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Hybridization of v-src sequences to human cellular DNA. Human placental DNA (25 jig per lane) was digested and analyzed by hybridization to 32P-labeled v-src probe II DNA after gel electrophoresis and Southern transfer. Lanes: A, BamHI; B, Hindlll; C, PvuII; D, 32P end-labeled, HindIll-cleaved lambda DNA markers.
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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-s...
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... It has been reported that retrovirus arrests the host proto-oncogene (c-src) at their replicative cycle and then gets transformed into the tumor cell. Out of these four genes, oncogenic src is the one that induces tumor formation and transforms cells (Bishop et al., 1979;Gibbs et al., 1985). Cellular src (csrc) has been found in human and shares sequence similarity with viral src (v-src) of RSV and with chicken c-src also. ...
... Cellular src (csrc) has been found in human and shares sequence similarity with viral src (v-src) of RSV and with chicken c-src also. Moreover, chicken's c-src shares sequence similarity with v-src of RSV 11 out of 12 exons and encodes phosphoprotein p60c-src and pp60v-src, respectively (Gibbs et al., 1985). However, pol gene encodes the reverse transcriptase enzyme that transcribes the RNA to cDNA (complementary DNA) molecule (Jacks and Varmus, 1985). ...
A small non-coding, evolutionarily conserved regulatory RNA molecule known as microRNA (miRNA) regulates various cellular activities and pathways. MicroRNAs remain evolutionarily conserved in different species of same taxa. They are present in all organisms including viruses. Viral miRNAs are small, less conserved and less stable and have higher negative minimal folding free energy than miRNAs of different organisms. The size of viral precursor miRNA is approximately 60–119 nucleotides in length. The structure of the mature miRNA sequences is predicted by using higher negative MFEI (ΔG) value. Rous sarcoma Virus (RSV), named after its inventor Peyton Rous, has been known for causing tumors in the chicken for which it is known as an oncogenic retrovirus. Using specific criteria we predicted 5 potential miRNAs in RSV which targeted 8 tumor suppressor genes in Gallus gallus. This study aims to predict the potential miRNAs, secondary structures and their targets for better understanding of the regulatory network of Rous sarcoma virus miRNA in forming sarcoma.
... This was followed by the discovery that v-SRC is a tyrosine kinase protein, essential for cell transformation by the Rous sarcoma virus, and derived from cellular chicken c-SRC, incorporated into Rous sarcoma virus via recombination [2]. Human proto-oncogene tyrosine-protein kinase SRC (SRC), the first proto-oncogene, was described as highly structurally similar to chicken c-SRC [3,4]. As v-SRC contains an altered carboxy terminus compared to SRC, these amino acids were expected to play an important role in the regulation of SRC activity and cellular transformation [5]. ...
Proto-oncogene tyrosine-protein kinase SRC (SRC), as other members of the SRC family kinases (SFK), plays an important role in regulating signal transduction by different cell surface receptors after changes in the cellular environment. Here, we reviewed the role of SRC in platelets and megakaryocytes (MK). In platelets, inactive closed SRC is coupled to the β subunit of integrin αIIbβ3 while upon fibrinogen binding during platelet activation, αIIbβ3-mediated outside-in signaling is initiated by activation of SRC. Active open SRC now further stimulates many downstream effectors via tyrosine phosphorylation of enzymes, adaptors, and especially cytoskeletal components. Functional platelet studies using SRC knockout mice or broad spectrum SFK inhibitors pointed out that SRC mediates their spreading on fibrinogen. On the other hand, an activating pathological SRC missense variant E527K in humans that causes bleeding inhibits collagen-induced platelet activation while stimulating platelet spreading. The role of SRC in megakaryopoiesis is much less studied. SRC knockout mice have a normal platelet count though studies with SFK inhibitors point out that SRC could interfere with MK polyploidization and proplatelet formation but these inhibitors are not specific. Patients with the SRC E527K variant have thrombocytopenia due to hyperactive SRC that inhibits proplatelet formation after increased spreading of MK on fibrinogen and enhanced formation of podosomes. Studies in humans have contributed significantly to our understanding of SRC signaling in platelets and MK.
