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At least 19 recordsLinked to original sources

PubMed1978

Avian myelocytomatosis and erythroblastosis viruses lack the transforming gene src of avian sarcoma viruses.

Using labeled cDNA specific for the detection of the src gene of avian sarcoma viruses, we find that avian myelocytomatosis virus strain MC29 and avian erythroblastosis virus strain ES4 lack nucleotide sequences related to the src gene. Furthermore, chicken fibroblasts as well as hematopoietic cells, infected and transformed with these viruses, show no enhanced level of transcription of the cellular nucleotide sequences related to the src gene of avian sarcoma viruses. These two viruses may thus contain their own transforming gene(s) or induce cellular genes unrelated to the src-like cellular sequences.

Alpharetrovirus↗

PubMed1987

Noncoding region between the env and src genes of Rous sarcoma virus influences splicing efficiency at the src gene 3' splice site.

Viral RNA and proteins in chicken embryo fibroblasts infected with different cloned variants of the Prague strain Rous sarcoma virus (RSV) were analyzed. The ratio of immunoprecipitated pp60src to the gag gene product p27 in Prague A (PrA) and Prague B (PrB) RSV-infected cells was two to three times that in Prague C (PrC) RSV-infected cells. A significant increase in the steady-state ratio of spliced 2.7-kilobase src gene mRNA to unspliced 9.3-kilobase genome-size RNA was observed in PrA- and PrB- compared with PrC-infected cells, consistent with the differences in the ratios of the gag to src gene protein products. Similar results were obtained when hybrid-selected RNA, which had been labeled for 3 h with [3H]uridine, was analyzed on formaldehyde-agarose gels, suggesting that the observed differences were due to splicing rather than RNA stability. Recombinant plasmids from infectious molecular clones of PrA and PrC were constructed to localize the regions responsible for the effects on src gene splicing. The substitution in place of the corresponding PrA region of the 262-base-pair region between the env gene and the src gene coding sequences from the PrC clone into the infectious PrA plasmid conferred the low src splicing efficiency of the PrC strain. The nucleotide sequence of this region of the PrA plasmid was determined and compared with the sequence of the PrC strain. Only four nucleotide differences were found; two changes were within the intron sequence, and two were in the exon sequence. The possible role of these differences in determining the extent of viral RNA splicing is discussed.

Animals↗

PubMed1985

Isolation and structural mapping of a human c-src gene homologous to the transforming gene (v-src) of Rous sarcoma virus.

We have utilized a lambda Charon 4A human genomic library to isolate recombinant clones harboring a highly conserved c-src locus containing nucleotide sequences homologous to the transforming gene of Rous sarcoma virus (v-src). Four overlapping clones spanning 24 kilobases of cellular DNA were analyzed by restriction endonuclease mapping. Human c-src sequences homologous to the entire v-src region are present in a 20-kilobase region that contains 11 exons as determined by restriction mapping studies utilizing hybridization to labeled DNA probes representing various subregions of the v-src gene and by preliminary DNA sequencing analyses. A considerable degree of similarity exists between the organization of the human c-src gene and that of the corresponding chicken c-src gene with respect to exon size and number. However, the human c-src locus is larger than the corresponding chicken c-src locus, because many human c-src introns are larger than those of chicken c-src. alu family repetitive sequences are present within several human c-src introns. This locus represents a highly conserved human c-src locus that is detectable in human cellular DNAs from various sources including placenta, HeLa cells, and WI-38 cells.

Avian Sarcoma Viruses↗

PubMed1990

Immunocytochemical localization of the neuron-specific form of the c-src gene product, pp60c-src(+), in rat brain.

Neurons express high levels of a variant form of the c-src gene product, denoted pp60c-src(+), which contains a 6 amino acid insert in the amino-terminal half of the c-src protein. We have determined the localization of pp60c-src(+) in neurons using an affinity-purified anti-peptide antibody, referred to as affi-SB12, that exclusively recognizes this neuron-specific form of the c-src gene product. Using affi-SB12, we examined the distribution of pp60c-src(+) by immunoperoxidase staining of sections through adult rat brains, pp60c-src(+) was widely distributed in rat brain and appeared to be differentially expressed in subpopulations of neurons. The majority of immunoreactive neurons was found in the mesencephalon, cerebellum, pons, and medulla. Telencephalic structures that contained substantial populations of pp60c-src(+)-immunoreactive neurons included layer V of the cerebral cortex and the ventral pallidum. Within individual neurons, pp60c-src(+) immunoreactivity was localized to the cell soma and dendritic processes, while labeling of axons and nerve terminals (puncta) was not as readily detected. Dense accumulations of immunoreactive axons were rare, being most prominent in portions of the inferior and superior olive, and in the spinal trigeminal nucleus. While the regional distribution of pp60c-src(+) immunoreactivity does not correlate with any specific neuronal cell type or first messenger system, this unique pattern of expression of pp60c-src(+) suggests the existence of a previously uncharacterized functional organization within the brain. Furthermore, the localization of this neuron-specific tyrosine kinase in functionally important areas of the nerve cell, namely, dendritic processes, axons, and nerve terminals, suggests that pp60c-src(+) may regulate pleiotropic functions in specific classes of neurons in the adult central nervous system.

