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Biomedical subjects

F Laigret

Publications and source records attributed to F Laigret.

At least 37 records · Page 2Linked to original sources
PubMed1996

Insusceptibility of members of the class Mollicutes to rifampin: studies of the Spiroplasma citri RNA polymerase beta-subunit gene.

In order to study the mechanism of insusceptibility of Spiroplasma citri to rifampin, we have cloned and sequenced its rpoB gene, which encodes the beta subunit of RNA polymerase. By comparison of the deduced amino acid sequence with sequences of beta subunits from susceptible and resistant bacteria, it was possible to identify several differences in the so-called Rif region (encompassing rpoB codons 500 to 575 in the Escherichia coli sequence). We constructed a chimeric rpoB gene made of the E. coli rpoB gene in which the Rif region was replaced by the equivalent region from S. citri. E. coli cells harboring this chimeric gene were resistant to rifampin. Subsequent experiments involving site-directed mutagenesis demonstrated that a single amino acid substitution (asparagine at position 526) was able to provide high-level rifampin resistance in E. coli.

Amino Acid Sequence↗
PubMed1995

Heterogeneity of genome sizes within the genus Spiroplasma.

Organisms belonging to the genus Spiroplasma are currently classified into 23 groups, 17 of which have been assigned species epithets. We determined the genome sizes of representatives of 20 groups by using pulsed-field gel electrophoresis. Each genome size was deduced from the mobility of linear nonrestricted DNA, as well as from the sum of the sizes of restriction fragments obtained after digestion with NotI, a restriction endonuclease with a limited number of restriction sites in spiroplasma DNA. The values which we obtained indicated that the genome sizes of members of the genus Spiroplasma range from 940 to 2,220 kbp.

Genome, Bacterial↗
PubMed1995

Integrative and free Spiroplasma citri oriC plasmids: expression of the Spiroplasma phoeniceum spiralin in Spiroplasma citri.

The replication region (oriC) of the Spiroplasma citri chromosome has been recently sequenced, and a 2-kbp DNA fragment was characterized as an autonomously replicating sequence (F. Ye, J. Renaudin, J. M. Bové, and F. Laigret, Curr. Microbiol. 29:23-29, 1994). In the present studies, we have combined this DNA fragment, containing the dnaA gene and the flanking dnaA boxes, with a ColE1-derived Escherichia coli replicon and the Tet M determinant, which confers resistance to tetracycline. The recombinant plasmid, named pBOT1, was introduced into S. citri cells, in which it replicated. Plasmid pBOT1 was shuttled from E. coli to S. citri and back to E. coli. In S. citri, replication of pBOT1 did not require the presence of a functional dnaA gene on the plasmid. However, the dnaA box region downstream of the dnaA gene was essential. Upon passaging of the S. citri transformants, the plasmid integrated into the spiroplasmal host chromosome by recombination at the replication origin. The integration process led to duplication of the oriC sequences. In contrast to the integrative pBOT1, plasmid pOT1, which does not contain the E. coli replicon, was stably maintained as a free extrachromosomal element. Plasmid pOT1 was used as a vector to introduce into S. citri the G fragment of the cytadhesin P1 gene of Mycoplasma pneumoniae and the spiralin gene of Spiroplasma phoeniceum. The recombinant plasmids, pOTPG with the G fragment and pOTPS with the spiralin gene, were stably maintained in spiroplasmal transformants. Expression of the heterologous S. phoeniceum spiralin in S. citri was demonstrated by Western immunoblotting.

Adhesins, Bacterial↗
PubMed1995

Preparation of high molecular weight genomic DNA from nuclei of woody plants.

