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PubMed2004

Italian metallo-beta-lactamases: a national problem? Report from the SENTRY Antimicrobial Surveillance Programme.

OBJECTIVES: As part of the SENTRY Antimicrobial Surveillance Programme, 383 non-replicative randomly collected Pseudomonas aeruginosa isolates were collected during 1999-2002. These strains originated from three geographically distinct hospitals within Italy: Genoa (Northern Italy); Rome and Catania (Sicily), and were further studied to identify the prevalence of metallo-beta-lactamase (MbetaL) alleles across Italy and to determine their genetic details. METHODS: Multidrug-resistant (MDR) strains were identified by MIC analysis followed by genotyping and PCR-based strategies. RESULTS: Initial MIC analysis identified 31 MDR isolates that displayed an Etest MbetaL-positive phenotype. Of these, 25 produced either the MbetaL VIM-1 or IMP-13 as detected by PCR and sequencing. VIM-1-producing isolates were found at all sites, whereas IMP-13-producing isolates were only found in Rome. MbetaL-producing isolates were found at all Italian SENTRY sites and together amounted to 6.5% of all P. aeruginosa isolates. Genetic analysis indicated that many strains contained multiple integrons and identified two novel MbetaL integrons, one from the site in Genoa and one from Sicily. Integrons identical in structure and sequence to In70, the first identified and characterized bla(VIM)-containing integron from Verona, were found in isolates with distinct ribotypes at the Roman and Sicilian sites indicating that this integron has recently disseminated across Italy. All 25 MbetaL-producing isolates were genetically linked in that all isolates contained Tn5051 sequences and all harboured the insertion sequence IsPa7 which may be involved in the mobilization of these resistance alleles. CONCLUSIONS: Taken together, these results indicate that Italy has a nationwide problem of MDR P. aeruginosa produced by mobile MbetaL genes.

Anti-Bacterial Agents↗

PubMed1980

The construction of a recombinant cDNA library representative of the poly(A)+ mRNA population from normal human lymphocytes.

A recombinant library has been constructed using the plasmid pAT153 and double stranded cDNA prepared from normal human lymphocyte poly(A)+ RNA. Transformation conditions were optimized to yield approximately 200,000 recombinants per microgram of double stranded cDNA. Statistical analysis as well as sequence complexity analysis of the inserted sequences indicates that the cDNA library is representative of > 99% of the poly(A)+ RNA present in the normal human lymphocyte.

Base Sequence↗

PubMed1987

Properties of Lactose Plasmid pLY101 in Lactobacillus casei.

A starter strain, Lactobacillus casei C257, was found to carry a lactose plasmid, pLY101. Restriction mapping showed that pLY101 DNA was 68.2 kilobases long. Since a non-lactose-utilizing variant of C257, MSK248, lost phospho-beta-galactosidase (P-beta-gal) activity and pLY101 DNA had a sequence(s) homologous to the streptococcal fragment including a P-beta-gal gene, pLY101 is likely to encode a P-beta-gal gene required for lactose metabolism in C257. MSK248 grew in galactose medium at a rate identical to that of C257 and retained phosphoenolpyruvate-dependent phosphotransferase system activity for lactose similar to that of C257. Therefore, the C257 chromosome appears to encode a complete set of genes for the lactose-phosphotransferase system and the predominant galactose metabolic pathway in C257. pLY101 DNA had a sequence homologous to a lactobacillus insertion sequence, ISL1, which mapped more than 12 kilobases from the sequence homologous to the streptococcal P-beta-gal fragment.

Journal Article↗

PubMed1990

Characterization of pXV10A, a Copper Resistance Plasmid in Xanthomonas campestris pv. vesicatoria.

