Mapping RNase T1-resistant oligonucleotides of avian tumor virus RNAs: sarcoma-specific oligonucleotides are near the poly(A) end and oligonucleotides common to sarcoma and transformation-defective viruses are at the poly(A) end

Wang, L H; Duesberg, Peter H.; Beemon, Karen; Vogt, Peter K.

doi:10.1128/jvi.16.4.1051-1070.1975

Cited by 239 publications

(120 citation statements)

References 28 publications

(90 reference statements)

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“…The genomic RNA of the td mutants is about 15% smaller than the parental RSV RNA, corresponding to the loss of ~ 1,500 nucleotides (7)(8)(9)(10). The analysis of oligonucleotides obtained after hydrolysis of the RNAs of parental RSV and of td mutants showed that the deletions occur in a specific portion of the RSV genome, located near the 3' terminus (11,12). Since this particular segment of the genome, deleted in td virus RNA, is also absent in naturally occurring nonsarcomagenic oncoviruses (avian leukosis viruses), it is thought to be responsible for sarcomagenic transformation, and was designated as the src gene (13)(14)(15).…”

mentioning

confidence: 99%

Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants.

Hanafusa¹,

Halpern²,

Buchhagen³

et al. 1977

The Journal of Experimental Medicine

147

101

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Transformation-defective (td) mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), which contains deletions in the gene responsible for transformation (src gene), are unable to transform chicken embryo fibroblasts in vitro. Injection of some of these td mutants into newborn chickens resulted in the formation of sarcomas from which sarcoma virus was unfailingly recovered. The possibility that transforming RSV was present in the td virus preparations was excluded by further purification of the td viruses. Morphology of the foci induced by the newly recovered sarcoma virus was distinct from that of foci induced by the parental Schmidt Ruppin strain of RSV. It is suggested that the new sarcoma virus was generated as a result of the genetic interaction between the genomes of td virus and chicken cells.

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mentioning

confidence: 99%

Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants.

Hanafusa¹,

Halpern²,

Buchhagen³

et al. 1977

The Journal of Experimental Medicine

147

101

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“…It was shown to be free from detectable DNase, RNases I and III, and exonucleases, as measured by polyacrylamide gel electrophoresis of bacteriophage VOL. 22,1977 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from T7 DNA and T7 RNA (9). The specific activity of the enzyme preparation was 17,000 U/mg of protein (1 U is defined as the hydrolysis of one nmol of RNA in a DNA-RNA hybrid in 1 h at 370C).…”

mentioning

confidence: 99%

New procedure for the direct analysis of in vitro reverse transcription of Rous sarcoma virus RNA

Darlix¹,

Bromley²,

Spahr³

1977

J Virol

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Based on the observation that in vitro transcription of Rous sarcoma virus (RSV) RNA by avian myeloblastosis virus DNA polymerase renders the RNA progressively more sensitive to Escherichia coli RNase H digestion, a new procedure for the in situ analysis of this process has been developed. In vitro transcription products of 32P-labeled RSV RNA are first treated with RNase H, the resistant fraction is then digested to completion with RNase T1, and the oligonucleotides are analyzed by a fingerprint technique. By using the established order of these oligonucleotides along the RNA molecule, a comparison of the yields of each oligonucleotide, before and after transcription, allows qualitative and quantitative in situ analyses of the transcription process. Using this new procedure, we find that upon transcription of purified RSV RNA, DNA synthesis occurs mainly at three.sites, one near the 5' end and two near the center of the subunit RNA molecule, and that most of these RNA molecules are competent templates for limited transcription at these specific sites. We also show that purified RSV 70S RNA contains a low-molecular-weight DNA hybridized to a nucleotide sequence near the center of the subunit molecule. Furthermore, we find that the low-molecular-weight nucleic acid fraction extracted from purified RSV virions contains DNA that can hybridize to RSV 70S RNA and that the virion DNA in such hybrids can function as a primer for an extensive in vitro reverse transcription.Studies on in vitro transcription of avian tumor virus RNA have been performed, using both detergent-disrupted virus particles and a reconstructed system consisting of purified viral 70S RNA and avian myeloblastosis virus (AMV) DNA polymerase. These studies have provided the following information. (i) Most of the in vitro synthesized DNA is of small size, with a mean length of about 400 to 700 nucleotides (20); however, under certain experimental conditions, a small fraction of the product has been found to be of a much larger size, possibly up to 10,000 nucleotides (6). (ii) Much of the in vitro synthesized DNA contains nucleotide sequences complementary to only a small fraction of that of the viral RNA subunit (11); however, the results of hybridization experiments using excess DNA over RNA have indicated that the in vitro synthesized DNA product contains all of the nucleotide sequences complementary to that of the viral RNA (11). (iii) A large part of the complementary Rous sarcoma virus (RSV) DNA synthesized in vitro has been found to be covalently linked to a tRNATr" (8,13), and this tRNA occupies a unique site near the 5' end of the RSV RNA subunit molecule (21).The precise mechanism of this in vitro tran-scription reaction remains to be elucidated. Most of our understanding of this process derives from experiments in which in vitro synthesized viral DNA is hybridized to viral RNA and the hybrids formed are characterized. Recently, a detailed analysis of these hybrids has been reported (3), based on the established map of large T. RNase oligonucl...

