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PubMed2006

A genomic library-based amplification approach (GL-PCR) for the mapping of multiple IS6110 insertion sites and strain differentiation of Mycobacterium tuberculosis.

Evidence suggests that insertion of the IS6110 element is not without consequence to the biology of Mycobacterium tuberculosis complex strains. Thus, mapping of multiple IS6110 insertion sites in the genome of biomedically relevant clinical isolates would result in a better understanding of the role of this mobile element, particularly with regard to transmission, adaptability and virulence. In the present paper, we describe a versatile strategy, referred to as GL-PCR, that amplifies IS6110-flanking sequences based on the construction of a genomic library. M. tuberculosis chromosomal DNA is fully digested with HincII and then ligated into a plasmid vector between T7 and T3 promoter sequences. The ligation reaction product is transformed into Escherichia coli and selective PCR amplification targeting both 5' and 3' IS6110-flanking sequences are performed on the plasmid library DNA. For this purpose, four separate PCR reactions are performed, each combining an outward primer specific for one IS6110 end with either T7 or T3 primer. Determination of the nucleotide sequence of the PCR products generated from a single ligation reaction allowed mapping of 21 out of the 24 IS6110 copies of two 12 banded M. tuberculosis strains, yielding an overall sensitivity of 87,5%. Furthermore, by simply comparing the migration pattern of GL-PCR-generated products, the strategy proved to be as valuable as IS6110 RFLP for molecular typing of M. tuberculosis complex strains. Importantly, GL-PCR was able to discriminate between strains differing by a single IS6110 band.

Chromosome Mapping↗
PubMed2006

Identification of a virulence-associated determinant, dihydrolipoamide dehydrogenase (lpd), in Mycoplasma gallisepticum through in vivo screening of transposon mutants.

To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.

Animals↗
PubMed2000

Sequence analysis of TnphoA insertion sites in Vibrio cholerae mutants defective in rugose polysaccharide production.

Vibrio cholerae can switch from a smooth to a wrinkled or rugose colony phenotype characterized by the secretion of a polysaccharide that enables the bacteria to survive harsh environmental conditions. In order to understand the genetic basis of rugosity, we isolated TnphoA-induced stable, smooth mutants of two O1 El Tor rugose strains and mapped the insertion sites in several of the mutants using a modified Y-adapter PCR technique. One of the TnphoA insertions was mapped to the first gene of the vps region that was previously shown to encode the rugose polysaccharide biosynthesis cluster. Three insertions were mapped to a previously unknown hlyA-like gene, also in the vps region. Five other insertions were found in loci unlinked to the vps region: (i) in the epsD gene (encodes the "secretin" of the extracellular protein secretion apparatus), (ii) in a hydG-like gene (encodes a sigma(54)-dependent transcriptional activator similar to HydG involved in labile hydrogenase production in Escherichia coli, (iii) in a gene encoding malic acid transport protein upstream of a gene similar to yeiE of E. coli (encodes a protein with similarities to LysR-type transcriptional activators), (iv) in dxr (encodes 1-deoxy-D-xylulose 5-phosphate reductoisomerase), and (v) in the intergenic region of lpd and odp (encode enzymes involved in the pyruvate dehydrogenase complex formation). These data suggest the involvement of a complex regulatory network in rugose polysaccharide production and highlight the general utility of the Y-adapter PCR technique described here for rapid mapping of transposon insertion sites.

Alkaline Phosphatase↗
PubMed1978

Characterization of an immunoglobin cDNA clone containing the variable and constant regions for the MOPC 21 kappa light chain.

Nucleotide sequence analysis and restriction endonuclease mapping have been used to characterize a cDNA copy of immunoglobulin MOPC 21 Kappa mRNA clones in the bacterial plasmid pMB9. Three regions of the inserted cDNA of plasmid pL21-1 have been sequenced and match the known protein sequence at amino acid residues 1-24, 128-138 and 171-179. With these sequences to provide absolute correlations between the restriction map and the structural gene sequence it has been possible to exactly deduce the positions of all 11 of the insert restriction sites mapped within the structural gene. The pL21-1 insert contains the complete variable and constant regions as well as parts of the 3' untranslated and polypeptide leader coding sequences.

