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Results for “transposon insertion sequencing”

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PubMed2003

[Comparison and analysis of the molecular character of breakpoints in introns of deletion hotspots of dystrophin gene].

OBJECTIVE: To study the exons deletion mechanisms for dystrophin gene, the molecular characters of breakpoints of junction fragments for deletion-type Duchenne muscular dystrophy (DMD) patients with 46 and 51 exons deletion were compared and analyzed. METHODS: Deletion-type DMD patients were detected by multiplex polymerase chain reaction(mPCR). The breakpoints of junction fragments with 46 and 51 exons deletions were cloned and sequenced respectively. RESULTS: Analysis of sequences of deletion-junction fragment of exon 46 showed that the 5'breakpoint was located in AT-rich region of intron 45 and the 3' breakpoint was in medium reiteration repeats (MER1) sequence. There existed 2 bp(ta) junction homology between two breakages. No small insertion, small deletion or point mutation was located near the junction point. Similarly, analysis of sequences of deletion-junction fragment of exon 51 showed that the 5 breakpoint was located in transposon-like human elements (THE1) of intron 50 and the 3' breakpoint was in L2 sequence. There existed 3 bp(cta) junction homology between two breakages. No small insertion, small deletion or point mutation was located near the junction point. By analyzing the secondary structure of junction fragments with 46 and 51 exons deletions, it was demonstrated that all breakpoints of junction fragments were located at the non-matching regions of single-strand hairpin. CONCLUSION: By comparing the junction fragments with 46 or 51 exons deletion, it was found that all of breakpoints were located in repeat sequences and the repeat sequences formed the single-strand hairpin which could make the introns instable and result in exon deletion.

Base Sequence↗

PubMed2002

Transposition of DEH, a broad-host-range transposon flanked by ISPpu12, in Pseudomonas putida is associated with genomic rearrangements and dehalogenase gene silencing.

Pseudomonas putida strain PP3 produces two hydrolytic dehalogenases encoded by dehI and dehII, which are members of different deh gene families. The 9.74-kb DEH transposon containing dehI and its cognate regulatory gene, dehR(I), was isolated from strain PP3 by using the TOL plasmid pWW0. DEH was fully sequenced and shown to have a composite transposon structure, within which dehI and dehR(I) were divergently transcribed and were flanked on either side by 3.73-kb identical direct repeats. The flanking repeat unit, designated ISPpu12, had the structure of an insertion sequence in that it was bordered by 24-bp near-perfect inverted repeats and contained four open reading frames (ORFs), one of which was identified as tnpA, putatively encoding an ISL3 family transposase. A putative lipoprotein signal peptidase was encoded by an adjacent ORF, lspA, and the others, ISPpu12 orf1 and orf2, were tentatively identified as a truncated cation efflux transporter gene and a PbrR family regulator gene, respectively. The orf1-orf2 intergenic region contained an exact match with a previously described active, outward-orientated promoter, Pout. Transposition of DEH-ISPpu12 was investigated by cloning the whole transposon into a suicide plasmid donor, pAWT34, and transferring the construct to various recipients. In this way DEH-ISPpu12 was shown to transpose in a broad range of Proteobacteria. Transposition of ISPpu12 independently from DEH, and inverse transposition, whereby the vector DNA and ISPpu12 inserted into the target genome without the deh genes, were also observed to occur at high frequencies in P. putida PaW340. Transposition of a second DEH-ISPpu12 derivative introduced exogenously into P. putida PP3 via the suicide donor pAWT50 resulted in silencing of resident dehI and dehII genes in about 10% of transposition transconjugants and provided a genetic link between transposition of ISPpu12 and dehalogenase gene silencing. Database searches identified ISPpu12-related sequences in several bacterial species, predominantly associated with plasmids and xenobiotic degradative genes. The potential role of ISPpu12 in gene silencing and activation, as well as the adaptation of bacteria to degrade xenobiotic compounds, is discussed.

Bacterial Proteins↗

PubMed1986

An inverted repeat sequence of the IncFI plasmid ColV2-K94 increases multimerization-mediated plasmid instability.

