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M Ouellette

Publications and source records attributed to M Ouellette.

At least 91 records · Page 5Linked to original sources
PubMed1990

Targeted insertion of the neomycin phosphotransferase gene into the tubulin gene cluster of Trypanosoma brucei.

Kinetoplastids are unicellular eukaryotes that include important parasites of man, such as trypanosomes and leishmanias. The study of these organisms received a recent boost from the development of transient transformation allowing the short-term expression of genes reintroduced into parasites like Trypanosoma brucei, the causative agent of African trypanosomiasis. We have obtained long-term stable transformants of T. brucei that have acquired the ability to grow in medium containing the drug G418, following the targeted insertion of the bacterial gene for neomycin phosphotransferase (neo(r) gene) into the trypanosome tubulin cluster. Plasmids in which part of the T. brucei tubulin gene cluster has been replaced by the neo(r) gene were used. Targeting efficiency was higher with a linearized than with a circular construct, and with 5 kilobases of tubulin gene cluster than with 0.9 kilobases. With these neo(r) constructs homologous recombination seems to be the preferred route for insertion of exogenous DNA into the trypanosome genome, allowing gene targeting without counter-selection.

Animals↗
PubMed1990

The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei.

The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5' ends of the major transcripts coming from the initiation region map at nucleotide sequences that do not strongly resemble rRNA transcriptional starts even though the transcripts are synthesized by an RNA polymerase highly resistant to alpha-amanitin. The cloned VSG gene 221 ES transcription initiation region promotes high CAT gene expression, when reintroduced by electroporation into T. brucei. We show that the activity of this expression site is controlled at or near transcription initiation in bloodstream trypanosomes. The 221 ES is inactivated without any sequence alteration within 1.4 kb of the transcription start site. This excludes mechanisms of promoter inactivation involving DNA rearrangements in the vicinity of the transcription start site, e.g. promoter inversion or conversion.

Animals↗
PubMed1990

The amplified H circle of methotrexate-resistant leishmania tarentolae contains a novel P-glycoprotein gene.

Acquired resistance to methotrexate in Leishmania species is often associated with the amplification of H circles, 68 kb duplex DNA circles containing a 30 kb inverted repeat. We report here that the H circle of Leishmania tarentolae contains an open reading frame, ltpgpA, that has the attributes of P-glycoproteins (large plasma membrane proteins known to extrude lipophilic drugs from mammalian cells). Although amplification of H circles is associated with proportionally increased levels of a 5.5 kb transcript of the ltpgpA gene, such methotrexate resistant mutants are not cross-resistant to any of the drugs extruded by mammalian multi-drug resistant cells. In Leishmania, ltpgpA is part of a gene family containing at least two other members. Sequences homologous to one of the nucleotide binding sites of ltpgpA are conserved in other kinetoplastida.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Increase in the relative abundance of preproenkephalin A messenger RNA in the ventricles of cardiomyopathic hamsters.

Preproenkephalin A messenger RNA was detected in hamster heart by Northern blot analysis using a human preproenkephalin A cDNA probe. Ventricular levels of this messenger were one order of magnitude lower than atrial levels, which were equivalent to brain levels. Furthermore, in the heart of cardiomyopathic hamsters, an animal model of cardiac hypertrophy and congestive heart failure, the relative abundance of the preproenkephalin A messenger RNA was found to increase three- to four-fold in ventricles while no change was seen in atria. These results support the hypothesis that the heart has the potential for locally synthesizing enkephalins and provide evidence that alterations in preproenkephalin A messenger RNA levels are associated with the development of cardiac hypertrophy and failure.

Animals↗
PubMed1988

Characterization of a plasmid isolated from Branhamella catarrhalis and detection of plasmid sequences within the genome of a B. catarrhalis strain.

