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At least 73 records · Page 4Linked to original sources
PubMed1984

Hematopoietic cell transformation by a murine recombinant retrovirus containing the src gene of Rous sarcoma virus.

A recombinant murine retrovirus (MRSV) containing the src gene of avian Rous sarcoma virus (RSV) was shown to induce hematopoietic colonies in infected mouse bone marrow. MRSV-induced colony formation followed single-hit kinetics and required mercaptoethanol in the agar medium. Cells from the colonies induced by MRSV could be established as continuous cell lines that demonstrated unrestricted self-renewal in vitro and tumorigenicity in vivo. The transformants, all of which expressed high levels of the Rous sarcoma virus transforming protein, pp60src, appeared to be at an early stage in lymphoid cell differentiation. They lacked Fc receptors and detectable immunoglobulin mu heavy chain synthesis, markers normally associated with committed B cells. The majority of the MRSV-transformed cell lines contained high levels of terminal deoxynucleotidyl transferase, an enzyme present in lymphoid progenitor cells committed to the T-cell lineage. One cell line expressed Thy-1 antigen, but none expressed Lyt-1 and Lyt-2, markers of more differentiated T cells. These findings demonstrate that the src gene is capable of transforming cells of hematopoietic origin.

Animals↗
PubMed2006

Formation of acellular cementum-like layers, with and without extrinsic fiber insertion, along inert bone surfaces of aging c-Src gene knockout mice.

To investigate the long-term effects of c-src deficiency on skeletal and dental tissues, we examined the lower jaws and long bones of c-src gene knockout (c-src KO) mice by histological and histochemical methods. Numerous multinucleated osteoclasts were distributed throughout the mandible in 5-wk-old c-src KO mice, but by 14 wk they had almost completely disappeared from the alveolar bone, leaving tartrate-resistant acid phosphatase (TRAP)-positive layers along the bone surface. Deposition of osteopontin-positive mineralized tissue, reminiscent of acellular afibrillar cementum (AAC), was confirmed along the TRAP-positive bone surface at 14 wk. The layer progressively thickened up to 21 months. A comparable mineralized layer was noted along the trabeculae of long bones as thickened cement lines. In the periostin-rich areas of jaw bones, but not in the long bones, portions of AAC-like mineralized layers were often replaced with and/or covered by acellular extrinsic fiber cementum (AEFC)-like tissue. These data suggest that the deposition of AAC-like mineralized tissue is a general phenomenon that may occur along inert or slowly remodeling bone surfaces under conditions characterized by reduced bone-resorbing activity, whereas the induction of AEFC-like tissue seems to be associated with the expression of certain molecules that are particularly abundant in the microenvironment of the periodontal ligament.

Aging↗
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↗
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↗
PubMed1984

Early release of the density-dependent inhibition of phosphate uptake and ATP synthesis after src gene expression in chick embryo fibroblasts.

Our results showed that the expression of the src gene in chick embryo fibroblasts (CEF) released the density-dependent inhibition (DDI) of phosphate metabolism (phosphate uptake and phosphorylation of small organic compounds). With increasing cell density, phosphate metabolism decreased by 58% in normal CEF and, in contrast, increased by 20% in Rous sarcoma virus (RSV)-transformed CEF. The same change in the DDI was observed in CEF infected by NY68 (a ts mutant for transformation of RSV) and maintained at the permissive temperature (37 degrees C) instead of the restrictive temperature (41.5 degrees C) for the expression of transformation. An interesting feature was that the release of the DDI of phosphate metabolism was an early event in the process of transformation, since it was almost concomitant with the stimulation of the pp60 src kinase activity following the shift from 41.5 to 37 degrees C of NY68 CEF. The phosphorylation of small organic compounds (Po) was more strongly increased by the change in temperature than was 32Pi accumulation. Furthermore, the percentage increases of Po and adenosine triphosphate (ATP) labelling with 32P were similar, suggesting that the expression of src gene enhanced ATP synthesis. In glucose-free medium, the stimulation of Po-labelling was still observed but was decreased. Therefore the activation of glycolytic activity is not an absolute requirement, but is necessary for the maximum effect of transformation on the release of DDI of phosphate metabolism. Oligomycin added in complete medium did not prevent the increase in Po-labelling. From these results, we assumed that ATP turnover was stimulated as a consequence of enhanced ATP degradation. We verified that the stimulation of Po phosphorylation was not a consequence of increased ATP utilization for RNA or protein synthesis. The stimulation of Po labelling was specifically abolished by quercetin. This drug inhibited the transformed cells more strongly than the non-transformed cells.

