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T Hunter

Publications and source records attributed to T Hunter.

At least 343 records · Page 19Linked to original sources
PubMed1982

Immunofluorescent localization of the transforming protein of Rous sarcoma virus with antibodies against a synthetic src peptide.

Antisera were raised against a synthetic peptide (src-c) containing the six COOH-terminal amino acids of p60src, the transforming protein of Rous sarcoma virus (RSV). Antibodies specific for the src-c peptide were purified by affinity chromatography and then used to study the location of p60src in transformed cells. The distribution of p60src was compared to that of vinculin, a candidate cytoskeletal substrate of p60src, by indirect double immunofluorescence microscopy. In RSV-transformed rat, mouse, and chicken cells, an extensive codistribution of p60src with vinculin was observed. Both proteins were concentrated in the few remaining focal adhesion plaques, in transformation-induced rosette clusters at the ventral cell surface, and in cell-cell contact areas. In addition, antibodies to both proteins stained the cytoplasm diffusely. In all cells examined, the immunofluorescent staining patterns produced by antibodies to the src-c peptide were indistinguishable from those obtained by immunolabeling of p60src with sera from RSV-infected tumor-bearing rabbits. The excellent agreement of the results obtained with two completely independent antibody preparations indicates strongly that the observed immunolabeling patterns correctly define the intracellular distribution of p60src. The significance of the intracellular location of p60src to the transforming activities of the protein is discussed.

Animals↗
PubMed1982

Expression of transforming region of Moloney murine sarcoma virus in Escherichia coli as a fusion protein with small tumor antigen of polyoma virus.

Bacterial expression of the transforming region of Moloney murine sarcoma virus, designated mos, was obtained as a fusion protein with a portion of the small tumor antigen of polyoma virus. This was accomplished by fusing the entire mos open reading frame, encoding a 41,000-dalton protein, with a plasmid that expresses a beta-galactosidase-polyoma fusion protein under lac operon control. The resulting plasmid directed synthesis of the predicted polyoma antigen-sarcoma virus fusion protein of 59,000 daltons. This protein was immunoprecipitated by an anti-polyoma tumor antigen antiserum that recognized polyoma determinants at the NH2 terminus of the hybrid protein. This protein was also immunoprecipitated by an antiserum directed against a synthetic peptide containing the 12 COOH-terminal amino acids encoded by the mos open reading frame. This work confirms the existence of a long open reading frame in the mos gene and resolves a discrepancy between different nucleotide sequences for its COOH-terminal coding region.

Animals↗
PubMed1982

Analysis of the sequence of amino acids surrounding sites of tyrosine phosphorylation.

We have identified the single phosphorylated tyrosine in p60src, the transforming protein of Rous sarcoma virus, as part of the sequence. NH2-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-COOH. Therefore, this is a sequence that is recognized efficiently by a tyrosine protein kinase in vivo. Phosphorylation of tyrosine in cellular proteins appears to play a role in malignant transformation by four classes of genetically distinct RNA tumor viruses. Phosphorylated tyrosines in several other proteins resemble of the tyrosine in p60src in that they are located 7 residues to the COOH-terminal side of a basic amino acid and either 4 residues to the COOH-terminal side of, or in close proximity to, a glutamic acid residue. Therefore it is possible that these features play a role in the selection of sites of phosphorylation by some tyrosine protein kinases. However, several clear exceptions to this rule exist.

Abelson murine leukemia virus↗
PubMed1982

Discrete primary locations of a tyrosine-protein kinase and of three proteins that contain phosphotyrosine in virally transformed chick fibroblasts.

We have studied the localization of three abundant cellular proteins which are substrates for tyrosine protein kinases in virally transformed chicken embryo fibroblasts. The primary location of each substrate is unaltered by transformation with Rous sarcoma virus (RSV). The tyrosine-phosphorylated species is localized with the nonphosphorylated species. Two of the proteins, of about 46,000 and 28,000 daltons, have a similar location. They are present in the high speed supernatant of cells homogenized in hypotonic buffer, and are soluble in nonionic detergent. The third protein, of about 39,000 daltons, is particulate when cells are homogenized in hypotonic buffer containing divalent cations, but approximately 30% is free in the high-speed supernatant when divalent cations are absent. This protein appears to be associated with the detergent-insoluble matrix when adherent cells are gently lysed in nonionic detergent in situ, but is soluble when the same cells are extracted with nonionic detergent in suspension. This suggests that one of the proteins are tightly associated with detergent-insoluble cytoskeletal structures, unlike the RSV transforming protein itself, which is the main tyrosine protein kinase known to be active in RSV-transformed cells.

Animals↗
PubMed1982

Expression of the PRC II avian sarcoma virus genome.

