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Avian reticuloendotheliosis virus-transformed lymphoid cells contain multiple pp59v-rel complexes. 总被引:25,自引:9,他引:16
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The v-rel oncogene of avian reticuloendotheliosis virus type T (REV-T) encodes a 59-kilodalton (kDa) phosphoprotein located principally in the cytosol of transformed lymphoid cells. All of the detectable pp59v-rel was present in high-molecular-weight complexes containing at least five cellular proteins (p124, p115, p75c-rel, p70hsc, and pp40). Antiserum was developed against the 40-kDa protein, the most abundant cellular protein associated with the complex. The 40-kDa phosphoprotein was complexed with pp59v-rel in REV-T-transformed lymphoid cell lines arrested at different stages of B-cell development as well as in lymphoid tumor cells and in fibrosarcomas. The half-life (8 h) of pp40 in REV-T-transformed lymphoid cells was the same as that of pp59v-rel. Antiserum against pp40 permitted the identification of two pp59v-rel complexes. The most abundant cytoplasmic complex contained approximately 75% of the pp59v-rel and all of the detectable pp40 in REV-T-transformed lymphoid cells. Twenty-five percent of the pp59v-rel was present in a minor complex that contained the majority of p75c-rel along with p115 and p124. In nuclear extracts of REV-T-transformed lymphoid cells, pp59v-rel was complexed with pp40. The two high-molecular-weight proteins (p115 and p124) and p75c-rel were not detected in the nuclear complex. In the cytosolic complexes, pp40 was heavily phosphorylated, whereas the nuclear form was much less extensively phosphorylated. 相似文献
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Different localization of the product of the v-rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T 总被引:29,自引:0,他引:29
Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus that transforms early lymphoid cells in vivo and in vitro, but REV-T does not transform chicken embryo fibroblasts (CEF). Using antisera to p59v-rel, the v-rel oncogene product of REV-T, we show that p59v-rel is expressed at equal levels and is a phosphoprotein in REV-T infected spleen cells and CEF. Biochemical fractionation and immunofluorescence of REV-T infected nontransformed CEF show that p59v-rel is loosely associated with the nucleus. However, in REV-T transformed spleen cells p59v-rel is primarily a cytoplasmic protein. MSB-1 cells, a Marek's disease virus transformed T cell leukemic line, and E26 virus transformed myeloid cells show nuclear staining of p59v-rel when they are infected by REV-T. Our results indicate that there is a correlation between a cytoplasmic localization of p59v-rel and transformation by REV-T, and they suggest that p59v-rel cannot transform cells in which it assumes solely a nuclear location. 相似文献
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Avian reticuloendotheliosis virus (REV-T) is the most virulent of all retroviruses, inducing an invariably fatal leukemia in chickens with a latent period of 7-10 days. Unlike avian cells transformed by other acutely transforming viruses, lymphoid cells transformed by REV-T are immortalized. Furthermore, in vitro derived, REV-T transformed cells which do not produce virus are tumorigenic and induce lethal reticuloendotheliosis when injected into histocompatible birds. Thus REV-T transforms its target cell both in vitro and in vivo. In addition this transformation is independent of any helper virus functions. Like other acute leukemia viruses, REV-T is replication-defective and must co-replicate with a reticuloendotheliosis associated virus (REV-A). During evolution, a substantial portion of its genome has been deleted and replaced with a host-derived genetic sequence, designated v-rel. Presumably, the v-rel oncogene was transduced from a normal turkey DNA locus, c-rel. There are 9 regions of homology between c-rel and v-rel, however, several differences exist between these genes, suggesting that transformation by REV-T results from the production of an altered v-rel protein. The v-rel sequence is distinct from other known oncogenes and encodes a 57-kDa phosphoprotein. In REV-T transformed cells, this pp57v-rel protein is localized in the cytoplasm. The product of the v-rel oncogene is present at a low level, representing only about 0.003% of total methionine-labelled protein. In addition, pp57v-rel is relatively stable, having an estimated half-life of 4-10 h. The v-rel protein when purified close to homogeneity is complexed with a 40-kDa cellular phosphoprotein in transformed lymphoid cells and possesses serine kinase activity. This review discusses the molecular aspects of transformation by REV-T in the context of other oncogene-encoded proteins. 相似文献
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Localization of the v-rel protein in reticuloendotheliosis virus strain T-transformed lymphoid cells. 总被引:10,自引:3,他引:7