Animals↗

PubMed1991

Functional aspects of a tyrosine kinase encoding protooncogene, the c-src gene.

To date the src gene family consists of at least 9 closely related protein tyrosine kinases belonging to the non-receptor type of kinases: c-src, c-yes, c-fgr, fyn, lyn, lck, hck, tkl and bkl. We have intensively studied the expression of the c-src gene during evolution and with respect to its possible functions in the processes of cellular differentiation and proliferation. From our results we conclude, that the c-src encoded tyrosine kinase could play a role in the development and/or maintenance of the multicellular organisation of primitive organisms like sponges or coelenterates. With respect to its tissue-specific expression pattern with neuronal cells always displaying elevated levels of pp60c-src and the observation that synaptophysin, the major constituent of the synaptic vesicle membrane protein, is phosphorylated by the c-src encoded tyrosinekinase in vitro and in intact synaptic vesicles, we suggest an essential role for pp60c-src in signal transduction pathways and/or axonal transport mechanisms in neurons.

Animals↗

PubMed1985

Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.

Amino Acid Sequence↗

PubMed1986

Genetic lesions involved in temperature sensitivity of the src gene products of four Rous sarcoma virus mutants.

The src genes of four Rous sarcoma virus (RSV) mutants temperature-sensitive (ts) for cell transformation were analyzed. The mutant src genes were cloned into a replication-competent RSV expression vector, and the contribution of individual mutations to the ts phenotype was assessed by in vitro recombination with wild-type src sequences. Three of the mutants, which were derived from the Schmidt-Ruppin strain of RSV, each encoded two mutations within the conserved kinase domain. In all three cases, one of the two mutations was an identical valine to methionine change at amino acid position 461. Virus encoding recombinant src genes containing each of these mutations alone were not ts for transformation, demonstrating that two mutations are required for temperature sensitivity. The sequence of the src gene of the Bryan high-titer strain of RSV was determined and compared with that of the fourth ts mutant which was derived from it, again revealing two lesions in the kinase domain of the mutant.

Amino Acid Sequence↗

PubMed1987

Isolation of a murine retrovirus with a temperature-sensitive src gene.

A murine retrovirus containing the src gene of avian sarcoma virus ts LA31A was generated. In contrast to the avian sarcoma virus, our recombinant murine retrovirus can efficiently infect mammalian cells. Transformation of NIH3T3 cells by the recombinant murine retrovirus is temperature sensitive. At the permissive temperature of 34 degrees, cells form foci of rounded cells. At the nonpermissive temperature of 39 degrees, the infected cells remain flat and exhibit contact inhibition. No disease was observed following infection of newborn NFS/n mice with the ts mutant virus. In contrast, infection of newborn NFS/n mice with a recombinant murine retrovirus containing the wild-type src gene causes fibrosarcomas and hepatosplenomegaly.

Animals↗

PubMed1985

Potential role of the src gene product in inhibition of gap-junctional communication in NIH/3T3 cells.

The effects of the src gene on the activity of protein kinase C and intercellular communication have been studied in transformed NIH/3T3 clones isolated from soft agar following transfection with the plasmid carrying the v-src gene (psrc-11). Six transformed clones that were studied contained newly incorporated v-src genes in the genome, had an increased amount of pp60src, and showed enhanced activities of protein kinase C. Intercellular communication, studied by observing with autoradiography the transfer of [3H]uridine nucleotide from prelabeled donor cells to recipient cells in contact, was found to be reduced in transformed clones as compared to parental NIH/3T3 cells. Treatment with phorbol 12-myristate 13-acetate was also found to increase protein kinase C activity and to reduce intercellular communication in normal NIH/3T3 cells. These results suggest that the v-src gene product, in a manner similar to some of the powerful tumor promoters, may directly or indirectly affect cell-cell communication.

Animals↗

PubMed1986

Investigations of the expression of the cellular src gene product.

We have examined the expression of the c-src gene product in a variety of embryonic and adult tissues, in peripheral blood cells, and in cells transformed by other tumor viruses, in an attempt to identify the types of cells in which pp60c-src might provide a specific function. These studies have indicated that c-src gene expression is regulated at multiple levels in different cell types and have suggested that pp60c-src is not exclusively involved in the regulation of cell proliferation. The lowest levels of pp60c-src were found in fibroblasts, which have previously served as the standard cell type for comparisons between pp60c-src and pp60v-src. The highest levels of pp60c-src-specific kinase activity were detected in three types of cells: neurons, platelets, and polyoma virus transformed cells. In this report, we will compare the expression of pp60c-src in fibroblasts to that in platelets, neurons, and polyoma virus transformed fibroblasts. In each of the three latter cell types, the c-src gene product displays a unique pattern of expression which can be distinguished from that in fibroblasts (see diagram Fig. 1).