We have developed a rapid and reliable method for preparation of high molecular weight genomic DNA from sweet orange (Citrus sinensis) suitable for subsequent digestion by rarely cutting restriction enzymes and then separation by pulsed-field gel electrophoresis (PFGE). Methods previously described for preparation of plant DNA prior to PFGE involved protoplast isolation, a procedure that can be inefficient and time-consuming for several plant species. Nuclei isolated from plant tissues were embedded into agarose blocks and treated to release DNA, which was cleaved by restriction enzymes and then submitted to PFGE. One gram of fresh leaves gave approximately 15 micrograms of high molecular weight genomic DNA (> 2000 kbp). Within-gel hybridizations were used instead of classical Southern blotting, and the resulting signals were adequate when they were compared with those obtained with DNA prepared from crude ground leaf tissues.

Cell Nucleus↗
PubMed1994

Cloning and sequencing of the replication origin (oriC) of the Spiroplasma citri chromosome and construction of autonomously replicating artificial plasmids.

A 5.6-kbp fragment of Spiroplasma citri DNA containing the dnaA gene has been cloned and sequenced. Nucleotide sequence analysis shows that this fragment harbors the genes for the replication initiator protein (dnaA), the beta subunit of DNA polymerase III (dnaN), and the DNA gyrase subunits A and B (gyrA and gyrB). The arrangement of these genes, dnaA-dnaN-gyrB-gyrA, is similar to that found in all Gram-positive bacterial genomes studied so far, except that no recF gene was found between dnaN and gyrB. Several DnaA-box consensus sequences were found upstream of dnaA and in the dnaA-dnaN intergenic region. The dnaA region with the flanking DnaA-boxes and the tetracycline resistance determinant, tetM, were linked into a circular recombinant DNA. This DNA was able to replicate autonomously when introduced by electroporation into S. citri cells. These experiments show that the dnaA region with the DnaA-boxes is the origin of replication of S. citri and can be used to construct gene vectors.

Amino Acid Sequence↗
PubMed1994

Cloning the ribosomal RNA operons of Mycoplasma flocculare and comparison with those of Mycoplasma hyopneumoniae.

In contrast to other mycoplasma species the 16S/23S rRNA and 5S rRNA operons of Mycoplasma flocculare and Mycoplasma hyopneumoniae map at least 150 kb apart (20% of the genome). Both operons from M. flocculare have been cloned and sequenced. The 23S rRNA gene sequence showed 96.7% homology with the corresponding gene of M. Hyopneumoniae, equalling that found earlier for 16S rRNA and confirming the close phylogenetic relationships of these organisms. A possible upstream promoter was identified. Sequence elements upstream and downstream from each structural gene could form a stem needed for maturation of the immature rRNA transcript to mature 16S and 23S rRNA. We also identified two possible stem-and-loop sequences 3' to the 23S rRNA gene. The 5S rRNA gene itself also showed high homology with the corresponding structural gene of M. hyopneumoniae, although the upstream and downstream sequences were highly heterologous.

Base Sequence↗
PubMed1993

Detection of mollicute contamination in cell cultures by 16S rDNA amplification.

A polymerase chain reaction (PCR) system was developed for the detection of mollicutes as contaminants of cell cultures. By using three oligonucleotides chosen in the 16S rDNA sequences, two sets of primers able to promote amplification of all Mycoplasma and Ureaplasma (molli1-molli2a) or all Acholeplasma (molli1-molli2b) species examined were determined. This PCR system, first applied to experimentally infected Vero cell lines, was then evaluated for the detection of mollicutes in 86 cell culture samples, comparatively to DNA staining, culture and ELISA. The results obtained by the four techniques were in agreement in 82 cases (36 positive, 46 negative). PCR allowed detection of contamination in one and two cases negative by ELISA and culture, respectively, and confirmed questionable results obtained by DNA staining. As described, PCR seems to be a very convenient tool for routine detection of cell culture contaminants.

Acholeplasma↗
PubMed1993

The genome of the non-cultured, bacterial-like organism associated with citrus greening disease contains the nusG-rplKAJL-rpoBC gene cluster and the gene for a bacteriophage type DNA polymerase.