We isolated a 12.2-kb plasmid from two clinical strains of Branhamella catarrhalis and evaluated its distribution among other B. catarrhalis strains by colony hybridization experiments using the whole plasmid as a probe. Homology was detected with the two plasmid-bearing strains and also with a third B. catarrhalis strain named E7, which is plasmidless. Southern transfer analysis of total digested E7 DNA using the purified plasmid as a probe revealed a single band of hybridization, different from those observed in plasmid-bearing strains, for each restriction enzyme used. The region of the plasmid hybridizing with DNA of strain E7 was located within a 4.5-kb SstI-EcoRV fragment. No homology was noted between our B. catarrhalis plasmid and about 20 other strains of different genera tested by colony hybridization.

Blotting, Southern↗
PubMed1987

Changes in the messenger RNA population from the heart of cardiomyopathic hamsters.

Messenger RNAs were extracted from the heart of cardiomyopathic hamsters at different phases of the disease and from age-matched control hamsters. They were translated into proteins in a rabbit reticulocyte lysate in the presence of [35S]methionine, the translational products were fractionated by two-dimensional polyacrylamide gel electrophoresis and analyzed by fluorography. No difference between cardiomyopathic and control hamsters could be detected when comparing the spots corresponding to the major contractile proteins. However, we observed that three translational products of minor abundance were reproducibly decreased in cardiomyopathic hamsters at 60 days (necrotic phase) and 200 days (final phase) but not at 30 days (prenecrotic phase). At 120 days (hypertrophic phase), the decrease could also be detected but was much less pronounced.

Animals↗
PubMed1987

Precise insertion of antibiotic resistance determinants into Tn21-like transposons: nucleotide sequence of the OXA-1 beta-lactamase gene.

Several plasmid-encoded beta-lactamases are on multiresistance transposable elements. The OXA-1 beta-lactamase gene is part of Tn2603, which is borne on the R plasmid RGN238. We report here the complete nucleotide sequence of the OXA-1 beta-lactamase gene and flanking sequences. The OXA-1 gene shows a greater than 50% sequence divergence from the OXA-2 gene, yet there is significant functional similarity at the peptide level. Analysis of 5' and 3' flanking sequences shows that Tn2603 differs from its probable precursor, Tn21, by a precise 1004-base-pair insertion, containing the OXA-1 structural gene, at the target sequence AAAGTT, which is located between the Tn21 streptomycin/spectinomycin (aadA) promoter and its structural gene. A 5- for 6-base repeat of the target sequence is found at the end of the insertion. The same precise insertion and repeat of the target sequence are found for the OXA-2 gene from R46. The 5' flanking regions of two other genes, the trimethoprim-resistance gene from R388 and the gentamicin resistance (aadB) gene from pDGO100, are greater than 98% homologous to the 5' flanking sequences of the OXA-1, OXA-2, and aadA genes until they diverge at the target sequence. From the available sequence data a recombinational hot spot is defined at the nucleotide level 5' of the aadA gene of Tn21, and a second potential hot spot is proposed 3' of this gene.

Amino Acid Sequence↗
PubMed1987

Isolation and molecular characterization of beta-lactamase-producing Haemophilus parainfluenzae from the genital tract.

Three Haemophilus parainfluenzae strains isolated from the urogenital tract harbored a beta-lactamase-coding 3.2-megadalton plasmid identical, by restriction endonuclease digestion and hybridization with radioactive and biotin-labeled probes specific for the TEM-1 beta-lactamase and TnA sequences, to the 3.2-megadalton "African-type" plasmid found in Neisseria gonorrhoeae.

DNA, Bacterial↗
PubMed1987

Acquisition by a Campylobacter-like strain of aphA-1, a kanamycin resistance determinant from members of the family Enterobacteriaceae.

A Campylobacter-like organism, BM2196, resistant to kanamycin and streptomycin-spectinomycin was isolated from the feces of a patient with acute enteritis. The kanamycin and streptomycin-spectinomycin resistances were not transferable to Camplylobacter sp. or to Escherichia coli, and no plasmid DNA was detected in this strain. The resistance genes were therefore tentatively assigned to a chromosomal locality. Analysis by the phosphocellulose paper-binding assay of extracts from BM2196 indicated that resistance to kanamycin and structurally related antibiotics was due to the synthesis of 3'-aminoglycoside phosphotransferase type I [APH(3')-I], an enzyme specific for gram-negative bacteria, and that resistance to streptomycin-spectinomycin was secondary to the presence of a 3",9-aminoglycoside adenylyltransferase. Homology between BM2196 and an APH(3')-I probe was detected by DNA-DNA hybridization. A 2.2-kilobase BM2196 DNA fragment conferring resistance to kanamycin was cloned in E. coli and was sequenced partially. The resistance gene appeared nearly identical to that of Tn903 from E. coli and was adjacent to IS15-delta, an insertion sequence widespread in gram-negative bacteria, thus indicating that Campylobacter species can act as a recipient for genes originating in members of the family Enterobacteriaceae.

