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1.
FBJ murine osteosarcoma virus: identification and molecular cloning of biologically active proviral DNA 总被引:125,自引:8,他引:117
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A 12.0-kilobase EcoRI restriction fragment containing FBJ murine osteosarcoma virus (FBJ-MSV) proviral DNA was identified in FBJ-MSV-transformed nonproducer rat cells and molecularly cloned in bacteriophage Charon 30 (lambda FBJ-1). A 5.8-kb HindIII fragment containing the entire FBJ-MSV proviral DNA was isolated from lambda FBJ-1 and subsequently subcloned in plasmid pBR322 (pFBJ-2). The DNA from recombinant plasmid pFBJ-2 was able to induce morphological transformation of rat fibroblasts in tissue culture. Transfected cells contained the p55 and p39 antigens specific for cells transformed by FBJ-MSV (T. Curran and N. M. Teich, J. Virol. 42:114-122, 1982). The organization of the FBJ-MSV provirus was analyzed by restriction endonuclease mapping, and a region of nonhomology with the helper virus was delineated. Sequences specific for this region (presumably the viral fos gene) were subcloned and used as a probe to identify related sequences present in the normal genomes of cells from a variety of mammalian species (cellular fos). A single-size (3.4 kilobases long) class of RNA hybridizing to the viral fos probe was identified in FBJ-MSV-transformed cells. 相似文献
2.
Candidate product of the FBJ murine osteosarcoma virus oncogene: characterization of a 55,000-dalton phosphoprotein 总被引:26,自引:4,他引:22
Sera from rat bearing tumors induced by inoculation of FBJ murine osteogenic sarcoma virus (FBJ-MSV) nonproducer rat cells precipitate two proteins with molecular weights of 55,000 (p55) and 39,000 (p39) from FBJ-MSV-transformed cells. These proteins cannot be precipitated from uninfected cells or cells transformed by other strains of murine sarcoma virus, nor can they be precipitated by sera specific for the viral structural proteins. A methionine tryptic peptide mapping analysis showed that p55 and p39 have little or no homology and that they are not related to the helper virus gag and env gene products. p55 could also be detected among the in vitro translation products of 70S RNA from FBJ murine leukemia virus plus FBJ-MSV virions but not among those from FBJ murine leukemia virus alone. This suggests that p55 is encoded by the FBJ-MSV genome, whereas p39, which was not detected among the in vitro translation products, may not be virus encoded. Another difference between p55 and p39 is that p55 is phosphorylated, with most of the phosphate on a serine residue(s), whereas p39 is phosphorylated to a much lesser extent, if at all. No protein kinase activity was associated with p55 and p39 immune complexes under standard conditions. Our data suggest that p55 is a strong candidate for the FBJ-MSV oncogene product. 相似文献
3.
Structure of the FBJ murine osteosarcoma virus genome: molecular cloning of its associated helper virus and the cellular homolog of the v-fos gene from mouse and human cells. 总被引:38,自引:10,他引:28
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The 8.2-kilobase (kb) unintegrated circular DNA form of the FBJ murine leukemia virus (FBJ-MLV) was linearized by cleavage at the single HindIII site, molecularly cloned into bacteriophage Charon 30, and subsequently subcloned into pBR322 (pFBJ-MLV-1). Both FBJ-MLV virion RNA and pFBJ-MLV-1 DNA were used to investigate the arrangement of helper virus sequences in the FBJ murine osteosarcoma virus genome (FBJ-MSV) by heteroduplex formation with cloned FBJ-MSV proviral DNA. The results showed that the FBJ-MSV genome contained 0.8 kb of helper virus sequence at its 5' terminus and 0.98 kb at its 3' terminus. Approximately 6.8 kb of helper virus sequence had been deleted, and 1.7 kb of unrelated sequence was inserted into the FBJ-MSV genome. This substituted region contains v-fos, the transforming gene of FBJ-MSV. Using a probe specific for v-fos, we have cloned homologous sequences (c-fos) from mouse and human chromosomal DNA. Heteroduplex analysis of FBJ-MSV DNA with these recombinant clones showed that both the c-fos(mouse) and the c-fos(human) sequences hybridized to the entire 1.7-kb v-fos region. However, five regions of homology of 0.27, 0.26, 0.14, 0.5, and 0.5 kb were separated by four regions of nonhomology of 0.76, 0.55, 0.1, and 0.1 kb from 5' to 3' with respect to the FBJ-MSV genome. The size of these sequences showed striking similarity in both c-fos(mouse) and c-fos(human). 相似文献
4.
