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PubMed1996

Strain typing with IS200 fingerprints in Salmonella abortusovis.

A collection of Salmonella abortusovis isolates was examined for the presence of insertion element IS200. All proved to contain three or four copies of the element. One IS200 hybridization band of approximately 9 kb was found in all isolates, indicating that all S. abortusovis strains carry an IS200 element in similar or identical locations; this band can be potentially useful for serovar identification. S. abortusovis collection isolates from distinct geographic areas were highly polymorphic, suggesting that IS200 fingerprints might provide information on the geographic origin of S. abortusovis strains. Isolates obtained from the same geographic area (the island of Sardinia, Italy) were less polymorphic: all shared three constant IS200 hybridization bands, indicating that they derive from a single ancestor. Most strains analyzed contained an additional copy of IS200 in the variable region of the virulence plasmid. Certain Sardinian flocks proved to be infected by only one S. abortusovis strain, while others harbored two strains. Strain typing with IS200 fingerprints proved to be more reliable than plasmid analysis, because the latter yielded a high degree of polymorphism, even among isolates from the same flock.

Abortion, Veterinary↗
PubMed1999

Molecular characterization of the PhoP-PhoQ two-component system in Escherichia coli K-12: identification of extracellular Mg2+-responsive promoters.

We identified Mg2+-responsive promoters of the phoPQ, mgtA, and mgrB genes of Escherichia coli K-12 by S1 nuclease analysis. Expression of these genes was induced by magnesium limitation and depended on PhoP and PhoQ. The transcription start sites were also determined, which allowed us to find a (T/G)GTTTA direct repeat in their corresponding promoter regions.

Adenosine Triphosphatases↗
PubMed1998

FISH investigation of 5q and 7q deletions in MDS/AML reveals hidden translocations, insertions and fragmentations of the same chromosomes.

We have studied, by fluorescence in situ hydridization (FISH), chromosomes 5 and 7 in a series of 11 cases with 5q deletion, as sole anomaly (four cases), or in association with 7q deletion (seven cases), in MDS/AML patients. We found that, in some cases, a part of the so-called 'lost' chromosome 5 and 7 material, was actually translocated. These translocations may be either end-arm or whole-arm, as well as small insertions. Chromosomes 5 or 7 may be broken in more than two segments, defining 'fragmentation', giving rise to marker chromosomes. FISH allowed the identification of small material insertion, which is totally unidentified by classical cytogenetics. Chromosome 5 and 7 translocations occur irrespectively of the 'de novo' or 'secondary' type of myelodysplastic syndrome (MDS)/acute myeloid leukaemia (AML) patients.

Acute Disease↗
PubMed1989

Molecular cloning of the outJ gene involved in pectate lyase secretion by Erwinia chrysanthemi.

The phytopathogenic enterobacterium Erwinia chrysanthemi secretes a number of enzymes involved in plant-tissue degradation, notably the five isoenzymes of pectate lyase. We have cloned a region involved in pectate lyase and cellulase secretion by complementation of non-secretory outJ mutants of E. chrysanthemi strain 3937 using the RP4::miniMu plasmid pULB110. The cloned region maps near the ade-22 marker on the E. chrysanthemi 3937 chromosome. An R-prime containing a chromosomal DNA insert of about 30kb was first obtained; subcloning into pBR325 permitted the isolation of a 4kb ClaI/SspI fragment able to complement outJ mutations in E. chrysanthemi. The isolation of phoA fusions in this fragment allowed us to determine the direction of transcription of the encoding region, which extends over about 2.5kb, and demonstrate that this region encodes exported protein(s). When the TnphoA insertions were transferred back into E. chrysanthemi chromosome, the recombined strains no longer secreted pectate lyases or cellulases. Identification of the products encoded by the ClaI/SspI fragment demonstrated that outJ encodes an 83 kD polypeptide which is processed to an 81 kD polypeptide by cleavage of a signal sequence. The cloned DNA fragment did not endow Escherichia coli with the ability to secrete pectate lyases.

Bacterial Proteins↗
PubMed2005

Combined evidence annotation of transposable elements in genome sequences.

Transposable elements (TEs) are mobile, repetitive sequences that make up significant fractions of metazoan genomes. Despite their near ubiquity and importance in genome and chromosome biology, most efforts to annotate TEs in genome sequences rely on the results of a single computational program, RepeatMasker. In contrast, recent advances in gene annotation indicate that high-quality gene models can be produced from combining multiple independent sources of computational evidence. To elevate the quality of TE annotations to a level comparable to that of gene models, we have developed a combined evidence-model TE annotation pipeline, analogous to systems used for gene annotation, by integrating results from multiple homology-based and de novo TE identification methods. As proof of principle, we have annotated "TE models" in Drosophila melanogaster Release 4 genomic sequences using the combined computational evidence derived from RepeatMasker, BLASTER, TBLASTX, all-by-all BLASTN, RECON, TE-HMM and the previous Release 3.1 annotation. Our system is designed for use with the Apollo genome annotation tool, allowing automatic results to be curated manually to produce reliable annotations. The euchromatic TE fraction of D. melanogaster is now estimated at 5.3% (cf. 3.86% in Release 3.1), and we found a substantially higher number of TEs (n = 6,013) than previously identified (n = 1,572). Most of the new TEs derive from small fragments of a few hundred nucleotides long and highly abundant families not previously annotated (e.g., INE-1). We also estimated that 518 TE copies (8.6%) are inserted into at least one other TE, forming a nest of elements. The pipeline allows rapid and thorough annotation of even the most complex TE models, including highly deleted and/or nested elements such as those often found in heterochromatic sequences. Our pipeline can be easily adapted to other genome sequences, such as those of the D. melanogaster heterochromatin or other species in the genus Drosophila.