Amino Acid Sequence↗
PubMed1989

Location of sites of transposon Tn916 insertion in the Mycoplasma mycoides genome.

The genome of Mycoplasma mycoides subsp. mycoides GC1176-2 was cleaved into six large fragments by the endonuclease KpnI which also cleaved the transposon Tn916 once. This has allowed genomic mapping of insertion sites for 50 transformants of GC1176-2 containing Tn916. Almost all of the mapped sites were clearly separate. The transformants provide a bank of genomes each with a KpnI site at a different position to facilitate mapping of gene loci.

Chromosome Mapping↗
PubMed2005

Large-scale enhancer detection in the zebrafish genome.

Murine retroviral vectors carrying an enhancer detection cassette were used to generate 95 transgenic lines of fish in which reporter expression is observed in distinct patterns during embryonic development. We mapped 65 insertion sites to the as yet unfinished zebrafish genome sequence. Many integrations map close to previously known developmental genes, including transcription factors of the Pax, Hox, Sox, Pou, Otx, Emx, zinc-finger and bHLH gene families. In most cases, the activated provirus is located in, or within a 15 kb interval around, the corresponding transcriptional unit. The exceptions include four insertions into a gene desert on chromosome 20 upstream of sox11b, and an insertion upstream of otx1. In these cases, the activated insertions are found at a distance of between 32 kb and 132 kb from the coding region. These as well as seven other insertions described here identify genes that have recently been associated with ultra conserved non-coding elements found in all vertebrate genomes.

Alleles↗
PubMed2002

A high-throughput Arabidopsis reverse genetics system.

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.

Agrobacterium tumefaciens↗
PubMed1995

Insertional mutation of the motor endplate disease (med) locus on mouse chromosome 15.

Homozygous transgenic mice from line A4 have an early-onset progressive neuromuscular disorder characterized by paralysis of the rear limbs, muscle atrophy, and lethality by 4 weeks of age. The transgene insertion site was mapped to distal chromosome 15 close to the locus motor endplate disease (med). The sequence of mouse DNA flanking the insertion site junctions was determined. A small (< 20 kb) deletion was detected at the insertion site, with no evidence of additional rearrangement of the chromosomal DNA. Noncomplementation of the transgene-induced mutation and med was demonstrated in a cross with medJ/+mice. The new allele is designated medTgNA4Bs (medtg). The homologous human locus MED was assigned to chromosome 12. Synaptotagmin 1 and contactin 1 were eliminated as candidate genes for the med mutation. The transgene-induced allele provides molecular access to the med gene, whose function is required for synaptic transmission at the neuromuscular junction and long-term survival of cerebellar Purkinje cells.

Animals↗
PubMed1999

A mouse chromosome 19 genetic map including the Lvis1 viral insertion site.

Somatic insertion mutations, such as those caused by the insertion of a proviral element, can contribute to abnormal cell growth by activating cellular proto-oncogenes or inactivating tumor suppressor genes. Lvis1 is a genomic locus frequently disrupted by viral insertion in AKXD B-cell lymphomas (manuscript submitted for publication). To determine whether insertion mutations at Lvis1 affect a known proto-oncogene or tumor suppressor gene, we mapped Lvis1 to distal mouse chromosome 19. A detailed molecular genetic map of this region was constructed, localizing Lvis1 relative to 20 gene and microsatellite markers, 3 of which have not been mapped in the mouse (Nfkb2, Nlz, and Wnt8b). This analysis revealed that Lvis1 maps between two previously identified viral insertion sites, His2 and Frat1, and does not cosegregate with known gene markers. In addition, our study refines the gene order for distal mouse chromosome 19 and expands the comparative map between mouse chromosome 19 and the human chromosome 10q23-q26 homology region.

Animals↗
PubMed2004

The distribution of transgene insertion sites in barley determined by physical and genetic mapping.

