Characterization of Rat Genetic Sequences of Kirsten

Transcription

1 JOURNL OF VIROLOGY, May 1976, p Copyright 1976 merican Society for Microbiology Vol. 18, No. 2 Printed in U.S.. Characterization of Rat Genetic Sequences of Kirsten Sarcoma Virus: Distinct Class of Endogenous Rat Type C Viral Sequences EDWRD M. SCOLNICK,* ROBERT J. GOLDBERG, ND DVID WILLIMS Tumor Virus Genetics Branch, National Cancer Institute, Bethesda, Maryland 214 Received for publication 13 November 1975 The nucleic acid sequences found in DN and RN from rat cells which are homologous to Kirsten sarcoma virus have been characterized. The homologous sequences are present in multiple copies per diploid rat cellular genome in a variety of different rat cellular DNs. In certain cells that constitutively express only low levels of sequences homologous to Kirsten sarcoma virus, bromodeoxyuridine treatment leads to the expression of high levels of these sequences in RN. Supernatants from cell lines producing the sequences homologous to Kirsten sarcoma virus contain high levels of these sequences which are purified to the same degree as the previously known rat type C viral nucleic acid sequences by type C particles being released from such cells. The results indicate that the sequences in rat cells homologous to Kirsten sarcoma virus have three characteristics of known mammalian type C viruses, and suggest that at least part of the Kirsten sarcoma virus rat-derived sequences represent a distinct class of endogenous rat type C virus that has no detectable homology to the other known class of endogenous rat type C virus. The isolation of type C sarcoma viruses with the biological potential to induce solid tumors in vivo and transform fibroblasts in vitro has been relatively rare. Two such viruses were identified after inoculation of inbred strain(s) of rats with nonsarcomagenic mouse type C viruses. The Kirsten sarcoma virus (Ki-sv) was derived by passage of Kirsten murine erythroblastosis virus (Ki-MuLV) in Wistar-Furth rats (2), whereas the Harvey sarcoma virus (Hasv) was isolated after inoculation of Chester- Beatty rats with Moloney leukemia virus (17). In each case, the fibroblast-transforming virus derived after rat inoculation was found to have a smaller RN subunit than helper type C viruses and to be defective with respect to replication in the absence of such a helper leukemia virus (2, 21, 23, 3). Some insight into the genetic composition of these replication-defective fibroblast-transforming viruses was provided by hybridization studies in which each virus was found to be a recombinant between portions of their respective mouse leukemia viruses and additional rat genetic information (3, 31). There is no direct information on which sequences in Ki-sv or Ha-sv code for the fibroblast-transforming function of the viruses. However, two lines of evidence support the hypothesis that rat-derived information in Ki-sv and Ha-sv plays an important role in the transforming function of each virus. First, the genesis of both fibroblast-transforming viruses involved the acquisition of at least partially homologous rat genetic sequences (3); secondly, the pathology of malignancies induced in vivo by pseudotypes of Ki-sv or Ha-sv is quite similar and is readily distinguishable from the types of malignancies induced by similar pseudotypes of other mammalian fibroblast-transforming viruses (28). However, direct proof of this model is still lacking because a transforming virus has not yet been isolated from appropriate rat cells in cell culture. The studies reported here have addressed themselves to a further characterization of the Ki-sv genome. In particular, we have attempted to determine the relationship between at least one portion of the rat-specific information of Ki-sv and Ha-sv and endogenous type C virus genetic information in rat cells. These studies were stimulated by earlier experiments that showed that (complementary)dn transcripts, synthesized from an endogenous type C virus (V-NRK), contained two distinct sets of sequences (29, 32). One set was present in a rat leukemia virus released from the RT21C Osborne-Mendel rat thymus cell line, whereas the second component was homologous to the Ki-sv and Ha-sv, but showed little genetic relatedness to the RT21c rat type C virus. The results 559 Downloaded from on June 3, 218 by guest