Adenosine Triphosphate↗
PubMed1987

Product of the src gene is not phosphorylated in avian sarcoma virus B77 infected but untransformed human cells.

Human embryo cells infected with the avian sarcoma virus B77 [Hu(B77)] but untransformed, contain the whole rescuable virus genome integrated in the host cell DNA. The cellular DNA induced the transformation of the infectious B77 virus after transfection of chicken cells. In the Hu(B77) cells the src gene product was expressed as a 58 kD protein which possessed phosphokinase activity but was not phosphorylated in comparison with the src production of B77 transformed rat cells RBI in which p60src is expressed. The addition of vanadate to tissue culture fluid reversibly elicited cell transformation in both control and virus infected human cells, but did not influence phosphorylation of the src gene product. The secretion of phosphoproteins in the investigated cells was different. Whereas transformed rat cells released a 62 kD transformation related phosphoprotein, the human cells did not. In tissue culture fluid from Hu(B77) cells an elevated amount of a 22 kD phosphoprotein was found in comparison with control cells. The implications of these findings are discussed with respect to the role of the v-src gene product in malignant cell transformation.

Animals↗
PubMed1980

The immune response against the ASV-coded src-gene product in syngeneic mice.

The antigenicity of the avian sarcoma virus (ASV)-coded src-gene product pp60src, which is responsible for fibroblast transformation after ASV infection, has been investigated in STU mouse fibrosarcoma cell lines and the corresponding immune response in syngeneic mice has been determined. The development of effective anti-pp60src antibody titres depends on the mode and stie of injection of tumour cells and parallels tumour growth. It was found that mouse immunoglobulin heavy chains are unable to serve as substrate for the protein kinase activity of pp60src. Therefore, an indirect protein kinase absorption (PKA) test was initiated to demonstrate recognition of the protein kinase activity associated with the src-gene product. The availability of syngeneic mice and the corresponding ASV-transformed tumour cells should facilitate studies designed to elucidate the possible relationship between the cytoplasmic pp60src and ASV-induced tumour-specific surface antigens (TSSA), for example, by allowing the production of stable mouse hybridomas synthesizing antibodies specific for pp60src and TSSA.

Alpharetrovirus↗
PubMed1978

Assay for early cytoplasmic effects of the src gene product of Rous sarcoma virus.

When microinjected into normal fibroblasts, cytoplasmic extracts of cells transformed by Rous sarcoma virus caused dissolution of microfilament bundles. This activity was not found in extracts of normal cells. The maximum effect was seen within 30 min of injection, and the activity could still be measured after a 10-fold dilution of the cytoplasmic extracts (14 mg/ml original protein concentration). The activity was trypsin sensitive and was destroyed by boiling, but was not RNase sensitive. Protein synthesis was not required for the disruption of actin-containing stress fibers by the injected activity. Microinjected cytoplasts prepared from normal 3T3 cells also showed dissolution of microfilament bundles, indicating that the cell nucleus was not required for expression of activity. Extracts made from fibroblasts transformed by Rous sarcoma virus having a temperature-sensitive mutation in the src gene were also temperature sensitive in the microinjection assay. Thus, the activity of extracts from cells infected with src mutant virus, but not from cells infected with wild-type virus, was destroyed either by in vitro incubation of the extract at the nonpermissive temperature before injection or by incubation of recipient cells at the nonpermissive temperature after injection. We conclude that the microinjection assay can detect a cytoplasmic activity coded for by the src gene of Rous sarcoma virus and that an early direct or indirect target of the src gene product is the cytoskeleton and cell motility system. This result is discussed in relation to the hypothesis that submembranous arrays of microfilaments, microtubules, and their associated proteins interact with cell surface receptors to form a surface modulating assembly that functions as a key regulator of cell growth.

