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J V Barnier

Publications and source records attributed to J V Barnier.

28 records · Page 2Linked to original sources
PubMed1992

Quail neuroretina c-Rmil(B-raf) proto-oncogene cDNAs encode two proteins of 93.5 and 95 kDa resulting from alternative splicing.

c-Rmil is the cellular allele of the v-Rmil oncogene transduced during in vitro passaging of Rous-associated virus type 1 in chicken embryonic neuroretina (NR) cells. The c-Rmil proto-oncogene is the avian homolog of the mammalian B-raf gene and belongs to the mil/raf oncogene family of serine/threonine protein kinases. The c-Rmil/B-raf gene is preferentially expressed in avian and mammalian neural tissues. Two c-Rmil cDNA species, resulting from an alternative splicing mechanism, were isolated from quail embryonic NR cDNA libraries. They encode two proteins of 767 and 807 amino acids that differ by the presence of an alternative exon, located upstream of the kinase domain. Expression of these cDNAs in COS-1 cells leads to the synthesis of two proteins with apparent molecular weights of 93.5 and 95 kDa, recognized by an Rmil-specific antiserum. Both proteins are phosphorylated in an immune complex kinase assay. A protein of 94 kDa is also immunoprecipitated in avian NR cells and is identical to the 93.5-kDa protein expressed in COS-1 cells, as shown by Staphylococcus aureus V8 protease mapping. The c-Rmil proteins contain the three conserved regions previously identified in mil/raf protein kinases. In addition, they contain amino-terminal sequences that are not present in the other mil/raf proteins identified to date. These additional sequences may define a novel functional domain for c-Rmil/B-raf and could play a role in signal transduction in neural cells.

Amino Acid Sequence↗
PubMed1992

Chromosomal assignment of two human B-raf(Rmil) proto-oncogene loci: B-raf-1 encoding the p94Braf/Rmil and B-raf-2, a processed pseudogene.

The B-raf gene is the human homolog of the avian c-Rmil proto-oncogene encoding a 94-kDa serine/threonine kinase detected in avian cells. We have previously shown that this protein contains amino-terminal sequences not found in other proteins of the mil/raf gene family. These sequences are encoded by three exons in the avian genome. We report that these three exons are conserved in the human B-raf gene and that they encode an amino acid sequence similar to that of the avian c-Rmil gene, indicating that in both avian and mammalian species the product of the B-raf/c-Rmil gene is a 94-kDa protein. We also identified two human B-raf loci: B-raf-1, located on chromosome 7q34, which encodes the functional B-raf/Rmil gene product, and B-raf-2, an inactive processed pseudogene located on chromosome Xq13.

Amino Acid Sequence↗
PubMed1991

Common mechanism of retrovirus activation and transduction of c-mil and c-Rmil in chicken neuroretina cells infected with Rous-associated virus type 1.

We previously described the isolation of the IC10 retrovirus which transduced the v-Rmil oncogene, a new member of the mil/raf gene family. This virus was generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina (NR) cells and was selected for its ability to induce proliferation of these nondividing cells. IC10 was isolated after six passages of culture supernatants but was not detected in proliferating NR cells during early virus passages. In this study, we molecularly cloned and sequenced another v-Rmil-containing provirus, designated IC11, from NR cells infected at the third virus passage of the same experiment. Both IC11 and IC10 transduced only the serine/threonine kinase domain of c-Rmil. Comparison of v-Rmil and c-Rmil sequences indicated that amino-terminal truncation is sufficient to activate the mitogenic properties of c-Rmil. IC11 and IC10 have identical 3' ends but differ by their 5' RAV-1-Rmil junctions. The 3' ends of both viruses were generated by recombination between Rmil and env genes, involving partial sequence identity. The 5' RAV-1-Rmil junction of IC11 was formed by a splicing process between the RAV-1 leader and a 37-bp c-Rmil exon located upstream of the kinase domain. NR cells infected with this virus synthesize a unique Rmil protein. IC10 contains most of the gag gene recombined with v-Rmil and encodes a gag-Rmil hybrid protein. Serial passaging of IC11 in NR cells led to the formation of a gag-Rmil-containing retrovirus. These results indicate that IC11 represents an early step in transduction and that this virus further recombined with RAV-1 to generate IC10. They confirm our previously proposed model for the multistep generation of v-mil-transducing retroviruses. Therefore, activation and transduction of c-mil and c-Rmil, in NR cells infected with RAV-1, result from a common mechanism.

