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At least 235 records · Page 13Linked to original sources
PubMed2003

Transposon-based strategies for microbial functional genomics and proteomics.

Transposons are mobile genetic elements that can relocate from one genomic location to another. As well as modulating gene expression and contributing to genome plasticity and evolution, transposons are remarkably diverse molecular tools for both whole-genome and single-gene studies in bacteria, yeast, and other microorganisms. Efficient but simple in vitro transposition reactions now allow the mutational analysis of previously recalcitrant microorganisms. Transposon-based signature-tagged mutagenesis and genetic footprinting strategies have pinpointed essential genes and genes that are crucial for the infectivity of a variety of human and other pathogens. Individual proteins and protein complexes can be dissected by transposon-mediated scanning linker mutagenesis. These and other transposon-based approaches have reaffirmed the usefulness of these elements as simple yet highly effective mutagens for both functional genomic and proteomic studies of microorganisms.

Artificial Gene Fusion↗
PubMed2006

Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway.

BACKGROUND: Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. RESULTS: A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC 3.1.3.25). Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC 3.1.3.15) in C. glutamicum. The cg0910 gene, renamed hisN, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes. CONCLUSION: The absence of regional and sequence preferences of IS6100-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain C.glutamicum ATCC 13032. The identification of the hisN gene encoding histidinol-phosphate phosphatase in C. glutamicum closed the last gap in histidine synthesis in the Actinobacteria. The system might be a valuable genetic tool also in other bacteria due to the broad host-spectrum of IS6100.

Actinobacteria↗
PubMed2004

Epigenetic control of CACTA transposon mobility in Arabidopsis thaliana.

Epigenetic mutation, heritable developmental variation not based on a change in nucleotide sequence, is widely reported in plants. However, the developmental and evolutionary significance of such mutations remains enigmatic. On the basis of our studies of the endogenous Arabidopsis transposon CACTA, we propose that the inheritance of epigenetic gene silencing over generations can function as a transgenerational genome defense mechanism against deleterious movement of transposons. We previously reported that silent CACTA1 is mobilized by the DNA hypomethylation mutation ddm1 (decrease in DNA methylation). In this study, we report that CACTA activated by the ddm1 mutation remains mobile in the presence of the wild-type DDM1 gene, suggesting that de novo silencing is not efficient for the defense of the genome against CACTA movement. The defense depends on maintenance of transposon silencing over generations. In addition, we show that the activated CACTA1 element transposes throughout the genome in DDM1 plants, as reported previously for ddm1 backgrounds. Furthermore, the CACTA1 element integrated into both the ddm1-derived and the DDM1-derived chromosomal regions in the DDM1 wild-type plants, demonstrating that this class of transposons does not exhibit targeted integration into heterochromatin, despite its accumulation in the pericentromeric regions in natural populations. The possible contribution of natural selection as a mechanism for the accumulation of transposons and evolution of heterochromatin is discussed.

Arabidopsis↗
PubMed2005

Mapping transposon insertion sites by touchdown PCR and hybrid degenerate primers.

A novel mapping method based on touchdown PCR was developed for identifying a transposon insertion site in genomic DNA using a hybrid consensus-degenerate primer in combination with a specific primer that anneals to the transposon. The method was tested using Xanthomonas citri transposon mutants. PCR products contained adjacent DNA regions that belonged to both X. citri genomic DNA and the transposon. Products were directly sequenced from PCRs using only the specific primer. Different PCR conditions were tested, and the optimized reaction parameters that increased product yields and specificity are described. Best results were obtained with the HIB17 hybrid primer, which is a 25-mer oligonucleotide having degenerate bases at 6 different positions within the last 12 bases at the 3' end. An X. citri mutants library was produced by random transposition using the EZ::TN transposon, and we identified the insertion sites within the genome of 90 mutants. Insertions were found within both the chromosomal and the plasmid DNA in these X. citri mutants. Restriction mapping and Southern blot analysis confirmed the insertion sites for eight randomly chosen mutants. This method is a very useful tool for large-scale characterization of mutants in functional genomics studies.

Chromosome Mapping↗
PubMed1995

Sequence of a transposon identified as Tn1000 (gamma delta).

We report the complete sequence of a transposon found in a cosmid clone of a human DNA sequence. The transposon is identified as the Escherichia coli transposon Tn1000 (also known as gamma delta) on the basis of the identity of the restriction map of the new sequence with that previously recorded for Tn1000 and homology between parts of the new sequence and that of published fragments of Tn1000 sequence. The transposon, which comprises 5,981 nucleotides including two 35 bp inverted terminal repeat sequences (ITRs), contains three open reading frames. The sequence of the resolvase coding region (tnpR) is identical to that published by others. A second reading frame can be identified as the tnpA gene, coding for the transposase, on the grounds of its strong homology with the corresponding gene from transposon Tn3. The third reading frame has the potential to code for a protein of unknown function containing 698 amino acids.