A detailed physical map of the region of the IncFI plasmid ColV2-K94 containing the Rep1 replicon, a Tn903 transposon, and an inverted repeat structure (X1) with unknown properties was prepared by cloning restriction fragments into pBR325. Inserts carrying the 1.2 kb repeated sequence of X1, but not the IS903 sequence of Tn903, had a destabilizing effect on pBR325 and pBR322 plasmid maintenance. One of these derivatives, pWS139, was studied further and was shown to have elevated levels of multimeric DNA forms; this resulted in decreased copy number and plasmid instability, as multimerization reduces the effective number of randomly segregating plasmids per cell. A ColV2-K94 miniplasmid, which has a copy number much lower than that of ColE1-derived vectors, was also less stably inherited if it contained the X1 structure. This destabilizing effect of the X1 repeat sequence was dependent on the RecA function, but not the RecB or the RecC functions of the host. These results suggest that the inverted repeat sequence of the X1 structure serves as a 'hot-spot' for generalized recombination. Thus, when present in cis, this sequence can generate plasmid instability because plasmid molecules are readily converted into multimeric forms through enhanced recombination at this site.

Chromosome Mapping↗

PubMed1987

A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum.

By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).

Amino Acid Sequence↗

PubMed1994

Evidence that coupling sequences play a frequency-determining role in conjugative transposition of Tn916 in Enterococcus faecalis.

The conjugative transposon Tn916 (encodes resistance to tetracycline), originally identified in Enterococcus faecalis, moves by an excision-insertion process in which the rate-limiting step is believed to be excision. Individual transposon-containing strains exhibit characteristic mating frequencies which range over several orders of magnitude; the basis of this phenomenon is addressed in the present study. We were able to generate independent single-copy insertions in identical target locations and with similar orientations within a plasmid hemolysin determinant (cylA); however, transposition from this site occurred at very different frequencies (10(-8) to 10(-4) per donor) depending on the individual isolate. DNA sequencing analyses showed that the coupling (junction) sequences differed between isolates and thus appeared to be responsible for differences in excision frequencies. Other experiments showed that inducible transcription into either end of the transposon had no significant effect on transfer.

Base Sequence↗

PubMed1991

The Escherichia coli htrP gene product is essential for bacterial growth at high temperatures: mapping, cloning, sequencing, and transcriptional regulation of htrP.

We identified and characterized a new Escherichia coli gene, htrP. The htrP gene was identified because its insertional inactivation by the Tn10 transposon results in the inability of E. coli to form colonies at temperatures above 37 degrees C and a slow growth phenotype at 30 degrees C. The htrP gene was cloned and mapped to 66.3 min on the E. coli genetic map, 4 kbp clockwise from the tolC gene. The htrP gene was sequenced and shown to code for an acidic, 27,471-Da polypeptide and to be transcribed counterclockwise with respect to the genetic map. The predicted HtrP protein has two potential transmembrane segments and shares an identity of 64.4% over a length of 210 amino acids with the LuxH protein. Despite the fact that the htrP gene is essential for E. coli growth exclusively at high temperatures, the levels of htrP-specific transcripts decrease with increasing temperature.

Amino Acid Sequence↗

PubMed2003

The foldback-like transposon Galileo is involved in the generation of two different natural chromosomal inversions of Drosophila buzzatii.

Chromosomal inversions are the most common type of genome rearrangement in the genus Drosophila. Although the potential of transposable elements (TEs) for generating inversions has been repeatedly demonstrated in the laboratory, little is known on their role in the generation of natural inversions, which are those effectively contributing to the adaptation and/or evolution of species. We have cloned and sequenced the two breakpoints of the polymorphic inversion 2q7 of D. buzzatii. The sequence analysis of the breakpoint regions revealed the presence in the inverted chromosomes of large insertions, formed by complex assemblies of transposons, that are absent from the chromosomes without the inversion. Among the transposons inserted, the Foldback-like element Galileo, that was previously found responsible of the generation of the widespread inversion 2j of D. buzzatii, is present at both 2q7 breakpoints and is the most likely inducer of the inversion. A detailed study of the nucleotide and structural variation in the breakpoint regions of six chromosomal lines with the 2q7 inversion detected no nucleotide differences between them, which suggests a monophyletic and recent origin. In contrast, a remarkable degree of structural variation was observed in the same six chromosomal lines. It thus appears that the two breakpoints of the inverted chromosomes have become genetically unstable hotspots, as was previously found for the 2j inversion breakpoints. The possibility that this instability is caused by structural properties of Foldback elements is discussed.