2 56 SCOLNICK ET L. of the current studies clearly show that at least part of the rat-specific information in the Ki-sv genome shares several characteristics with other mammalian endogenous type C viruses and represents a distinct class of endogenous rat type C viral sequences. MTERILS ND METHODS ll cells were grown in Dulbecco's modification of Eagle medium with 1% calf serum (Colorado Serum Co.). The normal rat kidney (NRK) cell line is derived from a strain of Osborne-Mendel rat, as originally described by Duc-Nguyen et al. (13); the RT21c cell line, derived from an inbred Osborne- Mendel rat, was originally described by Cremer et al. (12). The cell line, XC, is the cell used by Rowe et al. in the mixed cell cytopathogenicity assay for murine type C viruses (26) and is a rat cell transformed by the Prague strain of avian sarcoma virus. fibroblast cell line derived from an embryo of a Wistar-Furth rat, releasing rat type C virus, was obtained from Robert J. Huebner, National Cancer Institute. nonproducer mink lung fibroblast cell transformed by Ki-sv has been previously described (18, 29) and was obtained by limiting dilution of a mixture of Ki-sv and an amphitropic murine type C virus (amphitropic: grows in mouse cells and nonmouse cells); the virus mixture, containing a fivefold excess of amphitropic virus, was obtained by rescuing a Ki-sv nonproducer NIH 3T3 cell with the amphitropic strain of murine virus. The amphitropic murine type C virus has been found by Janet Hartley to grow in both NIH cells and mink cells, and was the gift of Janet Hartley and Wallace Rowe (National Institute of llergy and Infectious Diseases). The isolation of the transformed nonproducer cell was performed in Falcon microtest II plates as previously described (29). Viruses. subgroup C strain of feline type C virus was obtained from P. Sarma, National Cancer Institute. This virus was used to infect the Ki-svtransformed nonproducer mink cell to obtain a Kisv-feline leukemia (FeLV) virus mixture; this virus mixture was used to synthesize a probe containing the Ki-sv-specific rat sequences. rat type C virus was obtained from the supernatant of the RT21c cell line; this virus was not infectious for any other cell tested (unpublished data). Preparation of RN and DN. Total cellular RN was prepared from tissue culture cells by the cesium chloride procedure of Glison et al. (15). RN was extracted by disruption of cells in.1 M Trishydrochloride, ph 8., containing 4% sodium lauroyl sarcosinate (K & K Laboratories, Inc., Plainview, N. J.) and purified by centrifugation in cesium chloride. To obtain cytoplasmic RN, after washing the cells in phosphate-buffered saline, cells were suspended in approximately 5 volumes of.1 M Tris-hydrochloride, ph 7.5;.1 M sodium chloride;.6 M magnesium chloride; and.5% (vol/vol) Triton X-1. Cells were disrupted in a glass dounce homogenizer with approximately 1 strokes in a tight pestle. fter removal of nuclei by two successive centrifugations at 6 x g for 1 min at 4 C, the J. VIROL. supernatant was made 1% with sodium dodecyl sulfate (SDS), and extracted with equal volumes of buffered (ph 9.) redistilled aqueous saturated phenol and chloroform-isoamyl alcohol, 24:1. fter precipitation with ethanol and collection of the precipitate by centrifugation, the pelleted RN was dried under a nitrogen stream