We found that the genomic RNA of the replication-defective avian sarcoma virus PRC II was 4.0 kilobases long. A Northern blot analysis of the viral RNAs present in PRC II-transformed cells showed that the PRC II genome was expressed as a single 4.0 kilobase mRNA species. In vitro translation of polyadenylic acid-containing 70S virion RNA yielded two highly related proteins of 110,000 and 105,000 daltons (P110 and P105), which were synthesized from messenger activity that sedimented as expected for the 4.0 kilobase PRC II genome (at 25 to 27S). P110 and P105 were identified as in vitro translation products of the PRC II genome by immunoprecipitation and tryptic peptide mapping and were the only PRC II-specific polypeptides detected by in vitro synthesis. In addition, we found that immune complexes prepared from PRC II 70S virion RNA in vitro translation products contained a tyrosine-specific protein kinase activity. A comparison of the in vitro- and in vivo-synthesized proteins revealed that PRC II-transformed cells also contained 110,000- and 105,000-dalton proteins, which were indistinguishable from in vitro-synthesized P110 and P105 by electrophoretic mobility and tryptic peptide analysis. Both P110 and P105 were present in producer cells and in seven individual nonproducer clones. A pulse-chase analysis showed that P105 was the primary translation product of the PRC II genome and that P110 was derived from P105 by post-translational modification. Under conditions of long-term labeling with [35S]methionine, P110 and P105 were present in a molar ratio of approximately 1:1. These results indicated that the transformation-specific product of the PRC II genome, previously referred to as a single component (P105), actually consists of two polypeptides related by post-translational modification.

Alpharetrovirus↗
PubMed1982

Phosphorylation and metabolism of the transforming protein of Rous sarcoma virus.

p60src, the transforming protein of Rous sarcoma virus, was found to contain 0.5 to 0.9 mol of total phosphate per mol of polypeptide. The protein is known to be phosphorylated at two sites, a serine in the amino-terminal domain and a tyrosine in the carboxy-terminal domain. Because our indirect analysis suggests that the serine is phosphorylated to approximately twice the extent of the tyrosine, we estimate that p60src contains approximately 0.3 to 0.6 mol of phosphoserine and 0.2 to 0.3 mol of phosphotyrosine per mol of polypeptide. p60src was found to represent approximately 0.02% of the total incorporated radioactivity in Rous sarcoma virus-transformed chick cells labeled with [35S]methionine for 48 h. This corresponds to approximately 500,000 molecules of p60src per cell. Pulse-chase experiments revealed that the half-life of p60src ranged from 2 to 7 h, depending on the strain of virus examined. The P60src of the Schmidt-Ruppin strain was significantly more stable than that of the Prague strain.

Avian Sarcoma Viruses↗
PubMed1982

The transforming proteins of PRCII virus and Rous sarcoma virus form a complex with the same two cellular phosphoproteins.

P105 and P110, the presumptive transforming proteins of PRCII avian sarcoma virus, have been found to be present in transformed chicken cells in two forms: as monomers and as part of a complex which contains both a 50,000-dalton and a 90,000-dalton cellular phosphoprotein. The 90,000-dalton cellular protein was found to be identical to one of the proteins in chicken cells whose synthesis is induced by stress. The 50,000-dalton protein was found to contain phosphotyrosine when isolated from the complex and therefore may be a substrate for the tyrosine protein kinase activity which is associated with P105 and P110. These same two cellular phosphoproteins have previously been shown to be present in a complex with pp60src, the tyrosine protein kinase which is the transforming protein of Rous sarcoma virus. However, not all avian sarcoma virus transforming proteins with associated tyrosine protein kinase activities form a complex efficiently with these cellular proteins. Little if any of P90, the putative transforming protein of Yamaguchi 73 virus, was found in a complex with the 50,000-dalton and 90,000-dalton cellular phosphoproteins.

Alpharetrovirus↗
PubMed1982

Identification of proteins encoded by the Gazdar murine sarcoma virus genome by in vitro translation and comparison with Moloney murine sarcoma virus 124.

The gene products of Gazdar murine sarcoma virus (Gz-MuSV) were identified by in vitro translation of Gz-MuSV virion RNA. An overlapping set of proteins with approximate molecular weights of 37,000 (37K), 33K, 24K, and 18K were synthesized from the transforming gene of Gz-MuSV, v-mosGz. In addition, Gz-MuSV-specific RNA directed the in vitro synthesis of a 62K gag gene protein and a 37.5K env gene-related product. The Gz-MuSV-specific in vitro translation products were compared with the in vitro translation products of M-MuSV 124, an independent isolate with a similar v-mos gene. This analysis showed that the 62K Gz-MuSV gag gene protein and the 37K, 33K, 24K, and 18K v-mosGz proteins were almost identical to the M-MuSV 124 62K (gag) and 37K, 33K, 24K, and 18K (v-mosMo) proteins that we previously identified and characterized. The 37.5K env gene product from Gz-MuSV does not have a correlate in the M-MuSV 124 translation products. These results were analyzed in the context of expectations based on similarities and differences in genetic organization of these two viral genomes.