Actins↗
PubMed2000

Mutation of the SRC gene in endometrial carcinoma.

Recently, an activating mutation of the SRC gene has been implicated in about one-tenth of advanced colon cancers. The SRC 531 mutation results in truncation of SRC directly C-terminal to the regulatory Tyr 530 and appears to activate the Tyr 530. To investigate whether mutation of SRC plays an important role in the development and progression of gynecological tumors, we performed mutational analysis of the entire coding region of SRC in 70 ovarian carcinomas, 68 endometrial carcinomas and 3 endometrial stromal sarcomas by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis. We found one truncated mutation at codon 531 (Gln to Stop) in an endometrial carcinoma. However, we found no mutation of this gene in ovarian carcinoma or endometrial stromal sarcoma. Our results suggest that mutation of SRC may be implicated in a small proportion of endometrial carcinomas.

Adult↗
PubMed1991

V-src genes in two cell lines of so-called RSV-transformed human cells are defective and inactive.

Trial of Rous sarcoma virus (RSV) induction by cell fusion with chick embryo cells (CEC) and wing web test from so-called RSV-transformed human cells, KC and RSb cells, was unsuccessful. The loss of RSV inducibility was also confirmed by DNA transfection method. Southern blot and northern blot hybridization of DNA and RNA from those cells with the v-src probe revealed that the v-src genes in those cells were defective and not expressed. On the other hand, the v-src gene in RSV-transformed mouse and rat cells was complete and transforming virus was inducible from them.

Avian Sarcoma Viruses↗
PubMed1978

Comparison of the expression of the src gene of Rous sarcoma virus in vitro and in vivo.

We have compared the polypeptide products of the src gene of several strains of Rous sarcoma virus produced by in vitro translation of heat-denatured 70S virion RNA in the nuclease-treated reticulocyte lysate with those present in chick cells transformed by these viruses. We have done this by immunoprecipitation, using sera from rabbits injected at birth with Schmidt-Ruppin Rous sarcoma virus. In vitro translation results in the synthesis of at least nine polypeptides which appear to be encoded by the src gene. These range in size from 17,000 to 60,000 daltons. The sera from tumor-bearing rabbits precipitated these polypeptides arising from the in vitro translation of RNA from Schmidt-Ruppin Rous sarcoma virus of both subgroup A and subgroup D and from one stock of Prague Rous sarcoma virus of subgroup C. In each case, all of this family of related polypeptides could be precipitated except the smallest, the 17,000-dalton polypeptide. No precipitation of analogous polypeptides resulting from the translation of RNA from other strains of Rous sarcoma virus was observed. Cells transformed by these three strains of Rous sarcoma virus contain easily detectable amounts of a polypeptide, p60src, essentially identical to the 60,000-dalton in vitro product. With one exception, they do not contain significant amounts of polypeptides analogous to the smaller in vitro products which can be precipitated by these sera. Cells transformed by one stock of Schmidt-Ruppin Rous sarcoma virus of subgroup A did contain a 39,000-dalton polypeptide, which was related, by peptide mapping, to the 60,000-dalton polypeptide and was similar in size to a precipitable in vitro product. The 60,000-dalton polypeptide present in transformed cells appeared to be phosphorylated 10 to 25 min after its synthesis, metabolically very stable, and not derived from a precursor polypeptide. All immunoprecipitates from transformed cells which contained p60src also contained an 80,000-dalton phosphoprotein. This polypeptide is unrelated to p60src, as determined by peptide mapping, and may well be a host cell polypeptide which is specifically associated with p60src.

Avian Sarcoma Viruses↗
PubMed1977

Occurrence of partial deletion and substitution of the src gene in the RNA genome of avian sarcoma virus.

