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J Renaudin

Publications and source records attributed to J Renaudin.

At least 37 records · Page 2Linked to original sources
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

Expression in Spiroplasma citri of an epitope carried on the G fragment of the cytadhesin P1 gene from Mycoplasma pneumoniae.

We have previously described the use of the replicative form (RF) of Spiroplasma citri virus SpV1 as a vector for cloning and expressing foreign genes in S. citri, an organism which reads UGA as a tryptophan codon (C. Stamburski, J. Renaudin, and J.M. Bové, J. Bacteriol. 173:2225-2230, 1991). We now report cloning and expression in S. citri of the G fragment of cytadhesin P1 gene from Mycoplasma pneumoniae. The G fragment was inserted in the SpV1 RF downstream of a synthetic ribosome binding site and introduced into S. citri by electroporation. Northern (RNA) blot analyses showed that in S. citri, the G fragment was transcribed from an SpV1 RF promoter as a 1.2-kb mRNA. The translation product was detected by Western blotting (immunoblotting) with a rabbit antiserum raised against total proteins from M. pneumoniae (strain FH) and was proved to be P1 specific by using monoclonal antibodies specific for the G region of the P1 protein. The apparent molecular mass of the polypeptide (24.5 kDa) indicates that in S. citri, the G fragment was fully translated in spite of the seven UGA codons present in the reading frame.

Adhesins, Bacterial↗
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↗
PubMed1991

Nucleotide sequence of the Spiroplasma citri fibril protein gene.

Electron microscopic observation of spiroplasmas lysed by detergent (sodium deoxycholate) revealed the release of bundles of fibrils from the cells. Individual fibrils are 4 nm in diameter and possess a 9-nm periodicity along their length. These fibrils are thought to function as cytoskeletal structures involved in the shape and motility of spiroplasmas. Polyacrylamide gel electrophoresis of density gradient-purified fibrils showed a protein of approximately 55 kDa. Oligonucleotide probes were constructed from the N-terminal amino acid sequence of two peptides obtained after V8 protease hydrolysis of the fibril protein. The probes were used to identify the clones in a genomic DNA library of Spiroplasma citri that contained inserts carrying the probe sequence. Sequencing of a 3.3-kbp fragment yielded the full open reading frame of the fibril protein gene and the start of a second open reading frame of an unknown protein. The fibril protein is composed of 515 amino acids, which have a computed molecular mass of 59 kDa. Northern (RNA) blot hybridization and primer extension experiments showed that transcription of the fibril protein gene starts from a promoter located 100 nucleotides upstream of the initiation codon and stops at a rho-independent type terminator, leading to a 1.7-kbp transcript. Southern blot hybridization of genomic DNA using the fibril protein gene as the probe showed that a single copy of the gene is present in the chromosomes of both S. citri and Spiroplasma melliferum. The genotypic symbol fib is proposed for the spiroplasma fibril protein gene.

Amino Acid Sequence↗
PubMed1991

First step toward a virus-derived vector for gene cloning and expression in spiroplasmas, organisms which read UGA as a tryptophan codon: synthesis of chloramphenicol acetyltransferase in Spiroplasma citri.

Spiroplasmas are wall-less procaryotes in which the UGA codon serves not as a stop signal but as a code for the amino acid tryptophan. Spiroplasma genes that contain UGA codons thus cannot be studied in the usual Escherichia coli cloning and expression systems. Although this problem can be circumvented by using UGA-suppressor strains of E. coli, spiroplasmas themselves would provide a more efficient cloning and expression host. We have now successfully employed the replicative form (RF) of a filamentous spiroplasma virus (SpV1) to clone and express the E. coli-derived chloramphenicol acetyltransferase (CAT) gene in Spiroplasma citri. The CAT gene was inserted in one of the four intergenic regions of the SpV1 RF and introduced into cells by electroporation. Both the RF and the virion DNA produced by the transfected cells contained the CAT gene sequences. Northern blot analysis, primer extension, and S1 mapping showed that transcription of the CAT gene started from a promoter located on the SpV1 RF and was terminated downstream of the CAT gene, still within the viral RF. Expression of the CAT gene was demonstrated by acetylation of chloramphenicol by cell-free extracts from the transfected spiroplasmas.

Base Sequence↗
PubMed1990

Characterization of a promoter and a transcription terminator of Spiroplasma melliferum virus SpV4.