Animals↗

PubMed1989

Molecular characterization of two proteins involved in the excision of the conjugative transposon Tn1545: homologies with other site-specific recombinases.

Excision is probably the initial and rate-limiting step of the movements of conjugative transposons of Gram-positive bacteria such as Tn916 and Tn1545. We have shown, by molecular cloning and DNA sequencing, that a 2058 bp Sau3A right-junction fragment of transposon Tn1545 specifies two gene products that are involved in the excision of the element. The DNA sequence of these genes, designated orf1 and orf2, has been determined and the corresponding proteins, ORF1 and ORF2, have been identified in a bacterial cell-free coupled transcription-translation system. These proteins are freely diffusible since they are able to trans-complement in vivo a deletion derivative of Tn1545 defective for excision. Using an in vivo complementation assay, we have demonstrated that ORF2 alone is able to catalyse excision and that ORF1 strongly stimulates the activity of ORF2. We also found that ORF1 and ORF2 display local homology with, respectively, proteins Xis and Int from lamboid phages, which suggests that these excision systems have a common origin. Based on the functional properties of the integrase of bacteriophage lambda, on the analysis of the nucleotide sequence of the junction fragments and of the target before insertion and after excision, a model is proposed for ORF2-catalysed excision of Tn1545 and related conjugative transposons.

Amino Acid Sequence↗

PubMed2006

Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces.

DNA-based transposable elements, or DNA transposons, transpose in a cut-and-paste fashion, involving excision from the chromosome. If this process affects the function of a host gene and the excision rate is high, any gene associated with such an element would clearly be in a genetically "unstable" state, and there are many examples of unstable genes in various organisms. However, none have hitherto been reported in vertebrates. We here document the finding of an unstable mutant gene in the medaka fish, Oryzias latipes, a useful model animal for vertebrate genetics and evolutionary studies. In an inbred strain, excision of the Tol2 element inserted in a pigmentation gene occurs spontaneously, giving rise to different heritable phenotypes and new mutant genes that carry different excision footprint sequences. The phenotypic mutation rate is as high as 2% per gamete, representing a 1000-fold increase from spontaneous mutation rates so far determined with the same organism. With mutations caused by insertion, and then excision, of transposons, one can no longer recognize participation of transposons in their generation. Thus, the impact of DNA transposons on vertebrate genomes may be, and may have been, larger than commonly supposed.

Alleles↗

PubMed1992

Optimization of long-distance PCR using a transposon-based model system.

The ability to amplify routinely long PCR products (5-25 kb) with high specificity and fidelity, regardless of target template sequence or structure, would provide significant benefits to genome mapping and sequencing endeavors. Although occasional reports have described the generation of long PCR products, such results have been difficult to replicate and have frequently utilized probe hybridization to identify the specific product from nonspecific amplified DNA. Production of specific PCR products has generally been limited to target templates of less than 3 kb. To extend the effective range of standard PCR amplification, it may be necessary to utilize alternative reaction conditions and/or components, such as novel thermostable DNA polymerases or accessory proteins. We describe the use of a model system to evaluate systematically methodological changes that might enable efficient long-range PCR. Specifically, the transposon Tn5supF has been used to introduce randomly identical, known primer binding sites within separate isolates of phage clones carrying identical inserts. Transposon-based PCR allows us to study amplification of DNA fragments that vary in size and sequence using only a single set of primers. In the present studies, we describe conditions that enable PCR amplification of specific DNA templates ranging in size up to 9 kb. Some of the key features of our methodology include the use of recombinant Thermus thermophilus (rTth) DNA polymerase, the addition of gelatin to the reaction mixture, the use of wax-mediated "hot starts" and, lastly, the use of auto-segment extension thermocycling. These results also provide insights into additional approaches that might further enhance our ability to perform long-distance PCR.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1986

The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens.

