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At least 19 recordsLinked to original sources
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↗
PubMed1989

Analysis of insertion mutants reveals two new genes in the pNRC100 gas vesicle gene cluster of Halobacterium halobium.

The archaebacterium, Halobacterium halobium, achieves buoyancy through synthesis of intracellular gas-filled vesicles. The plasmid-encoded gene (gvpA) specifying the major structural gas vesicle protein has previously been cloned and sequenced allowing the analysis of high-frequency mutations to the vesicle negative phenotype. Among eighteen gas vesicle mutants analyzed, four were observed to contain insertion elements 0.2 to 2 kb upstream of the structural gene. To explain the phenotype of these mutants, the upstream area was analyzed by DNA sequencing and transcriptional mapping. This analysis showed the presence of two open reading frames, gvpD and gvpE, which are of opposite transcriptional orientation to gvpA (gene order gvpA-D-E). gvpD begins 201 nucleotides from the gvpA structural gene and is 1608 nucleotides long while gvpE begins two nucleotides from the 3'-end of gvpD and is 573 nucleotides long. Primer extension analysis showed the occurrence of divergent promoters in the gvpA-gvpD intergenic region with the transcription start sites separated by 109 nucleotides. The sites of three insertion sequences in gas vesicle mutants mapped within gvpE while the fourth insertion site mapped near the N-terminal coding region of gvpD. Homology between the gvpDE gene region and a chromosomal site in a H. halobium NRC-1 derivative and in several other Halobacterium strains was identified by Southern hybridization.

Amino Acid Sequence↗
PubMed2006

The DcMaster Transposon Display maps polymorphic insertion sites in the carrot (Daucus carota L.) genome.

DcMaster is a family of PIF/Harbinger-like class II transposable elements identified in carrot. We present a modified Transposon Display molecular marker system allowing amplification of genomic regions containing DcMaster elements. We scored 77 DcMaster Transposon Display (DcMTD) amplicons, of which 54 (70%) were segregating in the F(2) progeny from the cross between wild and cultivated carrot. Segregating amplicons were incorporated into a previously developed molecular linkage map of carrot. Twenty-eight markers were attributed to the wild parent, 23 originated from the cultivated parent, and three markers remained unlinked. The markers were evenly distributed among the nine linkage groups. However, differences in the distribution pattern of DcMaster insertion sites in the genomes of the wild and cultivated parent were observed. Specificity of the obtained amplicons was confirmed by sequencing and three putative DcMaster subfamilies, differing in the sequence of their terminal inverted repeats, were revealed.

Base Sequence↗
PubMed2001

Analyses of spontaneous mutations of cloned gene 49 of phage T4.

Holliday structure resolving enzyme endonuclease VII (endo VII) of phage T4 is highly toxic for E. coli when expressed outside of the phage infection environment. As a consequence, plasmids with a mutated gene 49, the gene which encodes for endo VII, can be easily isolated and characterised. We have isolated and characterised 400 survivors from independent transformations with a plasmid carrying gene 49 under the control of the T7 promoter. The majority had mutated gene 49 by IS10 insertions which almost exclusively mapped to a distinct site. When this site was mutated other insertion sites were observed as well as an increase in other mutational events including large deletions. Neither of the observed insertion sites mapped matched the consensus IS10 sequence completely. Additionally when the level of expression of gene 49 was altered the distribution of mutations was changed suggesting that other elements apart from the target sequence are necessary for determining IS10 insertion. The expression of gene 49 in E. coli provides a particularly useful tool for the analysis of mutational events.

Bacteriophage T4↗
PubMed2005

Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes.

