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M S Collett

Publications and source records attributed to M S Collett.

At least 73 records · Page 4Linked to original sources
PubMed1980

Transforming gene product of avian sarcoma viruses and its homolog in uninfected cells.

The product of the avian sarcoma virus (ASV) transforming gene is a 60,000-dalton phosphoprotein, pp60src. Sera from mice and from an occasional rabbit bearing ASV-induced tumors are capable of immunoprecipitating a phosphoprotein of similar, but not identical, structure from normal avian and mammalian cells. This protein is presumed to be the product of the cellular sarc gene, and has been tentatively designated pp60sarc. Analysis of the tryptic phosphopeptides reveals that the viral and avian proteins have an apparently identical phosphoserine-containing peptide. They evidently also both have an analogous phosphothreonine residue surrounded by a different amino acid sequence. Immunoprecipitates containing either the viral or normal cellular pp60 protein catalyze the transfer of radiolabeled phosphate from [gamma-32P]-ATP to the heavy chain of immune rabbit IgG, therefore suggesting that both viral and cellular phosphoproteins may be protein kinases.

Alpharetrovirus↗
PubMed1979

The src gene product of transformed and morphologically reverted ASV-infected mammalian cells.

Morphological revertants of avian sarcoma virus transformed vole cells contain the sarcoma gene product (pp60src) in an enzymatically active form, suggesting that the presence of pp60src protein kinase activity is infussicient to induce morphological transformation. Structural analyses of pp60src from infected vole cell clones suggest that in one of the revertant clones on alteration in pp60src may be responsible for morphological reversion while in a second clone, reversion may result from an alteration in a cell gene product with which pp60src must interact. As these morphological revertant cells are tumorigenic, different cell components are required to interact with pp60src to facilitate the two events.

Animals↗
PubMed1979

Evidence that the avian sarcoma virus transforming gene product is a cyclic AMP-independent protein kinase.

The avian sarcoma virus transforming gene product pp60src has been partially purified by using ion exchange or immunoaffinity chromatography. These preparations contain a cyclic AMP-independent protein kinase activity capable of transferring radiolabel from [gamma-32P]ATP to immune rabbit IgG, casein, and the heavy chain purified from normal rabbit IgG. The casein kinase activity is specifically inhibited by anti-pp60src IgG. Comparison of the pp60src-protein kinase isolated from cells transformed by a wild-type ASV or by an ASV temperature-sensitive transformation mutant revealed that the latter product had a half-life of 3 min at 41 degrees C whereas that of the wild-type product was 20 min.

Animals↗
PubMed1979

A normal cell protein similar in structure and function to the avian sarcoma virus transforming gene product.

This report extends our previous studies concerning the identification and characterization of a protein from normal cells that is closely related to the avian sarcoma virus (ASV) transforming gene product pp60src. This normal cellular protein, which we have found in both avian and mammalian cells and have tentatively designated pp60sarc, was detected by immunoprecipitation of radiolabeled cell extracts with serum derived from both mice and rabbits bearing ASV-induced tumors. The normal cell pp60sarc is a 60,000-dalton phosphoprotein that is structurally similar, but not identical, to viral pp60src. The phosphorylation patterns of the normal cell and viral proteins are also similar: both contain two major phosphorylated residues, a phosphoserine located on the NH2-terminal 60% of the polypeptide and a phosphothreonine present on the COOH-terminal 40% of the molecule. In addition, the normal cell pp60sarc from both chicken and mammalian cells appears to have an associated protein kinase activity analogous to that previously described for the viral pp60src. The possible roles played by the normal cell protein pp60sarc and the ASV transforming protein pp60src in normal cellular growth and neoplastic disease, respectively, are discussed.

Alpharetrovirus↗
PubMed1979

Structural analysis of the avian sarcoma virus transforming protein: sites of phosphorylation.

