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R R Friis

Publications and source records attributed to R R Friis.

At least 55 records · Page 3Linked to original sources
PubMed1982

Cytosolic malic dehydrogenase activity is associated with a putative substrate for the transforming gene product of Rous sarcoma virus.

A cellular protein of apparent Mr 34,000--36,000 was suggested as a possible physiological substrate for the protein kinase (EC 2.7.1.37) activity associated with the transforming gene product of Rous sarcoma virus. We find this protein to migrate with an apparent Mr of 38,000 in NaDodSO4/polyacrylamide gels. It was not separable from cytosolic malic dehydrogenase activity when purified by chromatography on DEAE-Sephacel, hydroxylapatite, poly(A)-Sepharose, and blue Sepharose, by gel filtration, and by isoelectric focusing. The Mr 38,000 protein as well as cytosolic malic dehydrogenase activity focused with a pI of 7.5. In gel filtration experiments, both displayed an apparent native Mr of 68,000. The male dehydrogenase activity contained in homogeneous preparations of the Mr 38,000 protein had a specific activity of up to 130 units/mg of protein. The recovery of the enzyme was 5--10% of the activity in the extract. Antiserum against the Mr 38,000 protein inactivated the malic dehydrogenase activity associated with the Mr 38,000 protein.

Animals↗
PubMed1982

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

The half-life of metabolically labeled pp60src of the Prague A strain of Rous sarcoma virus and of several transformation-defective, temperature-sensitive mutants was investigated by pulse-labeling infected cells with [35S]methionine, chasing for different times, and immunoprecipitating pp60src with tumor-bearing rabbit serum. These experiments showed that pp60src has a short half-life of approximately 60 min under normal physiological conditions and that the mutant pp60src proteins have similar half-lives to the wild type, irrespective of whether the cells are kept at the nonpermissive (42 degrees C) or permissive (35 degrees C) temperature. The half-life of the pp60src -associated kinase activity was determined by monitoring its decay by the immunoglobulin G heavy chain assay after the cells had been treated with several inhibitors of protein synthesis. In these experiments the kinase half-life was much longer than expected from the half-life of pp60src. The apparent contradiction between the half-lives of the kinase activity and the [35S]methionine-labeled pp60src protein could be resolved by the observation that treatment of cells with inhibitors of protein synthesis stabilized pp60src, resulting in a greatly extended half-life. Inhibitors of protein synthesis also extended the half-life of the gag precursor polypeptide, Pr76, suggesting that a host factor(s) may be required for the efficient intracellular processing of this polypeptide to the gag proteins.

Animals↗
PubMed1981

Early changes in the distribution and organization of microfilament proteins during cell transformation.

During the onset of transformation, Rous sarcoma virus-infected cells undergo characteristic morphological changes that reflect the biochemical events induced by the viral src gene. Temperature downshift experiments using chick embryo cells infected with transformation-defective temperature-sensitive viral mutants have shown two major morphological changes occurring at different times in the transformation process: ruffle-like flowers appear on the dorsal cell surface as early as 15 min after temperature shift, while later, between 6 and 12 hr, cytoskeletal stress fibers disappear and the cells round up. We report that flowers contain large accumulations of the cytoskeletal proteins actin, alpha-actinin, myosin and tropomyosin. Furthermore, since flowers stain very intensely with fluorescein-labeled phalloidin, a cyclopeptide that selectively binds to F-actin and not to G-actin, we suggest that these structures result from an early reorganization of microfilaments.

Animals↗
PubMed1981

Lack of correlation between pp60src kinase activity and transformation parameters in cells infected with temperature-conditional mutants of Rous sarcoma virus.