Pseudomonas aeruginosa is a major cause of nosocomial (hospital-derived) infections, is the predominant pathogen in chronic cystic fibrosis lung infections, and remains difficult to treat due to its high intrinsic antibiotic resistance. The completion of the P. aeruginosa PAO1 genome sequence provides the opportunity for genome-wide studies to increase our understanding of the pathogenesis and biology of this important pathogen. In this report, we describe the construction of a mini-Tn5-luxCDABE mutant library and a high-throughput inverse PCR method to amplify DNA flanking the site of insertion for sequencing and insertion site mapping. In addition to producing polar knockout mutations in nonessential genes, the promoterless luxCDABE reporter present in the transposon serves as a real-time reporter of gene expression for the inactivated gene. A total of 2519 transposon insertion sites were mapped, 77% of which were nonredundant insertions. Of the insertions within an ORF, -55% of total and unique insertion sites were transcriptional luxCDABE fusions. A bias toward low insertion-site density in the genome region that surrounds the predicted terminus of replication was observed. To demonstrate the utility of chromosomal lux fusions, we performed extensive regulatory screens to identify genes that were differentially regulated under magnesium or phosphate limitation. This approach led to the discovery of many known and novel genes necessary for these environmental adaptations, including genes involved in resistance to cationic antimicrobial peptides. This dual-purpose mutant library allows for functional and regulation studies and will serve as a resource for the research community to further our understanding of P. aeruginosa biology.

Antimicrobial Cationic Peptides↗
PubMed1998

Characterization of the phylogenetic distribution and chromosomal insertion sites of five IS6110 elements in Mycobacterium tuberculosis: non-random integration in the dnaA-dnaN region.

SETTING: Five IS6110 chromosomal insertion sites were characterized in the multidrug resistant Mycobacterium tuberculosis 'W' strain. OBJECTIVE: To use insertion site probes to study the phylogenetic distribution of IS6110 in the M. tuberculosis genome. DESIGN: A total of 722 M. tuberculosis isolates, previously genotyped using the standard IS6110 Southern blot hybridization methodology, were re-hybridized with the Region A insertion site probe and representative strains were further hybridized with the Region B and C probes. Strains were grouped on the basis of having IS6110 insertions in these different regions and their relatedness was further compared by sequencing the IS6110 insertion sites. RESULTS: The insertion site probes revealed that the collection of Chinese isolates previously grouped as the Beijing strain family shared IS6110 insertions in common with the W and other genotypic group 1 strains. Unexpectedly, we found that IS6110 integrated at least 10 independent times between the dnaA and dnaN genes encoding deoxyribonucleic acid replication proteins. CONCLUSIONS: IS6110 insertion site mapping is able to identify genetic relatedness among a collection of M. tuberculosis clinical strains representing the breadth of species diversity. The mapping data indicate that IS6110 insertion sites are not always random.

Blotting, Southern↗
PubMed2002

FLAGdb/FST: a database of mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants.

A large collection of T-DNA insertion transformants of Arabidopsis thaliana has been generated at the Institute of Agronomic Research, Versailles, France. The molecular characterisation of the insertion sites is currently performed by sequencing genomic regions flanking the inserted T-DNA (FST). The almost complete sequence of the nuclear genome of A.thaliana provides the framework for organising FSTs in a genome oriented database, FLAGdb/FST (http://genoplante-info.infobiogen.fr). The main scope of FLAGdb/FST is to help biologists to find the FSTs that interrupt the genes in which they are interested. FSTs are anchored to the genome sequences of A.thaliana and positions of both predicted genes and FSTs are shown graphically on sequences. Requests to locate the genomic position of a query sequence are made using BLAST programs. The response delivered by FLAGdb/FST is a graphical representation of the putative FSTs and of predicted genes in a 20 kb region.

Arabidopsis↗
PubMed2002

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana.

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation (Ds) transposon lines with insertions on all 5 Arabidopsis chromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsis genome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsis lines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.

Arabidopsis↗
PubMed1997

Cloning and characterization of the fmt gene which affects the methicillin resistance level and autolysis in the presence of triton X-100 in methicillin-resistant Staphylococcus aureus.

