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PubMed2002

Improvement of differentiation and interpretability of spoligotyping for Mycobacterium tuberculosis complex isolates by introduction of new spacer oligonucleotides.

The direct repeat (DR) region in Mycobacterium tuberculosis complex strains is composed of multiple well-conserved 36-bp DRs interspersed with nonrepetitive DNA spacer sequences of similar size. Clinical isolates show extensive polymorphism in this DR region, and this has led to the development of a 43-spacer reversed line blot methodology: spoligotyping. Although this method has contributed significantly to the molecular epidemiology of tuberculosis in the last decade, the discriminatory power and the readability of this method were not found to be optimal. In order to improve the discriminatory power, the usefulness of 43 redesigned oligonucleotides and the usefulness of 51 new spacer oligonucleotides were evaluated. For 314 M. tuberculosis complex strains isolated in the central part of The Netherlands over a 5-year period, 264 different IS6110 RFLP types could be distinguished, and 160 different spoligotype patterns were identified by traditional spoligotyping. After the introduction of 51 new spacer oligonucleotides, 14 additional spoligotypes were recognized. This enabled us to split 11 clusters of isolates identified by the traditional spoligotyping. Furthermore, on the basis of the new spacer oligonucleotides a dichotomy was found among the Beijing genotype isolates. Among 76 Mycobacterium bovis strains, 20 patterns were found by traditional spoligotyping and 30 patterns were found by novel probe spoligotyping, respectively. Nine M. bovis subsp. caprae isolates yielded six patterns by traditional spoligotyping and eight patterns by novel probe spoligotyping. A part of the redesigned oligonucleotides slightly improved the reading of spoligotype patterns. The reproducibility of spoligotyping, based on internal control probes, invariably yielded a high score; only 4 (1%) of the 314 patient isolates gave discrepant results. Analysis of a set of 31 duplicate M. tuberculosis complex strains demonstrated a 10% error rate for the identification of blinded duplicate samples. In a redundancy analysis, 40 essential spacer oligonucleotides of the 94-spacer sequences were selected, yielding the same number of spoligotype patterns. We propose to leave the traditional commercialized first-generation membrane for spoligotyping unchanged for current applications and to introduce a second-generation spoligotyping membrane whenever extended discrimination is required, e.g., for low-copy-number IS6110 strains or for phylogenetic studies of Beijing genotype strains.

Animals↗

PubMed1984

Identification of additional genes on transposon Tn10: tetC and tetD.

Two genes (tetC and tetD) were identified and located on transposon Tn10 between gene tetA and insertion sequence IS10R. Genes tetC and tetD encode proteins of apparent subunit molecular weights of 23,000 and 18,000, respectively. The TetD protein was found to be membrane associated. Tetracycline resistance levels promoted by transposon Tn10 were found to be unaffected in Escherichia coli K-12 when mutants lacking tetC or tetC and tetD were tested. The nucleotide sequence of genes tetC and tetD is reported in the accompanying article (K. Schollmeier and W. Hillen, J. Bacteriol. 160:499-503, 1984).

Bacterial Proteins↗

PubMed1990

Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium.

Transposon Tn5 insertions causing anaerobic cysteine auxotrophy were isolated from a Salmonella typhimurium cysI parent (auxotrophic under aerobic but not anaerobic conditions). Insertions in one mutant group appeared to be in cysG. A second group of insertions, designated asr (anaerobic sulfite reduction), were located near map unit 53 on the S. typhimurium chromosome. They did not cause aerobic or anaerobic auxotrophy in a cys1+ background but did prevent dissimilatory sulfite reduction. Plasmids containing asr DNA cloned from wild-type S. typhimurium conferred anaerobic prototrophy and the ability to produce hydrogen sulfide from sulfite on an Escherichia coli cys1 mutant.

Anaerobiosis↗

PubMed1993

Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology.

DNA fingerprinting of Mycobacterium tuberculosis has been shown to be a powerful epidemiologic tool. We propose a standardized technique which exploits variability in both the number and genomic position of IS6110 to generate strain-specific patterns. General use of this technique will permit comparison of results between different laboratories. Such comparisons will facilitate investigations into the international transmission of tuberculosis and may identify specific strains with unique properties such as high infectivity, virulence, or drug resistance.

Base Sequence↗

PubMed1987

Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis.

