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Query: UNIPROT:P06889 (Mol)
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The transforming protein of Rous sarcoma virus, pp60v-src, is covalently coupled to myristic acid by an amide linkage to glycine 2. Myristylation promotes the association of pp60v-src with cellular membranes, and this subcellular location is essential for transforming activity. The findings presented here, in conjunction with the previous reports of others, imply that the seventh amino acid encoded by v-src might be important in the myristylation reaction. Replacement of lysine 7 by asparagine greatly reduced the myristylation, membrane association, and transforming activity of pp60v-src. In contrast, substitution of arginine at residue 7 had no effect on any of these properties of pp60v-src. Addition of amino acids 1 to 7 encoded by v-src was sufficient to cause myristylation of a src-pyruvate kinase fusion protein. We conclude that the recognition sequence for myristylation of pp60v-src comprises amino acids 1 to 7 and that lysine 7 is a critical component of this sequence.
Mol Cell Biol 1988 Jun
PMID:The first seven amino acids encoded by the v-src oncogene act as a myristylation signal: lysine 7 is a critical determinant. 284 81

To identify mRNAs with altered expression in Rous sarcoma virus (RSV)-transformed cells, we screened a chicken embryo fibroblast (CEF) cDNA library by differential hybridization. One clone, designated R1H, showed markedly elevated mRNA expression in RSV-transformed cells. Nucleotide sequence analysis indicated that R1H mRNA encodes 78-kilodalton glucose-regulated protein (GRP78). Chicken GRP78 was found to be very highly conserved in comparison with rat GRP78 (96% identity between chicken and rat amino acid sequences). In contrast to previous observations, we found that GRP78 was induced in RSV-transformed cells in the absence of glucose deprivation. When cells were grown in glucose-supplemented medium, the level of GRP78 mRNA was approximately fivefold higher in RSV-transformed CEF than in transformation-defective virus-infected or uninfected CEF. Similar changes in GRP78 protein content were also found. Using a temperature-sensitive mutant of RSV and supplemental glucose, we found a gradual increase in the level of GRP78 mRNA beginning at 4 h after shiftdown to permissive temperature. Uridine supplementation did not block the induction seen in CEF infected with a temperature-sensitive mutant. These results indicate that GRP78 is induced by p60v-src in the absence of glucose deprivation.
Mol Cell Biol 1988 Jul
PMID:78-kilodalton glucose-regulated protein is induced in Rous sarcoma virus-transformed cells independently of glucose deprivation. 284 86

C-fos promoter activity was examined in FRTL5 rat thyroid cells transiently transfected with a chimeric gene containing the c-fos promoter region linked upstream of the receptor gene chloroamphenicol acetyl transferase (CAT). Thyroid-stimulating hormone (TSH) stimulation of transfected cells increased CAT activity 2- to 3-fold. TSH did not stimulate CAT activity driven by the Rous sarcoma virus (RSV) promoter. Both forskolin and 8-Br-cyclic AMP also stimulated CAT activity, and were not additive with TSH stimulation. The kinetics of c-fos-driven CAT activity in response to TSH and cyclic AMP inducers were similar, and stimulation was reversible. These data therefore provide the first evidence that TSH and cyclic AMP stimulate the activity of the c-fos promoter in FRTL5 cells.
Mol Cell Endocrinol 1988 Aug
PMID:TSH stimulates the activity of the c-fos promoter in FRTL5 rat thyroid cells. 285 Feb 50

Only a fraction of retroviral primary transcripts are spliced to subgenomic mRNAs; the unspliced transcripts are transported to the cytoplasm for packaging into virions and for translation of the gag and pol genes. We identified cis-acting sequences within the gag gene of Rous sarcoma virus (RSV) which negatively regulate splicing in vivo. Mutations were generated downstream of the splice donor (base 397) in the intron of a proviral clone of RSV. Deletion of bases 708 to 800 or 874 to 987 resulted in a large increase in the level of spliced RSV RNA relative to unspliced RSV RNA. This negative regulator of splicing (nrs) also inhibited splicing of a heterologous splice donor and acceptor pair when inserted into the intron. The nrs element did not affect the level of spliced RNA by increasing the rate of transport of the unspliced RNA to the cytoplasm but interfered more directly with splicing. To investigate the possible role of gag proteins in splicing, we studied constructs carrying frameshift mutations in the gag gene. While these mutations, which caused premature termination of gag translation, did not affect the level of spliced RSV RNA, they resulted in a large decrease in the accumulation of unspliced RNA in the cytoplasm.
Mol Cell Biol 1988 Nov
PMID:Regulation of Rous sarcoma virus RNA splicing and stability. 285 Apr 70