Journal Article↗
PubMed2002

Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes.

Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria. The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed against tuberculosis. In this work we describe the isolation and characterization of a Tn 5 transposon mutant of Mycobacterium smegmatis that is blocked in the production of mycothiol and accumulates its precursor, 1 D-myo-inosityl 2- L-cysteinylamido-2-deoxy-alpha-D-glucopyranoside (Cys-GlcN-Ins). Cys-GlcN-Ins isolated from this mutant was used to assay for acetyl-CoA:Cys-GlcN-Ins acetyltransferase (mycothiol synthase, MshD) activity, which was found in wild-type cells, but not in the mutant. Sequencing outward of the DNA of the mutant strain from the site of insertion permitted identification of the mshD gene in the M. smegmatis genome, as well as the orthologous gene Rv0819 in the M. tuberculosis genome. Cloning and expression of mshD from M. tuberculosis (Rv0819) in Escherichia coli gave a transformant with MshD activity, demonstrating that Rv0819 is the mshD mycothiol biosynthesis gene.

Acetyltransferases↗
PubMed2002

Enhancer trap expression patterns provide a novel teaching resource.

A collection of Arabidopsis enhancer trap transposants has been identified for use as a teaching tool. This collection serves to assist students in understanding the patterning and organization of plant tissues and cells, and will be useful in plant anatomy, morphology, and developmental biology courses. Each transposant exhibits reporter gene expression in a specific tissue, cell type, or domain, and these lines collectively offer a glimpse of compartments of gene expression. Some compartments correspond to classical definitions of botanical anatomy and can assist in anatomical identification. Other patterns of reporter gene expression are more complex and do not necessarily correspond to known anatomical features. The sensitivity of the beta-glucuronidase histochemical stain provides the student with a colorful and direct way to visualize difficult aspects of plant development and anatomy, and provides the teacher with an invaluable tool for a practical laboratory session.

Arabidopsis↗
PubMed1999

Low target site specificity of an IS6100-based mini-transposon, Tn1792, developed for transposon mutagenesis of antibiotic-producing Streptomyces.

To improve transposon mutagenesis of antibiotic-producing Streptomyces, a mini-transposon, Tn1792, was constructed, based on IS6100, originally isolated from Mycobacterium fortuitum. Easily manageable transposition assays were developed to demonstrate inducible transposition of Tn1792 into the Streptomyces genome from a temperature-sensitive delivery plasmid. Introduction of the selectable aac1 gene between the inverted repeats in Tn1792 allowed for both reliable identification of transposition events in Streptomyces, and also subsequent cloning of transposon-tagged sequences in Escherichia coli. This enabled the target site specificity of Tn1792 to be determined at nucleotide resolution, revealing no significant shared homology between different target sites. Consequently, Tn1792 is well suited for random mutagenesis of Streptomyces.

Anti-Bacterial Agents↗
PubMed2004

Identification of the Haemophilus influenzae tolC gene by susceptibility profiles of insertionally inactivated efflux pump mutants.

Isogenic strains containing insertional disruptions of 10 Haemophilus influenzae Rd genes were investigated for their effects on the susceptibility of the organism to various classes of antimicrobial compounds. MIC results show that HI1462, which encodes an Escherichia coli TolC homolog, is the third component of the H. influenzae AcrAB pump.

Bacterial Outer Membrane Proteins↗
PubMed2001

Identification of new genes involved in the virulence of Listeria monocytogenes by signature-tagged transposon mutagenesis.

Listeria monocytogenes is a gram-positive, facultative intracellular pathogen that can cause severe food-born infections in humans and animals. We have adapted signature-tagged transposon mutagenesis to L. monocytogenes to identify new genes involved in virulence in the murine model of infection. We used transposon Tn1545 carried on the integrative vector pAT113. Forty-eight tagged transposons were constructed and used to generate banks of L. monocytogenes mutants. Pools of 48 mutants were assembled, taking one mutant from each bank, injected into mice, and screened for those affected in their multiplication in the brains of infected animals. From 2,000 mutants tested, 18 were attenuated in vivo. The insertions harbored by these mutants led to the identification of 10 distinct loci, 7 of which corresponded to previously unknown genes. The properties of four loci involving putative cell wall components were further studied in vitro and in vivo. The data suggested that these components are involved in bacterial invasion and multiplication in the brain.