The genetic transformation of plant cells by Agrobacterium tumefaciens is mediated by the genes of the Ti plasmid vir region. To determine the genetic and transcriptional organization of the vir region of pTiA6, vir plasmid clones were saturated with insertion mutations of a Tn3-lacZ transposon. This element is both an insertion mutagen and a reporter for the expression of the sequences into which it has inserted. One hundred and twenty-four vir::Tn3-lac insertions were analyzed for their mutagenic effect on Agrobacterium virulence, and for their expression of beta-galactosidase activity, the lacZ gene product, in vegetative bacteria and in bacteria cocultivated with plant cells. These data in conjunction with genetic complementation results show that the pTiA6 vir region contains six distinct vir complementation groups: virA, virB, virC, virD, virE and virG. Mutations in these loci eliminate (virA, virB, virD and virG) or significantly restrict (virC and virE) the ability of Agrobacterium to transform plant cells. Each of the vir loci corresponds to a single vir transcription unit: virA is constitutively expressed and non-inducible; virB, virC, virD and virE are expressed only upon activation by plant cells; and virG is both constitutively expressed and plant-inducible. The two largest vir operons, virB and virD, are probably polycistronic. The pTiA6 vir region also contains plant-inducible loci (pin) which are non-essential for virulence.

Cloning, Molecular↗

PubMed2004

Disruption of the Aspergillus fumigatus argB gene using a novel in vitro transposon-based mutagenesis approach.

We disrupted the Aspergillus fumigatus argB gene, encoding ornithine transcarbamylase, using a novel in vitro transposon-based mutagenesis approach. This approach utilizes a modified transposon containing the Neurospora crassa pyr4 gene, which is randomly inserted in vitro into a target sequence of interest. Clones in which the gene of interest has been disrupted are identified by PCR and used to transform a pyrG-deficient strain of A. fumigatus. Using this approach, we obtained arginine auxotrophs of A. fumigatus. Full characterization of the argB insertion was performed by Southern blot analysis. These strains can be supplemented by addition of arginine into the culture medium and can be fully rescued to arginine prototrophy by transformation with the intact A. fumigatus argB gene.

Aspergillus fumigatus↗

PubMed2005

The biosynthetic genes for disorazoles, potent cytotoxic compounds that disrupt microtubule formation.

Disorazoles are polyketides produced by the myxobacterium Sorangium cellulosum So ce12. Their mode of action is to inhibit tubulin polymerization and destabilize microtubules. Using transposon mutagenesis, two mutant strains were identified that produced no disorazoles. Sequencing the DNA flanking the insertions revealed a polyketide synthase gene cluster that would encode three polypeptides, DszA, DszB, and DszC, with DszC containing both nonribosomal peptide synthetase and polyketide synthase modules. The disorazole polyketide synthase modules lack an acyltransferase domain. Instead, a separate gene, dszD, encodes an AT protein, thus revealing that the disorazole gene cluster falls into the trans-AT Type I family of PKS enzymes.

Amino Acid Sequence↗

PubMed2006

Transposon mutagenesis of Bacillus anthracis strain Sterne using Bursa aurealis.

Bacillus anthracis, a spore forming Gram-positive microbe, is the causative agent of anthrax. Although plasmid encoded factors such as lethal toxin (LeTx), edema toxin (EdTx), and gamma-poly-d-glutamic acid (PGA) capsule are known to be required for disease pathogenesis, B. anthracis genes that enable spore invasion, phagosomal escape and macrophage replication are still unknown. To establish transposon mutagenesis as a tool for the characterization of anthrax genes, we employed the mariner-based mini-transposon Bursa aurealis in B. anthracis strain Sterne 7702. B. aurealis carrying an erythromycin resistance cassette and its cognate transposase were delivered by transformation of two plasmids. B. aurealis transposition can be selected for by temperature shift to prevent plasmid replication and by screening colonies for erythromycin resistance. Using inverse polymerase chain reaction, DNA fragments of 129 random erythromycin-resistant transposon mutants were amplified and submitted to DNA sequence analysis. These studies demonstrate that B. aurealis inserts randomly into the genome of B. anthracis and can therefore be employed for finding genes involved in virulence.

Antigens, Bacterial↗

PubMed2003

Characterization of a plasmid-encoded adhesin of an avian pathogenic Escherichia coli (APEC) strain isolated from a case of swollen head syndrome (SHS).