The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcus faecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J. Christie and G. M. Dunny, Plasmid 15:230-241, 1986). When S. faecalis cells were mixed with Bacillus subtilis in broth, tetracycline resistance was transferred from S. faecalis. The tetracycline-resistant B. subtilis cells contained a 16-kb region of pCF10 (distinct from TRA) that carried the tetracycline resistance determinant (Tetr). This Tetr element was found to transfer between S. faecalis and B. subtilis strains in the absence of plasmids. Genetic and molecular techniques were used to establish locations of the element at several different sites on the B. subtilis chromosome. The Tetr element could be transferred in filter matings from B. subtilis to S. faecalis strains and between recombination-proficient and -deficient S. faecalis strains in the absence of any plasmid DNA. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The Tetr element was shown to transpose from the S. faecalis chromosome to various locations within the hemolysin plasmid pAD1. Together, the data indicate that the Tetr element, termed transposon Tn925, is very similar to the conjugative transposon Tn916 in both structure and function. A derivative of Tn925, containing transposon Tn917 inserted into a site approximately 3 kb from one end, exhibited elevated transfer frequencies and may provide a useful means for delivering Tn917 by conjugation into various gram-positive species.

Bacillus subtilis↗

PubMed1987

Cloning of Alu-containing cDNAs from human fibroblasts and identification of small Alu+ poly(A)+ RNAs in a variety of human normal and tumor cells.

Two clones have been selected from a human fibroblast cDNA bank. By DNA sequencing the clones were shown to contain Alu elements located near the ends of the cDNA inserts. DNA of the clones was used for Northern blot hybridization analysis of a number of poly(A)-containing RNAs from normal human tissues (brain, stomach, uterus, spleen, fibroblasts) and tumors (neurinoma, glioma, neuroblastoma, liposarcoma, adrenal cortex adenocarcinoma). All RNA samples reveal a heterodisperse distribution of Alu transcripts with discrete bands in the region of 7-12 S RNA. The majority of these small poly(A)+ Alu+ RNAs contain Alu sequences only in one (canonical) orientation with functional signals including the split promoter for RNA polymerase III.

Base Sequence↗

PubMed1998

Identification of a novel insertion element, IS1548, in group B streptococci, predominantly in strains causing endocarditis.

Hyaluronidase has been postulated to be a virulence factor in group B streptococci (GBS). No hyaluronidase activity was found in 15 of 50 GBS isolates from adults studied. Most of these hyaluronidase-negative strains belonged to serotype III. In strains lacking hyaluronidase activity, an insertion of 1317 nucleotides was found in the hyaluronidase gene. The fragment was cloned and sequenced and found to have characteristics of a novel insertion sequence, designated IS1548. As well as in GBS serotype III, this sequence was found in 3 of 6 serotype II isolates and in all 10 group A streptococcal strains (GAS) tested. Homologies were found with repeated sequences in Streptococcus pneumoniae and with H repeats in Escherichia coli. All GBS strains harboring IS1548 and some GAS strains had one copy of IS1548 located downstream of the C5a peptidase gene. IS1548 was present in 9 of 13 GBS isolates from blood in endocarditis patients and in 3 of 22 vaginally colonizing strains.

Adhesins, Bacterial↗

PubMed1999

Analysis of cDNA expressed sequence tags from Entamoeba histolytica: identification of two highly abundant polyadenylated transcripts with no overt open reading frames.

Upon analysis of 304 expressed sequence tags derived from the protozoan parasite Entamoeba histolytica, a number of novel protein encoding amoeba sequences were isolated. In addition, two unrelated, abundantly expressed transcripts were identified, and designated, ehapt1 and ehapt2. Although these transcripts do not contain any overt open reading frame, both are polyadenylated and together represent about 19% of total polyA+-RNA(11.6% for ehapt1 and 7.5% for ehapt2), thus being the most highly expressed polyA-containing transcripts so far identified in E. histolytica trophozoites. Northern blot and primer extension analyses revealed single-sized transcripts of 0.5 and 0.6 kb for ehapt1 and ehapt2, respectively, and Southern blot analysis suggests that both are encoded by multiple genes, which are distributed throughout the amoeba genome. Comparison between various ehapt1- and ehapt2-derived cDNAs indicated that both transcripts are highly polymorphic. Whereas nucleotide substitutions in ehapt2 are distributed throughout the sequence, variations in ehapt1 are mainly restricted to two regions, one of which comprises a deletion of variable length within an 8 nt tandem repeat unit. At present there is no convincing explanation for the possible role of ehapt1 and ehapt2 in E. histolytica, and analogous sequences have not been described so far for any other organism. Most likely they might represent regulatory RNAs or transcribed transposable elements.

Animals↗

PubMed1994

Catabolism of aromatics in Pseudomonas putida U. Formal evidence that phenylacetic acid and 4-hydroxyphenylacetic acid are catabolized by two unrelated pathways.