and resuspended in.1 M Tris-hydrochloride, ph 7.2. Poly()-containing RN was purified from cytoplasmic RN by dt(12-18)-cellulose chromatography as described by viv and Leder (3). Oligo(dT)-cellulose was obtained from Collaborative Research, Inc., Waltham, Mass. To obtain the RN from extracellular virus, varying volumes of virus were collected and clarified at 5, x g to remove cellular debris, and pelleted through a 35% glycerol column at 1, x g for 9 min at 5 C. The concentrated virus was suspended in 1 ml of.1 M Tris-hydrochloride, ph 7.2;.1 M sodium chloride; 13 M EDT; 1% (vol/vol) SDS, and then extracted with equal volumes of phenol and chloroform-isoamyl alcohol as indicated above for the extraction of cytoplasmic RN. Yeast RN (5 jig/ml) was added as carrier to the extraction procedure in cases where viral RNs were purified from extracellular volumes of 1 liter or less. DN was extracted from cells by the procedure of Marmur (24), and sheared in a French pressure cell (minco Co., Silver Spring, Md.) at approximately 36, to 4, lb/in2 to an average size of 5 to 7S. fter shearing, the DN was treated with.5 N sodium hydroxide for 5 to 7 h at 37 C, and dialyzed extensively before use. Synthesis of virus-specific cdn. Endogenous (no exogenous template or primer) reverse transcriptase reactions from sucrose density gradientbanded viruses were used as the source of all viral DN transcripts. ctinomycin D (Calbiochem, Los ngeles, Calif.) at 6,ug/ml was included in all reactions. Details of the preparation of the [3H]DN probes (2 x 17 counts/min per gg) from RT21c virus and the Ki-sv/FeLV mixture and of the purification of the Ki-sv-specific component of this mixture have been fully detailed in earlier publications (29). The same preparation of RT21c probe, and Ki-sv-specific probe was used in all experiments. Briefly, the [3H]cDN was hybridized to 7S RN from Kirsten murine leukemia virus and FeLV, and the nonhybridized portion was separated by hydroxylapatite chromatography (4). This probe represented the ratspecific portion of Ki-sv. The RT21c probe was prepared by standard procedures as detailed earlier (29). The representativeness of these probes has been fully documented in Table 2 in an earlier publication (29). Hybridization. To prepare unique sequence thymidine-labeled rat cellular DN, subconfluent NRK cells were labeled with tritiated thymidine (41 Ci/mmol) at 1,uCi/ml for 48 h. Labeled DN was extracted by the procedure of Marmur (24), and then sheared as indicated above. fter alkaline treatment and dialysis, the DN was reassociated to a Cot of approximately 2 to 3 mol/s per liter, and the single-stranded unique sequence DN was isolated by hydroxylapatite chromatography with.14 M sodium phosphate solution, ph 6.8, by procedures Downloaded from on June 3, 218 by guest

3 *~~~~~~~~~ VOL. 18, 1976 RT GENETIC SEQUENCES OF KI-SV 561 described previously (6, 7). This tritiated thymidine labeled unique sequence rat cellular DN was unique sequence rat cellular DN was used to assess the rate of reassociation of the various rat cellu- DN in parallel reactions. The results indicate, allowed to reassociate to the RT21c cellular lar DNs as a standard for approximately single copy DN. with both the RT21c probe and the Ki-sv-specific probe, that the rate of reassociation of Conditions for hybridization are given in the legends and figures; hybridization reactions are monitored with the use of S1 nuclease as has been previ- considerably faster than the reassociation of these two cdn's to RT21c rat cellular DN is ously reported. Cot or Crt values were calculated by the unique sequence rat cellular DN is to procedures of Britten and Kohne (1) and Birnsteil itself. lthough these experiments were performed with thymidine-labeled unique rat cel- et al. (9), respectively. In the analysis of the kinetics of hybridization