Animals↗

PubMed1990

Human cellular src gene product: identification of the myristoylated pp60c-src and blockage of its myristoyl acylation with N-fatty acyl compounds resulted in the suppression of colony formation.

A p60K protein in human colon adenocarcinoma tumor cell lines was identified as a myristoylated pp60c-src by fluorography and radioimmunoprecipitation analysis. Prevention of the myristoylation of pp60c-src was determined with N-fatty acyl glycinal compounds. Of the compounds tested, N-myristoyl glycinal diethylacetal, N-lauroyl glycinal diethylacetal, N-myristoyl glycyl glycinal diethylacetal, and N-myristoyl-4-aminobutyl-aldehyde diethylacetal strongly blocked the myristoylation, but N-decanoyl glycinal diethylacetal and N-palmitoyl glycinal diethylacetal did not. The myristoyl blocking compounds depressed colony formation, cell proliferation, and specific localization to the plasma membrane of pp60c-src. The results taken together suggest that myristoylation of the c-src oncogene product may be very important for tumorigenicity of c-src gene expressed cells.

Acetaldehyde↗

PubMedDate not supplied

Rous sarcoma virus does not induce sarcoma in early chick embryos by lack of the v-src gene expression.

The Schmidt-Ruppin or the B77 strain of Rous sarcoma virus (RSV) was inoculated into limb buds of 4.5-days-old avian embryos. No sarcoma but blister formation was observed in those RSV-inoculated embryos. Protein kinase activity of pp60v-src in RSV-inoculated embryos, even in the site of virus inoculation, was the same as that in mock-infected embryos. This indicated that the expression of the v-src gene did not attain superiority over that of the c-src gene in RSV-inoculated embryos. The v-src gene was detected in every DNA from tissues of RSV-inoculated embryos but not in DNAs from tissues of RSV-inoculated chicken except for the DNA from Rous sarcoma. Those results confirmed that the lack of sarcoma induction in early avian embryos by RSV was due to the lack of the expression of the v-src gene which was present in the target cells.

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↗

PubMed1992

Cloning and characterization of a thermolabile v-src gene for use in reversible transformation of mammalian cells.

The use of temperature-sensitive (ts) src mutants for studies of cell transformation and differentiation has been limited by the availability of cloned ts-src genes that are inactivated at temperatures compatible with growth of mammalian cells. In this report, we describe the cloning and characterization of the tsLA90src gene, which displays tight thermal sensitivity at 39.5 degrees C. Nucleotide sequence comparison of tsLA90 and wild-type src genes from the Schmidt-Ruppin subgroup A and D strains of Rous sarcoma virus (RSV) revealed four amino acid differences in tsLA90src. Substitution of one of these residues (Lys-280) from tsLA90src with its wild-type homolog (Glu-280) caused a reversion to a wild-type src phenotype. The cloned tsLA90 gene, designated tsUP1, was introduced into avian and mammalian retroviral vectors. Chicken embryo fibroblasts and immortalized mouse 3T3 cells infected with these viral vectors displayed a temperature-dependent transformed phenotype as assessed by cell morphology, secretion of plasminogen activator, transcriptional activation of the primary response genes, Egr-1 and TIS 10, and stimulation of tyrosine phosphorylation. In addition, chicken myoblasts (infected with RSVtsUP1) showed a temperature-dependent differentiation into myotubes. Thus, this cloned src gene should be ideally suited for inducing reversible transformation and differentiation of mammalian cells in culture.

3T3 Cells↗

PubMed1989

Deletions within the amino-terminal half of the c-src gene product that alter the functional activity of the protein.

