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PubMed1993

TnMax--a versatile mini-transposon for the analysis of cloned genes and shuttle mutagenesis.

A series of Tn1721-based mini-transposons (TnMax) has been developed which are suitable for the targeting of cloned genes, shuttle mutagenesis, identification of protein export signals and the rescue of chromosomal markers. TnMax mini-Tn consist of two 38-bp inverted repeats (IR) flanking a resolution site (res), a suicide replication origin (orifd), and a gene conferring resistance to either chloramphenicol (TnMax1) or erythromycin (TnMax2). Other versions of TnMax (TnMax3 and TnMax4) carry a promoterless alkaline phosphatase-encoding gene (phoA') useful for the identification of protein export signals. The various mini-Tn cartridges are fused on the same plasmid with a common transposition control unit (TCU) comprising the tnpA (transposase) and tnpR (resolvase) genes under control of the inducible Ptrc promoter. This configuration causes a high frequency of transposition in Escherichia coli (approx. 10(-2) events/copy of target plasmid) and a minimum size of the mini-Tn. Like Tn1721, TnMax variants prefer random insertion into plasmids rather than into the E. coli chromosome, thus representing superb tools for the insertion mutagenesis of cloned genes. The TnMax-borne orifd simplifies the identification of targeted plasmids and facilitates shuttle mutagenesis, i.e., suicide delivery of a mutated gene with subsequent allelic replacement of a corresponding resident gene, in a variety of microorganisms. Rescue of such targeted chromosomal genes is easily accomplished by the excision of TnMax plus flanking segments using appropriate restriction endonucleases, ligation, and transformation of an E. coli host permissive for orifd-directed replication.

Base Sequence↗
PubMed1989

Molecular analysis of a plasmid-encoded phenol hydroxylase from Pseudomonas CF600.

Pseudomonas strain CF600 is able to utilize phenol and 3,4-dimethylphenol as sole carbon and energy source. We demonstrate that growth on these substrates is by virtue of plasmid-encoded phenol hydroxylase and a meta-cleavage pathway. Screening of a genomic bank, with DNA from the previously cloned catechol 2,3-dioxygenase gene of the TOL plasmid pWW0, was used in the identification of a clone which could complement a phenol-hydroxylase-deficient transposon insertion mutant. Deletion mapping and polypeptide production analysis identified a 1.2 kb region of DNA encoding a 39.5 kDa polypeptide which mediated this complementation. Enzyme activities and growth properties of Pseudomonas strains harbouring this fragment on a broad-host-range expression vector indicate that phenol hydroxylase is a multicomponent enzyme containing the 39.5 kDa polypeptide as one component.

Bacterial Proteins↗
PubMed1985

Molecular tools for the mapping of the human genome.

The rapid progress in molecular cloning and DNA analysis techniques, together with the use of cloned DNA probes from specific chromosomes of the human genome might allow localisation and ultimately identification of genes defined by single mutations. The discrepancy between genetic dimensions expressed in centimorgans, each corresponding to millions of base pairs and distances easily accessible by molecular techniques amounting to at the most hundreds of kilobase pairs may be bridged with some new cloning techniques partially developed at the European Molecular Biology Laboratory. These techniques were designed to allow rapid cloning and analysis of large regions of mammalian genomes.

Animals↗
PubMed2026

Applications of transposon-insertion sequencing for understanding bacterial physiology.

Transposon-insertion sequencing (Tn-seq) couples transposon mutagenesis with next-generation sequencing to identify the transposon insertion site for thousands of mutants in parallel. It is a powerful technology with a myriad of uses beyond the identification of essential genes required for a cell to grow and divide. Tn-seq is particularly useful as a high-throughput method to assign function to function-unknown genes, which have increased steadily with the abundance of newly sequenced bacterial genomes. Tn-seq has now been adapted for use in over 100 bacterial species. Here, we summarize the applications of Tn-seq for querying bacterial physiology and discuss some of the possible applications for the future.