Amino Acid Sequence↗
PubMed1990

[Structure and stability of cointegrating plasmids mediated by the IS1 elements in transposon delta Tn9'].

In order to elucidate the structural features of the transposon Tn9', representative of the Tn9 family, which define the ability of the transposon to produce unstable cointegrates, we have obtained a derivative of this transposon carrying a deletion in its central region. The deletion in the obtained transposon delta Tn9' covers a DNA segment of about 50 bp in length, occupying the most distal position in relation to the cat gene, at its junction with the right copy of the IS1. The structure and stability of the IS1/delta Tn9'-mediated cointegrates between the plasmids pDK57.1 (pBR322::delta Tn9') and pRP3.1, a deletion derivative of RP1, have been studied. The three types of cointegrates were found. Those of the type I are predominantly formed, due to the left copy of the IS1 which in delta Tn9' occupies proximal position to the promoter of the cat gene. These cointegrates contain three copies of IS1 and are of high stability. The cointegrates of the type II contain two entire copies of delta Tn9' (i.e. four copies of IS1) as well as the structures of the type II, representing the cointegrate equivalent of inverse transposition and also containing four copies of IS1. Cointegrates of the type II and III dissociate efficiently in the rec+ cells but, in contrast to the cointegrates mediated by the original transposon Tn9', are unable to dissociate efficiently in the recA- cells. It was concluded that a DNA segment in the central region of Tn9' may be essential for the expression of the IS1-specific resolvase encoded by the right copy of IS1.

DNA Transposable Elements↗
PubMed1990

[The role of transposons in replication: Tn5 suppresses ts-mutation for plasmid RP1 replication in Escherichia coli cells].

Effect of restoration by transposon Tn5 of genetic damage in RP1 plasmid replication (named transposon suppression) was described. Hybrid plasmid, a derivative of RP1 and RP4, having ts mutation for replication--tsr12 and deletion in the aphA gene controlling kanamycin resistance, was constructed. Five of derivatives of this plasmid containing transposon Tn5 were made, and the strains containing both the Tn5 integrated into the chromosome and intact hybrid plasmid or the parental plasmid with the replication ts mutation, were constructed. It was shown that transposon Tn5 comprised within the hybrid plasmid or in the chromosome promotes maintenance of these replication defective plasmids in the bacterial culture at a non-permissive temperature and thus suppresses plasmid mutation tsr12. It was determined that the extent of suppression of plasmid replication ts mutation depends on the localization of transposon Tn5.

Conjugation, Genetic↗
PubMed1999

Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases.

DNA sequence database search revealed that most of Tn5053/Tn402 family transposons inserted into natural plasmids were located in putative res regions upstream of genes encoding various resolvase-like proteins. Some of these resolvase genes belonged to Tn3 family transposons and were closely related to the tnpR genes of Tn1721 and a recently detected Tn5044. Using recombinant plasmids containing fragments of Tn1721 or Tn5044 as targets in transposition experiments, we have demonstrated that Tn5053 displays striking insertional preference for the res regions of these transposons: more than 70% of Tn5053 insertion events occur in clusters inside the target res regions, while most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions. We demonstrate that Tn5053 insertions (both into and outside a res region of the target plasmid) require the presence of a functional cognate resolvase gene either in cis or in trans. To our knowledge, this is the first case when a site-specific recombination system outside a transposon has been shown to be involved in transposition.

Amino Acid Sequence↗
PubMed2004

The large resolvase TnpX is the only transposon-encoded protein required for transposition of the Tn4451/3 family of integrative mobilizable elements.

Chloramphenicol resistance in Clostridium perfringens and Clostridium difficile is often encoded by catP genes located within the 6.3 kb integrative mobilizable elements Tn4451 and Tn4453 respectively. This family of transposons is capable of being mobilized into a recipient cell in the presence of another conjugative element. Transposition is mediated by the large resolvase TnpX, which excises the element to produce a circular molecule that is the integrative intermediate. In this study, in vivo deletion analysis of the transposon-encoded tnpV and tnpY genes showed that they are not essential for excision or integration of this group of elements. Similar studies on tnpW suggested either that this gene is not essential for these functions or that TnpW does not function when provided in trans. Development and use of an in vivo insertion assay showed that TnpX is the only transposon-encoded protein required for the integration reaction. Subsequently, a TnpXLEH6 protein was purified and shown to catalyse excision in vitro in the absence of any other protein and preferentially to excise a supercoiled DNA substrate. In summary, these studies have shown that TnpX is the only transposon protein required in vivo and in vitro for the excision process and that, like excision, integration also occurs by a serine recombinase-mediated site-specific recombination mechanism.

