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At least 415 records · Page 23Linked to original sources
PubMed1996

A yeast artificial chromosome contig and NotI restriction map that spans the tumor suppressor gene(s) locus, 11q22.2-q23.3.

Human chromosome 11q22-q23 is a pathologically important region in which a high level of loss of heterozygosity has been reported for breast, ovary, cervical, colon, and lung carcinomas, malignant melanomas, and hematologic malignancies. This strongly indicates that one or more tumor suppressor genes reside within the deleted region. In this report, we report the development of a contig map that covers most of the deleted regions found in these malignancies. The map comprises a contig of 66 overlapping yeast artificial chromosomes (YACs) and spans a region of 17 Mb from the PGR gene at 11q22.2 to the MLL gene at q23.3. In the process of screening the YACs, 50 new sequence-tagged site markers were developed from the termini of the YAC inserts. These markers were used for chromosome walking, and the data were then integrated into the contig map. NotI restriction mapping of these YACs revealed the presence of at least 26 NotI sites in the region. Using 22 of them, a NotI restriction map of the region from PGR to D11S939 was developed. This YAC contig will provide efficient tools for identification of the putative tumor suppressor gene(s).

Base Sequence↗
PubMed1997

Construction of a Z-DNA-specific restriction endonuclease.

Novel restriction enzymes can be created by fusing the nuclease domain of FokI endonuclease with defined DNA binding domains. Recently, we have characterized a domain (Z alpha) from the N-terminal region of human double-stranded RNA adenosine deaminase (hADAR1), which binds the Z-conformation with high specificity. Here we report creation of a conformation-specific endonuclease, Z alpha nuclease, which is a chimera of Z alpha and FokI nuclease. Purified Z alpha nuclease cleaves negatively supercoiled plasmids only when they contain a Z-DNA forming insert, such as (dC-dG)13. The precise location of the cleavage sites was determined by primer extension. Cutting has been mapped to the edge of the B-Z junction, suggesting that Z alpha nuclease binds within the Z-DNA insert, but cleaves in the nearby B-DNA, by using a mechanism similar to type IIs restriction enzymes. These data show that Z alpha binds Z-DNA in an environment similar to that in a cell. Z alpha nuclease, a structure-specific restriction enzyme, may be a useful tool for further study of the biological role of Z-DNA.

Cloning, Molecular↗
PubMed1995

Selection of a remote cleavage site by I-tevI, the td intron-encoded endonuclease.

I-TevI, a double-strand DNA endonuclease involved in the mobility of the td intron of phage T4, is highly unusual in that it binds and cleaves intronless td alleles (td homing sites) in a site-specific but sequence-tolerant manner. The endonuclease binds to sequences flanking the intron insertion site and near the remote cleavage site, located 23 and 25 nucleotides away on the top and bottom strands, respectively. Mapping studies indicate that I-TevI has both sequence and distance sensors that function during cut-site selection. Although I-TevI cleavage of many insertion and deletion variants of the homing site is impaired, double-strand breaks are generated at positions that collectively span two turns of the helix, indicating that the interaction is extraordinarily flexible. However, the endonuclease does exhibit spacing preferences between its binding domains, and sequence preferences near the cleavage site, with the G:C pair at -23 implicated as a cleavage determinant. Furthermore, I-TevI appears to function through interactions across the minor groove at the cleavage site, as it does at the intron insertion site, and to be capable of cleaving sequentially, first on the bottom and then on the top strand. These properties of I-TevI are incorporated in a model wherein the endonuclease effects distant cleavage via a flexible hinge.

Bacteriophage T4↗
PubMed2003

Identification of 36 novel Jagged1 (JAG1) mutations in patients with Alagille syndrome.

Alagille syndrome (AGS) is an autosomal dominant disorder characterized by five major symptoms: cholestasis, vertebral deformity, heart malformations, ocular defects and peculiar facial appearance. The previously described Jagged1 (JAG1) gene on chromosome 20p12 has been identified as being responsible for AGS. JAG1 encodes a transmembrane protein acting as ligand for the evolutionarily conserved Notch signaling pathway. Here we report 36 novel mutations in the JAG1 gene. We identified 12 novel deletions, 4 insertions, 8 missense, 7 nonsense and 5 splice site mutations. All mutations map to the sequence encoding the extracellular part of the Jagged1 protein. The mutations spread over the entire gene with slightly increased rates in exons 2 to 6 and exon 23 and 24. Eight novel missense mutations map to the Delta-Serrate-Lag2 (DSL) domain and adjacent sequences which are important for ligand-receptor interaction. Inheritance was determined in 27 families. Sixteen mutations (55%) were de novo and eleven mutations (45%) were transmitted. Altogether 226 different JAG1 mutations have been described in association with AGS, including our novel 36 mutations. AGS variants are spread over the entire gene with only a few mutations in exon 26. A relatively high number of mutations are clustered in exons 2 to 6. This sequence region shows high interspecies conservation and encodes the Notch receptor-binding region (DSL domain).