The exact site of transgene insertion into a plant host genome is one feature of the genetic transformation process that cannot, at present, be controlled and is often poorly understood. The site of transgene insertion may have implications for transgene stability and for potential unintended effects of the transgene on plant metabolism. To increase our understanding of transgene insertion sites in barley, a detailed analysis of transgene integration in independently derived transgenic barley lines was carried out. Fluorescence in situ hybridization (FISH) was used to physically map 23 transgene integration sites from 19 independent barley lines. Genetic mapping further confirmed the location of the transgenes in 11 of these lines. Transgene integration sites were present only on five of the seven barley chromosomes. The pattern of transgene integration appeared to be nonrandom and there was evidence of clustering of independent transgene insertion events within the barley genome. In addition, barley genomic regions flanking the transgene insertion site were isolated for seven independent lines. The data from the transgene flanking regions indicated that transgene insertions were preferentially located in gene-rich areas of the genome. These results are discussed in relation to the structure of the barley genome.

Chromosome Mapping↗
PubMed1999

Insertional mutation of the collagen genes Col4a3 and Col4a4 in a mouse model of Alport syndrome.

Mice homozygous for the transgenic insertion in line OVE250 exhibit severe progressive glomerulonephritis. Ultrastructural changes in the glomerular basement membrane (GBM) at 2 weeks of age resemble those in Alport syndrome. The transgenic insertion site was mapped by FISH to mouse chromosome 1 close to Pax3. Genetic and molecular analyses identified a deletion of genomic DNA at the transgene insertion site. Exons 1 through 12 of the collagen IV gene Col4a4, exons 1 and 2 of the adjacent Col4a3 gene, and the intergenic promoter region are deleted. Transcripts of Col4a3 and Col4a4 are undetectable in mutant kidney, and both proteins are missing from the GBM. Persistent cellular proliferation in mutant kidneys suggests that interaction with the extracellular matrix may be important for cell maturation. Evolutionarily conserved sequence elements in the promoter regions of human and mouse Col4a3 and Col4a4 include a 19-bp element that was tandemly duplicated in the human lineage and a CTC box element common to several genes encoding extracellular matrix proteins. This new animal model of Alport syndrome, Col4Delta3-4, lacks both alpha3 and alpha4 chains of collagen IV and exhibits an earlier disease onset than mice lacking alpha3 only.

Animals↗
PubMed1995

Transposon mutagenesis and cloning of the genes encoding the enzymes of fengycin biosynthesis in Bacillus subtilis.

A total of 20 Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis. Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome. We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes. One of the clones obtained, pFC660, was 46 kb long. Eight transposon insertion sites were mapped within this plasmid. Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase. Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.

Adenosine Triphosphatases↗
PubMed1998

Sequence analysis of Tn10 insertion sites in a collection of Escherichia coli strains used for genetic mapping and strain construction.

The chromosomal insertion sites of Tn10-containing Escherichia coli strains were amplified by inverse PCR, and the nucleotide sequences of the junctions were determined. In 95 strains analyzed, 88 unique Tn10 positions were determined and matched to the E. coli chromosome sequence. Two gaps in insertion site positions were noted, one including the terminus of DNA replication and another bounded by recombination hot spots RhsA and RhsB.

Base Sequence↗
PubMed2006

Characterization of enteropathogenic Escherichia coli mutants that fail to disrupt host cell spreading and attachment to substratum.

Upon infection of host cells, enteropathogenic Escherichia coli (EPEC) delivers a set of effector proteins into the host cell cytoplasm via the type III secretion system (TTSS). The effectors subvert various host cell functions. We found that EPEC interferes with the spreading and ultimately with the attachment of suspended fibroblasts or epithelial cells, and we isolated mini-Tn10kan insertion mutants that failed to similarly affect host cells. In most mutants, the insertion sites were mapped to genes encoding TTSS components, including cesD, escC, escJ, escV, espD, sepL, espB, and escF. Other mutants contained insertions in micC or upstream of bfpP, yehL, or ydeP. The insertion upstream of ydeP was associated with a reduction in TTSS protein production and was studied further. To determine whether the apparent repression was due to constitutive expression of the downstream encoded genes, ydeP and ydeO expression vectors were constructed. Expression of recombinant YdeP, YdeO, or EvgA, a positive regulator of both ydeP and ydeO, repressed TTSS protein production. Our results suggest that upon activation of the EvgAS two-component system, EvgA (the response regulator) activates both ydeP and ydeO expression and that YdeP and YdeO act conjointly, directly or indirectly repressing expression of the TTSS genes.