An avian pathogenic Escherichia coli (APEC) strain designated SHS4, isolated from a chicken with clinical signs of swollen head syndrome (SHS), adhered to but did not invade Hep-2 and tracheal epithelial cells. The PCR amplified fimA, csgA and tsh gene sequences. It produced Ia, Ib, E1, E3, K, and B colicins, but not colicin V and aerobactin. It harboured two plasmids of 60 and 98MDa and was resistant to streptomycin and tetracycline. Conjugation with a nalidixic acid (Na) resistant K-12 recipient strain (MS101) showed that the 98MDa plasmid did not transfer, whereas transfer of the 60MDa plasmid resulted in concomitant transfer of adhesion to Hep-2 and tracheal epithelial cells, production of the colicins Ia, E1, E3, and K, and the tsh-related DNA sequence. Transposon (TnphoA) mutagenesis of strain TR4 gave rise to strain Mut23, which lost its adhesive capacities, but was still able to express the same colicins as did strain TR4. PCR was able to amplify the tsh-related DNA sequence in this strain and a molecular probe based on transposon TnphoA indicated that the transposon was inserted in the 60MDa plasmid. Based on these results, we suggest that the 60MDa plasmid have adhesion genes, which may be responsible for the initial colonization of the upper respiratory tract of chickens.

Adhesins, Escherichia coli↗

PubMed1997

Identification of Pseudomonas aeruginosa genes required for epithelial cell injury.

We have developed a simple, reproducible and rapid genetic screen for Pseudomonas aeruginosa-induced epithelial cell cytotoxicity in cultures of MDCK cells. This screen was used to isolate isogenic transposon-tagged non-cytotoxic mutants of a cytotoxic and lung-virulent strain of P. aeruginosa (PA103). The transposon-insertion site was determined by using an inverse polymerase chain reaction followed by DNA-sequence analysis. On the basis of phenotype and sequence analysis, these mutants fell into four classes. One class had absent or defective pill, based on their resistance to phage PO4 and/or loss of twitching motility (twt-). A second class exhibited decreased adherence. A third class of mutants exhibited probable defects in the machinery or targets of type III protein secretion. A final class of mutants exhibited decreased but not absent cytotoxicity. This class included members of the first three classes as well as other mutants. These results suggest that localized cytotoxicity is likely to require several steps and several components, including pili and other (unidentified) extracellular proteins. The type III protein-secretion apparatus appears to be involved in this process.

Amino Acid Sequence↗

PubMed1993

Autoimmune disease in mice due to integration of an endogenous retrovirus in an apoptosis gene.

The MRL-lpr/lpr mouse strain is a model of systemic autoimmune disease. In this model, intrinsic defects of intrathymic T cell development include defective deletion of self-reactive T cells and expression of endogenous retroviruses. Defective deletion of self-reactive T cells in the thymus has been proposed to be due to germline mutation in the Fas apoptosis gene. Using different fragments of a Fas cDNA probe, we determined that the lpr/lpr mutation is a 5.3-kb insertion of DNA within the second intron of the Fas gene. cDNA corresponding to this region was then derived from thymic RNA from MRL-lpr/lpr and MRL- +/+ mice using the polymerase chain reaction. All thymic RNA samples from MRL-lpr/lpr mice yielded a unique product that was 168 bp larger than that of MRL- +/+ mice. Complete sequence analysis indicated that this inserted sequence had 98% homology with a sequence from the 3' long terminal repeat of the early transposon (ETn). RNA analysis indicated higher expression of ETn RNA in the thymus of MRL-lpr/lpr than MRL- +/+ mice. The interdependence of ETn expression and abnormal Fas expression was then analyzed in a CD2-Fas transgenic mouse model in which a full-length murine Fas cDNA under the regulation of the CD2 promoter and enhancer was used to correct defective Fas expression in T cells of MRL-lpr/lpr mice. In these mice, reduced thymic ETn expression was observed, confirming that high ETn expression is related to abnormal Fas expression. These results establish a link between endogenous retrovirus expression, abnormal Fas expression, and autoimmune disease.

Amino Acid Sequence↗

PubMed1997

Transposon-disruption of a maize nuclear gene, tha1, encoding a chloroplast SecA homologue: in vivo role of cp-SecA in thylakoid protein targeting.

A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced > or = 40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-SecA function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.

Amino Acid Sequence↗