We have recently cloned three DNA fragments (In-2.6, In-1.0, and In-0.6) of the non-cultured, bacterial-like organism (BLO) associated with citrus greening disease. Nucleotide sequence determination has shown that fragment In-2.6 is part of the rplKAJL-rpoBC gene cluster, a well-known operon in eubacteria. The DNA fragment upstream of and partially overlapping with In-2.6 could be isolated and was shown to be the nusG gene. In Escherichia coli, nusG is also immediately upstream of rplKAJL-rpoBC. Fragment In-1.0 carries the gene for a bacteriophage type DNA polymerase. Fragment In-0.6 could not be identified. When In-2.6 was used, at high stringency, as a probe to detect greening BLO strains in infected plants, hybridization was obtained with all Asian strains tested, but not with the African strain examined. At lower stringencies, In-2.6 was able to detect also the African strain. The implications of these results in the taxonomical position of the greening BLO are discussed.

Amino Acid Sequence↗
PubMed1993

Evidence for a phosphoenolpyruvate dependent sugar-phosphotransferase system in the mollicute Acholeplasma florum.

In order to confirm the presence of a phosphoenolpyruvate (PEP)-dependent sugar-phosphotransferase system (PTS) in the mollicute Acholeplasma florum we studied the ability of cell free extracts of this organism to phosphorylate glucose and/or fructose in the presence of PEP. We also cloned and sequenced a DNA fragment coding for a putative polypeptide showing significant similarity with the enzyme II of the beta-glucoside PTS of Escherichia coli. Taken together, these results show that A florum possesses a fructose-PTS, but not a glucose-PTS, and that the amino acid sequence deduced from the DNA fragment is related to beta-glucoside and sucrose enzymes II of PTS from various bacteria.

Acholeplasma↗
PubMed1992

A physical and genetic map of the Spiroplasma citri genome.

A physical and genetic map of the Spiroplasma citri genome has been constructed using several restriction enzymes and pulsed field gel electrophoresis. A number of genes were subsequently localized on the map by the use of appropriate probes. The genome size of the spiroplasma estimated from restriction fragments is close to 1780 kbp, the largest of all Mollicutes studied so far. It contains multisite insertions of Spiroplasma virus 1 (SpV1) sequences. The physical and genetic map of the S. citri genome shares several features with that of other Mollicutes, especially those in the Mycoplasma mycoides cluster. This supports the finding that S. citri and these Mycoplasma spp. are phylogenetically related.

Bacteriophages↗
PubMed1992

Phylogenetic relationships of three porcine mycoplasmas, Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma hyorhinis, and complete 16S rRNA sequence of M. flocculare.

The nucleotide sequence of the 16S rRNA gene of Mycoplasma flocculare was determined and was compared with the sequence of a related porcine mycoplasma, Mycoplasma hyopneumoniae. While the overall level of DNA-DNA homology was approximately 11%, sequence alignment of the two 16S rRNA genes yielded a homology value of more than 95%, emphasizing the highly conserved nature of the 16S rRNA gene. Multiple sequence alignments with other mollicutes indicated that M. flocculare, M. hyopneumoniae, and Mycoplasma hyorhinis form a subcluster within the fermentans phylogroup, and this subcluster is distinct from the Mycoplasma pneumoniae phylogroup. Thus, the three mycoplasmas isolated from porcine respiratory systems exhibit phylogenetic similarities.

Animals↗
PubMed1992

[Mollicute Acholeplasma florum possesses a gene of phosphoenolpyruvate sugar phosphotransferase system and it uses UGA as tryptophan codon].

Acholeplasmas are mollicutes which do not require sterol for growth and do not possess a phosphoenolpyruvate-dependent sugar-phosphotransferase system. In contrast to spiroplasmas, mycoplasmas and ureaplasmas, they utilize UGA as a stop codon and not as a tryptophan codon. Acholeplasma florum, because of its metabolic properties and its close phylogenetic relationship to members of the genera Spiroplasma and Mycoplasma, does not seem to be an acholeplasma senso stricto. Here, we present molecular data to support this hypothesis. We have detected a gene coding for one of the components of the phosphotransferase system (PTS) in A. florum. In addition we demonstrate that the organism uses UGA as a tryptophan codon and not as a stop codon. These findings offer strong evidence, along with an earlier phylogenetic proposal, that A. florum is not a member of the genus Acholeplasma.