DNA Transposable Elements↗
PubMed2003

Evolutionary relationships among strains of Mycobacterium tuberculosis with few copies of IS6110.

Molecular typing of Mycobacterium tuberculosis by using IS6110 shows low discrimination when there are fewer than five copies of the insertion sequence. Using a collection of such isolates from a study of the epidemiology of tuberculosis in London, we have shown a substantial degree of congruence between IS6110 patterns and both spoligotype and PGRS type. This indicates that the IS6110 types mainly represent distinct families of strains rather than arising through the convergent insertion of IS6110 into favored positions. This is supported by identification of the genomic sites of the insertion of IS6110 in these strains. The combined data enable identification of the putative evolutionary relationships of these strains, comprising three lineages broadly associated with patients born in South Asia (India and Pakistan), Africa, and Europe, respectively. These lineages appear to be quite distinct from M. tuberculosis isolates with multiple copies of IS6110.

Africa↗
PubMed1995

Identification and characterization of the insertion element IS1070 from Leuconostoc lactis NZ6009.

A novel insertion sequence, designated IS1070, was identified on the lactose plasmid of Leuconostoc lactis NZ6009 by nucleotide sequence analysis. The 1027-bp sequence contains partially matched (24 of 28 bp) inverted repeats and has one long open reading frame. The deduced 305-amino-acid sequence demonstrated homology to transposases of IS30 from Escherichia coli, IS4351 from Bacteroides fragilis, IS1086 from Alcaligenes eutrophus, IS1161 from Streptococcus salivarius, ISAS2 from Aeromonas salmonicida and a putative protein encoded by ORF3 of virus SpV1-R8A2 B from Spiroplasma citri. At least fifteen IS1070-like sequences were detected in the genome of the parent Lc. lactis strain and five of these were situated on plasmids. Analysis of the direct repeats of two of these copies with that of IS1070 revealed differences in the target duplication lengths.

Amino Acid Sequence↗
PubMed1994

Identification and nucleotide sequence analysis of a transfer-related region in the streptococcal conjugative transposon Tn5252.

To obtain a functional map of Tn5252, a 47.5-kb streptococcal conjugative transposon, a series of defined deletion and insertion mutations were introduced within the transposon. Interruptions at several regions were found to affect the conjugal transposition functions of the element in filter-mating experiments. The nucleotide sequence of the left terminus of Tn5252 showed two open reading frames, ORF1 and ORF2, adjoining the att site. The organization of this region and the structure of the predicted integrase encoded by ORF1 were found to be similar to those of other site-specific recombination systems.

Amino Acid Sequence↗
PubMed1996

Identification and sequencing of a novel insertion sequence, IS1471, in Burkholderia cepacia strain 2a.

A novel insertion sequence (IS), IS1471, has been identified which is inserted into IS element IS1071 possessed by plasmid pIJB1 in Burkholderia cepacia strain 2a. Nucleotide sequencing has revealed that IS1471 is 1112 bp in length and is flanked by 22/21-bp imperfect inverted repeats with a 3-bp duplication of the target sequence IS1471 contains a single open reading frame encoding a putative polypeptide of 345 amino acids with molecular mass of 39406 Da. Searches of DNA and protein sequence databases did not result in the detection of any homologous IS elements, suggesting that IS1471 is novel and may not belong to any known IS groups.

Amino Acid Sequence↗
PubMed2004

Identification of an apolipoprotein A-I structural element that mediates cellular cholesterol efflux and stabilizes ATP binding cassette transporter A1.