The avian sarcoma virus (ASV) protein responsible for cellular transformation in vitro and sarcomagenesis in animals was studied structurally with special reference to the sites of phosphorylation on the polypeptide. The product of the ASV src gene, pp60src, is a phosphoprotein of 60,000 daltons. We found that pp60src contained two major sites of phosphorylation, one involving phosphoserine and the other involving phosphothreonine and possible addtional minor sites of phosphorylation. By using N-formyl[35S]methionyl-tRNAf as a radiolabeled precursor in the cell-free synthesis of the src protein in conjunction with partial proteolysis mapping, we determined that the major phosphoserine residue was located on the amino-terminal two-thirds of the molecule and that the phosphothreonine was located on the carboxy-terminal third. We further determined that the phosphorylation of pp60src in cell extracts involved at least two protein kinases, the one that phosphorylated the major serine site being cyclic AMP dependent and the other, acting on the threonine residue, being a cyclic nucleotide-independnet phosphotransferase. Finally, analysis of the pp60src isolated from cells infected with a temperature-sensitive src gene mutant of ASV revealed that phosphorylation of the major threonine residue was severely reduced when infected cells were grown at the nonpermissive temperature, whereas a phosphorylation pattern characteristic of the wild-type pp60src was observed at the permissive temperature. As pp60src has an associated protein kinase activity, the possible involvement of phosphorylation-dephosphorylation reactions in the functional regulation of ASV transforming protein enzymatic activity is discussed.

Alpharetrovirus↗
PubMed1979

Detection of the viral sarcoma gene product in cells infected with various strains of avian sarcoma virus and of a related protein in uninfected chicken cells.

Genetic analyses have defined a single gene (src) as that portion of the avian sarcoma virus (ASV) genome which encodes the protein directly responsible for ASV-induced neoplastic transformation. We have recently identified the polypeptide product of the src gene of the Schmidt-Ruppin (SR) strain of ASV, a 60,000-dalton phosphoprotein designated pp60(src), and have further determined that pp60(src) acts as a protein kinase. Essential to the identification and characterization of the pp60(src) protein of SR-ASV was the use of serum (TBR serum) from rabbits bearing SR-ASV-induced tumors. TBR serum was, however, strain specific, recognizing pp60(src) from SR-ASV-transformed cells only. We report here that sera from marmosets bearing tumors induced by the Bryan or SR strains of ASV (TBM sera) contain antibody which precipitates the transforming gene product from cells transformed by the SR, Bryan, Prague, or Bratislava strains of ASV. In contrast, rabbits bearing tumors induced by either the Bratislava or Bryan strains of ASV, or hamsters with SR-ASV-induced tumors did not produce antibody to pp60(src) from any strain of ASV. The 60,000-dalton polypeptides immunoprecipitated with TBM serum from cells transformed by each of the above virus strains are phosphoproteins. One-dimensional peptide mapping by limited proteolysis revealed that the pp60(src) proteins are structurally very similar, but not identical. Furthermore, all of the viral pp60(src) proteins have an associated phosphotransferase activity. In addition to detecting the viral src proteins, TBM serum was able to immunoprecipitate an antigenically related protein from normal uninfected avian cells.

Animals↗
PubMed1979

Initiation of DNA synthesis by the avian retrovirus reverse transcriptase in vitro: nature and location of the oligodeoxycytidylic acid primer binding site.

We have investigated the use of oligodeoxycytidylic acid [oligo(dC)] as a primer for the initiation of DNA synthesis by the avian retrovirus reverse transcriptase in vitro, employing the viral RNA genome as template. The addition of oligo(dC)(12-18) to viral 35S RNA results in a stimulation of DNA synthesis by the viral RNA-directed DNA polymerase comparable to that observed when oligo(dT) is employed as a primer. Under similar conditions neither oligo(dA)(12-18) nor oligo(dG)(12-18) was active as primer for transcription of the avian retrovirus genome. Several different approaches have been employed to localize the oligo(dC)(12-18) binding site on the viral genome, including isolation of poly(A)-containing fragments, competition hybridization, and RNase H hydrolysis. These analyses indicate that oligo(dC)(12-18) binds to a site approximately 2,000 to 3,000 nucleotides from the 3' terminus of the genome of transforming strains of avian sarcoma viruses and approximately 700 to 1,000 nucleotides from the 3' terminus of nontransforming avian retroviruses. Therefore, the major site of initiation of DNA synthesis by oligo(dC)(12-18) appears to be in the vicinity of the 3' end of the env gene and the 5' end of the src gene, although the presence of minor initiation sites located elsewhere on the viral genome cannot be excluded by these data. Characterization of oligonucleotides after pancreatic RNase hydrolysis and poly(C)-Sepharose chromatography of viral RNA directly demonstrates the presence of oligoguanylic acid residues in the avian sarcoma virus genome. DNA sequences transcribed from the oligo(dC) primer appear to be conserved in all of the avian leukosis-sarcoma viruses tested. The use of oligo(dC) as a tool for the production of specific complementary DNA probes is discussed.

