 biocide at each temperature. Columns (with inert glass bead support beds) were subjected to six consecutive incubation-leaching cycles, each consisting of 7.3-d incubation followed by 16-h leaching with synthetic acid rain. Glucose mineralization tests were used to assess overall microbial activity. Significant acidification and trace element leaching occurred when conditions favored microbial activity (16 and 28 degrees C). Extent of mobilization was element-specific with Zn, Ni, and Cu showing the greatest mobilization (99, 67, and 57%, respectively). Mobilization was reduced but still substantial at 4 degrees C. Conditions that best inhibited microbial activity (37 degrees C or biocide at any temperature) resulted in the least mobilization. Characterization of enrichments performed using thiosulfate as the sole energy source revealed the presence of both known and putative S-oxidizing bacteria in the sludge. The results suggest that microbial acidification via S oxidation can mobilize trace elements from sludge. Elemental mobility in field situations would also be governed by other factors, including the capacity of soil to buffer acidification and to adsorb mobilized elements.

Adsorption↗

Clusters of diverse genes existing as multiple, sequence-variable mosaics in a phytoplasma genome.

Phytoplasmas are cell wall-less prokaryotes living as obligate parasites and pathogens of plants and insects, making them attractive subjects for studies to gain a greater understanding of transkingdom parasitism and pathogenicity. During a study of two phytoplasma genomes, we obtained evidence for previously unreported clustering of genes, pseudogenes, mobile genetic elements, intergenic repeat units, and repetitive extragenic palindromes that occur in multiple, homologous clusters in some phytoplasma genomes. The clusters represent previously unrecognized mosaics, possibly assembled through multiple events of targeted mobile element attack, duplication, recombination, and rearrangement. Multiple clusters could conceivably afford potential for genome reduction through homologous recombination. Differences in the sizes and multiplicity of such clusters possibly account for some of the previously reported but unexplained variations in genome size among closely related phytoplasma strains.

Base Sequence↗

Structure analysis of a class II transposon encoding the mercury resistance of the Gram-positive Bacterium bacillus megaterium MB1, a strain isolated from minamata bay, Japan.

A unique transposon was found in the chromosome of Bacillus megaterium MB1, a Gram-positive bacterium isolated from mercury-polluted sediments of Minamata Bay, Japan. The transposon region of a 14.5kb DNA fragment was amplified by PCR using a single PCR primer designed from the nucleotide sequence of an inverted repeat of class II transposons. The molecular analysis revealed that the PCR-amplified DNA fragment encodes a transposition module similar to that of Tn21. The transposon also encodes a broad-spectrum mercury resistance region having a restriction endonuclease map identical to that of Bacillus cereus RC607, a strain isolated from Boston Harbor, USA. The result of a phylogenetic analysis of the amino acid sequence of putative resolvase of the transposon showed that the transposon is phylogenetically closer to the transposons of Gram-positive bacteria than those of Gram-negative bacteria. Besides the transposition module and mer operon, the transposon encodes a mobile genetic element of bacterial group II introns between the resolvase gene and mer operon. The intron, however, does not intervene in any exon gene. The discovery of this newly found combination of the complex mobile elements may offer a clue to understanding the horizontal dissemination of broad-spectrum mercury resistance among microbes.

Bacillus megaterium↗

Genetics of antimicrobial resistance.

Antimicrobial resistant strains of bacteria are an increasing threat to animal and human health. Resistance mechanisms to circumvent the toxic action of antimicrobials have been identified and described for all known antimicrobials currently available for clinical use in human and veterinary medicine. Acquired bacterial antibiotic resistance can result from the mutation of normal cellular genes, the acquisition of foreign resistance genes, or a combination of these two mechanisms. The most common resistance mechanisms employed by bacteria include enzymatic degradation or alteration of the antimicrobial, mutation in the antimicrobial target site, decreased cell wall permeability to antimicrobials, and active efflux of the antimicrobial across the cell membrane. The spread of mobile genetic elements such as plasmids, transposons, and integrons has greatly contributed to the rapid dissemination of antimicrobial resistance among several bacterial genera of human and veterinary importance. Antimicrobial resistance genes have been shown to accumulate on mobile elements, leading to a situation where multidrug resistance phenotypes can be transferred to a susceptible recipient via a single genetic event. The increasing prevalence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. The versatility with which bacteria adapt to their environment and exchange DNA between different genera highlights the need to implement effective antimicrobial stewardship and infection control programs in both human and veterinary medicine.

