Sarcoma-Virus-Related RNA Sequences in Normal Rat Cells

Abstract

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...

Distribution and virogenic effects of 5-bromodeoxyuridine in synchronized rat embryo cells.

“…Equally strong cytoplasmic immunofluorescence appeared in cells 12 hr after exposure to 0.1 mM BrdU (Table 2). DISCUSSION Although it has been shown repeatedly that information for endogenous type C virus is present in normal rat cells (1,12,13), the mechanism(s) by which BrdU elicits virus synthesis is unclear. At optimal virogenic BrdU concentrations (0.1 mM), [3H]BrdU is incorporated uniformly into nuclear DNA sequences (2,3,14), whereas [3H]BrdU at suboptimal concentrations (0.1 ,uM) is nonrandomly distributed as determined by DNA-DNA reassociation techniques (2,3).…”

Analysis of two divergent rat genomic clones homologous to the transforming gene of Harvey murine sarcoma virus.

“…Although similar in principle to that of other transforming retroviruses, the genetic organization of Ha-MuSV is more complex insofar as its 5.4-kilobase (kb) RNA genome is composed ofthree, rather than two, sets of sequences (8)(9)(10): (i) 0.1 kb at the 5' end and 0.9 kb at the 3' end, derived from the helper-independent virus Moloney murine leukemia virus; (ii) 3.5 kb sequences-highly homologous to a family of greatly reiterated rat genes, which are retroviral-like sequences expressed in many rat cellssometimes referred to as 30S sequences on the basis of the sedimentation coefficient of their RNA transcriptional product (28,29); and (iii) 1.0 kb of src sequences, completely bracketed by 30S sequences, that are required for transformation, encode the transformation-specific p21 protein, and originate from rat cellular sequences, which are highly conserved evolutionarily (10). As an initial step for investigating rat sarc sequences related to Ha-MuSV, we had defined a viral src probe from this latter set of sequences (10).…”

Proviral sequences that restrict retroviral expression in mouse embryonal carcinoma cells.

“…The donor splice sequence has been altered in the Ha-MSV genome and apparently no longer functions (25). The two sequences abruptly diverge at this point, and the remaining 210 bp included in the Ha-MSV element are related to rat VL30 sequences (13,52 If the lack of enhancer activity was responsible for the block in viral expression in infected EC cells, then the relatively high frequency of G418 resistance obtained after pMLV.WT transfection (Table 1) as compared with FVXMneo infection (1 to 10 G418-resistant colonies per 106 infected cells) was surprising. This is especially true if one considers that viral infection is reportedly a much more efficient means of introducing exogenous genetic material (7).…”