from small amounts of supernatant lular DN in parallel reactions and monitored fluid the convention of Vot was used as recently with S1 nuclease, a similar result was obtained suggested by Ringold et al. (25). Identical cultures were examined for supernatant RNs to insure that if the entire set of experiments was performed the supernatants accurately reflected the cell using hydroxylapatite as a method of analysis. numbers of the cultures examined. In studies using hydroxylapatite, the reassociation of the unlabeled rat cellular DN to itself RESULTS was monitored by absorbance measurements, Copy number of Ki-sv sequences. Earlier experiments had indicated that the rat sequences ture in which the viral [3H]cDN was also and was performed in the same reaction mix- homologous to Ki-sv could be detected in normal rat cellular DN in either NRK cells that the reassociation of either the RT21c or the Ki- allowed to hybridize. The one-half Cot value for were producing large amounts, or in RT21c sv-specific cdn is approximately 5 x 11 mol/s cells that produce small amounts of the homologous sequences in their RN (29). To character- Similarly in Fig. 1B, the same two cdn's per liter. ize the Ki-sv-specific sequences, DN-DN are hybridized to NRK cellular DN. gain, reassociation kinetics were performed with each probe hybridizes more rapidly to the NRK RT21c cellular DN, or NRK cellular DN cellular DN than the unique sequence thymidine-labeled DN hybridizes to the same NRK with cdn probes derived from the RT21c rat type C virus, or Ki-sv. The results are shown in cellular DN. In this case the Kirsten probe Fig. 1 and B. In Fig. 1, the RT21c and Kisv-specific cdn's are hybridized to RT21c cel- RT21c probe does; the one-half Cot for the Kir- hybridizes slightly faster to this DN than the lular DN. s a reference for the reassociation sten sequences is approximately 11 mol/s per of cellular DN to itself, tritiated thymidine- liter and approximately 4 x 11 for the RT21c S. KM a X - so Go ir 1 4 K- ; -S Kf Id ow IV Kr IT wo NY rf Cj (w4**- Mc/ liter) c.t (mon-ow/w) FIG. 1. DN-DN reassociation kinetics of rat DN and cdn probes. Each hybridization reaction was incubated at 66 C and contained in.5 ml:.2 M Tris-hydrochloride, ph 7.2;.75 M sodium chloride; 1-4 M EDT;.5% SDS; 2 pg of rat cellular DN; and approximately 2, trichloroacetic acid-insoluble counts/min of either cdn or tritiated thymidine-labeled "unique sequence" rat cellular DN. The reaction mixtures were heated at 95 C for 5 min, and then quenched at 4 C. Reactions were initiated by placing the hybridization tubes at 66 C; tubes were quick frozen in a dry ice-acetone bath at times varying from 5 min to 1 h, and analyzed for DN-DN hybridization with the use ofsi nuclease as previously described (4). The source of the cdn's and thymidine-labeled unique sequence cellular DN is given in Materials and Methods. The hybridization with the cdn probes has been normalized to 1%1; the actual values with the RT21c tritiated cdn was 75% and with the Ki-sv cdn 85%. Each cdn in the absence of added rat cellular DN had less than 2% of the trichloroacetic acid-insoluble counts/min resistant to Si nuclease and these values, approximately 4 counts/min, have been subtracted from each value. Hybridization with cellular DN was also normalized to 1%; actual values represent 82%. Symbols: () RT21c cdn versus RT21c cellular DN (); Ki-sv cdn versus RT21c cellular DN (-). (B) RT21c cdn versus NRK cellular DN (); Ki-sv cdn versus NRK cellular DN (); unique sequence rat cellular DN to itself (x). Downloaded from on June 3, 218 by guest