Acholeplasma↗
PubMed1991

Identification of a plant-derived mollicute as a strain of an avian pathogen, Mycoplasma iowae, and its implications for mollicute taxonomy.

Strain PPAV, a filamentous but nonhelical mollicute, was isolated from aborted apple seeds in France in late 1979. This organism grew well in SP-4 broth, fermented glucose, and required sterol for growth, and most of its properties suggested that it belonged to the genus Mycoplasma. However, it was serologically distinct; in addition, unlike other Mycoplasma species, genome measurements consistently yielded values of about 1,000 MDa (ca. 1,500 kbp), and the organism had a growth temperature optimum of 43 degrees C. A comparison of strain PPAV 16S rRNA sequences with those of other mollicutes revealed a high degree of sequence similarity to a strain of Mycoplasma iowae, which is commonly encountered in poultry. This relationship was confirmed by performing a restriction endonuclease pattern analysis and DNA-DNA hybridization tests. The genome size of type strain 695 of M. iowae was determined to be about 1,000 MDa (1,500 kbp) by renaturation kinetics, a value which is much higher than any other value known in the genus. Additional measurements by pulsed-field gel electrophoresis yielded values of 1,300 kbp for both strain PPAV and M. iowae. Subsequent phenotypic comparisons supported this relationship. Serologic tests with strain PPAV and other strains of M. iowae confirmed the findings of other investigators that this species is serologically heterogeneous. The high optimum temperature for growth of strain PPAV was also shared by a number of M. iowae isolates. Genome size is an inappropriate character for taxonomic assignment to the family Mycoplasmataceae because strain PPAV and other established species in this family are now known to have genomes ranging in size from 1,000 to 1,400 kbp.

Animals↗
PubMed1988

Identification of putative endogenous proviral templates for progenitor mink cell focus-forming (MCF) MuLV-related RNAs.

Murine leukemia virus (MuLV)-related RNAs exhibiting different env deletions are believed to participate in the generation of leukemogenic mink cell focus-forming (MCF) viruses. We have cloned an endogenous MuLV provirus from AKR/J mouse DNA, designated as A-2, which may serve as template for the env-deleted E2 MuLV RNA, expressed in GIX+ mice (D.E. Levy et al., J. Virol. 56, 691-700 (1985]. We have also isolated an endogenous MCF-related DNA, A-1, which shared close sequence homology with the 7.2-kb RNA expressed in AKR mice (F. Laigret et al., J. Virol. 62, 376-386 (1988] and sustained an identical env deletion. The data indicate that putative precursor MCF-related RNAs are transcribed from a heterogenous family of env-deleted endogenous MuLV DNAs.

Base Sequence↗
PubMed1988

Potential progenitor sequences of mink cell focus-forming (MCF) murine leukemia viruses: ecotropic, xenotropic, and MCF-related viral RNAs are detected concurrently in thymus tissues of AKR mice.