Synthetic peptides were used in this study to identify a structural element of apolipoprotein (apo) A-I that stimulates cellular cholesterol efflux and stabilizes the ATP binding cassette transporter A1 (ABCA1). Peptides (22-mers) based on helices 1 (amino acids 44-65) and 10 (amino acids 220-241) of apoA-I had high lipid binding affinity but failed to mediate ABCA1-dependent cholesterol efflux, and they lacked the ability to stabilize ABCA1. The addition of helix 9 (amino acids 209-219) to either helix 1 (creates a 1/9 chimera) or 10 (9/10 peptide) endowed cholesterol efflux capability and ABCA1 stabilization activity similar to full-length apoA-I. Adding helix 9 to helix 1 or 10 had only a small effect on lipid binding affinity compared with the 22-mer peptides, indicating that helix length and/or determinants on the polar surface of the amphipathic alpha-helices is important for cholesterol efflux. Cholesterol efflux was specific for the structure created by the 1/9 and 9/10 helical combinations, as 33-mers composed of helices 1 and 3 (1/3), 2/9, and 4/9 failed to mediate cholesterol efflux in an ABCA1-dependent manner. Transposing helices 9 and 10 (10/9 peptide) did not change the class Y structure, hydrophobicity, or amphiphilicity of the helical combination, but the topography of negatively charged amino acids on the polar surface was altered, and the 10/9 peptide neither mediated ABCA1-dependent cholesterol efflux nor stabilized ABCA1 protein. These results suggest that a specific structural element possessing a linear array of acidic residues spanning two apoA-I amphipathic alpha-helices is required to mediate cholesterol efflux and stabilize ABCA1.

ATP Binding Cassette Transporter 1↗
PubMed2004

Flight muscle-specific expression of act88F: GFP in transgenic Culex quinquefasciatus Say (Diptera: Culicidae).

A strategy to engineer a strain of Culex mosquitoes refractory to filarial transmission is described. A requirement for success of the strategy is identification of a flight muscle-specific promoter that functions in the mosquito. A GFP marker gene under the control of the promoter region of the Drosophila melanogaster act88F gene was inserted into the genome of Culex quinquefasciatus. Transformation was confirmed by Mendelian genetics. Hybridization of a genomic Southern blot to a radiolabeled probe verified that the entire donor plasmid integrated into the mosquito genome. GFP expression in the transgenic mosquitoes was restricted to the flight muscles.

Actins↗
PubMed2006

Two distinct regions of the large serine recombinase TnpX are required for DNA binding and biological function.

The large serine recombinase, TnpX, from the Clostridium perfringens integrative mobilizable element Tn4451, consists of three domains and has two known DNA binding regions. In this study random and site-directed mutagenesis was used to identify other regions of TnpX that were required for biological activity. Genetic and biochemical analysis of these mutants led to the identification of important TnpX residues in the N-terminal catalytic pocket. In addition, another region of TnpX (aa 243-261), which is conserved within large serine recombinases, was shown to be essential for both excision and insertion. Mutation of charged residues within this region led to a loss of biological activity and aberrant DNA binding. This phenotype was mediated by interaction with the distal DNA binding region (aa 598-707). In these mutants, removal of residues 598-707 resulted in loss of DNA binding, despite the presence of the primary DNA binding region (aa 533-583). Analysis of mutations within the aa 243-261 region indicated that different protein conformations were involved in the insertion and the excision reactions. In summary, we have shown that TnpX is a complex protein that has multiple intra- and intermolecular interaction sites, providing insight into the structural and functional complexity of this important enzyme family.

Amino Acid Sequence↗
PubMed1987

Cloning and genetic organization of the pca gene cluster from Acinetobacter calcoaceticus.