To examine how amino acid sequences outside of the catalytic domain of pp60c-src influence the functional activity of this protein, we have introduced deletion mutations within the amino-terminal half of pp60c-src. These mutations caused distinct changes in the biochemical properties of the c-src gene products and in the properties of cells infected with retroviruses carrying these mutant c-src genes. Cells expressing the c-srcNX protein, which contains a deletion of amino acids 15 to 89, displayed a refractile, spindle-shaped morphology, formed intermediate-sized, tightly packed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Thus, deletion of amino acids 15 to 89 can activate the kinase activity and transforming potential of the c-src gene product. Deletion of amino acids 112 to 225, however, did not increase the kinase activity or transforming ability of pp60c-src; indeed, deletion of these sequences in c-srcHP suppressed phenotypic alterations induced by pp60c-src. Cells expressing the c-srcNP or c-srcBS gene products (containing deletions of amino acids 15 to 225 and 55 to 169, respectively) displayed a fusiform, refractile morphology and formed diffuse colonies in soft agar; the mutant proteins displayed an increased in vitro protein-tyrosine kinase activity. However, only a few cellular proteins contained elevated levels of phosphotyrosine in vivo. Thus, deletions downstream of amino acid 89 severely restricted the ability of c-src to phosphorylate cellular substrates in vivo without affecting the intrinsic tyrosine kinase activity of the c-src gene product. These results suggest the existence of at least two modulatory regions within the amino-terminal half of pp60c-src that are important for the regulation of tyrosine kinase activity and for the interaction of pp60c-src with cellular substrates.

Amino Acid Sequence↗

PubMed1992

Differentiation of quail myoblasts transformed with a temperature sensitive mutant of Rous sarcoma virus. I. Relationship between differentiation and tyrosine kinase of src gene product.

Quail embryonic pectoral myoblasts fuse with each other at 35.5 degrees C and 41 degrees C to essentially equal extents. When the myoblasts were transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV), their fusion and biochemical processes of differentiation became temperature-sensitive: their fusion occurred at 41 degrees C, the non-permissive temperature, but not at 35.5 degrees C, the permissive temperature, suggesting that the fusion was regulated by the viral transforming gene. Fusion of the transformed cells proceeded more rapidly and synchronously than that of the parent cells at 41 degrees C, and was completely suppressed at the permissive temperature, unlike that of the parent cells. These transformed cells were used to examine the relationship between myogenic differentiation and the tyrosine kinase activity of the src gene product. In spite of the temperature sensitivity of transformation, results showed that expressions of the src gene at 35.5 degrees C and 41 degrees C were similar. However, the level of tyrosine-phosphorylated protein was decreased at 41 degrees C. Moreover, myoblast fusion could occur at 35.5 degrees C in the presence of herbimycin A, an inhibitor of the tyrosine kinase activity of the src gene product. These results indicate that the tyrosine kinase activity of the src gene product is closely associated with regulation of myogenic differentiation of the cells.

Animals↗

PubMed1990

Characterization of avian retroviruses carrying activated transforming human c-src genes and of steps involved in expression of activated src-PKases in vitro.

We have isolated four activated transforming human c-src mutants derived spontaneously from viruses carrying the normal human c-src (SRC) genes in the form of Rous sarcoma virus. These mutants induced transformed cell morphology distinguishable from each other in vitro as well as tumors in chicks, whereas normal SRC-carrying viruses did not. Analyses of the transforming SRC proteins together with the normal SRC protein showed that levels of the carboxy-terminal Tyr-phosphorylation were negatively correlated with both transforming ability and protein kinase (PKase) activity as determined by in vitro autophosphorylation. It was observed that two cell lysis methods, that is, NP-40 and RIPA, yielded two different phosphorylated forms of transforming SRC proteins: one possessed low levels of phosphorylation at the autophosphorylation site and the other possessed high levels of phosphorylation at this site. Using the two types of transforming SRC preparations, we have studied in vitro SRC-PKase reactions in relation to in vivo and in vitro autophosphorylation and in vitro phosphorylation of an exogenous substrate. A possible functional relationship between autophosphorylation and SRC-PKase expression is discussed.

Animals↗

PubMed1986

Activation of the cellular src gene by transducing retrovirus.

Newly isolated strains of avian sarcoma virus, S1 and S2, were shown to have the transduced cellular src gene as their viral transforming gene (Yamagishi et al., Virology 137:266-275, 1984). In this work, the S1 and S2 genomes were molecularly cloned, and the junction sequences between the viral genomes and the c-src genes and the complete nucleotide sequences of the v-src genes transduced in these viruses were determined. Data on the junction sequences suggested that 5' recombination had occurred between the 5'-noncoding region of c-src and the 5' region of the gag sequence encoding p19 in both viruses and that 3' recombination had occurred in the last coding exon of c-src with either the middle portion of the env sequence encoding gp85 for S1 or the 3' portion of pol coding for reverse transcriptase for S2. Comparison of the amino acid sequences of the S1 and S2 src products deduced from the nucleotide sequences (pp62S1-src and pp62S2-src with that of c-src protein (pp60c-src) indicated that in pp62S1-src the 8 carboxy-terminal amino acid residues of the total of 533 in pp60c-src are replaced by 43 residues translated from the env sequence at the wrong frame. In pp62S2-src, on the other hand, the 14 carboxy-terminal amino acids of pp60c-src are replaced by the 38 carboxy-terminal residues of reverse transcriptase. The mechanism of c-src transduction and the structural changes necessary for pp60c-src activation are discussed.

Animals↗