The genome size of 20 transformation-defective (td) viruses derived from different strains of Rous sarcoma viruses [Prague (subgroups A and C), Schmidt-Ruppin (subgroups A and D) (SR-D), Bratislava 77, and Carr-Zilber subgroup D)] was examined by polyacrylamide gel electrophoresis. All of the td viruses except td SR-D have 35S RNA of the same size-i.e., class b RNA. Two of five td SR-D viruses examined have a slightly larger RNA, corresponding to a td deletion that is about 25% smaller than that of class b RNA. However, the RNase T(1)-oligonucleotide fingerprints of all the td SR-D viruses are identical, lacking two sarcoma-specific oligonucleotides. The fingerprints of these viruses also showed a minor oligonucleotide present at very low concentration. A study of heteroduplex molecules formed between genome-length cDNA made from wild-type SR-D and 35S RNA of td SR-D showed a deletion loop of 2.0 and 1.5 kilobases, respectively, at the map position of the src gene for these two classes of td SR-D viruses, confirming the results of polyacrylamide gel electrophoresis. In addition, some heteroduplex molecules with a substitution loop of 0.6-0.7 kilobase at the same site as the deletion loop were observed in all five of the td SR-D viruses. We conclude that some of the td SR-D viruses have a partially deleted src gene and that all of the td SR-D viruses have incorporated heterologous sequences of distinct length in some RNA molecules at the position of the src gene. The nature and origin of these heterologous sequences are discussed.

Avian Sarcoma Viruses↗
PubMed1990

An alternative non-tyrosine protein kinase product of the c-src gene in chicken skeletal muscle.

While the c-src locus is expressed as a 4.0-kilobase (kb) mRNA coding for pp60c-src in various chicken tissues, including embryonic muscle, it is expressed as a novel 3.0-kb mRNA in adult skeletal muscle. We have analyzed the primary structure of this alternatively transcribed and spliced c-src mRNA. The sequence revealed three open reading frames, with the previously defined c-src exons 1 through 5 or 6 comprising the third, on the 3' untranslated region of this 3-kb mRNA. The exons coding for the tyrosine kinase domain of pp60c-src were excluded. On the 5' side, 2 kb of sequence upstream from the previously defined exon 1 of the c-src gene was included in this mRNA. The start site for the 3-kb mRNA probably lies downstream of that for the 4-kb mRNA. The first reading frame of the 3.0-kb mRNA, called sur (for src upstream region), encoded a 24-kilodalton (kDa) protein product rich in cysteine and proline residues. In vitro analysis indicated that the 24-kDa sur protein was membrane associated. Antibodies to sur protein detected in vivo a 24-kDa muscle-specific protein which was developmentally regulated and corresponded to the switch from the 4-kb to the 3-kb c-src mRNA. A striking kinetic pattern of appearance of sur protein and disappearance of pp60c-src suggests that the expression of these two proteins is inversely related.

Amino Acid Sequence↗
PubMed1982

Viral src gene products are related to the catalytic chain of mammalian cAMP-dependent protein kinase.

The transforming protein sequences translated from the Rous avian and Moloney murine sarcoma virus src genes are shown to be related to the catalytic chain of bovine cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The avian transforming protein, also a protein kinase, shows greatest homology with the bovine protein kinase in the carboxyl-terminal half, where the protein kinase activity is localized. Moreover, lysine occurs in the inferred transforming protein sequences at the position homologous with the proposed ATP-binding lysine of the bovine protein kinase. This relationship is consistent with the hypothesis that the src genes originated in the host genomes, in which they are members of a superfamily of distantly related protein kinases that are normal constituents of mammalian cells. In the host, these sequences are much more highly conserved than in the viruses.

Amino Acid Sequence↗
PubMed1988

The cellular src gene product regulates junctional cell-to-cell communication.