... Cellular oncogenes were originally identified based on their homology with viral oncogenes. For example, the viral src gene (v-src), the transforming gene of the Rous sarcoma virus (RSV), the first tumor virus identified (73), was found to have counterparts in cellular DNA, first in the chicken genome (85,94) and then in all mammalian genomes (27,67). Additional cellular versions of viral oncogenes, termed proto-oncogenes, were identified (34,93). ...
Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus.
... The IR mRNA protein-encoding region is 4.149 kb [7][8]. Human cSRC encodes 1.611 kb pp60cSRC, a non-receptor tyrosine kinase implicated in numerous growth receptor-mediated signaling pathways leading to cellular proliferation, differentiation and transformation [9][10]. ...
PCR amplification failure from cDNA libraries or RNA templates, under the optimal conditions is generally attributed to high GC content. Utilization of various additives without thorough analysis of secondary structures of the template as well as primers and subsequent PCR cycle conditions, generally leads to inadequate yields and/or truncated products. To address these concerns, we have examined two highly GC-rich human genes namely insulin receptor (IR) and cSRC kinase. In silico analysis of these genes revealed that their -5' and -3' sequences have > 80% GC content. Primers designed through these GC-rich regions had high self-dimer free energy values (DeltaG). Null mutations were introduced to bring down these DeltaG levels below -5.0 kcal/mol. Oligo(dT)18 primed cDNA was synthesized from HepG2 and HT29 total RNA to amplify IR and cSRC kinase ORFs, respectively. A multi-prong strategy including primer modifications, various DMSO-betaine combinations and high denaturing temperature conditions was pursued during cDNA synthesis to achieve optimal PCR amplification. The reported approach can be utilized to improve the amplification of templates with high GC content, which are otherwise relatively difficult to resolve.
... Human, chicken, and Drosophila c-src genomic DNAs covering the pp6Ocsrc coding region have been isolated and characterized (25,36,53,65,74,76). The pp60c-src coding sequences are distributed in 11 exons (75) and, in the case of neuronal c-src, in 12 to 13 exons (45,49,57). ...
To further characterize the gene structure of the proto-oncogene c-src and the mechanism for the genesis of the v-src sequence in Rous sarcoma virus, we have analyzed genomic and cDNA copies of the chicken c-src gene. From a cDNA library of chicken embryo fibroblasts, we isolated and sequenced several overlapping cDNA clones covering the full length of the 4-kb c-src mRNA. The cDNA sequence contains a 1.84-kb sequence downstream from the 1.6-kb pp60c-src coding region. An open reading frame of 217 amino acids, called sdr (src downstream region), was found 105 nucleotides from the termination codon for pp60c-src. Within the 3' noncoding region, a 39-bp sequence corresponding to the 3' end of the RSV v-src was detected 660 bases downstream of the pp60c-src termination codon. The presence of this sequence in the c-src mRNA exon supports a model involving an RNA intermediate during transduction of the c-src sequence. The 5' region of the c-src cDNA was determined by analyzing several cDNA clones generated by conventional cloning methods and by polymerase chain reaction. Sequences of these chicken embryo fibroblast clones plus two c-src cDNA clones isolated from a brain cDNA library show that there is considerable heterogeneity in sequences upstream from the c-src coding sequence. Within this region, which contains at least 300 nucleotides upstream of the translational initiation site in exon 2, there exist at least two exons in each cDNA which fall into five cDNA classes. Four unique 5' exon sequences, designated exons UE1, UE2, UEX, and UEY, were observed. All of them are spliced to the previously characterized c-src exons 1 and 2 with the exception of type 2 cDNA. In type 2, the exon 1 is spliced to a novel downstream exon, designated exon 1a, which maps in the region of the c-src DNA defined previously as intron 1. Exon UE1 is rich in G+C content and is mapped at 7.8 kb upstream from exon 1. This exon is also present in the two cDNA clones from the brain cDNA library. Exon UE2 is located at 8.5 kb upstream from exon 1. The precise locations of exons UEX and UEY have not been determined, but both are more than 12 kb upstream from exon 1. The existence and exon arrangements of these 5' cDNAs were further confirmed by RNase protection assays and polymerase chain reactions using specific primers. Our findings indicate that the heterogeneity in the 5' sequences of the c-src mRNAs results from differential splicing and perhaps use of distinct initiation sites. All of these RNAs have the potential of coding for pp60c-src, since their 5' exons are all eventually joined to exon 2.