Spiroplasma virus 4 (SpV4) is an isometric virus with single-stranded, circular DNA infecting the helical mollicute Spiroplasma melliferum, a honeybee pathogen. Previous studies in our laboratory led to the determination of the base sequence of the SpV4 DNA. Nine open reading frames and three promoterlike sequences (P1, P2, and P3) were identified. An inverted repeat leading to the formation of a hairpin structure on the transcription product was also found and predicted to be a transcription terminator (T). We have now studied the in vivo transcription of the SpV4 genome by Northern (RNA) blot analysis of the total RNAs extracted from SpV4-infected spiroplasma cells. Transcripts of 7.8, 4.4, 3.4, and 2.7 kilobases (kb) were detected. The 3.4-kb RNA was the major transcript. The 5' and 3' ends of this transcript were determined by S1 mapping and primer extension. Characterization of the 3' end by S1 mapping showed that the 3.4-kb transcript terminates within the stretch of uridine residues following the hairpin structure of terminator T. Characterization of the 5' end by S1 mapping indicated that transcription proceeds from a newly recognized promoter, P0, located 36 nucleotides upstream of P1. Primer extension resulted in two cDNA signals. The short cDNA was probably a primer extension artifact due to the presence of a hairpin structure on the transcript. When reverse transcriptase stopped at this hairpin or read through, the short or the long cDNA, respectively, was obtained. The size of the long cDNA identified P0 as the transcription promoter. Promoter P0 was also shown to be functional in Escherichia coli. Indeed, when inserted upstream of the chloramphenicol acetyltransferase gene of a promoter selection vector, it promoted transcription of this gene. As in the case of S. melliferum, two cDNAs were obtained by primer extension, the longer cDNA identifying P0 as the promoter.

Bacteriophages↗
PubMed1987

Spiroplasma virus 4: nucleotide sequence of the viral DNA, regulatory signals, and proposed genome organization.

The replicative form (RF) of spiroplasma virus 4 (SpV4) has been cloned in Escherichia coli, and the cloned RF has been shown to be infectious by transfection (M. C. Pascarel-Devilder, J. Renaudin, and J.-M. Bové, Virology 151:390-393, 1986). The cloned SpV4 RF was randomly subcloned and was fully sequenced by the dideoxy chain termination technique, using the M13 cloning and sequencing system. The nucleotide sequence of the SpV4 genome contains 4,421 nucleotides with a G+C content of 32 mol%. The triplet TGA is not a termination codon but, as in Mycoplasma capricolum (F. Yamao, A. Muto, Y. Kawauchi, M. Iwami, S. Iwagani, Y. Azumi, and S. Osawa, Proc. Natl. Acad. Sci. USA 82:2306-2309, 1985), probably codes for tryptophan. With these assumptions, nine open reading frames (ORFs) were identified. All nine are characterized by an ATG or GTG initiation codon, one or several termination codons, and a Shine-Dalgarno sequence upstream of the initiation codon. The nine ORFs are distributed in all three reading frames. One of the ORFs (ORF1) corresponds to the 60,000-dalton capsid protein gene. Analysis of codon usage showed that T- and A-terminated codons are preferably used, reflecting the low G+C content (32 mol%) of the SpV4 genome. The viral DNA contains two G+C-rich inverted repeat sequences. One could be involved in transcription termination and the other in initiation of cDNA strand synthesis. The SpV4 genome was found to contain at least three promoterlike sequences quasi-identical to those of eubacteria. These results fully support the bacterial origin of spiroplasmas.

Amino Acid Sequence↗
PubMed1987

Cloning and sequencing of the genome of spiroplasma virus 4.

Spiroplasma virus 4 (Sp V4) has a circular single-stranded DNA. The replicative form (RF) of Sp V4 has been purified from infected cells of Spiroplasma melliferum, strain G1, and cloned in Escherichia coli (HB101) using plasmid pBR328 as the vector. The cloned RF was shown to be infectious by transfection. The Sp V4 RF was randomly subcloned in E. coli (TG1) using the M1 3 mp8 RF as the vector and sequenced by the dideoxy chain termination technique. We found that UGA is probably not a termination codon, but codes for tryptophan. Eight open reading frames, including that for the 65,000-dalton capsid protein, have been detected; they involve all three reading frames.

Bacteriophages↗
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↗
PubMed1986

The spiroplasma virus 4 replicative form cloned in Escherichia coli transfects spiroplasmas.

The replicative form (RF) of spiroplasma virus 4 (SpV4) has been purified from infected cells of Spiroplasma melliferum strain G1 by alkaline lysis followed by low melting point agarose gel electrophoresis. A partial restriction map has been established. The circular RF was linearized by cutting at the unique ClaI restriction site and has been cloned in Escherichia coli HB101 using the plasmid pBR328 as a vector. The recombinant plasmid was purified by equilibrium centrifugation in ethidium bromide-cesium chloride gradient. After ClaI endonuclease digestion, the inserted SpV4 RF DNA was recovered by low melting point agarose gel electrophoresis and was recircularized by ligation. The cloned SpV4 RF DNA was demonstrated to be infectious by transfection.

