Search PubMed⌕ Search

Biomedical subjects

T Hunter

Publications and source records attributed to T Hunter.

At least 379 records · Page 21Linked to original sources
PubMed1980

Studies on the structure and function of the avian sarcoma virus transforming-gene product.

The protein kinase activity associated with pp60src of a mutant of RSV temperature sensitive for transformation was shown to be sixfold more labile than that of its wild-type parent at 45 degrees C when pp60src's synthesized in vitro were compared. Thus, a mutant that is temperature sensitive for transformation has a temperature-sensitive protein kinase activity. Analysis of the levels of protein kinase activity in immunoprecipitates from cells infected with four different temperature-sensitive mutants of RSV led to the surprising finding that two mutants had barely detectable levels of protein kinase activity even at the permissive temperature, whereas two others had levels of activity at the nonpremissive temperatures that were as great as 40% that of wild-type pp60src. Protein kinase activity of pp60src of NY68 was partially renatured when cells were shifted from 41 degrees C to 36 degrees C. This reactivation occurred in less than an hour and did not require protein synthesis. It was found that pp60src synthesized in vitro is phosphorylated. Finally, the methionine-containing tryptic peptides of pp60sarc immunoprecipitated from uninfected chick cells were very similar to those of viral pp60src.

Animals↗
PubMed1980

Temperature-sensitive transformation by Rous sarcoma virus and temperature-sensitive protein kinase activity.

The transforming protein of Rous sarcoma virus, p60src, has associated with it a protein kinase activity. We examined whether a correlation exists between the cellular concentration of enzymatically active p60src and the degree to which chick cells are transformed by mutants of Rous sarcoma virus which are temperature-sensitive for transformation. Such a correlation does exist, but cells infected with some mutants could be shown to contain, at the nonpermissive temperature, an amount of protein kinase activity equal to 30 to 40% of that in a wild-type transformed cell. We quantified the amount of virus-induced protein kinase activity by precipitation of p60src with an excess of antitumor antiserum. Our initial measurements of activity were serious underestimates, due to the lability of the protein kinase activity associated with p60src of at least four temperature-sensitive mutants. In fact, no activity at all was associated with p60src of tsLA90 when immunoprecipitation was performed by standard means. However, when immunoprecipitation was performed with procedures which minimize inactivation, it became apparent both that cells transformed by tsLA90 contained protein kinase activity and that cells infected with either NY68 or BK5 contained at the nonpermissive temperature, one-third to one-half as much activity as wild-type transformed cells. This level of activity was much more than that arising from p60sarc in uninfected cells. In uninfected cells we found an amount of protein kinase activity which varied from 3 to 5% as much as that in a virally transformed cell. The lability of the protein kinase activity of each of these mutants is a further demonstration that this activity is essential for the transformation of cells by Rous sarcoma virus. So as to explain the high protein kinase levels in cells infected with NY68 and BK5 at the nonpermissive temperature, the idea that transformation may be a response to a small quantitative change in the total activity of p60src and the possibility that there may be more than one viral function which is essential for transformation are discussed.

Animals↗
PubMed1979

Regions of the polyoma genome coding for T antigens.

The early region of the polyoma genome encodes three T antigens. We have analyzed the organization of the coding regions for the T antigens, using the nucleotide sequence of polyoma DNA and peptides derived from purified, radio-labeled T antigens, separated by two-dimensional electrophoresis and chromatography. We compared the peptides, predicted from the nucleotide sequence of the DNA, with those derived from the purified T antigens. We also compared chemically synthesized peptides, predicted from the DNA sequence, with observed peptides. The results show that the three polyoma T antigens are encoded in overlapping regions of the viral DNA, translated, in part, in two different reading frames.

Antigens, Viral↗
PubMed1979

Post-translational modification of actins synthesized in vitro.

It has been shown in two different ways that beta and gamma actins synthesized in vitro are acetylated and that the minor species of actin, delta and epsilon, are nonacetylated forms of beta and gamma actin, respectively. Firstly, additon of acetyl-CoA to the wheat germ system translating poly(A)-containing RNA from unfused rat L6 myoblasts, resulted in an increase in the synthesis of beta and gamma actins at the expense of delta and epsilon actins. Secondly, beta and gamma actins were labeled when synthesized in vitro in the presence of [3H]acetyl-CoA. No label was detectable in delta and epsilon actins. By extrapolation this indicates that beta and gamma actin are acetylated in vivo, probably at the N-terminus. beta and gamma actins synthesized in vivo contain a N tau-methylhistidine residue, but no methylation of beta and gamma actins synthesized in vitro was detectable, using S-[3H]adenosylmethionine as a methyl donor.

Acetyl Coenzyme A↗
PubMed1979

An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates.