Alagille Syndrome↗
PubMed1981

Integration specificity of an artificial kanamycin transposon constructed by the in vitro insertion of an internal Tn5 fragment into IS2.

IS2 has been marked genetically by the in vitro insertion into its HindIII site of a 3.3 Kb HindIII fragment of Tn5 conferring resistance to kanamycin. The transposition of the IS2::Km, thus obtained, to lambda has been found and insertion sites were characterised. Each of ten independent IS2::Km insertions were found at the same site at 61.2% of the lambda map, always in the same orientation (orientation II relative to the xis gene). The integration sites of IS2::Km in five of the kanamycin-transducing phages were determined by DNA sequence analysis, and were found to be identical at the nucleotide level. Further transposition of IS2::Km from lambda to the bacterial chromosome was demonstrated.

Bacteriophage lambda↗
PubMed1992

New IS10 transposition vectors based on a gram-positive replication origin.

We describe below a set of plasmid-based vehicles which can be used for delivery of IS10-derived transposons into Gram- bacteria. These vehicles replicate via a Gram+ plasmid origin that is inactive in Escherichia coli; they are easily maintained in Bacillus subtilis. Transposons are introduced by electroporation or transformation with the plasmid, and as in previous delivery systems, transpositions are selected with the appropriate antibiotic. This system should be particularly useful in situations where the standard delivery vehicles, based on bacteriophage lambda, are inappropriate. The system described incorporates a number of useful features: a variety of antibiotic markers (Er, Cm, Km or Tc), a polylinker containing restriction sites for rare-cutting endonucleases to facilitate physical mapping of chromosomal insertions, a mutant transposase that confers a relaxation in insertion specificity and positioning of the transposase-encoding gene outside of the transposing segment to ensure the stability of insertions once isolated.

Bacillus subtilis↗
PubMed1997

Insertional mutagenesis by a modified in vitro Ty1 transposition system.

Transposable elements are useful tools for insertional mutagenesis and have many potential applications in the characterization of complex genomes. Here we describe a system which facilitates the construction of large transposon insertion libraries useful for genome sequencing and functional genomic analysis. We developed two transposons, TyK and TyK'GFP+, which can be introduced into target DNAs by Ty1-mediated transposition in vitro, and several modifications which decrease the frequency of false transposition events and direct the recovery of transpositions into passenger rather than vector DNA. Insertions of TyK'GFP+ additionally may yield fusions to the Aequorea green fluorescent protein (GFP), useful in studies of gene expression and protein targeting. Transposition in vitro was obtained into target DNAs of up to 50 kb in size, restriction mapping showed insertion to be relatively random, and the sequence of 55 insertion sites showed neither strong site nor base compositional preference. Our data suggest that TyK-based artificial transposons will be suitable for a variety of genetic applications in many organisms.

DNA Transposable Elements↗
PubMed1999

Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a proteobacterial lipase family and an analogous lipase chaperone family.

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5kDa) and 347 aa (38.6kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.

Acinetobacter calcoaceticus↗
PubMed1997

Tn916 transposition in Haemophilus influenzae Rd: preferential insertion into noncoding DNA.

The availability of completely sequenced genomes has created an opportunity for high throughput mutational studies. Using the conjugative transposon Tn916, a pilot project was initiated to determine the efficiency of gene disruption in the first completely sequenced bacterium, Haemophilus influenzae Rd strain KW20. DNA was isolated from Tn916-mutagenized cells, and the point of transposon insertion was determined by inverse PCR, DNA sequencing, and mapping to the wild-type genome sequence. Analysis of the insertion sites at the nucleotide level demonstrated a biased pattern of insertion into regions rich in stretches of A's and T's. Although Tn916 integrated at multiple dispersed positions throughout the chromosome, 9 of 10 insertion events occurred in noncoding, intergenic DNA. It was determined that the intergenic DNA was over 5% more A + T-rich than that of protein coding sequences. This suggests that A + T-rich sequences similar to the Tn916 insertion site would be more likely to reside in the intergenic DNA. In an effort to identify other likely sites for transposon integration, a hidden Markov model of the consensus target insertion site was derived from the Tn916-H. influenzae junction fragments and searched against the entire genome. Eighty percent of the 30 highest-scoring predicted Tn916 target sites were from intergenic, nonprotein-coding regions of the genome. These data support the hypothesis that Tn916 has a marked preference for insertion into noncoding DNA for H. influenzae, suggesting that this mobile element has evolved to minimize disruption of host cell function on integration.