Phenylacetic acid (PhAcOH) and 4-hydroxyphenylacetic acid (4HOPhAcOH) are catabolized in Pseudomonas putida U through two different pathways. Mutation carried out with the transposon Tn5 has allowed the isolation of several mutants which, unlike the parental strain, are unable to grow in chemically defined medium containing either PhAcOH or 4HOPhAcOH as the sole carbon source. Analysis of these strains showed that the ten mutants unable to grow in PhAcOH medium grew well in the one containing 4HOPhAcOH, whereas four mutants handicapped in the degradation of 4HOPhAcOH were all able to utilize PhAcOH. These results show that the degradation of these two aromatic compounds in P. putida U is not carried out as formerly believed through a single linear and common pathway, but by two unrelated routes. Identification of the blocked point in the catabolic pathway and analysis of the intermediate accumulated, showed that the mutants unable to utilize 4HOPhAcOH corresponded to two different groups: those blocked in the gene encoding 4-hydroxyphenylacetic acid-3-hydroxylase; and those blocked in the gene encoding homoprotocatechuate-2,3-dioxygenase. Mutants unable to use PhAcOH as the sole carbon source have been also classified into two different groups: those which contain a functional PhAc-CoA ligase protein; and those lacking this enzyme activity.

Chromatography, High Pressure Liquid↗

PubMed2003

GABI-Kat SimpleSearch: a flanking sequence tag (FST) database for the identification of T-DNA insertion mutants in Arabidopsis thaliana.

SUMMARY: GABI-Kat SimpleSearch is a database of flanking sequence tags (FSTs) of T-DNA mutagenized Arabidopsis thaliana lines that were generated by the GABI-Kat project. Sequences flanking the T-DNA insertion sites were aligned to the A.thaliana genome sequence, annotated with information about the FST, the insertion site and the line from which the FST was derived. A web interface permits text-based as well as sequence-based searches for relevant insertions. GABI-Kat SimpleSearch aims to help biologists to quickly find T-DNA insertion mutants for their research. AVAILABILITY: http://www.mpiz-koeln.mpg.de/GABI-Kat/

Arabidopsis↗

PubMed1982

[Classification of R-factors. Possible errors and their elimination].

The necessity for classifying plasmids is emphasized. From our results the uniformity of a nosocomial infectious outbreak is only given by the R-factor and not by the infectious agent. Determination of the appropriate pilus groups by sexphages using the HFRT-system and identification of incompatibility as a further means of subdividing the pilus groups into incompatibility groups is described. A distinction is made between incoming- and resident incompatibility. To eliminate possible errors in plasmid classification the following points are considered: 1. Testing in recombination deficient rec A- -strains of E. coli K12 to avoid any recombination of the plasmids. 2. Trypsin addition to remove the possibility of plasmid coded colicin production. 3. A proper choice of the indicator plasmids. 4. Consideration of the spontaneous loss of the indicator plasmid and also an occurrence of transposons coding for an additional pilus type.

Anti-Bacterial Agents↗

PubMed1985

Identification of a telomeric DNA sequence in Plasmodium berghei.

A fragment of Plasmodium berghei DNA was cloned using a technique designed to select for telomeric sequences. The cloned fragment recognizes Bal31-sensitive bands in P. berghei genomic digests. It contains at its distal end at least 70 tandem repeats of the heptanucleotide sequence CCCTGAAA. The presence of natural single strand discontinuities in the telomeric regions of P. berghei DNA is demonstrated by the selective incorporation of deoxyribonucleoside triphosphates in the absence of DNase. The number of copies of the cloned sequence present in each genome agrees with an estimate of 6-12 chromosomes per nucleus.

Animals↗

PubMed1983

Identification of a transposon Tn3 sequence required for transposition immunity.

A plasmid containing transposon Tn3 is immune to further insertions of Tn3. This phenomenon works in cis and is referred to as transposition immunity. We have used the ability of Tn3 to form cointegrates between two plasmids to develop a quantitative assay to detect transposition immunity. Presence of Tn3 on both the plasmids reduces the cointegration frequency to less than 1/100 of parental. Using this assay, we have determined that (i) tnpR is not required for immunity, (ii) only the terminal 38 base pairs of Tn3 need be present to confer immunity, and (iii) other parts of Tn3 appear not to confer immunity.

Base Sequence↗

PubMed2000

Specific PCR identification of the T1 vaccine strains for contagious bovine pleuropneumonia.

A specific PCR test for the identification of the vaccine strains T1, T1/44 and T1sr, was developed. This PCR reaction is based on variations of DNA sequences in a region flanking one IS1296 copy. The specific primer pair MmmSCP1-T1M2 amplifies a 700-bp long DNA fragment in the T1 vaccine strains and gives no amplification with the 60 other Mycoplasma mycoides subsp. mycoides SC strains tested. This PCR will permit to distinguish the T1 strain from all other vaccine strains and therefore avoid possible confusions. In addition, it should enable better investigations of post-vaccinal reactions.