The efficacy of copper bactericides for control of Xanthomonas campestris pv. vesicatoria in eastern Oklahoma tomato fields was evaluated. Copper bactericides did not provide adequate control, and copper-resistant (Cu) strains of the pathogen were isolated. The Cu genes in these strains were located on a large indigenous plasmid designated pXV10A. The host range of pXV10A was investigated; this plasmid was efficiently transferred into 8 of 11 X. campestris pathovars. However, the transfer of pXV10A to other phytopathogenic genera was not detected. DNA hybridization experiments were performed to characterize the Cu genes on pXV10A. A probe containing subcloned Cu genes from X. campestris pv. vesicatoria E3C5 hybridized to pXV10A; however, a subclone containing Cu genes from P. syringae pv. tomato PT23 failed to hybridize to pXV10A. Further DNA hybridization experiments were performed to compare pXV10A with pXvCu plasmids, a heterogenous group of Cu plasmids present in strains of X. campestris pv. vesicatoria from Florida. These studies indicated that the Cu genes on pXV10A and pXvCu plasmids share nucleotide sequence homology and may have a common origin. Further experiments showed that these plasmids are distinctly different because pXV10A did not contain sequences homologous to IS476, an insertion sequence present on pXvCu plasmids.

Journal Article↗

PubMed1996

On the nature and use of randomly amplified DNA from Staphylococcus aureus.

Various DNA-based methods have been introduced to genetically type Staphylococcus aureus strains but not a single technique is universally applicable. In order to search for DNA probes suitable for differentiating strains, randomly amplified polymorphic DNA patterns were generated for 243 S. aureus strains and a single isolate of Staphylococcus intermedius. All fingerprints were examined for unique amplicons, and the nature of 42 of these DNA fragments was investigated. Partial DNA sequences were determined, and several homologies were discovered with known S. aureus sequences (plasmid pSH6 DNA with insertion sequences, agrA and agrB sequences, hld genes, the gene for 23S rRNA, the lysyl tRNA synthetase gene, and the threonyl tRNA synthetase gene) and with genes from other species (Haemophilus influenzae bexA and Bacillus subtilis spoF and ctrA). Thirty fragments were of previously unknown origin. In Southern blots containing Eco RI-digested DNA from S. aureus strains and the S. intermedius strain, nine probes demonstrated the capacity to differentiate strains on the basis of the presence or absence of the sequence element in the staphylococcal genome involved. The remainder of the probes displayed restriction fragment length polymorphisms (n = 12), hybridized in a homogeneously positive fashion (n = 13) or hybridized only with their source strains (n = 8) (four of the latter were specific to S. intermedius). Three of the nine strain-specific probes were overlapping, and two of the others were found to display a high level of inconsistency among epidemiologically related strains. Thus, five strain-specific probes remained that, in a 5-digit typing system, accurately distinguished epidemiologically related and unrelated strains of S. aureus. We conclude that application of strain-specific DNA probes, selected on the basis of differing randomly amplified polymorphic DNA patterns, promises to become a technically simple, robust, and reproducible tool that may significantly facilitate the study of the epidemiology of S. aureus infections.

Bacterial Typing Techniques↗

PubMed2005

First nosocomial outbreak of vancomycin-resistant Enterococcus faecium expressing a VanD-like phenotype associated with a vanA genotype.