Base Composition↗
PubMed1987

Oligonucleotide probes for the detection of TEM-1 and TEM-2 beta-lactamase genes and their transposons.

We describe the use of molecular probes to detect the TEM-type beta-lactamase genes. As a general probe, we prepared a 656 base pair restriction fragment, entirely within the TEM structural gene. This probe was specific for the TEM family, hybridizing only with TEM-1 and TEM-2. The TEM-1 and TEM-2 beta-lactamases differ by only one amino acid. We synthesized two oligonucleotides whose central bases correspond to this difference. The use of these oligonucleotides enables us to discriminate between TEM-1 and TEM-2 genes. Using oligonucleotides homologous to parts of Tn3, we also monitored the presence of TnA-like transposons in bacteria harboring different beta-lactamase genes. Only the TEM-1 and TEM-2 genes were found to be on transposons with terminal sequences identical to those of Tn3. All hybridization experiments were performed with both dot-blot and colony-hybridization techniques, and the suitability of these two methods for epidemiological studies is compared.

DNA Transposable Elements↗
PubMed1986

Analysis by using DNA probes of the OXA-1 beta-lactamase gene and its transposon.

From recombinant clone pTY27, encoding an OXA-1 beta-lactamase gene, we performed subcloning experiments to more precisely delimit the gene. We describe the use as probes of six different restriction fragments known from subcloning experiments to be within the structural gene or part of the transposable element, Tn2603, flanking the OXA-1 determinant. We showed that the OXA-1 structural gene is slightly related to the OXA-2 determinant and also that sequences within Tn2603 are common to all the OXA- and PSE-producing strains tested. For epidemiological purposes, we began nucleotide sequencing of the OXA-1 determinant, and from preliminary sequence data we synthesized an oligonucleotide 15 bases in length, corresponding to a sequence within the OXA-1 gene. This oligonucleotide was found to be specific for the OXA-1 determinant, because it hybridized only to bacteria producing that beta-lactamase.

Cloning, Molecular↗
PubMedDate not supplied

Drug resistance and P-glycoprotein gene amplification in the protozoan parasite Leishmania.

Amplification of the H circle is often associated with methotrexate (MTX) selection in Leishmania species. We have shown that the H circle of Leishmania tarentolae contains an open reading frame, ItpgpA, that has the attributes of P-glycoproteins (large plasma membrane proteins known to extrude lipophilic drugs from mammalian cells). H region amplification was also noted in some mutants selected for resistance to arsenite and vinblastine. Mutants having the complete 68-kb circles were cross-resistant to MTX, but two arsenite mutants having only part of the H region amplified, but including ItpgpA, were not cross-resistant to MTX. These results suggest that the putative determinant for MTX resistance present on the H circle is not ItpgpA. We have also determined how ItpgpA-containing plasmids were generated from the chromosomal copy. The H circle contains a 30-kb inverted duplication separated by two unique DNA segments. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We have shown that the two unique segments in chromosomal DNA are flanked by inverted repeats suggesting that H circles could be formed by a foldback mechanism (see fig. 2). Unexpectedly, a plasmid present in cells selected for arsenite resistance lacked part of the H region and the long inverted repeats. It appears to have been formed by intrachromosomal recombination between two P-glycoprotein genes, ItpgpA and ItpgpB, located adjacent to the H region. Our results show that under drug pressure, the same P-glycoprotein-encoding region in Leishmania may be amplified by very different mechanisms and yield different amplicons.

Animals↗