Viral RNA of murine sarcoma virus produced by a hamster-mouse somatic cell hybrid

Tsuchida, Nobuo; Long, Cedric; Hatanaka, Masakazu

doi:10.1016/0042-6822(74)90377-8

Cited by 19 publications

(15 citation statements)

References 12 publications

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“…In relation to the main theme of this communication, the retention of 30S M-MSV-specific RNA in the original nonproducer rat tumor parallels the situation found in M-MSV hamster tumors and KiSV-transformed mouse cells where 30S virus-specific RNAs are also found (7,22,27).…”

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confidence: 89%

“…KiSVspecific [3H]DNA (58-2TS DNA) was prepared as follows: 58-2T [3H]DNA (7,27) Preparation of High-Molecular-Weight RNA of 32P-Labeled RaLV. RaLV was labeled with 32p and high-molecular-weight was purified as described (22). Since high-molecular-weight RNA of [32P]RaLV forms a heterogeneous peak at 60-70 S (27), RNA sedimenting at 50-80 S was used for subunit RNA analysis.…”

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confidence: 99%

“…Increasing amounts of RNA were incubated with 500 cpm of [3H]DNA in 100 jsl of twice the concentration of standard saline-citrate at 660 for 20 hr. The extent of hybridization was determined with a single-strand-specific S1 nuclease as described (22 Fig. 2B and C).…”

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Sarcoma-Virus-Related RNA Sequences in Normal Rat Cells

Tsuchida

Gilden

Hatanaka

1974

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

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A rat type C virus spontaneously activated from the NRK (normal rat kidney) cell line was found to have two major size classes of viral RNA subunits sedimenting at 35 and 30 S. Virus-producing cells contained both RNA species, while normal "virus-free" rat cells contained primarily virus-specific 30S RNA species. A DNA transcript, specific for Kirsten sarcoma virus, pre- Three isolates, previously considered to be murine sarcoma viruses, have been studied by nucleic acid hybridization techniques during the past 2 years. Two of these isolates, the Harvey (1) and Kirsten (2) strains, arose on rat passage of mouse leukemia viruses (Moloney and Kirsten strains, respectively), and the third, the Moloney strain (3), arose on mouse passage of the Moloney leukemia virus (M-MuLV). Based on minimal hybridization between rat and mouse type C viruses (4, 5), it was possible to show that the Kirsten and Harvey sarcoma viruses derived their sarcoma-specific nucleic acid sequences from the rat type C virus (4, 6), while the Moloney strain (M-MSV) was exclusively of mouse origin (4, 5).We have previously reported preparation of KiSV-specific DNA transcripts that did not hybridize with RNA of helper mouse virus (7,27 spontaneously virus-producing rat cells has now shown KiSVspecific sequences associated with a 30S RNA in both cells, whereas virus-producing rat cells produce an additional 35S RNA subunit that is not related to KiSV. MATERIALS AND METHODSCells and Media. The following rat cell lines were used: NRK-9 (RaLV-NRK), a normal rat kidney (NRK) cell line producing endogenous rat type C virus (RaLV) (8); MSB-1 (9), a rat cell transformed by M-MSV producing M-MSV-(RaLV) (5); a Fisher rat embryo cell (5053) The following mouse cells were used: BALB/3T3 (A-31), and a derivative K-234, a BALB/3T3 cell line transformed by KiSV (NP cell) (12, 13); M-58-2, a mutant cell line derived from the NP cell after short-term treatment with 5'-bromodeoxyuridine (BrdU) (14, 15); 58-2T, a cell line established from a slowly growing tumor in a BALB/c mouse that was inoculated with M-58-2 cells (7); Ki-MuLV-producing NIH/ 3T3 cell (Ki-MuLV-NJH/3T3), obtained from Dr. V. Klement (Ki-MuLV had not been passaged in rat); and JLS-V9 infected with Rauscher strain of MuLV (R-MuLV) (16).A nonproducer hamster cell transformed by M-MSV (HT-1) was also used (17).All cells were grown in Eagle's medium with 10% (v/v) fetal bovine serum and antibiotics.Preparation of RNA. Cell RNA was prepared as described (18), by the hot phenol method. The concentration was determined by the orcinol reaction (19). Synthesis of Virus-Specific DNA. Viral [3H]DNA was prepared by the endogenous RNA-directed DNA polymerase reaction with RaLV, R-MuLV, M-MSV(MuLV), Ki-MuLV, and the virus produced by 58-2T cells, 58-2T virus. These viruses were purified as described (11). Reaction mixtures, generally 5 ml, contained 1-2 ml of purified virus solution (0.1-1.0 mg/ml of protein), 0.1 M glycine-NaOH buffer, (pH 8.0), 10 mM dithiothreitol, 0.05 mM each of dATP, dGTP, and d...