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The protein (p59rel) encoded by the transforming gene of reticuloendotheliosis virus strain T (REV-T) has been identified in REV-T-transformed avian lymphoid cells by using antisera raised against synthetic peptides whose sequences were derived from three nonoverlapping regions of v-rel (N. R. Rice, T. D. Copeland, S. Simek, S. Oroszlan, and R. V. Gilden, Virology 149:217-229, 1986). To obtain polyclonal antibodies directed against a larger number of p59rel epitopes, a 262-amino acid segment was expressed in bacteria. Antisera raised against this fusion protein (v-delta-rel) precipitated p59rel from lysates of [35S]methionine-labeled REV-T-transformed cells, thus confirming previous results obtained with the peptide antisera. We used this new antiserum to localize p59rel in REV-T-transformed cells by subcellular fractionation using differential centrifugation and by indirect immune fluorescent staining. After fractionation and immune precipitation, the majority of p59rel was found in the cytosolic fraction. Indirect immunofluorescence experiments also gave results consistent with the cytoplasmic localization of the v-rel protein in transformed lymphoid cells. In previous studies (Rice et al., Virology 149:217-229, 1986) it was shown that immune precipitates formed with one of the three p59rel peptide antisera possessed in vitro protein kinase activity. Immune precipitates formed with the fusion protein antiserum also showed kinase activity in the in vitro assay. Most of this activity was found in the soluble cytoplasmic fraction, indicating that the kinase may be p59rel or a protein closely associated with it. 相似文献
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Avian reticuloendotheliosis virus: identification of the hematopoietic target cell for transformation 总被引:24,自引:0,他引:24
R B Lewis J McClure B Rup D W Niesel R F Garry J D Hoelzer K Nazerian H R Bose 《Cell》1981,25(2):421-431
Non-virus-producing hematopoietic cells transformed in vitro by reticuloendotheliosis virus (REV-T) induce lethal "reticuloendotheliosis" when inoculated into histocompatible chickens. This is the first direct demonstration that an in vivo target cell of an avian acute leukemia virus can be transformed in vitro. The tumorigenic, REV-T-transformed non-virus-producing cells fail to express helper-virus-coded proteins. REV-T transformed tumorigenic cells therefore do not require helper-virus functions. Cells transformed in vivo or in vitro by REV-T have lymphoblastoid morphology and express low levels of terminal-deoxynucleotidyl-transferase activity and bursal-cell determinants. One clone synthesized Ig mu. The preferred target cells for REV-T transformation are therefore immature lymphoid cells that express B-cell determinants. We propose that the unique transforming sequence of REV-T be designated rel (lymphoid). 相似文献
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Oncogenic transformation by vrel requires an amino-terminal activation domain. 总被引:44,自引:22,他引:22
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J Kamens P Richardson G Mosialos R Brent T Gilmore 《Molecular and cellular biology》1990,10(6):2840-2847
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H Y Tung W J Bargmann H R Bose 《Biochemical and biophysical research communications》1988,152(1):441-448
The transforming protein encoded by the v-rel oncogene of reticuloendotheliosis virus has been purified from REV-T transformed lymphoid cells by sequential DEAE-Sepharose and immunoaffinity chromatography. The purified preparation consisted of pp59v-rel and the 40 kDa cellular protein which is complexed with the v-rel oncogene product in transformed cells as well as some minor proteins. Incubation of this purified preparation in the presence of Mg2+ and (gamma-32P)ATP resulted in phosphorylation of both pp59v-rel and the 40 kDa protein. This preparation was also able to phosphorylate casein on serine residues. Immunoprecipitates obtained from extracts of REV-T transformed lymphoid cells labeled with 32P-orthophosphate contained 59 and 40 kDa phosphoproteins. Both pp59v-rel and the 40 kDa protein were phosphorylated on serine residues in transformed cells. 相似文献
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Cloning of the p50 DNA binding subunit of NF-kappa B: homology to rel and dorsal 总被引:189,自引:0,他引:189
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Expression of v-rel induces mature B-cell lines that reflect the diversity of avian immunoglobulin heavy- and light-chain rearrangements. 总被引:9,自引:3,他引:6
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The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte. 相似文献
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J H Chen 《Molecular and cellular biology》1985,5(11):2993-3000
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Characterization of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning. 总被引:45,自引:24,他引:21