The pp60src kinase activity of cells infected with temperature-conditional mutants of Rous sarcoma virus (RSV), which induce only a partial transformation, was compared to the pattern of transformation parameters induced by these mutants. The tsGI251-infected cells were thermosensitive for hexose transport, fibrinolysis, morphological alterations and pp60src kinase activity, but cold-sensitive for loss of growth control. The tsGI253-infected cells were similar to tsGI251-infected cells except that they were only slightly cold-sensitive for loss of growth control and had a very low pp60src kinase activity which was temperature-insensitive. Thus, the pp60src kinase activity measured in vitro with IgG as a substrate did not correlate in a consistent way with any of the measured parameters of transformation.

Animals↗
PubMed1981

In vitro transformation with Rous sarcoma virus and the pp60src-associated protein kinase.

Fifteen transformation defective sensitive mutants of Rous sarcoma virus have been investigated to see if the expression of the pp60src-associated protein kinase activity correlated with other parameters of transformation such as altered growth control, morphological changes, increased hexose transport, and increased plasminogen activator protease synthesis. The expression of a protein kinase activity paralleled or preceded the onset of other parameters of transformation with but one exception: altered control of cell growth. The stability of the pp60src molecule in mutant-infected cells at the nonpermissive temperature was investigated with the finding that mutant pp60src did not show an increased turnover at the nonpermissive temperature as compared to wild type virus pp60src. Furthermore, it could be shown that pre-existing pp60src in mutant-infected cells maintained at the non-permissive temperature became activated after temperature shift to the permissive temperature. Temperature shift performed under conditions of inhibition of new protein synthesis with cycloheximide, puromycin, or emetine was followed by greatly increased protein kinase activity, and a parallel phosphorylation of pp60src itself in tyrosine residues. Morphological features of transformation could be demonstrated likewise under conditions of inhibition of protein synthesis.

Animals↗
PubMed1980

Integration sites of adenovirus type 12 DNA in transformed hamster cells and hamster tumor cells.

The patterns and sites of integration of adenovirus type 12 (Ad12) DNA were determined in three lines of Ad12-transformed hamster cells and in two lines of Ad12-induced hamster tumor cells. The results of a detailed analysis can be summarized as follows. (i) All cell lines investigated contained multiple copies (3 to 22 genome equivalents per cell in different lines) of the entire Ad12 genome. In addition, fragments of Ad12 DNA also persisted separately in non-stoichiometric amounts. (ii) All Ad12 DNA copies were integrated into cellular DNA. Free viral DNA molecules did not occur. The terminal regions of Ad12 DNA were linked to cellular DNA. The internal parts of the integrated viral genomes, and perhaps the entire viral genome, remained colinear with virion DNA. (iii) Except for line HA12/7, there were fewer sites of integration than Ad12 DNA molecules persisting. This finding suggested either that viral DNA was integrated at identical sites in repetitive DNA or, more likely, that one or a few viral DNA molecules were amplified upon integration together with the adjacent cellular DNA sequences, leading to a serial arrangement of viral DNA molecules separated by cellular DNA sequences. Likewise, in the Ad12-induced hamster tumor lines (CLAC1 and CLAC3), viral DNA was linked to repetitive cellular sequences. Serial arrangement of Ad12 DNA molecules in these lines was not likely. (iv) In general, true tandem integration with integrated viral DNA molecules directly abutting each other was not found. Instead, the data suggested that the integrated viral DNA molecules were separated by cellular or rearranged viral DNA sequences. (v) The results of hybridization experiments, in which a highly specific probe (143-base pair DNA fragment) derived from the termini of Ad12 DNA was used, were not consistent with models of integration involving true tandem integration of Ad12 DNA or covalent circularization of Ad12 DNA before insertion into the cellular genome. (vi) Evidence was presented that a small segment at the termini of the integrated Ad12 DNA in cell lines HA12/7, T637, and A2497-3 was repeated several times. The exact structures of these repeat units remained to be determined. The occurrence of these units might reflect the mechanism of amplification of viral and cellular sequences in transformed cell lines.

Adenoviridae↗
PubMed1979

Src Gene product from different strains of avian sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective, temperature-sensitive mutant and wild-type virus.