Although enterococci expressing acquired vancomycin resistance phenotype have been reported increasingly worldwide, they have been rarely reported in France. From August to December 2004 we faced an outbreak of vancomycin-resistant Enterococcus faecium (VRE) isolates in the nephrology department at Bicêtre Hospital (K.-Bicêtre, France). The expression of the glycopeptide resistance varied among the 26 VRE isolates, with vancomycin MICs ranging from 12 to >256 microg/ml, whereas teicoplanin MICs ranged from 4 to 48 microg/ml. However, several strains appeared to be susceptible to glycopeptides according to disk diffusion testing and expressed resistance only after subculture with glycopeptides. In addition, a heterogeneous expression of glycopeptide resistance was also observed. This so-called VanD-like phenotype of resistance (low-level resistance to vancomycin and mostly susceptibility to teicoplanin) was surprisingly associated with a vanA gene. Plasmid extraction and mating-out experiments indicated that the vanA gene was located on a 200-kb self-transferable plasmid. Pulsed-field gel electrophoresis identified mostly dissemination of a single clone, whereas diffusion of the VanA-positive plasmid in different genomic backgrounds had also occurred. The vanA gene was part of a vanA-type operon for expression of resistance located on a Tn1546-like transposon. Sequencing of this transposon identified insertion of insertion sequence IS16 in the vanY gene that encodes a d,d-carboxypeptidase that might explain in part the peculiar VanD-type phenotype of resistance. This report is the first description of a VRE outbreak in France and underlines the difficulty in detecting this organism due to variability on the expression of the glycopeptide resistance trait, if any.

Adult↗

PubMed2000

Cloning and expression of MXR7 gene in human HCC tissue.

AIM:To clone and identify the whole cDNA of MXR7 gene and to find out its expression in human HCC, and normal tissues.METHODS:The DNA primers were designed and synthesized according to the whole cDNA sequence of MXR7 gene. The cDNA of human HCC was taken as the template while the cDNA of MXR7 gene was synthesized by polymerase chain reaction (PCR). Recombinant DNA conforming to reading frame was constructed by connecting purified PCR product of the cDNA of MXR7 gene with expression vector pGEX-5X-1 of fusion protein. The plasmid MXR7/pGEX-5X-1 was identified by sequencing. Using (32)P labeled MXR7 cDNA as probe, MXR7 mRNA expression was detected by Northern blot analysis in 12 different human normal tissues, 7 preoperatively untreated non-liver tumor tissues, 30 preoperatively untreated HCC, the paracancerous liver tissues and 12 normal liver tissues samples.RESULTS:Restriction enzyme and sequence analysis confirmed that the insertion sequence in vector pGEX-5X-1 was the same as the cDNA sequence of MXR7 gene. Northern blot analysis showed no expression of MXR7 mRNA in 12 kinds of normal human tissues including liver, 7 tumor tissues in other sites and 12 normal liver tissues, the frequencies of MXR7 mRNA expression in HCC and paracancerous liver tissues were 76.6% and 13.3%, respectively. The frequency of MXR7 mRNA expression in HCC without elevation of serum AFP and in HCC <5cm was 90% (9/10) and 83.3% (5/6), respectively.CONCLUSION:MXR7 mRNA is highly expressed in human HCC, which is specific and occurs at an early stage of HCC, suggesting MXR7 mRNA can be a tumor biomarker for HCC. The detection of MXR7 mRNA expression in the biopsied liver tissue is helpful in discovering early subclinical liver cancer in those with negative serum AFP.

Journal Article↗

PubMed2000

In situ-PCR for the detection of Mycobacterium paratuberculosis DNA in paraffin-embedded tissues.

Molecular biological techniques have permitted the rapid and sensitive detection of the Mycobacterium paratuberculosis genome in infected tissues, most commonly by polymerase chain reaction amplification of sequences in the IS900 DNA insertion sequence. The aim of this work was the detection of M. paratuberculosis DNA in ovine tissues by in situ-polymerase chain reaction, which is sensitive and localises the signal within the tissue sample. Paraffin embedded tissues from three acid-fast positive ovine guts with classical lesions of paratuberculosis, and from negative control samples were tested. A 413-bp fragment of the IS900 sequence was amplified in-situ and hybridised to an internal PCR-synthesised digoxygenin-labelled probe. The samples from sheep affected by paratuberculosis clearly showed cell-specific cytoplasmic signals in mucosal and submucosal macrophages. This technique could be useful both in the diagnosis and study of the pathogenesis of infections in which involvement of M. paratuberculosis is suspected.

Animals↗

PubMed1992

Unexpected heterogeneity in E2A/PBX1 fusion messenger RNA detected by the polymerase chain reaction in pediatric patients with acute lymphoblastic leukemia.