4 562 SCOLNICK ET L. J. VIROL. sequences; the one-half Cot for the unique sequence DN is 13 mol/s per liter. The results indicate that the Ki-sv-specific sequences are present in multiple copies (2 to 4) in diploid Osborne-Mendel rat cellular DN. Recently, Benveniste and Todaro (7) demonstrated that all endogenous mammalian type C viruses are present in multiple copies in the DN of their natural hosts. Thus, by this criterion, the Ki-sv sequences are like other endogenous mammalian type C viruses. Thus, although the Ki-sv and RT21c sequences have no detectable homology to each other, they are both present in multiple copies in uninfected rat cells. In similar experiments on DN obtained from the liver of other strains of Rattus norvegicus, namely Wistar-Furth rats, and Fisher rats, or from a separate species of Rattus, namely Rattus rattus, both sets of sequences were also found to be present in multiple copies per diploid genome in the liver of each of these species of rats. Purification of Ki-sv-specific sequences. Recent experiments have indicated that the encapsidation of nucleic acid sequences by type C viruses is a highly specific event (16). lthough other non-type C nucleic acids can be found in type C virus nucleic acid preparations (14, 19), type C viruses are able to purify from cells with specificity only other type C viral RNs. Therefore, the RT21c cell was examined for its constitutive intracellular and extracellular levels of both RT21c sequences and Ki-sv-specific sequences, to determine if the Ki-sv-specific sequences were purified to the same degree as the RT21c sequences in supernatants from RT21c cells. The cytoplasmic RN, the poly()-con- 4a so ~4 ~2 taining cytoplasmic RN, and the supernatants from RT21c cells were analyzed by RN- [3H]DN reassociation kinetics for the RT21c sequences and Ki-sv-specific sequences; the results are shown in Fig. 2. s previously reported (29), the RT21c sequences are present in high amounts in this cell in the cytoplasmic RN, with a one-half Crt of approximately 8 x 11 mol/s per liter. pproximately a 1-fold purification is achieved by purifying for the poly()-containing cytoplasmic RN, and approximately another 1-fold purification of the sequences is achieved in the crude supernatant of this cell. s previously noted, the Ki-svspecific sequences in the same cell are present in much lower quantities in the cytoplasmic RN (or in other studies total cellular RN); the one-half Crt for hybridization with this probe is approximately 4 x 12 to 8 x 12 mol/s per liter, indicating that there is approximately a 1- to 2-fold less concentration of these sequences than the RT21c-like sequences in the cytoplasmic RN. The RN contains poly () sequences since it is again purified approximately 1-fold by oligo(dt)-cellulose chromatography. Importantly, the Ki-sv sequences are purified an additional 1-fold, to a similar degree to which the RT21c sequences are purified, in the supernatant from these cells. Thus, although the Ki-sv-specific sequences are present in relatively lower concentrations in RT21c cells, they are purified in parallel to the RT21c sequences in the supernatant of these cells releasing type C virus. Levels of Ki-sv sequences in uninduced and induced rat cells. Since bromodeoxyuridine (BUdR) has been observed to induce type C o o ~~ D om* I i * Downloaded from on June 3, 218 by guest *. I I I Crt (mole sec * Iiter') FIG. 2. Simultaneous purification ofrt21c and Ki-sv sarcoma sequences from RT21c cells. Each.5-ml reaction mixture was incubated at 66 C for varying periods and contained.1 M tris-hydrochloride, ph 7.2;.3 M NaCi; 5 x 1-5 M EDT;.5% SDS; 2 pg ofyeast RN; 1 pg of calf thymus DN; and tritiated DN probes prepared from RT21c virus (2,3 counts/min) or Ki-sv (3,2 counts/min). Background (Si nuclease-resistant fraction of each probe in the absence of added cellular or viral RN) was less than 5% of either [3H]DN. Ki-sv [3H]DN versus cytoplasmic (-), oligo(dt)-cellulose-purified (), and supernatant (U) RNs; RT21c [3H]DN versus cytoplasmic (), oligo(dt)-cellulose-purified (), and supernatant (El) RNs.