We previously described a recombinant Moloney murine leukemia virus (Mo-MuLV) carrying the v-src oncogene, Mo-MuLV(src). Mo-MuLV(src) encodes a gag-src fusion protein, transforms cells in culture, and induces fibrosarcomas in vivo. To compare transforming properties of the gag-src fusion protein to pp60src encoded by Rous sarcoma virus, we constructed a new recombinant virus, Mo-MuLV(+ src). Mo-MuLV(+ src) encodes pp60src in the context of Mo-MuLV. Cells transformed by Mo-MuLV(+ src) were round and formed colonies in soft agar, whereas Mo-MuLV(src)-infected cells were fusiform and did not grow in suspension. Thus, the extent of transformation induced by Mo-MuLV(+ src) was greater than that induced by Mo-MuLV(src). Subcutaneous inoculation of either virus into neonatal NIH Swiss mice resulted in fibrosarcomas at the site of injection. Further studies indicated that tumors induced by Mo-MuLV(+ src) grew rapidly but rarely metastasized. In contrast, tumors induced by Mo-MuLV(src) grew somewhat more slowly but metastasized with a high frequency (60%). These viruses may provide a useful model system for tumor metastasis. Another src-containing virus was also studied, MRSV (constructed by Anderson and Scolnick). MRSV also encodes pp60src but in the context of amphotropic MuLV. When injected intravenously into six-week-old mice, MRSV induced splenomegaly and spleen foci but no solid tumors, as reported previously. In contrast, Mo-MuLV(src)-induced fibrosarcomas mostly in the spleen under the same inoculation protocol. These results suggest that the v-src oncogene was the major pathogenic determinant in neonatal mice for all three src-containing viruses; however, variations in the nature of the transforming protein modulated the behavior of the induced tumors. In adult mice, greater differences in pathogenicity were observed.
Mol Carcinog 1988
PMID:Comparison of three recombinant murine leukemia viruses carrying the v-src oncogene of avian sarcoma virus: differences in in vitro transformation and in vivo pathogenicity. 285 3

We have developed a convenient and sensitive assay of eucaryotic gene expression which uses the Escherichia coli lacZ gene product, beta-galactosidase, as a nonselectable marker. This system has been applied to the analysis of Rous sarcoma virus replication and gene expression. Avian cells were transfected with plasmids encoding in-frame gene fusions of the N-terminal portion of the gag gene to a 'lacZ gene, which requires both transcriptional and translational initiation signals; these were supplied by the virus long terminal repeat and leader region. Readily detectable quantities of beta-galactosidase were synthesized in transfected cells; it was demonstrated that the levels of enzyme activity induced in such cultures increased linearly with the input DNA concentration and also correlated with mRNA levels. By using a Rous sarcoma virus-derived vector containing the src gene and a related virus as a helper, it was shown that lac sequences were compatible with all phases of the virus life cycle. gag-lacZ fusion proteins were immunoprecipitable from cultures which stably expressed lacZ as well as src. Virus rescued from stably transfected cultures resulted in continued lac and src expression in recipient cells. One particular construction was efficiently transmitted as virus, although it lacked sequences thought to be important for encapsidation of RNA into virions. The data presented here demonstrate the use of lacZ as a marker of retrovirus gene expression and replication.
Mol Cell Biol 1985 Feb
PMID:Bacterial beta-galactosidase as a marker of Rous sarcoma virus gene expression and replication. 298 87