Amino Acid Sequence↗
PubMed2000

Identification of seven genes in the major histocompatibility complex class I region of the zebrafish.

Physical linkage of genes whose products are involved in similar physiological pathways may have functional significance. The identification of conserved gene linkage in distantly related organisms can therefore strengthen the hypothesis of selection acting towards keeping genes on a chromosome. We used the cDNA selection technique and the polymerase chain reaction (PCR) with generic primers for the identification of new genes on the genomic clones bearing the major histocompatibility complex (Mhc) class I genes of the zebrafish (Danio rerio). We found six new genes (BING1, DAXX, TAPBP, KNSL2, TAP2B and KE6) whose orthologues are known to be linked to the Mhc class II region in humans and mice. In addition, a new zebrafish Mhc class I gene, termed Dare-UFA, was detected. By contrast, a search for the human leucocyte antigen (HLA)-linked BING3, KE3 and SACM2L genes revealed that these loci are not located on the class I clones of the zebrafish. The zebrafish class I region contains repetitive elements with similarity to the DANA, SATA and LINE repeats, as well as Tc1 transposable elements. Our findings indicate a high degree of linkage conservation between the zebrafish class I and the mammalian class II regions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

The raspberry locus of Drosophila melanogaster includes an inosine monophosphate dehydrogenase like coding sequence.

DNA from the raspberry gene of Drosophila melanogaster has been cloned through P-element tagging of a dysgenically induced lethal allele. A transcribed DNA segment adjacent to the P insert encodes an amino acid sequence that is similar to known inosine monophosphate dehydrogenase (IMPDH) sequences. Further dysgenically induced lethals and all four known spontaneous ras eye-colour mutations have changes in the DNA either within or just upstream from the transcribed region. Identification of IMPDH as a probable product of the ras gene is compatible with the finding of an allele that requires dietary guanosine (GR), since the enzyme mediates the first of two biosynthetic steps leading from inosine monophosphate (IMP) to guanosine monophosphate (GMP). However, other auxotrophic mutants at the locus remain unexplained by the finding. The results further suggest that GMP synthesis from IMP is an essential process, despite the capacity of the fly for salvage synthesis of GMP from GR. Consideration of the phenotypes associated with mutants at the ras locus suggests that IMPDH activity is regulated in a tissue-specific manner.

Alleles↗
PubMed2006

Identification and characterization of a major Zn(II) resistance determinant of Mycobacterium smegmatis.

A zinc ion-sensitive mutant of Mycobacterium smegmatis was isolated. The transposon insertion was located in zitA (MSMEG0750), a gene coding for a cation diffusion facilitator family protein. Zinc ions specifically induced expression of zitA. In silico analysis revealed that environmental and opportunistic pathogenic species contain higher numbers of cation diffusion facilitator genes than do obligate pathogens.

Anti-Bacterial Agents↗
PubMed1998

[Identification of polygenes in the system achaete-scute in Drosophila melanogaster using cluster analysis].

Data on location of mobile elements mdg1, Dm412, copia, and B104 in 33 isogenic lines of Drosophila melanogaster has been processed by means of cluster analysis to reveal the relationship between the penetrance for bristle reduction and the distribution of mobile elements. The presence of two groups of sites specific for lines with contrasting penetrance levels have been demonstrated. The specificity suggests that the sites can be associated with the location of corresponding polygenes, affecting the penetrance level in mutant lines.

Animals↗
PubMed1993

Identification of a cryptic gene associated with an insertion sequence not previously identified in Bacillus thuringiensis.

After screening several Bt strains with a cryII toxin probe, clones from two strains were found to contain a cryptic cryIIB gene associated with an insertion sequence element belonging to the IS2/IS3 family. The lack of expression of this gene appears to result from mutation of the upstream orf2 gene which has been shown to be necessary for cryII expression.

Amino Acid Sequence↗
PubMed2006

Identification of specific gene sequences conserved in contemporary epidemic strains of Salmonella enterica.

Genetic elements specific to recent and contemporary epidemic strains of Salmonella enterica were identified using comparative genomic analysis. Two epidemic multidrug-resistant (MDR) strains, MDR Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) and cephalosporin-resistant MDR Salmonella enterica serovar Newport, and an epidemic pansusceptible strain, Salmonella serovar Typhimurium DT160, were subjected to Salmonella gene microarray and suppression subtractive hybridization analyses. Their genome contents were compared with those of coexisting sporadic strains matched by serotype, geographic and temporal distribution, and host species origin. These paired comparisons revealed that epidemic strains of S. enterica had specific genes and gene regions that were shared by isolates of the same subtype. Most of these gene sequences are related to mobile genetic elements, including phages, plasmids, and plasmid-like and transposable elements, and some genes may encode proteins conferring growth or survival advantages. The emergence of epidemic MDR strains may therefore be associated with the presence of fitness-associated genetic factors in addition to their antimicrobial resistance genes.

Animals↗