5 IVOL. 18, 1976 RT GENETIC SEQUENCES OF KI-SV 563 co Z K!- 6 N 4 E 2C m CIS z 6C 2c- 2. B. * *. --D. o-.~~~ I * M I 1 (2 1 2 Crt ( mole sec Iiter-' ) FIG. 3. Hybridization of RT21c [3H]DN with total RN from various rat cell lines uninduced and induced with BUdR. Cultures were treated with medium containing 6 pg of BUdR per ml for 36 h at 37 C. The BUdR was then removed and the cultures were incubated for an additional 48 h at 37 C before extraction of total cellular RN. Control cultures were treated identically with the exception of the addition ofbudr. Hybridization conditions and analysis by S1 nuclease are as described in Fig. 2 except that.6 M sodium chloride was used. Each.5-ml reaction contained 1 pg of cellular RN. Each reaction mixture contained approximately 3, trichloroacetic acid counts/min of RT21c [3H]DN. Closed symbols, BUdR-treated cultures; open symbols, control, untreated cultures. () NRK cells; (B) RT21c cells; (C) XC cells; (D) WIFu cells. viruses in murine cells (22), several rat cell lines were examined for the ability of BUdR to induce either the RT21c sequences or the Kisv-specific sequences. The results are presented in Fig. 3 for the RT21c probe and in Fig. 4 for the Ki-sv-specific probe. In Fig. 3, the results with the RT21c probe indicate that NRK cells can be induced with BUdR to make increased levels of the RT21c sequences. fter BUdR treatment only a small increase is noted, twoto threefold, in the Ki-sv homologous sequences in NRK cells. In contrast, in the RT21c cell (Fig. 4B) after BUdR induction, levels of the Ki-sv-specific sequences rise dramatically; the one-half Crt before induction cannot be achieved even at values of 5 x 12 mol/s per liter, whereas after induction it is 4 x 11 mol/s per liter. The RT21c sequences rise only slightly after BUdR treatment (Fig. 3B). Thus, in the induced cells, the one-half Crt for each component is now approximately the same in contrast to the uninduced cells where there is a large excess of RT21c sequences. In Fig. 3C and 4C, the RN from XC cells was examined with and without BUdR treatment for both sets of sequences. In Fig. 3C, the RT21c-like sequences can be seen to rise approximately four- to fivefold after BUdR induc- I_ tion, from a.4 Crt of approximately 4 x 12 mol/s per liter to a.4 Crt of approximately 6 x 11 mol/s per liter. For the Ki-sv sequences, the uninduced XC cell has a one-half Crt of approximately 5 x 11 mol/s per liter, and the induced XC cell RN about 3 x 11 mol/s per liter. Thus, the XC cell resembles more the NRK cell in that the Kirsten sequences are in excess over the RT21c sequences in the uninduced cell, and rise only slightly with BUdR treatment. In Fig. 3D and 4D the Wistar-Furth cell was examined with and without BUdR. In Fig. 3D, it can be seen that the RT21c-like sequences in the Wistar-Furth cells change very little after BUdR induction; the one-half Ct in both cases is approximately 2 x 12 mol/s per liter. However, again after BUdR induction, in Fig. 4D the Ki-sv sequences, which are present in very low levels in uninduced W/Fu cells, rise dramatically. The one-half Crt is approximately 5 x 11 mol/s per liter in the induced cell. Thus, in two cell lines, RT21c and W/Fu, making low constitutive levels of Ki-sv sequences, BUdR treatment results in high levels of RN in the cell. In two cell lines, NRK and XC cells, making high constitutive levels of Ki-sv-specific sequences, BUdR induction changes these levels only somewhat. Thus, the Ki-sv-specific sequences, like other type C sequences in mammalian cells, are inducible with BUdR. Levels of Ki-sv sequences in uninduced and induced supernatants. Next, the supernatant from uninduced and induced RT21c cells was cc 8C N 4 E 2C m Z 8C 6C QL 4 2C. B.. 6 )-8~~~~~ )-C. :. o r -D. *. ~~~~ - 1 O 1' 12 Crt (mole sec liter-' ) FIG. 4. Hybridization with Ki-sv probe and various uninduced and induced rat cellular RNs. Induction with BUdR and each hybridization reaction was carried out as indicated in the legend to Fig. 3 except the Ki-sv-specific tritiated cdn was present instead of the RT21c tritiated cdn with 2,5 trichloroacetic acid counts/min per assay. Each.5- ml hybridization reaction mixture contained 1 pg of total cellular RN. Closed symbols, BUdR-treated culture; open symbols, uninduced cultures. () NRK cells; (B) RT21c cells; (C) XC cells; (D) WIFu cells. Downloaded from on June 3, 218 by guest