Animals↗

The nature of unstable insertion mutations and reversions in the locus cut of Drosophila melanogaster: molecular mechanism of transposition memory.

The segment of the locus cut containing the mobile genetic element mdg4 (gypsy) insertions which induce unstable ct and ct mutations has been cloned. Both mutations depend on the insertion of mdg4 into the same sequence, which coincides with that in ct allele. The ct mutation differs from ct by additional insertion of a novel mobile element jockey into mdg4. Jockey is 2.8 kb long, represented by 2-100 copies per genome, very homogeneous and lacks long terminal repeats (LTRs). The excision of mdg4 takes place in stable ct reversions. On the other hand, a complete single LTR is retained in the case of unstable ct reversions characterized by a high level of reverse directed transpositions of mdg4 into the locus cut. The LTR serves as a guide for reinsertion of mdg4 itself or mdg4 with jockey into the same site of the genome. A possible mechanism of transposition memory (homologous recombination with extrachromosomal circular DNA) is discussed.

Journal Article↗

Complete genome sequence of an antibiotic-resistant and virulent Escherichia marmotae isolate recovered from a urinary tract infection.

OBJECTIVES: We aimed at analyzing the whole genome of the antibiotic-resistant Escherichia marmotae isolate 23-MO01035-0, which was obtained from a urinary tract infection of a male patient in Germany. The study focused on the characterization of mobile genetic elements and genetic factors that contribute to antibiotic resistance and pathogenicity. METHODS: Phenotypic antimicrobial susceptibility testing was performed and genomic DNA was sequenced using Illumina NextSeq (2 × 151 bp) and a MinION Mk1C device followed by de novo hybrid assembly using Unicycler v0.4.8. Resistance genes, virulence factors, plasmids, and mobile elements were identified with the bakcharak pipeline and PathogenFinder. RESULTS: The isolate belonged to sequence type ST133 and the 5,041,084 bp whole genome, consisting of one chromosome and five plasmids with an average GC content of 50.4%, revealed resistance genes for various antibiotic classes on an IncFII plasmid that had not yet been described. The isolate was predicted to be a potential human pathogen due to the presence of multiple virulence genes. CONCLUSION: Here, we report one of the first detailed genomic characterization of an antibiotic-resistant and virulent E. marmotae isolate from Germany. Considering that this bacterium was isolated from the urinary tract and possibly belonged to the faecal microbiota of the patient at that time, E. marmotae might contribute to the spread of antibiotic resistance in humans. This underscores the importance of genomic surveillance of E. marmotae, which causes human infections, but may be misidentified as E. coli.

Escherichia marmotae↗

Sex and the spread of retrotransposon Ty3 in experimental populations of Saccharomyces cerevisiae.

Mobile genetic elements may be molecular parasites that reduce the fitness of individuals that bear them by causing predominantly deleterious mutations, but increase in frequency when rare because transposition increases their rates of transmission to the progeny of crosses between infected and uninfected individuals. If this is true, then the initial spread of a mobile element requires sex. We tested this prediction using the yeast retrotransposon Ty3 and a strain of Saccharomyces cerevisiae lacking Ty3. We infected replicate isogenic sexual and asexual populations with a galactose-inducible Ty3 element at an initial frequency of 1%. In two of six asexual populations, active Ty3 elements increased in frequency to 38 and 86%, due to the spread in each population of a competitively superior mutant carrying a new Ty3 insertion. Ty3 frequencies increased above 80% in all sexual populations in which transposition was induced in haplophase or in diplophase. Ty3 did not increase in frequency when active during both haplophase and diplophase, apparently because of selective sweeps during adaptation to galactose. Repressed Ty3 elements spread in sexual populations, by increasing sexual fitness. These results indicate that active Ty3 elements are more likely to become established in sexual populations than in asexual populations.