Animals↗

PubMed1994

Cloning of the nupC gene of Escherichia coli encoding a nucleoside transport system, and identification of an adjacent insertion element, IS 186.

Escherichia coli is known to contain more than one active transport system for nucleoside uptake. In the present study we report the sequence of a gene encoding a second nucleoside transport system, nupC (in addition to nupG). An open reading frame (ORF) of 1200 bp was identified that codes for a hydrophobic polypeptide of 43,560 Da and an NupC fusion protein was shown to be membrane associated. The native NupC protein is also identified, following over-expression. NupC exhibits short regions of homology to several membrane-associated proteins, including LacY and Cyd. Analysis of the nupC promoter region revealed the presence of at least two putative CRP-binding sites, centred at -40bp and -89bp, which probably flank a CytR-binding site. In addition, an adjacent IS 186 element was identified and found to reside within a putative terminator structure, downstream from the nupC ORF. This arrangement is shown to reflect the previously established gene order on the E. coli chromosome.

Amino Acid Sequence↗

PubMed2004

Identification of two mariner-like elements in the genome of the mosquito Ochlerotatus atropalpus.

Two distinct mariner-like elements, Atmar-1 and Atmar-2, were isolated from the genome of the mosquito Ochlerotatus atropalpus. Full-sized Atmar-1 elements, obtained by screening a genomic library, have a 1293-bp consensus sequence with 27-bp inverted terminal repeats and a 1047-bp open reading frame (ORF) encoding the transposase. The Atmar-2 elements were amplified by polymerase chain reaction from genomic DNA and contain the central part of the transposase ORF. Individual clones of both mariner elements contain deletions, frameshifts, and stop codons. The Atmar-1 elements are present in 370-1200 copies, while the Atmar-2 elements are present in approximately 100-300 copies per haploid genome. One of the Atmar-1 elements, Atmar-1.33, could be mobilized, suggesting the presence of functional Atmar-1 elements elsewhere in the genome. Phylogenetic analysis demonstrated that Atmar-1 elements belong to the irritans subfamily and Atmar-2 elements to the cecropia subfamily of mariner elements.

Amino Acid Sequence↗

PubMed2006

Study of an alternate glyoxylate cycle for acetate assimilation by Rhodobacter sphaeroides.

Organisms, which grow on organic substrates that are metabolized via acetyl-CoA, are faced with the problem to form all cell constituents from this C(2)-unit. The problem was solved by the seminal work of Kornberg and is known as the glyoxylate cycle. However, many bacteria are known to not contain isocitrate lyase, the key enzyme of this pathway. This problem was addressed in acetate-grown Rhodobacter sphaeroides. An acetate-minus mutant identified by transposon mutagenesis was affected in the gene for beta-ketothiolase forming acetoacetyl-CoA from two molecules of acetyl-CoA. This enzyme activity was missing in this mutant, which grew on acetoacetate and on acetate plus glyoxylate. A second acetate/acetoacetate-minus mutant was affected in the gene for a putative mesaconyl-CoA hydratase, an enzyme which catalyses the hydration of mesaconyl-CoA to beta-methylmalyl-CoA. Beta-methylmalyl-CoA is further cleaved into glyoxylate and propionyl-CoA. These results as well as identification of acetate-upregulated proteins by two-dimensional gel electrophoresis lead to the proposal of a new pathway for acetate assimilation. In a first part, affected by the mutations, two molecules of acetyl-CoA and one molecule CO(2) are converted via acetoacetyl-CoA and mesaconyl-CoA to glyoxylate and propionyl-CoA. In a second part glyoxylate and propionyl-CoA are converted with another molecule of acetyl-CoA and CO(2) to l-malyl-CoA and succinyl-CoA.

Acetates↗

PubMed1992

Identification of a complete P-element in the genome of Drosophila bifasciata.

A full-size P-element (IbifM3) was isolated from a genomic library of Drosophila bifasciata. The sequence has a length of 2935 bp and is flanked by 8 bp duplications of the target site. The termini are formed by 31 bp inverted repeats. The four exons have intact reading frames and possess the coding capacity for a protein of 753 amino acids and a molecular weight of 86.4 kd. The sections of the D. melanogaster transposase presumed to be functionally important (three leucine zippers and a helix turn helix motif) are conserved in the D. bifasciata P-element. Copy number and genomic distribution resemble the situation in true P-strains of D. melanogaster. Both findings support the idea that IbifM3 represents an active transposon. The sequence comparison between the P-elements of D. bifasciata, D. melanogaster and Scaptomyza pallida reveals relationships not in accordance with the phylogeny of the species. This result suggests a further case of horizontal transmission involving mobile elements in the genus Drosophila.

Amino Acid Sequence↗