Cell Adhesion↗
PubMed2004

Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR.

BACKGROUND: Insertion sequences (IS) are small DNA segments capable of transposing within and between prokaryotic genomes, often causing insertional mutations and chromosomal rearrangements. Although several methods are available for locating ISs in microbial genomes, they are either labor-intensive or inefficient. Here, we use vectorette PCR to identify and map the genomic positions of the eight insertion sequences (IS1, 2, 3, 4, 5, 30, 150, and 186) found in E. coli strain CGSC6300, a close relative of MG1655 whose genome has been sequenced. RESULTS: Genomic DNA from strain CGSC6300 was digested with a four-base cutter Rsa I and the resulting restriction fragments ligated onto vectorette units. Using IS-specific primers directed outward from the extreme ends of each IS and a vectorette primer, flanking DNA fragments were amplified from all but one of the 37 IS elements identified in the genomic sequence of MG1655. Purification and sequencing of the PCR products confirmed that they are IS-associated flanking DNA fragments corresponding to the known IS locations in the MG1655 genome. Seven additional insertions were found in strain CGSC6300 indicating that very closely related isolates of the same laboratory strain (the K12 isolate) may differ in their IS complement. Two other E. coli K12 derivatives, TD2 and TD10, were also analyzed by vectorette PCR. They share 36 of the MG1655 IS sites as well as having 16 and 18 additional insertions, respectively. CONCLUSION: This study shows that vectorette PCR is a swift, efficient, reliable method for typing microbial strains and identifying and mapping IS insertion sites present in microbial genomes. Unlike Southern hybridization and inverse PCR, our approach involves only one genomic digest and one ligation step. Vectorette PCR is then used to simultaneously amplify all IS elements of a given type, making it a rapid and sensitive means to survey IS elements in genomes. The ability to rapidly identify the IS complements of microbial genomes should facilitate subtyping closely related pathogens during disease outbreaks.

Bacterial Typing Techniques↗
PubMed2002

Functional genomics on potato virus A: virus genome-wide map of sites essential for virus propagation.

Transposition-based in vitro insertional mutagenesis strategies provide promising new approaches for functional characterization of any cloned gene or genome region. We have extended the methodology and scope of such analysis to a complete viral genome. To map genome regions both essential and nonessential for Potato virus A propagation, we generated a genomic 15-bp insertion mutant library utilizing the efficient in vitro DNA transposition reaction of phage Mu. We then determined the proficiency of 1125 mutants to propagate in tobacco protoplasts by using a genetic footprinting strategy that simultaneously mapped the genomic insertion sites. Over 300 sites critical for virus propagation were identified, and many of them were located in positions previously not assigned to any viral functions. Many genome regions tolerated insertions indicating less important sites for virus propagation and thus pinpointed potential locations for further genome manipulation. The methodology described is applicable to a detailed functional analysis of any viral nucleic acid cloned as DNA and can be used to address many different processes during viral infection cycles.

3' Untranslated Regions↗
PubMed2001

Requirement of DNA repair mechanisms for survival of Burkholderia cepacia G4 upon degradation of trichloroethylene.

A Tn5-based mutagenesis strategy was used to generate a collection of trichloroethylene (TCE)-sensitive (TCS) mutants in order to identify repair systems or protective mechanisms that shield Burkholderia cepacia G4 from the toxic effects associated with TCE oxidation. Single Tn5 insertion sites were mapped within open reading frames putatively encoding enzymes involved in DNA repair (UvrB, RuvB, RecA, and RecG) in 7 of the 11 TCS strains obtained (4 of the TCS strains had a single Tn5 insertion within a uvrB homolog). The data revealed that the uvrB-disrupted strains were exceptionally susceptible to killing by TCE oxidation, followed by the recA strain, while the ruvB and recG strains were just slightly more sensitive to TCE than the wild type. The uvrB and recA strains were also extremely sensitive to UV light and, to a lesser extent, to exposure to mitomycin C and H(2)O(2). The data from this study establishes that there is a link between DNA repair and the ability of B. cepacia G4 cells to survive following TCE transformation. A possible role for nucleotide excision repair and recombination repair activities in TCE-damaged cells is discussed.

Bacterial Proteins↗