... In this paper, we have used an avian retroviral expression system to introduce and to express at high levels normal and mutated forms of the human c-src gene product in chicken * Corresponding author. embryo fibroblasts (CEF) in the absence of polyomavirus middle T antigen (1,10,15,16). The mutant human c-src genes used here were derived from retroviral vector constructs either spontaneously (WO CST-2 and WO CST-5) or by in vivo mutagenesis (CY CST103), as described elsewhere (8; Tanaka et al., submitted for publication). ...
Chicken embryo fibroblast cells overexpressing activated mutant forms of human pp60c-src, but not those overexpressing normal
human pp60c-src, exhibited high levels of type I phosphatidylinositol (PI) kinase activity associated with pp60c-src. Levels
of PI kinase activity were positively correlated with src tyrosine protein kinase activity and not with absolute levels of
pp60c-src. Our results suggest that a linkage exists between certain forms of pp60c-src and the PI signal transduction pathway.
... The v-src protein induces cell transformation, whereas the c-src protein does not under usual conditions (21,22,32,40,47). The human c-src gene (c-src-l), localized on chromosome 20, contains 11 coding exons spanning a distance of 19.5 kilobases, more than three times that of the chicken c-src coding regions (6 kilobases) (1, 16,31,45). The DNA sequence of exons 6 to 12, corresponding to the 3' two-thirds of the human c-src coding region, was determined previously (1). ...
... Previously, we studied the exon-intron structure of the human c-src gene by hybridization to v-src DNA probes and also by comparison with that of the chicken c-src gene (1, 16). For exon 3, however, its tentative localization was ambiguous (16). ...
... Previously, we studied the exon-intron structure of the human c-src gene by hybridization to v-src DNA probes and also by comparison with that of the chicken c-src gene (1, 16). For exon 3, however, its tentative localization was ambiguous (16). To localize exon 3 more precisely, we used the calf thymus DNA random primer method (35) to obtain a human c-src cDNA probe prepared from 70S virion RNA of a retrovirus (WO CVS virus) whose genome contains a chimeric, intronless form of human c-src sequence (47). ...
We sequenced the 5'-coding region of the human c-src gene, exons 2 through 5, corresponding to one-third of the human c-src
protein consisting of 536 amino acids. Sequence analysis of the src type of protein kinases revealed that the amino-terminal
region encoded by exon 2 contains sequences specific for the src proteins and raised the possibility that this region is involved
in the recognition of a src-specific substrate(s) or receptor(s).
... These studies have shown that most of the chicken c-src coding sequences are very homologous to those of v-src and are contained within 11 exons (numbered 2 through 12) that span approximately 6 kilobases (kb) of cellular DNA; in addition another exon (exon 1) codes for a 5' untranslated region of mRNA (55). At this time, relatively little is known about the properties of human c-src loci and human pp60C src.Our laboratory has recently characterized, by restriction enzyme mapping, molecular clones of a highly conserved human c-src gene isolated from a lambda phage genomic library (14). This c-src locus spans 20 kb of cellular DNA and contains at least 11 exons homologous to v-src (14). ...
... At this time, relatively little is known about the properties of human c-src loci and human pp60C src.Our laboratory has recently characterized, by restriction enzyme mapping, molecular clones of a highly conserved human c-src gene isolated from a lambda phage genomic library (14). This c-src locus spans 20 kb of cellular DNA and contains at least 11 exons homologous to v-src (14). This gene has been called human c-src locus 1 (c-src-l), because another somewhat less homologous c-src-related gene (locus 2) has been detected by others (14,34). ...
... This c-src locus spans 20 kb of cellular DNA and contains at least 11 exons homologous to v-src (14). This gene has been called human c-src locus 1 (c-src-l), because another somewhat less homologous c-src-related gene (locus 2) has been detected by others (14,34). c-src-1 is transcriptionally active and codes for a polyadenylated mRNA species of ca. 5 kb (34; J. Radul and D. J. Fujita, unpublished results). ...