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Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus which causes a rapid neoplastic disease of the lymphoreticular system. Upon infection, this virus gives rise to two species of unintegrated linear viral DNA, which are 8.3 and 5.5 kilobase pairs long and represent the helper virus (REV-A) and the oncogenic component (REV-T), respectively. Restriction endonuclease cleavage maps of these two DNA components indicate that REV-T DNA has a large portion of the genome deleted with respect to REV-A DNA and a substitution about 0.8 to 1.5 kilobase pairs long that is unrelated to REV-A DNA. These additional sequences comprise the putative transforming region of REV-T (rel). A chicken spleen cell line transformed by REV-T produced virus which upon infection gives rise to three species of unintegrated linear viral DNA (8.3, 5.5, and 3,3 kilobase pairs). We isolated the proviruses of the 8.3- and 3.3-kilobase pair species from this cell line by cloning in the phage vector Charon 4A. Restriction enzyme mapping showed that the two proviral clones are proviruses of REV-A and a variant of REV-T, respectively. A subclone of the variant REV-T provirus specific for the rel sequences of REV-T was used as a hybridization probe to demonstrate that the rel sequences are different from the putative transforming sequences of Schmidt-Ruppin Rous sarcoma virus strain A, avain myelocytomatosis virus, avian myeloblastosis virus, avian erythroblastosis virus, Abelson murine leukemia virus, and Friend erythroleukemia virus. In addition, the rel-specific hybridization probe was used to identify a specific set of sequences which are present in uninfected avian DNAs digested with several restriction enzymes. The corresponding cell sequences are not arranged like rel in REV-T. 相似文献
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Reticuloendotheliosis virus strain T (Rev-T) induces a lethal lymphoma in young birds and transforms avian lymphoid cells in vitro. The transforming gene of Rev-T, v-rel, was derived from the turkey proto-oncogene c-rel. Comparison of the nucleotide sequences of v-rel and c-rel indicates that in addition to several internal amino acid changes relative to c-rel, p59v-rel has amino acid sequences at both ends derived from the reticuloendotheliosis virus strain A-related virus env gene (K. C. Wilhelmsen, K. Eggleton, and H. M. Temin, J. Virol. 52:172-182, 1984). In this report, the v-rel sequences important for transformation were defined by constructing recombinant retroviruses in which c-rel sequences replaced the analogous v-rel sequences. These recombinant viruses expressing chimeric proteins were tested for their ability to transform spleen cells in vitro and to induce tumors in young chickens. Activation of the oncogenicity of c-rel in Rev-T required alteration of the amino terminus and the central region of the protein. Deletion of the noncoding sequences 3' to c-rel and of most of the helper virus-related env sequences was necessary for the formation of Rev-T. 相似文献
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p59v-rel, the transforming protein of reticuloendotheliosis virus, is complexed with at least four other proteins in transformed chicken lymphoid cells 总被引:21,自引:3,他引:18
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Previous studies have identified the protein product of v-rel, the oncogene carried by reticuloendotheliosis virus (REV), as a 59,000-dalton phosphoprotein located predominantly in the cytosol of transformed chicken lymphoid cells. In immune precipitates of p59v-rel, there is a closely associated protein kinase activity. In chicken lymphoid cells that do not contain REV, p68c-rel is found free in the cytosol not associated with other proteins and not detectably phosphorylated. In this study, we found that immune precipitates of 59v-rel from REV-transformed cells contain at least four other proteins, of approximate molecular weights 124, 115, 68, and 36 kilodaltons (kDa). The 124-, 115-, and 36-kDa proteins are apparently unrelated to p59v-rel in sequence, and their coprecipitation suggests that they are complexed with p59v-rel. The coprecipitating 68-kDa protein was found to be p68c-rel, which, like the other three proteins, precipitates by virtue of its association with p59v-rel. Glycerol gradient analysis suggested the presence of more than one type of complex: one containing p115, p68c-rel, p59v-rel, and p36, and another containing p124, p115, p59v-rel, and possibly p68c-rel. In vitro kinase activity was found in all size classes, coinciding with the distribution of p115 and p59v-rel. The complex(es) was stable under a variety of conditions, including a wide range of ionic strengths, chelators, and detergents, and through multiple cycles of immune precipitation and elution. This suggests a specific and functionally significant interaction among the members that may be of direct relevance to the mechanism of REV-induced transformation. 相似文献