Sera from certain rabbits bearing Schmidt-Ruppin strain Rous sarcoma virus (RSV)-induced tumors precipitated p60(src) from chicken cells transformed by the homologous virus as well as by other strains [Prague strain RSV, Bryan high-titer strain RSV, and Bratislava 77 strain of avain sarcoma virus (ASV)], the molecular weights (M(r)s) ranging from 60,000 to 64,000. The p60(src) immunoprecipitated from cells transformed by each of these strains incorporated [gamma-(32)P]ATP into the M(r) 53,000 subunit of IgG, though with differing activities. No such protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) was observed when the following immunoprecipitates were used: from uninfected cells, from untransformed cells infected by Rous-associated virus, or from cells transformed by acute leukosis viruses, avian erythroblastosis virus, or myelocytoma virus 29. The kinase reaction had a pH optimum at pH 5.9 and an apparent K(m) for ATP of 4.9 +/- 2 muM, and was dependent on Mg(2+) (K(b) = 46 +/- 12 mM), for which Ca(2+) was no substitute. The kinase was cyclic AMP independent. In order to test whether the protein kinase reaction is directly catalyzed by p60(src), we compared the in vitro temperature sensitivities of the kinase activities from cells infected by transformation-temperature-sensitive mutant and parental wild-type virus. The first-order rate constant for the inactivation of the kinase from extracts of cells infected by the mutant virus was 2-fold greater than that from cells infected by wild-type virus. This result implicates the protein kinase as an enzymatic activity of the src gene product, the p60(src). Concomitant with the loss of the kinase activity by heat inactivation, p60(src) loses 60-70% of its phosphate content. The kinetics of dephosphorylation exactly parallel those for the inactivation of the kinase activity, suggesting that the p60(src) kinase is itself dependent on phosphorylation for its activity.

Animals↗
PubMed1979

Antigenic determinants specific to the envelope glycoprotein of exogenous avian tumor viruses.

Antibody in the sera of 15B X 7(2) chickens was measured for reactivity to determinants of the envelope glycoprotein of endogenous and exogenous avian tumor viruses. The pattern of reactivity of animals infected with exogenous sarcoma virus served to operationally define a class of determinants present on the envelope glycoprotein of exogenous viruses and absent from that of the endogenous virus Rous-associated virus-0.

Animals↗
PubMed1979

Two avian sarcoma virus mutants with defects in the DNA polymerase-RNase H complex.

Two mutants of avian sarcoma virus which exhibit different phenotypes have been analyzed for the properties of their RNA-dependent DNA polymerase and RNase H activities. LA 338 is a complex multiple mutant with at least one lesioneach in transformation and replication functions. The purified RNA-dependent DNA polymerase-RNase H complex from the mutant is twofold more thermolabile than that from the wild-type parent. A peculiarity of this mutant is that the ability of the enzyme to respond to synthetic template-primers is lost more rapidly than is the response to native RNA as template. The mutant enzyme cannot be protected from inactivation by the addition of synthetic template-primers. LA 672 represents a different phenotype among reverse transciptase mutant, showing a "late"-acting block in replication which affects only production of progeny by infected cells grown at the nonpermissive temperature. The purified DNA polymerase-RNase H complex of LA 672 is not thermolabile; rather, progeny grown at the nonpermissive temperature yield purified enzyme with a 20-fold-reduced specific activity in both DNA polymerase and RNase H. The content of reverse transcriptase protein in such noninfectious progeny, furthermore, did not appear to be significantly diminished since immunologically active enzyme could be demonstrated in a competition test for anti-reverse transcriptase antibody and since beta and alpha subunits of reverse transcriptase could be identified after polyacrylamide gel electrophoresis of partially purified enzyme preparations. The amounts of beta and alpha from the mutant were about twofold lower.

Avian Sarcoma Viruses↗