The beta-ketoadipate pathway of Acinetobacter calcoaceticus comprises two parallel metabolic branches. One branch, mediated by six enzymes encoded by the cat genes, converts catechol to succinate and acetyl coenzyme A (acetyl-CoA); the other branch, catalyzed by products of the pca genes, converts protocatechuate to succinate and acetyl-CoA by six metabolic reactions analogous or identical to those of the catechol sequence. We used the expression plasmid pUC18 to construct expression libraries of DNA from an A. calcoaceticus mutant strain from which the cat genes had been deleted. Immunological screening with antiserum to the pcaE gene product, beta-ketoadipate:succinyl-CoA transferase I, resulted in the isolation of a cloned 11-kilobase-pair (kbp) fragment which inducibly expressed all six pca genes under control of the lac promoter on pUC18. The induced Escherichia coli cells formed the six pca gene products at levels 10- to 30-fold higher than found in fully induced A. calcoaceticus cultures, although protocatechuate 3,4-dioxygenase (the iron-containing product of the pcaA gene) from the recombinant strain possessed a relatively low turnover number. An E. coli culture expressing the cloned pca genes quantitatively converted protocatechuate to beta-ketoadipate; failure of the organism to metabolize the latter compound can be most readily ascribed to relatively low pool levels of succinyl-CoA, a required substrate for beta-ketoadipate:succinyl-CoA transferase, in E. coli. The gene order and direction of transcription were determined to be pcACBDFE by identification of enzymes expressed in subclones, by using natural transformation to identify subclones carrying DNA corresponding to dysfunctional alleles in mutant A. calcoaceticus strains, and by restriction mapping of both the 11-kbp fragment and derivatives of the 11-kbp fragment containing Tn5 in the pcaA, pcaB, pcaD, and pcaE genes. The fragment containing the pca gene hybridized strongly and specifically to a previously cloned fragment containing A. calcoaceticus cat genes.

Acinetobacter↗
PubMed1998

The use of restriction fragment length polymorphism (RFLP) analysis for epidemiological studies of tuberculosis in developing countries.

DNA fingerprinting, of which restriction fragment length polymorphism (RFLP) typing is the most common method used, has permitted novel investigations of the epidemiology and pathogenesis of tuberculosis. The use of IS6110, an insertion sequence which is present in Mycobacterium tuberculosis, is generally considered to be the standard RFLP method, but other molecular typing techniques may be used as adjuncts in selected circumstances. A number of epidemiologic studies using RFLP typing have been done in both industrialized and developing countries. The major findings include the confirmation or identification of chains of transmission (of both drug-sensitive and drug-resistant strains of tuberculosis), distribution of strain clusters within populations, differentiation of relapse from exogenous reinfection, better understanding of the pathogenesis of tuberculosis, identification of laboratory cross-contamination, and insight into the molecular evolution of the species. For developing countries, where the burden of tuberculosis is greatest, three major areas of investigation for the use of RFLP analysis in epidemiologic studies of tuberculosis have been identified: 1) community transmission, 2) nosocomial transmission, and 3) human immunodeficiency virus (HIV)-related tuberculosis. Elements of protocols are suggested which can be used by investigators to perform well-designed epidemiologic studies which will be relevant to developing countries and which are likely to have an impact on control programmes in these settings.

AIDS-Related Opportunistic Infections↗
PubMed2005

Random insertional mutagenesis of Leptospira interrogans, the agent of leptospirosis, using a mariner transposon.

The recent availability of the complete genome sequences of Leptospira interrogans, the agent of leptospirosis, has allowed the identification of several putative virulence factors. However, to our knowledge, attempts to carry out gene transfer in pathogenic Leptospira spp. have failed so far. In this study, we show that the Himar1 mariner transposon permits random mutagenesis in the pathogen L. interrogans. We have identified genes that have been interrupted by Himar1 insertion in 35 L. interrogans mutants. This approach of transposon mutagenesis will be useful for understanding the spirochetal physiology and the pathogenic mechanisms of Leptospira, which remain largely unknown.

DNA Transposable Elements↗
PubMed1987

Identification of Tn4451 and Tn4452, chloramphenicol resistance transposons from Clostridium perfringens.