Polyoma T antigen immunoprecipitates contain a protein kinase-like activity which preferentially phosphorylates material of 50-60,000 daltons molecular weight. Phosphorylation is not diminished in extracts of polyoma tsA mutant-infected cells shifted to the nonpermissive temperature late in infection, conditions which inactivate the large T antigen. Phosphorylation is reduced or absent in cells infected with polyoma host range nontransforming (hr-t) mutants, which have defective small and medium T antigens. The major acceptor of phosphate is not the heavy chain of immunoglobulin, but appears to be the polyoma medium T antigen. The large T antigen is also phosphorylated, but usually to a lower specific activity. In terms of acid and alkali sensitivity and electrophoretic and chromatographic mobility in one and two dimensions, the phosphorylated residue behaves identically to phosphotyrosine and differently than phosphorylated serine, threonine, lysine and histidine.

Animals↗
PubMed1979

Association of simian virus 40 T antigen with simian virus 40 nucleoprotein complexes.

Viral nucleoprotein complexes were extracted from the nuclei of simian virus 40 (SV40)-infected TC7 cells by low-salt treatment in the absence of detergent, followed by sedimentation on neutral sucrose gradients. Two forms of SV40 nucleoprotein complexes, those containing SV40 replicative intermediate DNA and those containing SV40 (I) DNA, were separated from one another and were found to have sedimentation values of 125 and 93S, respectively. [(35)S]methioninelabeled proteins in the nucleoprotein complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to VP1, VP3, and histones, a protein with a molecular weight of 100,000 (100K) is present in the nucleoprotein complexes containing SV40 (I) DNA. The 100K protein was confirmed as SV40 100K T antigen, both by immunoprecipitation with SV40 anti-T serum and by tryptic peptide mapping. The 100K T antigen is predominantly associated with the SV40 (I) DNA-containing complexes. The 17K T antigen, however, is not associated with the SV40 (I) DNA-containing nucleoprotein complexes. The functional significance of the SV40 100K T antigen in the SV40 (I) DNA-containing nucleoprotein complexes was examined by immunoprecipitation of complexes from tsA58-infected TC7 cells. The 100K T antigen is present in nucleoprotein complexes extracted from cells grown at the permissive temperature but is clearly absent from complexes extracted from cells grown at the permissive temperature and shifted up to the nonpermissive temperature for 1 h before extraction, suggesting that the association of the 100K T antigen with the SV40 nucleoprotein complexes is involved in the initiation of SV40 DNA synthesis.

Antigens, Neoplasm↗
PubMed1979

Structural relationship between the 100,000- and 17,000- molecular-weight T antigens of simian virus 40 (SV40) as deduced by comparison with the SV40-specific proteins coded by the nondefective adenovirus type 2-SV40 hybrid viruses.

The two-dimensional peptide maps of the methionine-containing tryptic peptides of the 100,000-molecular-weight (100K) and 17K T antigens of simian virus 40 (SV40) have been compared. The two proteins share nine methionine-containing tryptic peptides in common. The 17K T antigen has two peptides not found in the 100K T antigen, and the 100K T antigen has 14 unique peptides. The peptide maps of the 100 K and 17K T antigens were also compared with those of the SV40-specific proteins found in cells infected by the nondefective adenovirus type 2-SV40 hybrid viruses, which we have previously shown are encoded by defined sequences within the early region of SV40 (K. Mann, T. Hunter, G. Walter, and H.K. Linke, J. Virol. 24:151-169, 1977). This comparison shows that the 100K and 17K T antigens share common N-terminal sequences coded for between 0.65 and 0.59 map units on the SV40 genome. Furthermore, none of the sequences in the 17K T antigen arises from the region between 0.54 and 0.18 map units. We deduce that the sequences unique to the 17K T antigen originate between 0.59 and 0.54 map units. This type of structural relationship between the 100K and 17K T antigens fits well with the proposed model (L.V. Crawford, C.N. Cole, A. E. Smith, E. Paucha, P. Tegtmeyer, K. Rundell, and P. Berg, Proc. Natl. Acad. Sci. U.S.A. 75:117-121, 1978) for the expression of the early region of SV40.

Adenoviridae↗
PubMed1979

Polymorphism of avian sarcoma virus src proteins.

The src gene products of seven different avian sarcoma viruses were compared. In vitro translation of virion RNA yielded products identified unambiguously as p60src in the case of two stocks of the Schmidt-Ruppin strain, three stocks of the Prague strain, the Bryan strain, and the Bratislava 77 strain of avian sarcoma virus. Differences in the electrophoretic mobility of these seven p60src proteins in sodium dodecyl sulfate-polyacrylamide gels, corresponding to variation in the apparent molecular weights ranging from 56,000 to 60,500, were observed. Antigenic variability was also found; only three of the seven viruses tested encoded a p60src, which was precipitated by antisera derived from rabbits bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Examination of the methionine-containing tryptic peptides of the seven ;60src proteins by two-dimensional mapping revealed four common peptides but marked variability in the five to eight other peptides in each protein. Clear differences in the peptide maps of p60src were observed, both between different strains of virus and within strains. In the three cases examined, p60src synthesized in transformed cells was found to be essentially identical to that synthesized in vitro. We conclude that there is significant polymorphism in the p60src proteins of the avian sarcoma viruses.

Avian Sarcoma Viruses↗
PubMed1979

Product of in vitro translation of the Rous sarcoma virus src gene has protein kinase activity.