The t(1;19)(q23;p13) is the most common recurring chromosomal translocation in childhood acute lymphoblastic leukemia (ALL) and has been associated with adverse prognosis. It involves the rearrangement of two genes, PBX1 and E2A, resulting in the production of transforming chimeric DNA-binding proteins. In all previous reports in which the presence of a chimeric transcript was described, the fusion point between the coding sequences of E2A and PBX1 was found to be constant at the RNA level. We have used RNA-based polymerase chain reaction (PCR) for the detection of E2A/PBX1 messenger RNAs (mRNAs) in children with ALL at the time of diagnosis. Of 21 patients exhibiting this rearrangement, 3 (14%) expressed a variant E2A/PBX1 transcript in addition to the expected one. The relative amounts of the two chimeric mRNAs varied between the patients, but remained constant in the same patient during different stages of the disease. Sequence analysis showed an identical insertion of 27 bp at the E2A/PBX1 junction of the variant RNA species, the translation of which would result in the replacement of Val478 by 10 amino acids. The inserted sequence has not been detected in any other human transcript besides the variant E2A/PBX1 RNA species and probably represents a splicing variant of the chimeric RNA. We conclude that a subset of pediatric patients with ALL that carry the E2A/PBX1 rearrangement express two types of the chimeric mRNA. The biologic significance of this additional E2A/PBX1 transcript is discussed.

Adolescent↗

PubMed2006

Comparison of the virulence plasmid genomes of two strains of Shigella which lost the ability to bind Congo red.

We determined and analyzed the Shigella flexneri serotype 5 (pSF5) and S. dysenteriae serotype 1 (pSD1) virulence plasmid genomes. The total length of pSF5 is 136513 bp, including 165 open reading frames (ORFs). Of these ORFs, 133 were identified and 32 of those had no significant homology to proteins with known functions. The length of pSD1 is 182545 bp, including 224 ORFs, of which we identified 181. The remaining 43 ORFs were not significantly homologous to proteins with known functions. The insertion sequence (IS) elements are 53787 bp in pSF5, and 49616 bp in pSD1, which represents 39.4% and 27.1% of the genome, respectively. There are 22 IS element types in pSF5 and pSD1, among which we report ISEc8 and ISSbo6 for the first time in the Shigella virulence plasmid. Compared to pCP301, there are a large number of deleted genes and gene inversions in both pSF5 and pSD1. The ipa-mxi-spa locus in pSF5 is completely absent, and the genes related to the O-antigen biosynthesis are partially missing. In contrast, the above genes in pSD1 are integral, with the exception of virF. The whole genome analysis of the two plasmids shows that the loss of genes related to gene invasion or regulation also obliterates the ability of pPF5 and pSD1 to bind Congo red (Crb). Whether these genes determine the Crb function requires continued investigation.

Acyltransferases↗

PubMed1991

Illegitimate integration of non-replicative vectors in the genome of Rhodococcus fascians upon electrotransformation as an insertional mutagenesis system.

Electrotransformation of Rhodococcus fascians by non-replicating plasmids containing a suitable resistance marker resulted in stable transformants by integration of these constructs at various sites in the genome, thereby generating different mutations. Tagged genes could be isolated in Escherichia coli owing to the presence of a CoIE1 replicon and an ampicillin resistance gene in the inserted sequences. Southern analysis and nucleotide sequencing revealed that recombination can occur at defined locations in the plasmid, while no site preference for target sequences could be detected. Low homology between the recombining sequences indicates illegitimate recombination. The specificity of the plasmid sites could be explained by assuming a linear recombination intermediate, generated by cleavage of the transformed plasmid.

Base Sequence↗

PubMed1985

Repeated sequences similar to insertion elements clustered around the nif region of the Rhizobium japonicum genome.