Base Sequence↗
PubMed1992

A system of transposon mutagenesis for bacteriophage T4.

We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deleted phage. The transposition occurred at a frequency of only 10(-7) per progeny phage, even though a dam- host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.

Base Sequence↗
PubMed1992

vacB, a novel chromosomal gene required for expression of virulence genes on the large plasmid of Shigella flexneri.

Shigellae, the causative agents of bacillary dysentery, are capable of adhering to and invading epithelial cells and spreading into adjacent cells. A chromosomal mutant of Shigella flexneri 2a YSH6000 with reduced invasive capacity was isolated by Tn5 insertion mutagenesis. The linkage of the mutant phenotype to the Tn5 insertion was determined by P1 phage transduction. The site of the Tn5 insertion was assigned to a NotI chromosomal restriction map, confirming that the virulence-associated locus, designated vacB, is a new locus on the chromosome. In the vacB mutant, production of the four plasmid-encoded virulence antigens, IpaB, -C, and -D and VirG, decreased to a low level compared with that in the wild type. In contrast, levels of transcription of the operons for virG, ipa, region-3.4, region-5, virF, and virB on the large plasmid, as determined by Northern dot blotting, were unaffected in the vacB mutant. Furthermore, transcriptional activation of the ipa operon by exploiting a tac promoter could not restore the vacB mutant to production of the same levels of the IpaB, -C, and -D proteins as those in the wild type, indicating that the vacB locus is involved in expression of the vir genes on the large plasmid at the posttranscriptional level. Cloning followed by nucleotide sequencing of the vacB region showed it to contain a 2,280-bp open reading frame encoding an 86.9-kDa protein located 669 bp downstream from the 3' end of the open reading frame for the purA gene. Disruption of the vacB gene of other serotypes of Shigella spp. and enteroinvasive Escherichia coli (EIEC) resulted in reduced expression of virulence phenotypes, indicating that the vacB gene encodes a novel type of virulence-associated gene required for the full expression of the virulence phenotype of Shigella spp. and EIEC.

Amino Acid Sequence↗
PubMed2005

Natural transposon mutagenesis of clinical isolates of Mycobacterium tuberculosis: how many genes does a pathogen need?

Transposable elements can affect an organism's fitness through the insertional inactivation of genes and can therefore be used to identify genes that are nonessential for growth in vitro or in animal models. However, these models may not adequately represent the genetic requirements during chains of human infection. We have therefore conducted a genome-wide survey of transposon mutations in Mycobacterium tuberculosis isolates from cases of human infection, identifying the precise, base-specific insertion sites of the naturally occurring transposable element IS6110. Of 294 distinct insertions mapped to the strain H37Rv genome, 180 were intragenic, affecting 100 open reading frames. The number of genes carrying IS6110 in clinical isolates, and hence apparently not essential for infection and transmission, is very much lower than the estimates of nonessential genes derived from in vitro studies. This suggests that most genes in M. tuberculosis play a significant role in human infection chains. IS6110 insertions were underrepresented in genes associated with virulence, information pathways, lipid metabolism, and membrane proteins but overrepresented in multicopy genes of the PPE family, genes of unknown function, and intergenic sequences. Population genomic analysis of isolates recovered from an organism's natural habitat is an important tool for determining the significance of genes or classes of genes in the natural biology of an organism.

Adolescent↗
PubMed1986

Enhancement of bacterial gene expression by insertion elements or by mutation in a CAP-cAMP binding site.

The regulatory region (bglR) of the cryptic bgl operon was characterized by DNA sequence analysis and transcription mapping. Bgl(-)-specific transcription was found to occur in both the wild-type Bgl- and mutant Bgl+ cells. However, the steady-state level of bgl RNA was much higher in the Bgl+ mutant than in the wild-type. Activation of the bgl operon by insertion sequence-mediated bglR mutations or point mutations in bglR is therefore the result of increased transcription. The ethylmethane sulfonate-induced point mutations in bglR are alterations in a single base in the cAMP binding protein (CAP) binding site, leading to a stronger binding of the CAP-cAMP complex. The IS1 and IS5-mediated bglR mutations analyzed show that the insertion sequences can activate the bgl operon by integration 78 to 125 base-pairs upstream from the transcription initiation site. The role of the insertion sequences in activation of the bgl operon is discussed.

Base Sequence↗
PubMed1995

New tools for integrated genetic and physical analyses of the Escherichia coli chromosome.