In methicillin-resistant Staphylococcus aureus (MRSA) strains, Triton X-100 reduced the oxacillin resistance level, although the degree of reduction varied from strain to strain. To study the responses of MRSA strains to Triton X-100, we isolated a Tn551 insertion mutant of the COL strain that became more susceptible to oxacillin in the presence of 0.02% Triton X-100. The Tn551 insertion of the mutant was transduced back to the parent strain, other MRSA strains (strains KSA8 and NCTC 10443), and methicillin-susceptible strain RN450. All transductants of MRSA strains had reduced levels of resistance to oxacillin in the presence of 0.02% Triton X-100, while those of RN450 did not. Tn551 mutants of KSA8 and NCTC 10443 also had reduced levels of resistance in the absence of 0.02% Triton X-100. The autolysis rates of the transductants in the presence of 0.02% Triton X-100 were significantly increased. Amino acid analysis of peptidoglycan and testing of heat-inactivated cells for their susceptibilities to several bacteriolytic enzymes showed that there were no significant differences between the parents and the respective Tn551 mutants. The Tn551 insertion site mapped at a location different from the previously identified fem and llm sites. Cloning and sequencing showed that Tn551 had inserted at the C-terminal region of a novel gene designated fmt. The putative Fmt protein showed a hydropathy pattern similar to that of S. aureus penicillin-binding proteins and contained two of the three conserved motifs shared by penicillin-binding proteins and beta-lactamases, suggesting that fmt may be involved in cell wall synthesis.

Bacteriolysis↗
PubMed2003

Generation and flanking sequence analysis of a rice T-DNA tagged population.

Insertional mutagenesis provides a rapid way to clone a mutated gene. Transfer DNA (T-DNA) of Agrobacterium tumefaciens has been proven to be a successful tool for gene discovery in Arabidopsis and rice ( Oryza sativa L. ssp. japonica). Here, we report the generation of 5,200 independent T-DNA tagged rice lines. The T-DNA insertion pattern in the rice genome was investigated, and an initial database was constructed based on T-DNA flanking sequences amplified from randomly selected T-DNA tagged rice lines using Thermal Asymmetric Interlaced PCR (TAIL-PCR). Of 361 T-DNA flanking sequences, 92 showed long T-DNA integration (T-DNA together with non-T-DNA). Another 55 sequences showed complex integration of T-DNA into the rice genome. Besides direct integration, filler sequences and microhomology (one to several nucleotides of homology) were observed between the T-DNA right border and other portions of the vector pCAMBIA1301 in transgenic rice. Preferential insertion of T-DNA into protein-coding regions of the rice genome was detected. Insertion sites mapped onto rice chromosomes were scattered in the genome. Some phenotypic mutants were observed in the T1 generation of the T-DNA tagged plants. Our mutant population will be useful for studying T-DNA integration patterns and for analyzing gene function in rice.

Base Sequence↗
PubMed1997

Complete sequence and transposon mutagenesis of the BamHI J fragment of Cydia pomonella granulosis virus.

The BamHI J fragment of Cydia pomonella granulosis virus was subcloned and subjected to transposon mutagenesis in Escherichia coli using a Tn3 derivative. After screening by restriction endonuclease digestion and polymerase chain reaction (PCR), 44 clones were selected representing insertions every 100 to 300 bp. The complete sequence was compiled and the transposon insertion sites mapped precisely by sequencing. Analysis of the sequence revealed the presence of 7 potential open reading frames (ORFs). The BamHI J fragment was already known to encode IAP and OPDV-E6. Three other ORFs encode products similar to known proteins, viz. an Autographa californica nuclear polyhedrosis virus 8.6 kDa protein, a Lymantria dispar nuclear polyhedrosis virus 34 kDa protein, and vertebrate reovirus omega-1 proteins. The ORF with similarity to omega-1 is also similar to baculovirus p10 proteins. In both cases, the similarity occurs in regions likely to form a coiled-coil structure.