Two different repeated sequences (RSs) were discovered in the Rhizobium japonicum genome: RSRj alpha is 1126 base pairs long and is repeated 12 times; RSRj beta is approximately 950 base pairs long and is repeated at least 6 times. Their arrangement in root nodule bacteroid DNA is the same as in DNA from bacteria grown in culture. Deletion analysis showed that many copies of alpha and beta are clustered around the nitrogenase genes nifDK and nifH, or, in general, they are found within a genomic region harboring genes that are nonessential for growth. One copy each of alpha and beta are located upstream of nifDK and are adjacent to each other. Neither of them, however, is involved in the expression of nifDK. Nucleotide sequence analysis of three copies of RS alpha revealed many characteristics of procaryotic insertion sequence elements: potential inverted repeats at their ends, potential target site duplication, and large open reading frames. Despite this, their genomic positions appear to be stable. One possible function of these RSs is in deletion formation probably via recombination between them.

DNA Transposable Elements↗

PubMed1989

In situ detection of a heat-shock regulatory element binding protein using a soluble synthetic enhancer sequence.

In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also it was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer.

Cell Line↗

PubMed1999

Identification of soluble interleukin-4 receptor in rat glomerular epithelial cells(1).

Interleukin (IL)-4, a pleiotropic cytokine involved in many glomerular diseases, is regulated positively by membrane-bound IL-4R (mIL-4R) and negatively by soluble IL-4R (sIL-4R). Because natural sIL-4R has been documented only in mice, we undertook this study in rats to determine whether they, too, express sIL-4R, particularly in kidney cells. A pair of IL-4R primers was designed for this purpose and used in the polymerase chain reaction. As a result, sIL-4R was found not only in rats spleen cells but also in their glomerular epithelial cells (GEC). Sequence analysis revealed that the mRNA of rat sIL-4R has a 75-bp insert sequence. This insert generated a termination TGA codon upstream from the transmembrane region, resulting in formation of the sIL-4R. Subsequent screening of the kidney cDNA library enabled us to obtain the whole 3605-bp cDNA of sIL-4R; the full-length 3530-bp mIL-4R cDNA was also identified as a much longer sequence than previously published. Among the total 39 clones positive for IL-4R, two were confirmed as sIL-4R, and 37 clones were positive for mIL-4R. Next, the translated portion of sIL-4R cDNA was constructed into an expression vector, enabling us to obtain a recombinant sIL4R-myc fusion protein. By using this recombinant sIL-4R, we proved that sIL-4R can antagonize the IL-4-induced proliferation of spleen cells. Present study demonstrated that sIL-4R is expressed in kidney cells and antagonistically functional.

Animals↗

PubMed2002

Organization of the membrane domain of the human liver sodium/bile acid cotransporter.

Mammalian sodium/bile acid cotransporters (SBATs) are glycoproteins with an exoplasmic N-terminus, an odd number of transmembrane regions, and a cytoplasmic C-terminus. Various algorithms predict eight or nine membrane-embedded regions derived from nine hydrophobic stretches of the protein (H1-H9). Three methods were used to define which of these were transmembrane or membrane-associated segments in the liver bile acid transporter. The first was in vitro translation/insertion scanning using either single hydrophobic sequences between the N-terminal domain of the alpha-subunit of the gastric H,K-ATPase and the C-terminal domain of the beta-subunit that contains five N-linked glycosylation exoplasmic flags or using constructs beginning with the N-terminus of the transporter of various lengths and again ending in the C-terminus of the H,K-ATPase beta-subunit. Seven of the predicted segments, but not the amphipathic H3 and H8 sequences, insert as both individual signal anchor and stop transfer sequences in the reporter constructs. These sequences, H3 and H8, are contained within two postulated long exoplasmic loops in the classical seven-transmembrane segment model. The H3 segment acts as a partial stop transfer signal when expressed downstream of the endogenous H2. In a similar manner, the other amphipathic segment, H8, inserts as a signal anchor sequence when translated in the context with the upstream transporter sequence in two different glycosylation constructs. Alanine insertion scanning identified regions of the transporter requiring precise alignment of sequence to form competent secondary structures. The transport activity of these mutants was evaluated either in native protein or in a yellow fluorescent protein (YFP) fusion protein construct. All alanine insertions in H3 and H8 abolished taurocholate uptake, suggesting that both these regions have structures with critical intramolecular interactions. Moreover, these insertions also prevented trafficking to the plasma membrane as assessed by confocal microscopy with a polyclonal antibody against either the C-terminus of the transporter or the YFP signal of the YFP-transporter fusion protein. Two glycosylation signals inserted in the first postulated loop region and four of five such signals in the second postulated loop region were not recognized by the oligosaccharide transferase, and the L256N mutation exhibited 10% glycosylation and was inactive. These findings support a topography with nine membrane-spanning or membrane-associated segments.