Bacteriophages↗
PubMed1984

Spiroplasmas and the transfer of genetic material by transformation and transfection.

Two plasmids, pMH1 with 7 kbp and pM41 with 8 kbp were purified from Spiroplasma citri strains MH and M4 respectively. On the basis of guanine + cytosine content and restriction enzyme mapping, the two plasmids are different. The linearized pMH1 plasmid was introduced into Escherichia coli plasmid vector pBR328 and could be cloned in E. coli. Using radioactive probes specific for each plasmid, we found that pM41 was present in three additional S. citri strains and in three other spiroplasmas not belonging to the S. citri species. pMH1 was found as a free 7-kbp plasmid only in the S. citri strain MH. However, the pMH1 probe hybridized strongly with high molecular weight DNA of several S. citri strains and strains of spiroplasmas other than S. citri. The major membrane protein of S. citri, spiralin, is strongly antigenic and rabbit antibodies against whole S. citri cells strongly react with spiralin. Thus, the enzyme-linked immunosorbent assay (ELISA) has been used to screen E. coli clones that were transformed with HindIII-generated S. citri DNA fragments inserted into the HindIII site of pBR328. One E. coli transformant strongly reacted in ELISA with S. citri polyclonal antiserum. The same transformant also gave a positive reaction with monospecific antiserum against spiralin. These results demonstrate that a gene from S. citri, the spiralin gene, could be expressed in a bacterium. The isometric virus SV4, infecting honeybee spiroplasmas of Group I-2, was shown to possess circular single-stranded DNA of molecular weight 1.7 X 10(6) Da. Transfection of spiroplasma G1 with purified DNA of SV4 was achieved. These experiments open the way to the introduction of foreign genes into spiroplasmas.

Bacterial Outer Membrane Proteins↗
PubMed1984

Characterization of spiroplasma virus group 4 (SV4).

Spiroplasma virus Group 4 was propagated, purified and characterized. The isometric capsid is made from one major protein of 60,000 Da and contains a circular single-stranded DNA molecule of 1.7 X 10(6) Da. The host-range of SV4 is limited to Subgroup I-2 spiroplasmas. The virions are released from the infected cells by a lytic process. Transfection of spiroplasmas of Subgroup I-2 by the viral DNA was demonstrated. A high efficiency of transfection was routinely obtained in the presence of polyethyleneglycol. These experiments indicate that spiroplasmas can absorb viral DNA, opening the way for cloning.

Bacteriophages↗
PubMed1983

Inappropriate antidiuretic hormone syndrome in craniofacial surgery.

Three instances of inappropriate ADH syndrome following craniofacial operations are reported. The cornerstone to diagnosis is careful fluid and electrolyte monitoring. Treatment consists of fluid restrictions in the acute phase and demeclocycline for refractory cases. Seizures should be symptomatically treated. Surgeons involved in the care of craniofacial anomalies must be aware of this syndrome because the symptoms closely mimic those commonly observed following intracranial procedures. If unrecognized, the consequence is potentially lethal.

Child↗
PubMed1979

Superficial temporal arteriovenous fistula.

A case of post-traumatic arteriovenous fistula involving the superficial temporal artery is reported. The traumatic episode antedated the clinical manifestation and diagnosis by 28 months. The salient feature of the physical examination was the presence of a pulsatile mass with detectable bruit. This lesion, which was confirmed by angiography and computerized tomography, was treated by direct excision.

Adult↗
PubMed1978

Spinal intramedullary glioblastoma with intracranial seeding. Report of a case.

Approximately one year after partial removal of an intramedullary glioblastoma at T12, a patient was admitted to Wadsworth Veterans Administration Hospital with signs and symptoms of an intracranial tumor. We describe the unusual occurrence of intracranial seeding from spinal glioblastoma and review this rare condition and the possible mechanism involved.

Brain Neoplasms↗
PubMed1978

Chip fracture through the superior articular facet with compressive cervical radiculopathy.

Four patients with cervical radiculopathy secondary to a chip fracture through the superior articular facet have been treated in the past 2 years. These small-element fractures may occasionally pass unrecognized. Although oblique X-rays are helpful, lateral tomography has provided the most precise detail. The pathological process is a compressive radiculopathy, explicable on the basis of the foraminal anatomy. The authors feel myelography is not essential. The preferred surgical approach is posterior: the results have been excellent.

Adult↗