Genetic and biophysical techniques have traditionally been applied to genome mapping independently of one another. We present a series of Escherichia coli mini-Tn10 insertions that contain the rare-cutting polylinker 1 (RCP1) of rare restriction sites [including BlnI/AvrII, SpeI, NheI, XbaI, NotI, PacI and SfiI; Mahillon and Kleckner, Gene 116 (1992) 69-74] which allows them to be used not just for genetic mapping, but also for rapid physical mapping and integrated physical and genetic mapping of the E. coli chromosome. Their isolation and their physical and genetic coordinates in K-12 strain MG1655 are presented. Also, their use in purifying insertion-delimited DNAs from E. coli K-12 and in macrorestriction mapping of a pathogenic strain's chromosome is demonstrated. These insertions allow integration of (i) different macrorestriction patterns of a single strain's chromosome, (ii) the physical map of a single strain's chromosome with the genetic map of the species, and (iii) the physical maps of different strains' chromosomes.

Chromosome Mapping↗
PubMed1996

Rapid restriction mapping of cosmids by sequence-specific triple-helix-mediated affinity capture.

A simple and rapid strategy for restriction mapping based on sequence-specific triple-helix affinity capture (TAC) was developed. The strategy was applied to the analysis of cosmid clones by the construction of a new cosmid vector, ScosTriplex-II, containing two different triple-helix-forming sequences flanking the cloning site of the original SuperCos-1 cosmid vector. For restriction mapping, the recombinant cosmid DNA is digested with NotI restriction enzyme or with one of four intron-encoded endonucleases for excision of intact inserts followed by controlled partial digestion with a mapping enzyme used in conjunction with the corresponding methyltransferase. The partial digestion products are combined with biotinylated triple-helix-forming oligonucleotides to form a triple-helical complex. The triple-helix complexes are immobilized on streptavidin-coated magnetic beads, washed, and eluted with pH 9 buffer solution. The fragments are separated and directly sized by agarose gel electrophoresis. Bidirectional maps are obtained simultaneously by binding to the two different triple-helix-forming oligonucleotides. No probe labeling, gel drying, blotting to membranes, hybridization, or autoradiography is necessary. Also, TAC conditions that permit gel-free isolation of the terminal restriction fragments from cosmid inserts were found. These advantages afforded by ScosTriplex-II should facilitate the automation of cosmid restriction site fingerprinting needed for large-scale mapping and sequencing projects.

Base Sequence↗
PubMed1983

Analysis of tetracycline resistance encoded by transposon Tn10: deletion mapping of tetracycline-sensitive point mutations and identification of two structural genes.

Deletions in the tet genes derived from Tn10 were formed from different tet::Tn5 insertion mutations by removing DNA sequences located between a HindIII site in Tn5 and a HindIII site adjacent to the tet genes. Tetracycline-sensitive point mutations were mapped in recombination tests with the deletions and were thus aligned with the genetic and physical map of the tet region. Plasmids carrying point mutations were tested for complementation with derivatives of pDU938, a plasmid carrying cloned tet genes derived from Tn10 which had been inactivated by Tn5 insertions. Complementation occurred between promoter-proximal tet point mutations and distal tet::Tn5 insertions, suggesting the existence of two structural genes, tetA and tetB. These results, together with the analysis of polypeptides in minicells harboring pDU938tet::Tn5 mutants, suggested that tetA and tetB are expressed coordinately in an operon. The tetB gene encodes the previously characterized 36,000-dalton cytoplasmic membrane TET protein, but the product of tetA was not identified. Point mutations in either tetA or tetB led to the defective expression of the resistance mechanism involving tetracycline efflux. It is suggested that the tetA and tetB products interact cooperatively in the membrane to express resistance.

Biological Transport↗
PubMed1982

Tn7 and Tn501 Insertions into Pseudomonas aeruginosa plasmid R91-5: mapping of two transfer regions.

We constructed a restriction endonuclease map of the Pseudomonas aeruginosa narrow-host-range plasmid R91-5. Insertions of transposons Tn7 and Tn501 into the plasmid DNA were characterized physically and genetically. The distribution of sites of insertion showed some regional specificity for the insertion of these transposons, especially TN501. The insertion of Tn7 was unusual in that all 42 of 43 insertions were in the same orientation. By relating phenotypic changes to the site of insertion, the Tn1 transposon that was already present on R91-5 and coded for carbenicillin resistance was mapped, and its orientation was determined. Two major transfer regions were identified. We believe that Tra1 is involved in conjugal DNA metabolism, whereas Tra2 is involved mainly in production of the sex pili.

Adsorption↗