Leukemogenic mink cell focus-forming (MCF) viruses of AKR mice are believed to originate in thymic tissue via recombination between ecotropic, xenotropiclike, and endogenous MCF-related murine leukemia virus (MuLV) sequences. We have previously used a synthetic 16-base-pair MCF env-specific oligomer probe to identify subgenomic MCF-related mRNAs present in the thymus tissues of AKR mice prior to the appearance of full-length (8.4-kilobase [kb]) recombinant MCF viral RNAs (A. S. Khan, F. Laigret, and C. P. Rodi, J. Virol. 61:876-882, 1987). These potential MCF env precursors consisted of 7.2-, 3.0-, and 1.8-kb RNA species. In this study, we have determined the structure of the MCF-related mRNAs on the basis of Northern (RNA) blot hybridization analyses by using 10 different MuLV subgenomic DNA probes, determined the nucleotide sequence of a cloned cDNA segment representing the 3' portion of the 7.2-kb mRNA, and studied the expression of ecotropic and xenotropic MuLV sequences by using env-specific DNA probes. The results indicated that ecotropic, xenotropic, and MCF-related transcripts were constitutively and concurrently expressed exclusively in thymus tissue of 2-month-old AKR mice prior to detection of MCF viral RNAs. We have molecularly characterized these thymic MuLV RNAs, which may participate in formation of recombinant MCF viruses; a novel recombinant ecotropic viral RNA was identified as a putative intermediate in the stepwise generation of leukemogenic MCF MuLVs. We have also described the unique structure of the 6.0-kb MCF-related RNAs which were expressed specifically in liver and kidney tissues of AKR mice; these RNAs contained an upstream non-MuLV transcriptional regulatory element.

Amino Acid Sequence↗
PubMed1987

Expression of mink cell focus-forming murine leukemia virus-related transcripts in AKR mice.

We used a synthetic 16-base-pair mink cell focus-forming (MCF) env-specific oligomer as radiolabeled probe to study MCF murine leukemia virus (MuLV)-related transcripts in brain, kidney, liver, spleen, and thymus tissues of AKR mice ranging from 5 weeks to 6 months (mo) of age. Tissue-specific expression of poly (A) + RNAs was seen: 6.0-kilobase (kb) transcripts were detected in the liver and kidney; 7.2- and 1.8-kb RNA species were present in the thymus. In addition, all the tissues tested contained 3.0-kb messages. The transcription of these MCF-related mRNAs was independent of the presence of ecotropic and xenotropic MuLVs. In general, expression of the MCF env-related transcripts appeared to peak at 2 mo of age; these messages were barely detectable in brain, kidney, liver, and spleen tissues after 2 mo and in thymus tissue after 4 mo of age. All of the subgenomic MCF env-related mRNAs (6.0, 7.2, 1.8, and 3.0 kb) appeared to contain the 190-base-pair cellular DNA insert, characteristic of the long terminal repeats associated with endogenous MCF env-related proviruses (A. S. Khan and M. A. Martin, Proc. Natl. Acad. Sci. USA 80:2699-2703, 1983). No genomic-size (8.4-kb) transcripts corresponding to endogenous MCF-related proviruses were detected. An 8.4-kb MCF env-related mRNA was first seen at 3 mo of age, exclusively in thymus tissue. This species most likely represents the first appearance of a recombinant MCF-related MuLV genome. The transcripts which were detected in thymus tissue might be involved in the generation of leukemogenic MCF viruses.

Age Factors↗
PubMed1987

Spiroplasmas: gene structure and expression.

Upon sequencing of the SpV4 genome, eight putative open reading frames (ORFs) including that for the 65-kilodalton (kDa) capsid protein were detected. They involve all three reading frames. Three promoter sequences were found, as well as a transcription terminator and the initiation site for complementary strand synthesis. Ribosome binding sites and regulatory sequences are closely related to those of Eubacteria. Codon usage analysis showed that A and T terminated codons are preferably used. UAA is the major termination codon. Upon cloning of the full-size SpV4 replicative form, the capsid protein gene could not be expressed in Escherichia coli, whereas the spiralin gene cloned in the same bacterium is expressed. These results suggest that in spiroplasmas, as in Mycoplasma capricolum, UGA is not a termination codon, but very probably codes for tryptophan. Spiralin contains no tryptophan. Hence, its gene contains no UGA codons and can thus be expressed in E. coli. On the other hand, the gene for capsid protein has nine UGA codons and cannot be fully expressed in the bacterium. Our results fully support the bacterial origin of spiroplasmas.

Bacteria↗