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“…One utilized serum from mammals bearing virus-induced tumors for immunoprecipitation of radiolabeled proteins from transformed cells; a technique that had proved successful in the identification of nonstructural virus-encoded polypeptides of DNA tumor viruses (11,12). The other was cell-free translation of the subgenomic 3'-third of the RSV genome, the region of the genome that had previously been shown to contain the src gene (13) . These techniques led to the identification of a transformation-specific antigen in all RSV-transformed cells examined (14,15) and to the identification of a polypeptide among the products of cellfree translation that was synthesized only when src-containing RNA was translated (16) .…”

Section: Identification Of the Src Gene Productmentioning

confidence: 99%

Molecular events in cells transformed by Rous Sarcoma virus.

Erikson¹,

Purchio²,

Erikson³

et al. 1980

The Journal of Cell Biology

117

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The Rous sarcoma virus (RSV) transforming gene product has been identified and characterized as a phosphoprotein with a molecular weight of 60,000, denoted pp60sr°. Partially purified pp60" displays a closely associated phosphotransferase activity with the unusual specificity of phosphorylating tyrosine residues in a variety of proteins . That the enzymatic activity observed is actually encoded by the RSV-transforming gene is indicated by the comparison of the pp60"-protein kinase isolated from cells transformed by a wild-type RSV or by a RSV temperature-sensitive transformation mutant ; these experiments revealed that the latter enzyme had a half-life of 3 min at 41°C, whereas that of the wild-type enzyme was 20 min . Evidence is now beginning to accumulate showing that viral pp60"expresses its protein kinase activity in transformed cells as well as in vitro because at least one cellular protein has been identified as a substrate for this activity of pp60".Although the protein kinase activity associated with pp60" is itself cyclic AMP (CAMP) independent, the molecule contains at least one serine residue that is directly phosphorylated by the cellular CAMP-dependent protein kinase, thus suggesting that the viral transforming gene product may be regulated indirectly by the level of CAMP. The significance of this latter observation must be regarded from the point of view that the RSV src gene is apparently derived from a normal cellular gene that seemingly expresses in normal uninfected cells a phosphoprotein structurally and functionally closely related to pp60". This cellular protein, found in all vertebrate species tested, also is a substrate for a CAMP-dependent protein kinase of normal cells, and, therefore, may have evolved to function in a regulatory circuit involving CAMP .Rous sarcoma virus (RSV) causes rapid and efficient transformation of cells in culture and tumor formation in several species . The malignant changes are the consequence of the expression of a single viral gene termed src for sarcoma (1, 2). The src gene is not required for virus replication ; spontaneous deletions of the src gene can occur in the viral genome resulting in the generation of nonconditional transformation-defective viruses that replicate normally (3) . Furthermore, a number of RSV temperature-sensitive (ts) mutants that have an altered capacity to transform cells have also been isolated (4-6) . Avian or mammalian cells infected with a is mutant can be reversibly switched from the normal to the transformed phenotype by the appropriate shift in the temperature at which the cells are maintained . These and other features of this tumor virus-host system make it useful for the study of the biochemistry of one form of malignant transformation .

show abstract

Mapping RNase T1-resistant oligonucleotides of avian tumor virus RNAs: sarcoma-specific oligonucleotides are near the poly(A) end and oligonucleotides common to sarcoma and transformation-defective viruses are at the poly(A) end

Cited by 239 publications

References 28 publications

Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants.

Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants.

New procedure for the direct analysis of in vitro reverse transcription of Rous sarcoma virus RNA

Molecular events in cells transformed by Rous Sarcoma virus.

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