Amino Acid Sequence↗
PubMed1986

Molecular cloning and characterization of sequences from the regulatory cluster of the Pseudomonas plasmid alk system.

Alkane oxidation functions encoded by the Pseudomonas plasmid CAM-OCT are positively regulated by one or more products of a locus designated alkR. To characterize this locus in greater detail, molecular cloning and restriction mapping of sequences covering the alkR region have been carried out in Escherichia coli, followed by mobilization to Pseudomonas recipients for analysis of genetic content. Inserts from Pseudomonas (CAM-OCT) strains were cloned into vectors pLAFR1, the pLAFR1::Tn7S derivative pXJS5403, and the transposon vector Tn3 delta 596. This has made it possible to: (1) construct a detailed restriction map of cloned fragments and the alkR region of CAM-OCT; (2) map insertion sites of the transposon Tn7S into alkR cistrons; and (3) analyze the ability of cloned sequences to complement or effect marker rescue of alkR nitrosoguanidine- and Tn7S-induced mutations. In addition, transcription of an alkB'-lacZ transcription fusion in the presence of a cloned 18.5 kb EcoRI alkR fragment and an inducer of the alk system confirmed that our cloned sequences contain functional alkR cistrons. The complementation/marker rescue results indicate that alkR is a complex locus and that the products of at least three cistrons are required for the complete AlkR+ phenotype. One of these cistrons is identified by mutations which alter a component of the inducer recognition system.

Alkanes↗
PubMed1993

Avian herpesvirus as a live viral vector for the expression of heterologous antigens.

Control of Marek's disease in the poultry industry has been successfully achieved for several decades by large-scale vaccination of day-old chickens with live herpesvirus of turkeys (HVT) strains. Several features of this virus including lack of pathogenicity and long-term immune protection due to a persistent viraemic infection made us decide to use HVT as a live viral vector for the expression of foreign antigens. Potential sites for the integration of foreign DNA in the unique short region of the HVT genome were identified by the insertion of a beta-galactosidase expression cassette. Vaccination trials with recombinant virus strains indicated that the marker gene was expressed and stably maintained during animal passage. Based on an insertion site mapping in one of the open reading frames of the unique short region, a general recombination vector was designed for the integration of foreign genes into HVT. Recombinant virus-directed expression of individual antigens from Newcastle disease virus was driven by a strong promoter element derived from the lung terminal repeat sequence of Rous sarcoma virus.

Amino Acid Sequence↗
PubMed2002

Identification and evolution of an IS6110 low-copy-number Mycobacterium tuberculosis cluster.

A cohort of 56 patients infected with related strains of Mycobacterium tuberculosis, the S75 group, was identified in a New Jersey population-based study of all isolates with a low number of copies of the insertion element IS6110. Genotyping was combined with surveillance data to identify the S75 group and to elucidate its recent evolution. The S75 group had similar demographic and geographic characteristics. Seventeen persons (30%) were linked epidemiologically. The S75 group was segregated from other low-copy-number isolates on the basis of several independent molecular methods. This group included 3 IS6110 genotype variants: BE, H6, and C28, containing 1, 2, and 3 IS6110 insertions, respectively. IS6110 insertion site mapping and comparative sequence analysis strongly suggest a stepwise acquisition of IS6110 elements from BE to H6 to C28. S75 represents a locally produced strain cluster that has recently evolved. The combination of multiple molecular tools with traditional epidemiology provides novel insights into dissemination, local transmission, and evolution of M. tuberculosis.

DNA Transposable Elements↗
PubMed2004

Clonal expansion of a globally disseminated lineage of Mycobacterium tuberculosis with low IS6110 copy numbers.