Amino Acid Sequence↗

PubMed2006

Detection of a new mutation in KIT exon 9 in a gastrointestinal stromal tumor.

Gastrointestinal stromal tumors are mesenchymal tumors arising in the stomach and small bowel and more rarely in the rectum, esophagus, peritoneum and retroperitoneum. These tumors are characterized by KIT or PDGFRA mutations. KIT mutations are all in frame and lead to constitutive tyrosine kinase domain activation without ligand binding. Mutations concern four exons (9, 11, 13 and 17) but mainly exon 11. We report a new mutation in exon 9, since only AY 502-503 duplication/insertion, FAF 506 insertion and P456S substitution have been previously reported. This mutation consists of a large deletion of 43 nucleotides and an insertion of 25 nucleotides. More surprisingly, the sequence inserted corresponds to the complementary sequence of the wild allele but in the opposite sense. To our knowledge, this mutation has never been previously described.

Aged↗

PubMed2000

ETn insertion in the mouse Adcy1 gene: transcriptional and phylogenetic analyses.

Early retrotransposons (ETn) are murine transposable elements, bearing some structural similarity to integrated proviruses, and can be insertional mutagens. We have recently identified the causative mutation of the barrelless (Adcy1brl) phenotype as an integration of a 5.7-kb ETn in an intron of the adenylyl cyclase type I (Adcy1) gene. In the present study, Northern blot analysis shows that the ETn insertion results in loss of the normal Adcy1 transcript, a finding consistent with the loss-of-function Adcy1brl mutation, and generation of shorter transcripts. These aberrant transcripts are the products of abnormal RNA splicing and termination owing to the inserted sequence, and transcription initiation within the 3' long terminal repeat (LTR) of the ETn. The DNA sequences of the LTRs were compared in phylogenetic analyses with LTRs from 22 other ETn-related sequences. Three distinct families of ETn sequences can be identified on the basis of their LTRs. The ETn found in Adcy1brl is a member of a family that includes all classified ETn elements known to have recently transposed. Further, of the four known solitary (solo) LTRs, we have identified two that show evidence of recombination between LTRs from different ETn families.

Adenylyl Cyclases↗

PubMed1991

Evolutionarily recent transfer of a group I mitochondrial intron to telomere regions in Saccharomyces cerevisiae.

The junctions between X and Y' subtelomeric repeats in Saccharomyces cerevisiae usually contain a stretch of telomere sequences, (G1-3T)n. Two of three cloned X-Y' junctions from strain YP1 have a replacement of about 200 bp of X, the internal telomere sequence, and 49 bp of Y' by a 292 bp sequence. The first 227 bp of this insertion sequence are 100% identical to the fourth intron of cytochrome b. The rest of the insertion has homology to an unknown dispersed nuclear sequence. Recombination among subtelomeric regions can explain the nuclear distribution of this sequence and why telomeres can trap and maintain sequences that would otherwise be lost.

Base Sequence↗