Alpharetrovirus↗
PubMed1978

Evidence for splicing of avian sarcoma virus 5'-terminal genomic sequences into viral-specific RNA in infected cells.

The 5'-terminal nucleotide sequences of the avian sarcoma virus (ASV) genome are transcribed by the reverse transcriptase in vitro into a DNA transcript that represents the entire distance ( approximately 100 nucleotides) between the tRNA(Trp) primer molecule and the 5' terminus. We have used these DNA(100) transcripts in hybridization reactions with ASV-specific RNA from infected avian cells and find nucleotide sequences complementary to these transcripts on all of the various size classes of viral mRNA identified. Similar hybridization results were obtained with a specific DNA transcript complementary to viral genomic nucleotide sequences between the tRNA(Trp) primer molecule and up to, but not including, the terminal redundant sequences (DNA(70)), indicating that the observed hybridization of DNA(100) to all size classes of viral RNA in infected cells did not reflect hybridization of DNA(100) to the terminal redundant sequences at the 3' end of the viral genome. Escherichia coli RNase H hydrolysis of RNA.DNA hybrids consisting of genomic 35S RNA obtained from virus and DNA(100) transcripts indicated that viral genomic sequences complementary to these DNA transcripts were not present at sites distal to the ends of the RNA genome and therefore not adjacent to the corresponding gene sequences representing the various species of viral mRNA from infected cells. These studies suggest that the 5'-terminal genomic nucleotide sequences, or a portion thereof, are somehow added or "spliced" onto each ASV-specific mRNA species in infected cells either during or after transcription of proviral DNA for some as yet undetermined purpose.

Avian Sarcoma Viruses↗
PubMed1978

Protein kinase activity associated with the avian sarcoma virus src gene product.

Incorporation of phosphorus from [gamma-32P]ATP into protein was catalyzed by specific immunoprecipitates from avian sarcoma virus (ASV)-transformed avian and mammalian cells. This incorporation was observed only when antiserum from tumor-bearing rabbits able to specifically precipitate the ASV sarcoma gene product, p60src, was used to immunoprecipitate antigens from transformed cell lysates. Immunoprecipitates of extracts from normal cells or cells infected with a transformation-defective ASV mutant showed no activity in this assay, nor did any immune complexes formed with normal rabbit serum and any of the cell extracts tested. The expression of the protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) was growth temperature-dependent in cells infected with an ASV mutant temperature-sensitive for the transformation. These results on an enzymatic activity associated with the ASV transforming protein are discussed in terms of protein phosphorylation as a mechanism for viral transformation.

Alpharetrovirus↗
PubMed1978

Unwinding-like activity associated with avian retrovirus RNA-directed DNA polymerase.

The avian retrovirus RNA-directed DNA polymerase contains an activity that is capable of removing hydrogen bonds from duplex nucleic acid molecules. This "unwinding-like" activity appears to be specific in its action, affecting RNA.DNA and DNA.DNA duplex molecules but not RNA.RNA duplexes. Studies with defined RNA.DNA hybrid molecules (e.g., Rous sarcoma virus RNA and complementary DNAs representing specific regions of the Rous sarcoma virus genome) and DNA.DNA duplexes indicate that, although this activity can remove a portion of the hydrogen bonds from these double-stranded structures, complete separation of complementary strands is not accomplished. The unwinding-like activity exhibits sensitivities to temperature and monovalent and divalent cation concentrations. It can also remove a specific large oligonucleotide from the 5' end of the viral genome subsequent to RNase H hydrolysis of viral RNA complexed to DNA present at that terminus. This reverse transcriptase-associated unwinding-like activity is discussed with respect to recently proposed models of retrovirus proviral DNA synthesis.

Avian Leukosis Virus↗