In vitro association of unique species of cellular 4S RNA with 35S RNA of RNA tumor viruses

Waters, Larry C.; Mullin, Beth C.; Ho, Ti; Yang, Wei‐Pang

doi:10.1016/0006-291x(74)90267-8

Cited by 10 publications

(11 citation statements)

References 12 publications

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“…2A). This result is very similar to that observed when 4S RNA of AKR mouse embryo cells is hybridized with 35S RNA of either AKR murine leukemia virus or AMV (9). Comparison of Fig.…”

Section: Methodssupporting

confidence: 76%

“…We have recently reported that distinct species of tRNA from a variety of mammalian cells in culture will hybridize with 35S RNA of AKR murine leukemia virus and of AMV in vitro (9). We now report that distinct species of 4S RNA of chick embryo cells will hybridize in vitro with AMV 35S RNA and that a major component of this RNA is tryptophan tRNA.…”

mentioning

confidence: 89%

“…In the absence of 35S RNA no radioactivity sediments in the region of fractions 1-13 (see ref. 9). The results shown represent one-half of the hybridization mixture.…”

Section: Methodsmentioning

confidence: 99%

“…The cells were grown in medium 199 in either petri plates or in roller bottles. 32p labeling was in medium 199 (containing 5% fetal calf serum) with 10% of the usual phosphate content, and RNA extractions were the same as reported (9). RPC-5 column packing was prepared as described (10).…”

Section: Methodsmentioning

confidence: 99%

“…The 35S RNA was prepared from AMV-containing chicken plasma (kindly provided by Dr. J. W. Beard of Life Sciences, Inc., Gulfport, Fla.) as described (9). 4S RNA of chick embryo cells, both radioactively labeled and nonlabeled, was prepared from primary cultures.…”

Section: Methodsmentioning

confidence: 99%

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Ability of tryptophan tRNA to hybridize with 35S RNA of avian myeloblastosis virus and to prime reverse transcription in vitro.

Waters¹,

Mullin²,

Ho³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

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Selected species of 4S RNA of chick embryo cells will hybridize in vitro with 35S RNA of avian myeloblastosis virus. A major tRNA component of the hybridizable 4S RNA is tryptophan tRNA. A hybrid prepared from purified tryptophan tRNA and 35S RNA of avian myeloblastosis virus in vitro is an efficient templateprimer for DNA synthesis catalyzed by reverse transcriptase (RNA-dependent DNA polymerase).Initiation sites or primers are required for reverse transcription of 35S RNA of RNA tumor viruses in vitro. It has been reported that in avian myeloblastosis virus (AMV) (1) and in Rous sarcoma virus (2, 3) the primer is a low-molecularweight RNA. In AMV, 4S RNA with structural (4) and functional (5) properties of transfer RNA has been shown to be closely associated with the viral 70S RNA. A 4S RNA capable of priming DNA synthesis in Rous sarcoma virus has been isolated from the virus and has been shown to be a single species (6, 7). A tRNA with similar properties has been demonstrated in both avian and mammalian cells and was reported to be specifically aminoacylatable with tryptophan (8).We have recently reported that distinct species of tRNA from a variety of mammalian cells in culture will hybridize with 35S RNA of AKR murine leukemia virus and of AMV in vitro (9). We now report that distinct species of 4S RNA of chick embryo cells will hybridize in vitro with AMV 35S RNA and that a major component of this RNA is tryptophan tRNA. Furthermore, this tRNA, when hybridized to AMV 35S RNA, will prime the synthesis of DNA in the presence of purified AMV RNA-dependent DNA polymerase. MATERIALS AND METHODSThe 35S RNA was prepared from AMV-containing chicken plasma (kindly provided by Dr. J. W. Beard of Life Sciences, Inc., Gulfport, Fla.) as described (9). 4S RNA of chick embryo cells, both radioactively labeled and nonlabeled, was prepared from primary cultures. The cells were grown in medium 199 in either petri plates or in roller bottles. 32p labeling was in medium 199 (containing 5% fetal calf serum) with 10% of the usual phosphate content, and RNA extractions were the same as reported (9). RPC-5 column packing was prepared as described (10). Chromatographic conditions were optimized for the separation of eukaryotic tRNAs by C. K. solution of 10 mM Tris-HCl (pH 7.6); 0.1 M NaCl; 0.1% sodium dodecyl sulfate; and 1 mM EDTA. The total mixture, 385 JAl, was contained in 1-ml ampules, which were flushed with nitrogen before sealing. The contents were heated to 800 for 5 min and quickly chilled in ice water. The sample was then heated at 650 for 30 min, and the temperature of the water bath reset and maintained at 550 for an additional 14-16 hr (approximately 75 min were required for the temperature to reach 550). After hybridization the samples were quickly chilled in ice water, and the hybridized 4S RNA was separated from the free 4S RNA by sucrose density gradient centrifugation at 50,000 rpm in an SW 56 rotor through a 10-30% sucrose gradient in a solution of 10 mM Tris-HCl (pH 7.6); 10 mM NaCl; 0.1% sodium dod...

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“…2A). This result is very similar to that observed when 4S RNA of AKR mouse embryo cells is hybridized with 35S RNA of either AKR murine leukemia virus or AMV (9). Comparison of Fig.…”

Section: Methodssupporting

confidence: 76%

mentioning

confidence: 89%

“…In the absence of 35S RNA no radioactivity sediments in the region of fractions 1-13 (see ref. 9). The results shown represent one-half of the hybridization mixture.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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