The recombinant plasmids pJIR45 and pJIR97 contain the chloramphenicol resistance determinants derived from the Clostridium perfringens R plasmids pIP401 and pJIR27, respectively. Escherichia coli cultures which harbored these recombinant plasmids rapidly became chloramphenicol sensitive when grown in the absence of chloramphenicol. The loss of resistance was associated with the loss of 6.2-kilobase (kb) segments from both plasmids. Detailed restriction analysis of E. coli- and C. perfringens-derived deletion plasmids indicated that deletion of these segments was essentially precise. Transposition of the 6.2-kb segments was demonstrated by cloning the determinants into a temperature-sensitive plasmid, curing the recombinant plasmids, and selecting chloramphenicol-resistant, plasmid-free clones. Southern hybridization analysis of chromosomal DNA isolated from these recA E. coli clones indicated that the 6.2-kb segments had transposed to different sites on the chromosome. Heteroduplex analysis and restriction mapping indicated that the transposons, Tn4451 (pIP401) and Tn4452 (pJIR27), were closely related and did not contain large inverted or directly repeated sequences. These transposons represent the first transposable elements from the clostridia to be identified and characterized.

Chloramphenicol↗
PubMed2005

Strategies for characterising cis-regulatory elements in Xenopus.

Understanding the cis-regulatory architecture of metazoan organisms is the greatest challenge facing genome biology today. In vertebrate organisms, distinct sequence elements mediate transcriptional regulation and are scattered throughout the genome, either proximal or distal to promoters. The identification of transcriptional enhancers has proven rather difficult by conventional experimental approaches. In the past decade, the rapid generation of genomic sequences for multiple vertebrate organisms, accompanied by sophisticated comparative tools, has facilitated the identification of non-coding evolutionarily conserved regions that may encode cis-regulatory elements. Validating computational predictions and characterising cis-regulatory elements in vivo, however, has been a major bottleneck, mainly because the most commonly used organism for these experiments has been the mouse, and generating transgenic mice or modifying the mouse genome continues to be a labour-intensive, low-throughput, expensive process. This has led to the use of Xenopus, which holds great promise for high-throughput interrogation of putative cis-regulatory elements. In particular, Xenopus tropicalis may become particularly powerful for elucidating regulatory networks, chiefly because it is amenable to genetic manipulations, and its genome is being sequenced.

Animals↗
PubMed2004

The repetitive landscape of the chicken genome.

Cot-based cloning and sequencing (CBCS) is a powerful tool for isolating and characterizing the various repetitive components of any genome, combining the established principles of DNA reassociation kinetics with high-throughput sequencing. CBCS was used to generate sequence libraries representing the high, middle, and low-copy fractions of the chicken genome. Sequencing high-copy DNA of chicken to about 2.7 x coverage of its estimated sequence complexity led to the initial identification of several new repeat families, which were then used for a survey of the newly released first draft of the complete chicken genome. The analysis provided insight into the diversity and biology of known repeat structures such as CR1 and CNM, for which only limited sequence data had previously been available. Cot sequence data also resulted in the identification of four novel repeats (Birddawg, Hitchcock, Kronos, and Soprano), two new subfamilies of CR1 repeats, and many elements absent from the chicken genome assembly. Multiple autonomous elements were found for a novel Mariner-like transposon, Galluhop, in addition to nonautonomous deletion derivatives. Phylogenetic analysis of the high-copy repeats CR1, Galluhop, and Birddawg provided insight into two distinct genome dispersion strategies. This study also exemplifies the power of the CBCS method to create representative databases for the repetitive fractions of genomes for which only limited sequence data is available.

Animals↗
PubMed1998

Identification of a DNA cytosine methyltransferase gene in conjugative transposon Tn5252.

The nucleotide sequence of the 3.5-kb right junction fragment of the streptococcal conjugative transposon Tn5252 was obtained. The DNA fragment was found to carry four putative genes one of which displayed a high degree of similarity to prokaryotic 5C-cytosine methyltransferases carrying multiple sequence specificities. No cognate endonuclease gene was detected in the sequenced DNA. Purified methylase polypeptide synthesized in a T7 promoter-controlled overexpression system was found to lack methylase activity while the cell extracts of host cells containing the recombinant plasmid carrying the methylase gene were active. In vivo mutations in the methylase gene did not seem to affect the transferability of the element.

Amino Acid Sequence↗