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Sarcoma-Virus-Related RNA Sequences in Normal Rat Cells

Tsuchida

Gilden

Hatanaka

1974

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

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“…"2P-Labeled viral RNA (70 S) of JLS-V9 was a gift of Dr. N. Tsuchida (11). "2P-Labeled ribosomal RNA, 18 S and 28 S, used as the marker in the sedimentation analysis, was prepared from a culture of JLS-V9 in MEM containing 2% FBS and 100 uCi/ml [82p]-phosphate (carrier-free, New England Nuclear) followed by phenol extraction of the cytoplasmic fraction after separation by the method described above.…”

Section: Methodsmentioning

confidence: 99%

Fate of viral RNA of murine leukemia virus after infection.

Takano¹,

Hatanaka²

1975

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

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ABSTRACT['H]Uridine-labeled Rauscher leukemia virus was used to infect mouse embryo fibroblasts. After the infected cells were separated into nuclear and cytoplasmic fractions nucleic acid was extracted by sodium dodecyl sulfate-phenol-chloroform treatment and analyzed by C82SO4 and sucrose density gradient centrifugation. Between 45 and 70 min after infection a transient and synchronized shift of the acid-insoluble radioactive peak toward the RNA-DNA hybrid region occurred in both the nuclear and cytoplasmic fractions. The density of the cytoplasmic hybrid shifted to 1.56 g/ml (RNA = about 50%), while the sedimentation rate decreased from 36 S to 14 S; however, the density of the nuclear hybrid shifted to 1.58-1.48 g/ml (RNA = 57-17%, respectively), while its sedimentation rate remained about 65 S. The hybrids in both the nuclear and the cytoplasmic fractions still showed hybrid density after heat denaturation. The processes of the early stages of RNA tumor virus infection are discussed with regard to the functions of viral RNA-dependent DNA polymerase (reverse transcriptase) and a possible integration of viral genetic information into the host chromosome.DNA synthesis in cells in the early stages of infection by an RNA tumor virus appears to play an important role in both the expression of transformation (1, 2) and the production of the progeny viruses (3). DNA copies homologous to the viral RNA have been found in virus-producing (4, 20) as well as virally transformed cell lines (5). These results suggest that reverse transcription of the viral information into DNA and the integration of this DNA into the host chromosome may be necessary for the maintenance and the expression of the viral information (6).The process of the integration of the viral genetic information, however, is entirely obscure. In order to study these processes, we followed the fate of ['H] mmol, New England Nuclear). After 10-to 14-hr incubation at 370, the radioactive medium was discarded and an equal volume of fresh MEM containing 3% FBS was added to each bottle. The virus was collected for 90 min after the medium change. DNase I (Worthington Biochemical Corp.; RNasefree) was added to give a final concentration of 10 ,g/ml for the last 30 min of each collection period. This collection procedure was repeated after another feeding with nonradioactive medium. The combined medium containing radioactive virus was purified by sucrose gradient centrifugation. Virus was pelleted onto a 60% sucrose cushion and then banded in a 15-50% sucrose gradient. The peak fraction of radioactivity in the final sucrose gradient (density = 1.16 g/ml) was used as a preparation of purified virus. The purified virus was diluted with 5 volumes of Earles' balanced salt solution (BSS, Flow Laboratories) and used for infection on the same day the purification was done.Infection with 3H-Labeled Viruses. Monolayers of MEF were plated 8-12 hr before infection at a density of 1.5 X 106 cells per 60-mm plastic petri dish with 5 ml of MEM supplemented with 10% FBS, and ...

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“…The defective mammalian sarcoma viruses appear to be deletion mutants which have lost coding capacity for some essential proteins. The 30-40 S RNA of MSV contains a component which is smaller than the RNA of murine leukemia virus, and this component is thought to represent the fibroblast-transforming information of the defective sarcoma virus plus sequences coding for some but not all replicative functions such as structural proteins (Maisel et al, 1973;Tsuchida et al, 1974). The larger component of the 30-40 S RNA obtained from MSV pseudotypes probably represents the RNA of the MuLV helper virus.…”

Section: Replication and Coordinately Defective Mammalian Sarcoma Virmentioning

confidence: 97%