In vitro translation of Rous sarcoma virus virion RNA resulted in the synthesis of a protein kinase which, when immunoprecipitated with antitumor serum, phosphorylated the immunoglobulin heavy chain. Even though in vitro translation of virion RNA resulted in the synthesis of a number of polypeptides which were recognized by antitumor serum, control experiments demonstrated that an immunoprecipitable protein kinase activity was found only when an immunoprecipitable p60src, the polypeptide product of the src gene, was synthesized. A protein kinase with similar properties was therefore intimately associated with p60src which was synthesized in vitro in the reticulocyte lysate, just as it is with p60src which is obtained from transformed chick and mammalian cells. It is therefore highly unlikely that this association is artifactual. ts NY68 is a mutant of Rous sarcoma virus which is able to transform cells at 36 but not at 41 degrees C. In vitro translation of ts NY68 virion RNA at 30 degrees C resulted in efficient synthesis of immunoprecipitable p60src, but very inefficient synthesis of an immunoprecipitable protein kinase. The p60src obtained by in vitro translation of wild-type virion RNA was more than 20-fold more active as a protein kinase than was that obtained from ts NY68 RNA. The correlation in the case of ts NY68 of a deficiency in protein kinase activity with an inability to transform cells at high temperature suggests that the protein kinase activity associated with p60src is indeed critical to cellular transformation.

Avian Sarcoma Viruses↗
PubMed1978

Characterization of T antigens in polyoma-infected and transformed cells.

Polyoma-infected 3T6 cells contain a number of proteins precipitable by serum from rats carrying polyoma-induced tumors. The virus codes for three species having apparent molecular weights of 90,000, 60,000 and 22,000 daltons, as determined by polyacrylamide gel electrophoresis (90K, 60K and 22k). The 90K and 22K species produced by a large plaque and a small plaque wild-type polyoma have similar mobilities, but the 60K species produced by the large plaque wild-type. In cells infected by each of seven polyoma tsA mutants, the 90K species is unstable at the nonpermissive temperature, while the 60K and 22K species are stable. In cells infected by a mutant carrying a deletion between roughly 98 and 3 map units in the early region of the viral genome, the 22K species is present, but the 90K and 60K species are absent. Tryptic peptide analysis of the isolated 90K, 60K and 22K species shows that the three species have common N terminal regions. The 60K and 22K species contain amino acid sequences not found in the 90K species , and the 60K species has several unique, methionine-containing peptides not found in either the 22K or 90K species. Two polyoma-transformed BHK cell lines do not have detectable amounts of the 90K protein.

Antigens, Neoplasm↗
PubMed1978

Translation of polyoma virus T antigens in vitro.

Polyoma virus-specific RNA isolated from the cytoplasm of lytically infected cells can be translated in vitro to yield three T antigens, of Mrs approximately 90,000, 60,000, and 22,000. The tryptic peptide patterns of the T antigens synthesized in vitro are similar or identical to the patterns of the corresponding proteins in polyoma-infected cells. All three proteins incorporate methionine donated from initiator tRNA in vitro. Polyoma cRNA codes for a protein that is slightly larger than the 22,000 T antigen and that, by other criteria, is similar to the 22,000 T antigen. Translation of cRNA does not yield the 90,000 and 60,000 T antigens, suggesting that the generation of the mRNAs for these T antigens requires the removal of intervening sequences. The mRNA for the 90,000 T antigen is smaller than the mRNAs for the 22,000 and 60,000 proteins. All three proteins share common NH2-terminal sequences, and the 60,000 T antigen may be translated partially in a different reading frame from sequences also coding for the 90,000 T antigen. The demonstration that polyoma virus codes for three different T antigens raises the possibility that all three proteins may be involved in cell transformation.

Antigens, Neoplasm↗
PubMed1978

Characterization of the mRNA's for the polyoma virus capsid proteins VP1, VP2, and VP3.

Polyadenylated cytoplasmic RNA from polyoma virus-infected cells can be translated in the wheat germ system to yield all there polyoma virus capsid proteins, VP1, VP2, and VP3. The translation products of RNA selected from total cytoplasmic RNA of infected cells by hybridization to polyoma virus DNA showed a high degree of enrichment for VP1, VP2, and VP3. The identity of the in vitro products with authentic virion proteins was established in two ways. First, tryptic peptide maps of the in vitro products were found to be essentially identical to those of their in vivo counterparts. Second, the mobilities of the in vitro products on two-dimensional gels were the same as those of viral proteins labeled in vivo. VP1, VP2, and vp3 were all labeled with [35S] formylmethionine when they were synthesized in the presence of [35S] formylmethionyl-tRNAfmet. We determined the sizes of the polyadenylated mRNA's for VP1, VP2, and VP3 by fractionation on gels. The sizes of the major mRNA species for the capsid proteins are as follows: VP2, 8.5 X 10(5) daltons; VP3, 7.4 X 10(5) daltons; and VP1, 4.6 X 10(5) daltons. We conclude that all three viral capsid proteins are synthesized independently in vitro, that all three viral capsid proteins are virally coded, and that each of the capsid proteins has a discrete mRNA.

Capsid↗