Knowledge of the clonal expansion of Mycobacterium tuberculosis and accurate identification of predominant evolutionary lineages in this species remain limited, especially with regard to low-IS6110-copy-number strains. In this study, 170 M. tuberculosis isolates with </=6 IS6110 insertions identified in Cape Town, South Africa, were characterized by principal genetic grouping, restriction fragment length polymorphism analysis, spoligotyping, IS6110 insertion site mapping, and variable-number tandem repeat (VNTR) typing. These analyses indicated that all but one of the isolates analyzed were members of principal genetic group 2 and of the same low-IS6110-copy-number lineage. The remaining isolate was a member of principal genetic group 1 and a different low-IS6110-copy-number lineage. Phylogenetic reconstruction suggests clonal expansion through sequential acquisition of additional IS6110 copies, expansion and contraction of VNTR sequences, and the deletion of specific direct-variable-repeat sequences. Furthermore, comparison of the genotypic data of 91 representative low-IS6110-copy-number isolates from Cape Town, other southern African regions, Europe, and the United States suggests that certain low-IS6110-copy-number strain spoligotypes and IS6110 fingerprints were acquired in the distant past. These clones have subsequently become widely disseminated and now play an important role in the global tuberculosis epidemic.

DNA Transposable Elements↗
PubMed2004

Rapid, large-scale generation of Ds transposant lines and analysis of the Ds insertion sites in rice.

Rapid, large-scale generation of a Ds transposant population was achieved using a regeneration procedure involving tissue culture of seed-derived calli carrying Ac and inactive Ds elements. In the F(2) progeny from genetic crosses between the same Ds and Ac starter lines, most of the crosses produced an independent germinal transposition frequency of 10-20%. Also, many Ds elements underwent immobilization even though Ac was expressed. By comparison, in a callus-derived regenerated population, over 70% of plants carried independent Ds insertions, indicating transposition early in callus formation. In the remaining population, the majority of plants carried only Ac. Most of the new Ds insertions were stably transmitted to a subsequent generation. An exceptionally high proportion of independent transposants in the regenerated population means that selection markers for transposed Ds and continual monitoring of Ac/Ds activities may not necessarily be required. By analyzing 1297 Ds-flanking DNA sequences, a genetic map of 1072 Ds insertion sites was developed. The map showed that Ds elements were transposed onto all of the rice chromosomes, with preference not only near donor sites (36%) but also on certain physically unlinked arms. Populations from both genetic crossing and tissue culture showed the same distribution patterns of Ds insertion sites. The information of these mapped Ds insertion sites was deposited in GenBank. Among them, 55% of Ds elements were on predicted open-reading frame (ORF) regions. Thus, we propose an optimal strategy for the rapid generation of a large population of Ds transposants in rice.

Chromosome Mapping↗
PubMed2000

Mapping of IS6110 insertion sites in two epidemic strains of Mycobacterium tuberculosis.

A widely distributed strain designated 210 was identified in a study of the diversity of Mycobacterium tuberculosis DNA fingerprints from three geographically separate states in the United States. This strain is characterized by a 21-band fingerprint pattern when probed with IS6110, and the pattern is similar to that displayed by strains designated W. Intracellular growth of strain 210 isolates in human macrophages is significantly faster than that of isolates from other clusters or nonclustered isolates. The purpose of this study was to identify the sites of IS6110 insertions in strain 210 and compare these to IS6110 insertion sites in strain W. Our hypothesis is that an IS6110 insertion site(s) could possibly be responsible for a strain's increased capacity for transmission and/or replication. In this report, the insertion sites in strains 210 and W are described and referenced to their location in the M. tuberculosis H37Rv genome sequence. The W and 210 strains have 17 identical sites of IS6110 insertion and additional sequence not found in H37Rv but present in other clinical isolates. The IS6110 insertion site in the 36-bp direct repeat (DR) region of strains 210 and W has 15 spacers in the left flanking region. The DR region on the right side of IS6110 has been deleted. Five sites of insertion in strain 210 not found in strain W are described, as well as two unique sites in strain W. One copy of IS6110 was found to reside 55 bp in the ctpD gene. This gene is expressed, indicating that IS6110 can provide a promoter sequence for the transcription of genes.

Base Sequence↗