We have recently demonstrated that an NH2-terminal sequence required for myristylation and membrane association of the Rous sarcoma virus transforming protein, p60src, is contained within amino acids 2-14 [Cross, F.R., Garber, E. A., Pellman, D. & Hanafusa, H. (1984) Mol. Cell. Biol. 4, 1834-1842]. This sequence is also required for cell transformation. We have now constructed five mutants of Rous sarcoma virus that contain alterations in the src sequence coding for these 14 amino acids. Mutants encoding src proteins with a peptide insertion between amino acids 1 and 2, or peptide substitutions for amino acids 2-4, 3-4, or 7-15, were transformation-defective. The src proteins of these mutants differed from the wild-type protein in that they were not myristylated and did not fractionate with the plasma membrane of infected cells. The fifth mutant encoded a src protein with a short peptide substituted for amino acids 11-15. This protein was myristylated and plasma membrane associated, and the virus transformed cells. We therefore conclude that a sequence required for myristylation and membrane association of p60src is located within the first 7-10 amino acids of the src protein, and that p60src myristylation and membrane association are required for cell transformation. Consistent with this idea, we have isolated four transforming revertants from one of the transformation-defective mutants. The src proteins of all four revertants were found to be myristylated and membrane associated.
 ...
PMID:Fine structural mapping of a critical NH2-terminal region of p60src. 298 63

We used several quantitative assays of in vivo transient gene expression to dissect the elements within the Rous sarcoma virus long terminal repeat (LTR) which constitute the retroviral transcription control region. Site-directed deletion mutagenesis was used to locate and define the enhancer and promoter elements within the LTR. In addition, we inserted exogenous DNA fragments into the LTR to examine the effects of position and sequence on the activity of these LTR transcriptional elements. The Rous sarcoma virus enhancer element, which we propose is located entirely within the LTR, was shown to activate both the beta-globin and retroviral LTR promoters when located in cis. We observed a striking correlation between the degree of activation and the distance between the retroviral promoter and enhancer elements. The LTR promoter element mediated the activation effect of the enhancer element, as LTR deletion mutants containing only the enhancer and TATA box region expressed little activity. The promoter region encoded a low but significant level of transcriptional activity even in the absence of an enhancer. Overall LTR transcriptional activity declined sharply with increasing distance between the LTR promoter and initiator elements. These results shed light on both the importance of the spatial arrangement of the sequence elements within this eucaryotic transcription control region and on the functional interrelationship between these elements.
Mol Cell Biol 1985 Mar
PMID:Functional analysis of the transcription control region located within the avian retroviral long terminal repeat. 298 53

We have analyzed the effects of transformation by Rous sarcoma virus on expression of types I and II collagen and fibronectin genes in vertebral chondrocytes and compared them with expression of these genes in skin fibroblasts. Transformed chondrocytes display a dramatically decreased amount of type II collagen RNA, which can account fully for the decreased synthetic rate of this protein. Paradoxically, these cells also display greatly increased amounts of type I collagen RNAs, which are translated efficiently in vitro, but not in the intact cells. We show here that the type I collagen RNAs in transformed chondrocytes are nearly indistinguishable from those found in skin fibroblasts, and that they clearly differ from the type I collagen RNAs found in normal chondrocytes. Transformed chondrocytes also display an increased amount of fibronectin RNAs, which can account fully for the increased synthetic rate of this protein. Thus, the effects of transformation by Rous sarcoma virus on type I collagen and fibronectin RNAs in chondrocytes are the opposite of those observed in fibroblasts, which display decreased amounts of these three RNAs. These data indicate that the effects of transformation on the genes encoding type I collagen and fibronectin must be modulated by host cell-specific factors. They also imply that the types I and II collagen genes may be regulated by different mechanisms, the type I genes being controlled at both transcriptional and posttranscriptional levels, and the type II gene being controlled primarily at the transcriptional level.
Mol Cell Biol 1985 May
PMID:Control of types I and II collagen and fibronectin gene expression in chondrocytes delineated by viral transformation. 298 70

The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.
Mol Cell Biol 1985 May
PMID:Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins. 298 71


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