Overexpression of the cellular src gene in NIH 3T3 cells causes reduction of cell-to-cell transmission of molecules in the 400- to 700-dalton range. This down-regulation of gap junctional communication correlates with the activity of the gene product, the protein tyrosine kinase pp60c-src. The down-regulation was enhanced by point mutation of Tyr527 (a site that is phosphorylated in pp60c-src and that inhibits kinase activity) or by substitution of the viral-src for the cellular-src carboxyl-terminal coding region. Mutation of Tyr416 (a site phosphorylated upon Tyr527 mutation) suppresses both the down-regulation of communication by Tyr527 mutation and that by gene overexpression. The regulation of communication by src may be important in the control of embryonic development and cellular growth.

Animals↗
PubMed1981

A brain membrane protein similar to the rat src gene product.

We report the purification to homogeneity of a 20,000-dalton, transformation-related, rat cell membrane protein. This protein, p20, was originally identified in preparations of a defective woolly monkey leukemia virus pseudotype of Kirsten sarcoma virus. The chromatographically purified p20 was an acidic hydrophobic protein, capable of specifically binding GTP (dissociation constant = 15 microM). This nucleotide binding property and other previously reported characteristics were similar to properties ascribed to the Harvey sarcoma virus src gene product. p20 also appeared similar to this src gene product when immunoprecipitates of both proteins were directly compared by one- and two-dimensional NaDodSO4 gel electrophoreses. However, the proteins were not identical, because their tryptic maps differed. Using a competition radioimmunoassay, we have measured the concentration of p20 in cells, viruses, and rat tissues: p20 was not encoded by rat sarcoma viruses because it was increased only slightly after Kirsten sarcoma virus transformation of rat cells and was not increased in nonrat cells transformed by the Kirsten or Harvey sarcoma virus. Remarkably, of 10 rat tissues examined, p20 was found predominantly in brain, specifically in the membranes.

Animals↗
PubMed1979

Immunofluorescence on avian sarcoma virus-transformed cells: localization of the src gene product.

The localization of the avian sarcoma virus src gene product (termed p60src) was examined by indirect immunofluorescence in cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D (SR-RSV-D). Antiserum to p60src was obtained from rabbits bearing SR-RSV-D-induced tumors, and immunofluorescence was performed on chicken embryo fibroblasts (CEF) transformed with SR-RSV-D, as well as normal rat kidney (NRK) cells transformed by the same virus (termed SR-RK cells). Both acetone and formaldehyde fixation were used for the immunofluorescence tests. The specificity of the anti-tumor serum was first demonstrated in both cell systems by gel electrophoresis of immunoprecipitates prepared from 35S--methionine-labeled cells. Anti-tumor serum precipitated p60src from SR-RSV-D-transformed CEF but not from CEF infected with a transformation-defective mutant of SR-RSV-D. All viral structural proteins and precursors contained in these immunoprecipitates could be eliminated by competition with unlabeled virus. Similar experiments on SR-RK cells indicated that no viral proteins other than p60src were expressed in these cells, and this observation was supported by immunofluorescence tests using antiserum to whole virus. For immunofluorescence localization of p60src, reactions with viral structural proteins were blocked with unlabeled virus. This presaturation step, obligatory for p60src detection in the SR-RSV-D-transformed CEF, was unnecessary when antitumor serum was tested on SR-RK cells, since p60src was the only viral protein detectable in these cells. With acetone-fixed cells, p60src-specific immunofluorescence revealed a characteristic fluorescence pattern which was similar in both cell systems. The principal pattern was diffuse and situated in the cytoplasm. A clear nuclear fluorescence was never observed. Immunofluorescence on formaldehyde-fixed cells also indicated the cytoplasmic location of p60src and revealed a specific subcytoplasmic concentration of the fluorescence. With both fixation methods, an additional fluorescence pattern was seen between cells in contact, and was also found in both SR-RK cells and SR-RSV-D-transformed CEF. Immunofluorescence on viable cells suggested that p60src was not on the surface of these transformed cells. The fluorescence patterns were specific for avian sarcoma virus-transformed cells and were not found in uninfected cells, cells infected with a transformation-defective mutant of SR-RSV-D or cells transformed by an antigenically unrelated murine sarcoma virus. Furthermore, anti-tumor serum did not contain antibodies to proteins of the microtubules or intermediate filaments.

Animals↗