6 564 SCOLNICK ET L. examined with both probes, and that result is shown in Fig. 5. The supernatant derived in this experiment is from the identical culture shown in Fig. 3B and 4B where the cells were examined at the same time as this supernatant was collected for the RT21c and Ki-sv sequences. In Fig. 5, it can be seen that the onehalf Vot for the RT21c sequences decreases only somewhat from approximately 5 x 1' mls-h to approximately 1.5 x 11 mls-h after BUdR induction. This rise parallels the two- to threefold increase in the intracellular levels of RT21c RN after BUdR induction. In Fig. 5B, it can be seen that with the Ki-sv probe, the.2 Vot is between 5 x 1' to 5 x 12 mls-h in uninduced supernatants, and approximately 2 x 1 mls-h in induced supernatants. This ex- 5 so 46 D' le, J. VIROL. tracellular rise of approximately 2-fold parallels again the rise seen intracellularly in Fig. 4B after BUdR induction. Thus, after induction with BUdR the Ki-sv sequences in RT21c cells rise both intracellularly and in parallel extracellularly and are now present extracellularly in approximately equal concentrations with the RT21c sequences. ccordingly, after BUdR, the RT21c supernatant resembles the V-NRK culture. Next, we analyzed the uninduced and induced W/Fu supernatant for the same two sets of sequences. These results are shown in Fig. 6. In this case, the supernatant examined was not from the identical culture examined in Fig. 3B and 4B for intracellular RN, and therefore no direct comparison in this particular study can OF w OD IV Vot (mb-hrs) Vot (Moo-gw) FIG. 5. Hybridization with supernatant from control and induced RT21c supernatant. pproximately 1 ml of supernatant fluid was collected 48 h after a medium change from the RT21c uninduced and induced culture at the time the cells were extracted for RN as indicated in Fig. 3 and Fig. 4. The culture fluids were clarified at 3, rpm at 4 C to remove cells, and centrifuged at 1, x g for 75 min at 4 C. Total RN, sedimentable at 1, x g, was extracted with phenol and chloroform as indicated in Materials and Methods in the presence of5 pg ofyeast RN per ml. Hybridization was performed as a function of Vot (25) in.5-ml reaction mixtures and contained ionic components as indicated in the legend to Fig. 3. Either RT21c probe (1,3 trichloroacetic acid counts/min) or Ki-sv cdn (2,5 trichloroacetic acid counts/min) was included in the reactions. () RT21c probe versus () control supernatant or (4) induced supernatant. (B) Ki-sv probe versus () control supernatant or () induced supernatant. z 4 m z xx 6C cr we 2.. I ~~~I I I I - ~~~ & O -.f _ f I Ā ~~~~~~~~ ~~~~~~~~~ a I I I 1- ~~~~~ G O 1 a * i I, p*. e Downloaded from on June 3, 218 by guest L I a 11 1J,I 1 Kcr KY IV ID VP ' DP Vot (mls-ha) Vol (m - hrs) FIG. 6. Hybridization with supernatant from control and induced W/Fu supernatant. W/Fu cells were treated with either deoxycytidine alone (12 pg/ml) or a combination of BUdR (12 ug/ml) and 12 pg of deoxycytidine per ml (8). Treatment was continued for 72 h. The medium was removed and replaced with fresh medium containing either deoxycytidine or deoxycytidine and BUdR. Twenty-four hours later, 5 ml offluid was collected from control or induced cultures. Supernatant fluids were then processed and hybridization performed as indicated in the legend to Fig. 5. () RT21 cdn (1,3 trichloroacetic acid counts/min) versus () uninduced or () induced supernatant. (B) Ki-sv cdn (2,5 trichloroacetic acid counts/min) versus () uninduced or () induced supernatants.