An avian transforming retrovirus isolated from a nephroblastoma that carries the fos gene as the oncogene. 总被引:16,自引:3,他引:13
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A new avian transforming retrovirus, NK24, was isolated from a chicken with a nephroblastoma. This transforming virus induced fibrosarcomas with osteogenic cell proliferation and nephroblastomas in vivo and transformed fibroblast cells in vitro. From extracts of NK24-transformed cells, anti-gag serum immunoprecipitated a 100-kilodalton nonglycosylated protein with no detectable protein kinase activity. An NK24 provirus present in infected quail cells was molecularly cloned and subjected to nucleotide sequence analysis. The genome of NK24 was 5.3 kilobases long and had a 1,126-base-pair sequence of cellular origin in place of a viral sequence of avian leukosis virus containing the 3' half of the gag gene and the 5' half of the pol gene. Although the entire env gene was retained, it appeared to be inactive, possibly owing to the loss of function of its splice acceptor site as a result of a second deletion of 1,598 bases in the 3' half of the pol gene that extended to the acceptor site. Nucleotide sequence analysis revealed that the NK24 virus contained the fos gene, previously identified as the oncogene of FBJ and FBR murine osteosarcoma viruses. Unlike the v-fos gene products of FBJ and FBR, which suffer a structural alteration at their carboxyl termini, the NK24 v-fos gene product seemed to have the same carboxyl-terminal structure as the chicken c-fos gene product. A comparison of the structures of the products of the NK24 v-fos and mouse c-fos genes suggested that the fos gene product consists of highly conserved regions and relatively divergent regions. 相似文献
5.
Extended life span and tumorigenicity of nonestablished mouse connective tissue cells transformed by the fos oncogene of FBR-MuSV 总被引:16,自引:0,他引:16
We have analyzed the transforming potential of two fos oncogene products in nonestablished cultures of mouse connective tissue cells: p55fos of FBJ-MuSV and p75gag-fos of FBR-MuSV. Although both proteins induced morphological transformation and colony formation at low cell density in a G418 resistance selection assay, p75gag-fos exhibited more pronounced transforming potential than p55fos. In addition, p75gag-fos-transformed cells overcame crisis with a high probability and were tumorigenic in syngenic mice. These properties of the FBR-MuSV appear to be linked to structural alterations in the p75gag-fos oncogene product. Polyoma virus large T protein complemented the transforming potential of fos, in that it not only increased the probability of establishment of fos-transformed cells but also enhanced fos-induced morphological transformation. Our results suggest that different oncogenes affect morphological transformation, low cell density growth, establishment, and tumorigenicity to various degrees. 相似文献
6.
S L Giardina S W Evans L Gandino F A Robey E Bonvini D L Longo L Varesio 《Analytical biochemistry》1987,161(1):109-116
A hybridoma producing a monoclonal antibody (MoAB) recognizing both the cellular and viral forms of fos has been generated by somatic cell hybridization techniques from spleen cells of mice immunized with a synthetic peptide corresponding to amino acids 128-152, a consensus region, of both the v-fos and c-fos oncogene products. Three proteins with molecular weights of 55,000, 44,000, and 42,000 were detected by immunoblotting. While MoAB 2G9C3 failed to immunoprecipitate fos from Finkel-Biskis-Jenkins murine osteosarcoma-virus-infected fibroblasts, both the 55,000 v-fos protein and the 39,000 cellular protein were coprecipitated using polyvalent rabbit antibodies to the same peptide. Whereas no cell surface membrane expression of fos was detected, after membrane permeabilization by a brief exposure to lysolecithin it was possible to specifically detect internal fos by immunofluorescence flow cytometry. Immunohistochemical staining of FBJ virus-infected cells revealed intense, nuclear staining. 相似文献
7.