Chloramphenicol Resistance↗
PubMed1987

Tn2501, a component of the lactose transposon Tn951, is an example of a new category of class II transposable elements.

Tn2501 is a cryptic class II transposon found as part of the lactose transposon Tn951. Insertional inactivation and nucleotide sequence analysis of Tn2501 allowed us (i) to localize the transposase (tnpA) and the resolvase (tnpR) genes as well as the resolution site (res) of Tn2501 and (ii) to compare Tn2501 with other well-known elements of the two subgroups of class II transposons (Tn3, gamma delta, Tn951, IS101; and Tn21, Tn501, Tn1721). The genetic organization of Tn2501 is similar to that of Tn3 with divergent transcription of the tnpA and tnpR genes away from the intervening res site. The tnpR gene of Tn2501 shows weak homology with that of Tn3 and even less with those of Tn21 and Tn501. However, the tnpA gene and the inverted repeat sequences of Tn2501 present more homology with those of Tn21 and Tn501 than with those of Tn3. Complementation studies showed that TnpA- mutants of Tn2501 can be complemented, at a low frequency, by the Tn21 transposase. None of the tested transposons complemented TnpR- mutants of Tn2501.

Amino Acid Sequence↗
PubMed2003

Structure of haloacetate-catabolic IncP-1beta plasmid pUO1 and genetic mobility of its residing haloacetate-catabolic transposon.

The self-transmissible plasmid pUO1 from Delftia acidovorans strain B carries two haloacetate-catabolic transposons, TnHad1 and TnHad2, and the mer genes for resistance to mercury. The complete 67,066-bp sequence of pUO1 revealed that the mer genes were also carried by two Tn402/Tn5053-like transposons, Tn4671 and Tn4672, and that the pUO1 backbone regions shared 99% identity to those of the archetype IncP-1beta plasmid R751. Comparison of pUO1 with three other IncP-1beta plasmids illustrated the importance of transposon insertion in the diversity and evolution of this group of plasmids. Mutational analysis of the four outermost residues in the inverted repeats (IRs) of TnHad2, a Tn21-related transposon, revealed a crucial role of the second residue of its IRs in transposition.

Acetates↗
PubMed2026

When Homing Endonuclease Meets Transposon: The OMEGA System.

Sequence-specific DNA endonucleases have made significant contributions to biology, biotechnology, and medicine; restriction enzymes and homing endonucleases are among classic examples. The demonstration of programmable genome editing using Cas9 in the CRISPR-Cas system, in which the target DNA sequence is recognized by base pairing with a guide RNA, revolutionized the field of genome engineering, making target selection more flexible and convenient. The OMEGA (Obligate Mobile Element-Guided Activity) system, considered a precursor to Cas12, and likely to Cas9, in the CRISPR-Cas system, is an RNA-guided DNA endonuclease composed of a TnpB, IscB, IsrB, or Fanzor protein, and a structural RNA designated reRNA or ωRNA. The OMEGA system is present in the three domains of life as an auxiliary component of transposons. The OMEGA system cuts DNA in an allele from which a transposon is excised and triggers recombination to reinstate the transposon at the same position. This "transposon restorative homing" redefines the OMEGA system as a homing endonuclease. In this review, the selfish aspects of the OMEGA system are discussed in the historical context of homing endonuclease research.

Cas12↗
PubMed2000

Identifying tagged transposon insertion sites in yeast by direct genomic sequencing.

Tagged transposons are powerful tools for large-scale studies of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae. The current techniques used to identify transposon insertion sites in the yeast genome require a DNA amplification step that can be time-consuming and problematic. We show that the DNA amplification step can be bypassed. Insertion sites can be identified rapidly and reliably by direct genomic sequencing using a transposon-specific primer, BigDye-labelled terminators, and an automated sequencer. Direct genomic sequencing can also save time on the genetic analysis phase of transposon-based projects.

Base Sequence↗
PubMed1984

Transposition and transposition immunity of transposon Tn3 derivatives having different ends.

Novel Tn1/3 derivatives that contained either two left- or two right-hand ends of the transposon were constructed in a small plasmid. Both transposed at reasonable frequencies to give normal transposition products, suggesting that only the 38-bp inverted repeats of Tn3 are essential for transposition. Plasmids containing transposon derivatives with only one end (either left or right) undergo transposase-dependent transposition between replicons at much lower frequencies, resulting in co-integrate molecules in which there is no substantial duplication of transposon DNA and that appear to be simple fusions of the two plasmids. Both the right and left halves of the transposon are separately able to confer transposition immunity to the plasmid, this immunity being inseparably linked to transposition proficiency and specificity.