7 VOL. 18, 1976 be made between the relative intracellular and extracellular levels of these two sets of sequences. In Fig. 6 it can be seen that, with BUdR treatment, the supernatant from the W/ Fu cell actually decreases in RT21c-like sequences. (This is probably due to the fact that fewer cells were present in the BUdR-treated culture [see legend to Fig. 6]); one-half Vot increased from approximately 2 x 11 mls-h to 12 mls-h, a drop of about fivefold in concentration after BUdR induction. Despite the fewer cells, after BUdR treatment (Fig. 6B), the Ki-sv sequences rise from a one-half Vot of approximately 13 mls-h before BUdR, to 12 mls-h after BUdR, a rise of 1-fold. Thus, in the supernatant from the W/Fu cells after BUdR induction, the Ki-sv sequences are present in roughly equal concentrations with the RT21clike sequences, whereas before induction the RT21c-like sequences are present in approximately a 1-fold excess. DISCUSSION The current studies have characterized the nucleic acid sequences found in the DN and RN of normal rat cells which are homologous to the RN sequences found in the two oncogenic viruses, the Kirsten and Harvey strains of murine sarcoma virus. Since both of these viruses acquired the ability to transform fibroblasts concomitantly with a gain of rat genetic sequences, it has been of interest to attempt to determine if the genetic sequences acquired were of type C rat origin, or of rat cellular origin. lthough it is not known with certainty that the rat genetic sequences in Ki-sv and Hasv code for proteins(s) responsible for the viruses' ability to cause fibroblast transformation, this hypothesis (31) has seemed like a reasonable one to explain the observed biological history. By three criteria -(i) presence in multiple copies in diploid rat DN; (ii) the fact that expression can be regulated by BUdR induction; (iii) the fact that sequences can be specifically purified and reverse transcribed (29) by type C viruses released from rat cells - at least some of the sequences found in normal rat cells homologous to Ki-sv or Ha-sv have properties that allow them to be classified as type C viral nuclei acid sequences. Thus, although the sequences homologous to Ki-sv and Ha-sv have no detectable homology by hybridization to the RT21c rat type C viral sequences present in the same rat cells, they appear to be a distinct class of endogenous rat type C viral sequences. Perhaps this is not surprising since other examples exist where mammalian species RT GENETIC SEQUENCES OF KI-SV 565 harbor partially or completely different endogenous type C viral sequences. In one case, in mice, BLB virus 1 and BLB virus 2 (1), both endogenous viruses found in BLB/C mice, share only approximately 3% genetic homology (11). In the case of cat cells, two distinct endogenous viral genomes have been found in normal cat cellular DN, the FeLV genome and the RD114 genome (6, 27). In this case, no detectable homology between these genomes by nucleic acid hybridization has been found. In the case of the distinct endogenous mouse viruses, presumably the different endogenous viruses evolved from a common mouse virus progenitor (11). In the case of the cat, recent evidence (6) has suggested that the mechanism of acquisition of these two distinct endogenous viruses might be the result of horizontal infection of cats. It will be of interest in future studies to attempt to trace the evolution and origin of these two distinct endogenous rat type C viruses to see which model for such wide divergence fits the rat system. lthough certain rat cells contain high levels of the sequences homologous to Ki-sv, no rat cell yet found releases high levels of Ki-sv sequences in the absence of RT21c sequences. Thus, as yet we have no protein markers for the sequences in rat cells homologous to Ki-sv, and cannot say whether these sequences represent a complete infectious type C virus, or a defective endogenous type C virus. In this regard, it is interesting that the largest size of the Ki-sv sequences found in V-NRK cells in 3S, which is smaller than 35S RN of the RT21c-like virus (32) also produced by these cells. It is thus tempting to speculate that these Ki-sv homologous sequences represent a class of replicationdefective endogenous mammalian type C viruses. In rats or also in other mammalian species, such a class of type C viral sequences might only be detectable by encapsidation of these sequences by a heterologous helper virus (29), or after they had become a stable part of a transmissible virus through recombination. lternately, if assays become available that will detect proteins coded for by these rat type C sequences, it will be of interest to examine other mammalian cells for possible cross-reacting proteins. LITERTURE CITED 1. aronson, S.., and J. R. Stephenson Independent segregation of loci for activation of biologically distinguishable RN C-type viruses in mouse cells. Proc. Natl. cad. Sci. U.S.. 7: aronson, S.., and C. Weaver Characterization of murine sarcoma virus (Kirsten) transformation of mouse and human cells. J. Gen. Virol. 13: viv, H., and P. Leder Purification of biologically Downloaded from on June 3, 218 by guest

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