Genes, Viral↗
PubMed1982

Two structurally and functionally different forms of the transforming protein of PRC II avian sarcoma virus.

The primary translation product of the PRC II avian sarcoma virus genome is a protein of 105,000 daltons (P105), and we have previously shown that approximately 50% of the P105 molecules are converted to molecules of 110,000 daltons (P110) by posttranslational modification. Fractionation of PRC II-infected cells showed that P105 was contained primarily in a nonionic detergent-soluble compartment, whereas P110 partitioned almost exclusively with a nonionic detergent-insoluble or crude cytoskeletal fraction. The tyrosine-specific protein kinase activity previously observed in immunoprecipitates which presumably contained both P110 and P105 was found predominantly in the P110-containing immunoprecipitates made from the cytoskeletal fraction and was essentially absent from the P105-containing immunoprecipitates prepared from the soluble fraction. Individual analysis of 32P-labeled P110 and P105 prepared by this fractionation technique revealed that P110 contained more phosphotyrosine per mole of protein than did P105. Examination of the tryptic peptide maps of 32P-labeled P110 and P105 suggested that the additional phosphotyrosine in P110 resulted from phosphorylation at discrete sites within the protein. From these experiments, we conclude that PRC II-infected cells contain two discrete forms, P105 and P110, of the transforming protein and that each of these proteins exhibits distinct structural and functional characteristics.

Alpharetrovirus↗
PubMed1982

Effect of duration and temperature of storage on serum analyte stability: examination of 14 selected radioimmunoassay procedures.

We determined appropriate temperatures for sample storage and the resulting stability of 14 analytes commonly radioimmunoassayed in the clinical laboratory. Serum specimens to be tested for concentrations of cholylglycine, cortisol, digoxin, ferritin, follitropin, immunoglobulin E, lutropin, prolactin, thyroxin (also blood-spot thyroxin), triiodothyronine, and triiodothyronine uptake could be stored for up to two weeks at room temperature, refrigerated, or frozen without any loss of analyte activity. Specimens for insulin testing require freezing or refrigeration, and specimens for gastrin testing should be stored at -70 degrees C for optimal results.

Blood Chemical Analysis↗
PubMed1981

Structural comparison of fibronectins from normal and transformed cells.

Comparative study of the structures of fibronectins from normal and transformed cells by partial proteolysis as well as by tryptic peptide fingerprinting and analysis of phosphorylation show that: 1) fibronectin molecules from normal and transformed cells probably have very similar primary structures; 2) the phosphorylation of fibronectin is a highly specific and conserved phenomenon; 3) fibronectin from both normal and transformed cells is phosphorylated only on serine residues; 4) although the major sites of phosphorylation in fibronectin are the same in normal and transformed cells, fibronectin from transformed cells appears to be phosphorylated to a much higher extent than that from normal cells.

Amino Acids↗
PubMed1981

Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells.

Addition of EGF to A431 cells at physiological concentrations causes a rapid three- to four-fold increase in the abundance of phosphotyrosine in cellular protein. The increase is essentially complete within 1 min and is maintained for several hours. No change in phosphotyrosine levels is found with fibroblast growth factor or insulin. Two phosphoproteins (molecular weights of 39 and 81 kd) containing phosphotyrosine appear de novo upon administration of EGF to A431 cells. The EGF receptor itself is a phosphoprotein containing phosphotyrosine as well as phosphoserine and phosphothreonine. Changes in the phosphorylation pattern of the EGF receptor are seen upon treatment of A431 cells with EGF. Increased phosphorylation of tyrosine is the most rapid response of cells to EGF known, and may play an important role in the biological effects of EGF.

Cell Division↗
PubMed1981

Analysis of transforming gene products from Moloney murine sarcoma virus.

We previously showed that in vitro translation of M-MuSV virion RNA yielded a 62 kd gag gene product and an overlapping set of four proteins with approximate molecular weights of 37,000, 33,000, 24,000 and 18,000. In this paper we show, by use of hybrid arrest translation with cloned recombinant DNAs containing M-MuSV v-mosMo sequences, that the 37, 33, 24 and 18 kd proteins are synthesized in their entirety from the v-mosMo gene. Analysis of the primary sequence of these proteins shows that each one is initiated independently from AUG codons within the v-mosMo gene and utilizes the long open reading frame predicted from the v-mosMo DNA sequence. Antisera against synthetic peptides corresponding to the C terminus of the predicted v-mosMo gene product precipitate all four in vitro v-mosMo proteins.

Amino Acid Sequence↗