DNA Transposable Elements↗
PubMedDate not supplied

Functional exploration of the yeast (Saccharomyces cerevisiae) genome: use of a mini-Mu transposon to analyse randomly cloned DNA sequences.

The development of mega-sequencing techniques requires new methods for global functional analysis of cloned DNA fragments. We have developed a mini-Mu transposon adapted to yeast cloned DNA fragment analysis. This transposon allows us to do the following in a single construction: (i) to probe yeast cells for the presence of expressed open reading frames (ORFs) in the cloned DNA fragment; (ii) to localize these ORFs in the fragment and determine their transcription orientation; (iii) to use beta-galactosidase protein fusions to study regulation of these ORFs; and (iv) to disrupt the corresponding chromosomal genes. On a 5-kb yeast DNA sequence, we have verified the reliability of this new tool by comparing the data obtained with the mini-Mu transposon to those obtained by classical methods. This transposon should be of immediate use in the yeast genome sequencing programme.

Amino Acid Sequence↗
PubMed1994

Tc1 transposon-like sequences are widely distributed in salmonids.

Transposon-like elements flanked by inverted repeats, although common in invertebrates, have only recently been found in vertebrates. We report the presence of Tc1 transposon-like sequences in salmon, trout and charr species and find that these elements belong to several families that do not follow phylogenetic lines. As many as 15,000 copies reside in the Atlantic salmon haploid genome. The complete DNA sequence of one of these transposon-like elements (SALT1) is 1535 base-pairs long, including 35 nucleotide-long terminal inverted repeats. It contains a degenerate open reading frame (ORF) of 1273 nucleotides whose inferred amino acid sequence shares sequence similarity with the "D,D35E" family of transposases, particularly those from Caenorhabditis sp. and Drosophila sp. Southern blot analysis indicated that Tc1 transposon-like sequences are present in other lower vertebrates, including several fish species and amphibians, but the copy number can vary significantly in different lineages.

Amino Acid Sequence↗
PubMed1996

Discovery of amphibian Tc1-like transposon families.

We have discovered transposase sequences in the bull frog (Rana catesbeiana) and in the clawed frog (Xenopus laevis), which demonstrates that there are DNA-mediated transposons in Amphibia. The DNA sequences of 11 new Xenopus elements describe two new vertebrate transposon families. Phylogenetic analysis, using these sequences along with previously defined vertebrate and invertebrate elements, reveals at least five families of Tc1-like elements in Vertebrata. Some of these families co-exist in the same genome. Furthermore, the grouping of one of the amphibian transposon families with a branch of the teleost transposons raises the possibility of horizontal transfer.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of transposon-induced mutants of Porphyromonas gingivalis deficient in fimbriation.

Fimbriae are considered to be an important virulence factor of Porphyromonas gingivalis. In order to identify genes essential for fimbriation, other than fimA which encodes the major subunit protein of fimbriae, transposon mutagenesis and immunological screening techniques were used to isolate fimbria-deficient mutants. R751::*Omega4, a suicide vector that carries Tn4351, was transferred from Escherichia coli to P. gingivalis by conjugation. Twenty-two independent fimbria-deficient mutants were identified among the resulting transformants. Southern hybridization analysis with pBlue 4351, a transposon-specific probe, and R751 indicated that 45% of the mutants resulted from single transposon insertions and that the remaining 55% of the mutants resulted from cointegration of R751 sequences. Southern hybridization analysis with pUCBg12.1, a probe for the fimA region, indicated that nine of the mutants contained insertions within the 2.5 kb SacI DNA fragment of P. gingivalis that contains fimA, ORF1 (which encodes a 15 kDa protein), and the C-terminal portion of ORF5 (which encodes a 63 kDa protein). Polymerase chain reaction (PCR) analysis and further Southern hybridization analysis indicated that the insertion site(s) for all nine of these mutants was within the fimA gene. Southern hybridization analysis also indicated that the remaining thirteen mutants contained insertions somewhere outside the 10 kb fimA region. Analysis by pulsed field gel electrophoresis (PFGE) revealed that insertions for most of the thirteen mutants mapped to a 300 kb NotI fragment and are located at least approximately 200 kb away from fimA. These results identify genetic loci other than fimA, that are required for fimbriation of P. gingivalis. Future cloning and characterization of these genetic loci should be straightforward since they are now marked by antibiotic resistance genes carried by the transposon.

Bacterial Proteins↗