8.
R J Vosatka A Hermanowski-Vosatka R Metz E B Ziff 《Journal of cellular physiology》1989,138(3):493-502
The c-fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v-fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c-fos is a member of a family of structurally related proteins which includes the fos-related antigens (fra). We have studied the dynamic state of the c-fos protein with an antibody prepared by immunizing rabbits with a plasmid-encoded fos fusion protein. In serum-stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS-PAGE as a 60-68-kD group of bands corresponding to c-fos, a doublet at 44-45-kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50-kD band corresponding to a fra. We show that although c-fos protein synthesis is only transiently induced by serum, the c-fos protein persists within the cell after its synthesis has ceased, and it decays with a half-life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune-precipitated complex even at times when c-fos is no longer synthesized. These kinetics indicate that even following shutoff of c-fos protein synthesis, p39 is newly synthesized and can complex with c-fos protein or a fos-related antigen. During this time, c-fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c-fos and/or the fos-related antigens over a period of 4-8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c-fos and fra protein interactions for the function of these proteins. 相似文献
9.
A retrovirus carrying the K-fgf oncogene induces diffuse meningeal tumors and soft-tissue fibrosarcomas. 总被引:2,自引:1,他引:2
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The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family and transforms cells through an autocrine mechanism which requires extracellular activation of its receptor(s). To identify the cell and tissue targets of K-fgf oncogenic potential in vivo, we constructed a recombinant retrovirus carrying the human K-fgf cDNA and injected it, together with helper Moloney murine leukemia virus, into immunocompetent as well as nude mice. The original construct was highly transforming in tissue culture but produced no detectable pathologies in vivo with the exception of a single fibrosarcoma which arose after a long latency. The virus produced by this tumor appears to have undergone a complex series of recombination events involving the helper Moloney murine leukemia virus. It encodes an Env/K-FGF fusion protein whose expression is under the control of a hybrid long terminal repeat. This virus (designated MFS, for meningeal fibrosarcoma) induces tumors in mice with high frequency and short latency. These neoplasms consist of aggressive fibrosarcomas of soft tissue as well as diffuse meningeal tumors originating from the dura mater that surround the whole central nervous system and cause severe hydrocephalus. The Env/K-FGF fusion protein expressed by the MFS virus has retained all of the biological properties of native K-FGF, including secretion, mitogenic activity, heparin binding, and neutralization by anti-K-FGF antibodies. These and other results indicate that the tumors induced by the MFS virus result from the oncogenic potential of K-FGF. 相似文献
10.
Viral and cellular fos proteins: a comparative analysis 总被引:46,自引:0,他引:46
The FBJ murine osteosarcoma virus (FBJ-MuSV) induces osteosarcomas in mice and transforms fibroblasts in vitro. It contains an oncogene termed v-fos derived from a normal cellular gene by recombination with an associated helper virus. The product of the v-fos gene is a 55,000 dalton protein, p55v-fos. This protein was found in the nuclei of cells containing amplified levels of the v-fos gene, and also in the nuclei of virus-transformed cells. The c-fos protein was localized in the nuclei of normal mouse amnion cells and in the nuclei of cells transformed by a recombinant plasmid that expresses the c-fos gene product. However, p55c-fos undergoes more extensive post-translational modification in the nucleus than p55v-fos. Immunofluorescence data indicate that the level of p55c-fos in normal mouse amnion cells is similar to that found in fibroblasts transformed by the v-fos or c-fos proteins. 相似文献
11.