Amino Acid Sequence↗
PubMed1990

Transformation-defective mutants with 5' deletions of the src gene are frequently generated during replication of Rous sarcoma virus in established quail fibroblasts.

Replication of Rous sarcoma virus (RSV) in avian fibroblasts leads to the generation of replication-competent variants that are defective for cell transformation (td virus). These td variants contain deletions affecting various portions of the v-src gene. We compared the rate of td virus production in Q3B cells, a quail cell line established by mutagen treatment, and in normal quail fibroblasts. Twenty-five days after infection with an RSV stock containing only transforming virions, Q3B cells harbor similar amounts of v-src-containing and v-src-deleted proviruses. However, these cells synthesize very low levels of p60v-src and generate large excess of td variants, as determined by biological assays. Unlike Q3B cells, normal quail fibroblasts infected with the same virus stock produce td variants only after multiple passages of undiluted virus on fresh cells. Restriction analysis showed that the td virus produced by Q3B cells is composed of two types of genomes: one lacking the entire v-src gene and the other carrying partial deletions of this gene predominantly located in the amino-terminal portion of the coding region of v-src. To study the mechanisms of these partial deletions, we molecularly cloned and sequenced the v-src genes of several td proviruses. We show that these mutants carry single or multiple v-src deletions of limited size, presumably generated by multiple mechanisms. Two deletions of 170 and 112 bp located in the 5' portion of v-src are frequently generated during RSV replication in Q3B cells and may represent preferential sites for v-src deletion in these cells.

Animals↗
PubMed1989

High constitutive transcription of HSP86 gene in murine embryonal carcinoma cells.

In order to investigate HSP86 heat-shock gene expression in embryonal carcinoma cell lines (EC), a partial mouse HSP86 cDNA clone was isolated and characterized. As observed for the corresponding protein, HSP86 RNA is shown to be constitutively more abundant in PCC4 and undifferentiated F9 EC cells than in fibroblasts, while its amount decreases upon F9 differentiation. Although mRNA stabilization is suggested to account in part of the high constitutive expression of the heat-shock-like protein HSC73 in F9 cells, HSP86 RNA appears as stable in fibroblasts as in F9 cells. Using run-on experiments we have established that high HSP86 expression in undifferentiated F9 cells in mainly due to enhanced transcription of the gene. Possible mechanisms responsible for this high level of transcription are discussed.

Animals↗
PubMed1989

Transduction of the cellular src gene and 3' adjacent sequences in avian sarcoma virus PR2257.

When injected into chickens, a transformation-defective mutant of the Prague C strain of Rous sarcoma virus induced tumors at low incidence and after a long latency. One such tumor released a replication-defective virus designated PR2257. We molecularly cloned and sequenced the proviral DNA from quail fibroblasts transformed by PR2257. Comparison of PR2257 sequence with that of Prague C, cellular src, and 3' adjacent cellular DNA showed that the spliced version of the c-src gene and about 950 base pairs (bp) of 3'-flanking cellular DNA were transduced into PR2257. This transduction eliminated nearly all replicative genes, since the gag gene splice donor site was linked to the splice acceptor site of the src gene and, on the 3' side, recombination occurred in the end of env gene. Insertion of two extra cytosines 23 bp before and 19 bp after the c-src stop codon resulted in an extension of the coding portion up to 587 amino acids, divergence of sequences after Pro-525 and replacement of Tyr-527 by a valine residue. In addition, it appears that the 5' and 3' untranslated regions of PR2257 result from multiple recombinations between exogenous and endogenous virus genomes. Limited digestion of p66src encoded by PR2257 with Staphylococcus aureus V8 protease yielded a V2 peptide (C-terminal moiety) with an apparent molecular mass of 31 kilodaltons, consistent with the 5.7-kilodalton increase expected from the DNA sequence. The structure of PR2257 suggests that the first step in the capture of c-src gene by avian lymphomatosis viruses is the trans splicing of the viral leader mRNA to exon 1 of c-src.