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.
... The human SRC gene is comprised of 15 exons Bonham and Fujita, 1993;Bonham et al., 2000;Gibbs et al., 1985;Parker et al., 1985) (Fig 1.5). ...
The human SRC gene encodes pp60c-Src (or c-Src), a 60 kDa, non-receptor tyrosine kinase frequently activated in colon and other tumors. Many studies have demonstrated c-Src activation can be accounted for by overexpression of c-Src protein, and that this overexpression is important for the fully transformed phenotype of cancer cells. The general goal of this thesis, therefore, was to determine the mechanism of this overexpression in human cancer cells. Examination of c-Src expression and activity in human colon cancer cell lines showed that c-Src activation was due to transcriptional activation of the SRC gene. SRC transcription is directed by the ubiquitous, Sp1 regulated SRC1A promoter, and the HNF-1alpha regulated, tissue restricted SRC1alpha promoter. To study the mechanism of SRC transcriptional activation in human cancer cell lines, a dual SRC promoter reporter construct was generated with both these promoters in their natural, physiologically linked context. Very low activity of the SRC1alpha promoter, relative to SRC1A, was consistently observed from this construct, leading to the conclusion that an enhancer element elevates SRC1alpha promoter activity. Interestingly, the HNF binding site in the SRC1alpha promoter enhanced SRC1A promoter activity in the dual promoter construct, but only in a colon cancer cell line with activated SRC. These results therefore suggest SRC transcriptional activation results from enhancer action and/or SRC promoter cross-talk in subsets of human cancer cells.
This study has also determined that histone deacetylase inhibitors (HDIs), compounds with documented anti-neoplastic properties, repress transcription from both SRC promoters in various cancer cell lines. To identify the mechanism of this repression, various deletion and mutant SRC promoter constructs were assayed, but HDI response elements were not identified. However, it was discovered that both promoters shared a common requirement for functional TAF1/TAF(II)250, a component of the general transcription factor TFIID. Compromised TAF1 function impaired SRC transcription, but also blocked SRC repression by HDIs. Experiments with SRC:WAF1 promoter chimeras showed the SRC promoters' TAF1 requirement could be conferred on the heterologous, TAF1-independent promoter for the WAF1 gene, which encodes the cell cycle inhibitor p21. These chimeras were also repressed by HDIs, despite WAF1 normally being strongly induced by these agents. These results therefore provide a potential functional link between promoter architecture, TAF1 dependence, and HDI mediated transcriptional repression.
... Introns within the human c-src gene are all significantly larger than the corresponding introns within the chicken c-src gene ( Table 2). The increased intron size in the human c-src gene accounts for its much larger size (20 kb) relative to that of the chicken c-src gene (6 kb) (14). ...
Thenucleotide sequence ofthe3'two-thirds ofahighly conserved, molecularly cloned humancellular src gene(c-src) hasbeendetermined. Thisregion ofthec-src geneencodes thetyrosine kinase domainofthe cellular srcprotein (pp60csrc) andcorresponds toexons6through 12ofthechicken c-src gene, aswellas nucleotides 545to1542oftheRoussarcoma virus srcgene(v-src). Thehumanc-src sequence isverystrongly conserved withrespect toboththechicken c-src andtheRoussarcoma virus v-src genes, withnearly 90% nucleotide homology observed inthis region. Aminoacid sequence conservation inthis region isevengreater; 98%oftheaminoacids areconserved between humanandchicken c-src. Furthermore, theexonsizes andthe locations oftheexon-intron boundaries areidentical inthehumanandchicken c-src genes. However, sequences within theintrons havenotbeenconserved, andtheintrons within thehumanc-src genearesignificantly larger thanthecorresponding introns within thechicken c-src gene. Thestrong aminoacid conservation between the carboxy-terminal two-thirds ofpp60`csrc ofspecies asdivergent ashumansandchickens suggests thatthis portion ofthepp60`csrc protein specifies one ormore functional domains that areofgreat importance tosome aspect ofnormal cellular growth ordifferentiation.