A novel oncogene, v-ryk, encoding a truncated receptor tyrosine kinase is transduced into the RPL30 virus without loss of viral sequences.
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The RPL viruses are acute oncogenic avian retroviruses isolated from chicken tumors. We carried out a genetic analysis of three of the viruses, RPL25, RPL28, and RPL30. While RPL25 and RPL28 were shown to contain the erbB oncogene, RPL30 appeared to contain a novel protein tyrosine kinase oncogene. This gene, v-ryk, was cloned and sequenced. The v-ryk oncogene contains a 1.39-kb nonretroviral sequence that includes a tyrosine kinase domain which was inserted into the viral envelope protein gp37-coding region and fused in frame with upstream gp37 to generate a P69gp37-ryk fusion oncoprotein. Unlike that of other acutely transforming retroviruses, transduction of the v-ryk gene into RPL30 did not result in deletion of viral sequences. Sequence analysis suggested that v-Ryk is more homologous to receptor-type tyrosine kinases than to nonreceptor-type kinases. By reconstitution of a virus from its cDNA, the v-ryk oncogene has been shown to be fully responsible for the transforming activity of the RPL30 virus. Antibodies specific to v-Ryk immunoprecipitated the v-Ryk oncoprotein from cells transformed by the RPL30 virus. The v-Ryk protein was shown to be first synthesized as a 150-kDa precursor and then cleaved into the mature 69-kDa gp37-Ryk fusion protein, both parts of which were found to be localized to the membrane fraction. As expected from the sequence of v-Ryk, immunoprecipitates of v-Ryk from RPL30-transformed cells were found to display a protein tyrosine kinase activity in vitro, and the levels of tyrosine-phosphorylated proteins are elevated in v-ryk-transformed cells. 相似文献
12.
本文将克隆于pBR322的人胃癌Ha-ras基因(PGC6.6)和带有上游区片段的Ha-ras基因(PGC9.1)的CC~*GG位点甲基化后,转化NIH3T3细胞。发现pGC6.6甲基化与非甲基化对转化效率无明显影响,而pGC9.1甲基化后转化效率明显低于非甲基化pGC9.1者,甲基化/非甲基化pGC9.1的转化效率均明显高于甲基化/非甲基化pGC6.6者。本文又对人胃癌组织及癌旁组织DNA中Ha-ras基因的HpaⅡ、Msp Ⅰ限制性内切酶图谱作了比较,并同对比较了癌及癌旁组织中Ha-ras基因的mRNA水平,发现一例病人癌组织中Ha-ras基因的CC~*GG位点甲基化程度较癌旁组织中者低,且该例中Ha-ras基因表达水平在癌组织中明显地高。这些结果,结合我们以前的研究表明:在人胃Ha-ras癌基因上游区可能存在一增强子样作用的区域,对Ha-ras基因起调控作用。该上游区CC~*GG位点的甲基化能降低这种调控作用。仅Ha-ras结构基因的CC~*GG位点甲基化不足以明显影响其转化活性。在体内,Ha-ras基因甲基化水平降低可能与其达表水平升高以至诱发癌症有关。 相似文献
13.
Neuregulin-regulated gene expression in mammary carcinoma cells 总被引:3,自引:0,他引:3
14.
豚鼠气道及肺组织原癌基因表达与哮喘的相关研究 总被引:4,自引:0,他引:4
目的:为研究原癌基因在哮喘发病中的作用。方法:以卵白蛋白致敏豚鼠建立哮喘模型,用Dot-blot、North-ern-blot分子杂交及免疫组化技术,分别观察正常及哮喘发作后豚鼠气道上皮及肺组织中原癌基因c-fos、c-myc的表达水平。结果:正常豚鼠气道及肺组织中c-fos及c-mycmRNA无或很少表达,哮喘发作后豚鼠气道上皮及肺组织中c-fos和c-mycmRNA表达明显增强,30min达高峰,发作后4h降至正常水平。地塞米松对c-fos及c-mycmR-NA有部分抑制作用。结论:原癌基因c-fos及c-myc在哮喘发病过程中可能起一定作用。 相似文献
15.