Amino Acid Sequence↗
PubMed1987

Mouse 89 kD heat shock protein. Two polypeptides with distinct developmental regulation.

Unstressed early mouse embryos have been previously shown [1] to synthesize at very high rates 70 and 89 kD proteins belonging to the heat shock protein (HSP) family. But it was not clear whether expression of heat shock-inducible or non-inducible (cognate) genes accounted for this spontaneous synthesis. In this report we show that the 89 kD mouse HSP can be separated into two proteins by high resolution PAGE. These two components show distinct but related peptide pattern after limited proteolysis. They are synthesized from distinct mRNAs. One of these proteins--HSP89f--is synthesized at a high rate by unstressed cells and its synthesis is rather insensitive to stress, whereas synthesis of the other protein--HSP89s--is strongly stimulated by heat shock in fibroblasts. Both HSP89f and HSP89s are major proteins synthesized in unstressed mouse preimplantation embryos and embryonal carcinoma (EC) cells. After in vitro differentiation of the EC cells the spontaneous synthesis of HSP89s decreases. Thus spontaneous expression of a mammalian inducible HSP is developmentally regulated.

Animals↗
PubMed1987

N-terminal deletion in the src gene of Rous sarcoma virus results in synthesis of a 45,000-Mr protein with mitogenic activity.

Expression of the v-src gene of Rous sarcoma virus in avian embryo neuroretina cells results in transformation and sustained proliferation of these normally resting cells. Transformed neuroretina cells are also tumorigenic upon inoculation into immunodeficient hosts. We have previously described conditional mutants of Rous sarcoma virus encoding p60v-src proteins which induce proliferation of neuroretina cells in the absence of transformation and tumorigenicity. These results suggest that p60v-src is composed of functionally distinct domains which may interact with multiple cellular targets. In this study, we describe a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion of 278 base pairs in the 5' portion of the v-src gene but which has retained the ability to induce proliferation of quail neuroretina cells. The deleted v-src gene encodes a 45,000-molecular-weight phosphoprotein which contains both phosphoserine and phosphotyrosine, is myristylated, and possesses tyrosine kinase activity indistinguishable from that of wild-type p60v-src. Molecular cloning and sequence analysis of the mutant v-src gene have shown that this deletion extends from amino acid 33 to 126 of the wild-type p60v-src. Therefore, this portion of the v-src protein is dispensable for the mitogenic activity of Rous sarcoma virus in neuroretina cells.

Animals↗
PubMed1984

gamma-Hexachlorocyclohexane inhibition of the calcium fluxes at the desensitized mouse neuromuscular junction.

The peripheral neurotoxicity of lindane was studied in vitro on mouse phrenic-diaphragm preparation. The experiments were performed on normal and denervated preparations. Twitches evoked by direct or indirect electrical stimulations and contractions evoked by external application of cholinergic agonists were compared in the presence or absence of lindane in the bath medium. Histochemical localizations of calcium were made on fibers after chemical stimulations. The results indicated that, at the normal endplate or at extrajunctionnal sites after denervation, there was a change in the intracellular Ca2+ distribution in response to external application of concentrated cholinergic agonists. This modification caused a transient contracture and could be visualized either by 45Ca autoradiography or by Alizarin Red S intracellular precipitations. Lindane inhibited the ACh-dependent contracture (I50 less than 10(-5) M) and suppressed the histochemical localizations of calcium. These lindane effects were observed on both normal and denervated muscles. It was concluded that lindane inhibited the Ca2+ influx presented by the desensitized ACh-sensitive areas of the muscle.

Acetylcholine↗