Crosses, Genetic↗

The mobilization regions of two integrated Bacteroides elements, NBU1 and NBU2, have only a single mobilization protein and may be on a cassette.

Bacteroides conjugative transposons can act in trans to excise, circularize, and transfer unlinked integrated elements called NBUs (for nonreplicating Bacteroides units). Previously, we localized and sequenced the mobilization region of one NBU, NBU1, and showed that this mobilization region was recognized by the IncP plasmids RP4 and R751, as well as by the Bacteroides conjugative transposons. We report here that the single mobilization protein carried by NBU1 appears to be a bifunctional protein that binds to the oriT region and catalyzes the nicking reaction that initiates the transfer process. We have also localized and sequenced the mobilization region of a second NBU, NBU2. The NBU2 mobilization region was 86 to 90% identical at the DNA sequence to the oriT-mob region of NBU1. The high sequence similarity between NBU1 and NBU2 ended abruptly after the stop codon of the mob gene and about 1 kbp upstream of the oriT region, indicating that the oriT-mob regions of NBU1 and NBU2 may be on some sort of cassette. A region on NBU1 and NBU2 which lies immediately upstream of the oriT region had 66% sequence identity to a region upstream of the oriT region on a mobilizable transposon, Tn4399, an element that had previously appeared to be completely unrelated to the NBUs.

Amino Acid Sequence↗

Single-element and competitive metal mobility measured with column infiltration and batch tests.

The distribution coefficient, Kd, is often used to quantify heavy metal mobility in soils. Batch sorption or column infiltration tests may be used to measure Kd. The latter are closer to natural soil conditions, but are difficult to conduct in clays. This difficulty can be overcome by using a laboratory centrifuge. An acceleration of 2600 gravities was applied to columns of London Clay, an Eocene clay sub-stratum, and Cu, Ni, and Zn mobility was measured in centrifuge infiltration tests, both as single elements and in dual competition. Single-element Kd values were also obtained from batch sorption tests, and the results from the two techniques were compared. It was found that Kd values obtained by batch tests vary considerably depending on the metal concentration, while infiltration tests provided a single Kd value for each metal. This was typically in the lower end of the range of the batch test Kd values. For both tests, the order of mobility was Ni>Zn>Cu. Metals became more mobile in competition than when in single-element systems: Ni Kd decreased 3.3 times and Zn Kd 3.4 times when they competed with Cu, while Cu decreased only 1.2 times when in competition with either Ni or Zn. Our study showed that competitive sorption between metals increases the mobility of those metals less strongly bound more than it increases the mobility of more strongly bound metals.

Adsorption↗

IS5: a mobile enhancer of transcription in Escherichia coli.

The cryptic bgl operon of Escherichia coli is activated by the spontaneous insertion of mobile DNA elements. Screening of a collection of such mutations revealed insertion of the 1195-base-pair element IS5 into various positions both upstream and downstream of the bgl promoter P0. Activation of the operon was in all cases attributable to enhancement of P0 activity. Introduction of internal deletions into IS5 almost completely abolished P0 enhancement, demonstrating that enhancement is not simply the result of mutational inactivation of some inhibitory sequences. Intact copies of IS5 in trans restored the enhancing activity of the deletion derivatives. The trans-activator is encoded by IS5 gene ins5A, an essential transposition function. Activation of gene expression by means of interaction of a defective mobile element in cis with functions encoded by a nondefective element in trans has so far been described only for a maize controlling element.

Base Sequence↗

The molecular analysis of brown eye color mutations isolated from geographically discrete populations of Drosophila melanogaster.

A large proportion of spontaneous mutations in Drosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessive brown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caught Drosophila melanogaster females, bw mutations were isolated from seven separate geographic sites distributed among Japan, California. Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in the bw gene. The remainder are inseparable from wild-type bw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneous bw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.

Alleles↗