16.
We have succeeded in establishing a method to reproducibly immortalize human T cells by oncogene(s) transfection (Alam, 1997).
This study was based on our previous discoveries that these immortalized T cell lines contained T cells which showed cytotoxicity
against K562 cells in MHC-nonrestricted manner. Then we attempted to obtain human T cell clones exhibiting natural killer-like
activity. Here, we tried to establish clones from these immortalized T cell lines by limiting dilution after stimulation with
K562 cells, and then obtained 16 T cell clones. Two clones among them maintained their stability and showed vigorous growth
phenotype. Thus we selected these two clones for further analysis. One is derived from the T cell line transfected with oncogenes
ras and fos, the other is from the T cell line transfected with myc and fos. Both clones were demonstrated to be CD4+ T cells, indicating that CD4+ T cells were preferably expanded from T cell lines immortalized by oncogene transfection. These two clones showed cytotoxicity
against K562 cells, indicating that these two T cell clones still retain a natural killer-like activity of killing target
cells of K562 cells in a MHC-nonrestricted manner. The natural killer-like activity of the T cell clones was shown to be stable
for more than 2 yr when cultured in the presence of IL-2, indicating that introduction of two oncogenes such as ras/fos or
myc/fos resulted in the acquisition of infinite replicative life-span but not in transformational alteration of cellular function.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
17.
18.
LMP1 of Epstein-Barr virus induces proliferation of primary mouse embryonic fibroblasts and cooperatively transforms the cells with a p16-insensitive CDK4 oncogene 总被引:10,自引:0,他引:10
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The latent membrane protein LMP1 of Epstein-Barr virus (EBV) is often present in EBV-associated malignancies including nasopharyngeal carcinoma and Hodgkin's lymphoma. Previous work demonstrates that the LMP1 gene of EBV is sufficient to transform certain established rodent fibroblast cell lines and to induce the tumorigenicity of some human epithelial cell lines. In addition, LMP1 plays pleiotropic roles in cell growth arrest, differentiation, and apoptosis, depending on the background of the target cells. To examine the roles of LMP1 in cell proliferation and growth regulation in primary culture cells, we constructed a recombinant retrovirus containing an LMP1 gene. With this retrovirus, LMP1 was shown to stimulate the proliferation of primary mouse embryonic fibroblasts (MEF cells). It has a mitogenic activity for MEF cells, as demonstrated by an immediate induction of cell doubling time. In addition, it significantly extends the passage number of MEF cells to more than 30 after retroviral infection, compared with less than 5 for uninfected MEF cells. Furthermore, LMP1 cooperates with a p16-insensitive CDK4(R24C) oncogene in transforming MEF cells. Our results provide the first evidence of the abilities of the LMP1 gene, acting alone, to effectively induce the proliferation of primary MEF cells and of its cooperativity with another cellular oncogene in transforming primary cells. 相似文献
19.
Nucleotide sequence of avian sarcoma virus UR2 and comparison of its transforming gene with other members of the tyrosine protein kinase oncogene family. 总被引:45,自引:15,他引:30
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The genome of avian sarcoma virus UR2 was completely sequenced and found to have a size of 3,165 nucleotides. The UR2-specific transforming sequence, ros, with a length of 1,273 nucleotides, is inserted between the truncated gag gene coding for p19 and the env gene coding for gp37 of the UR2AV helper virus. The deduced amino acid sequence for the UR2 transforming protein P68 gives a molecular weight of 61,113 and shows that it is closely related to the oncogene family coding for tyrosine protein kinases. P68 contains two distinctive hydrophobic regions that are absent in other tyrosine kinases, and it has unique amino acid changes and insertions within the conserved domain of the kinases. These characteristics may modulate the activity and target specificity of P68. 相似文献