A candidate rat-specific gene product of the kirsten murine sarcoma virus

VIROLOGY

99,

31-48 (1979)

A Candidate Rat-Specific Gene Product Kirsten Murine Sarcoma Virus

of the

GARTH R. ANDERSON,’ KEITH R. MAROTTI, AND PATRICIA A. WHITAKER-DOWLING Department of Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Accepted July 16, 1979 Anaerobic stress has been shown to induce normal, uninfected rat cells to express high concentrations of those rat cell RNA sequences (MSV-rat RNA) also present in the Kirsten murine sarcoma virus (KiMSV) genome. We find this stress-induced RNA as complete, functional mRNA. We have reacted antibodies directed against uninfected rat cells induced to express this genetic information, with non-rat cells expressing this same (rat) genetic information as a result of KiMSV infection. An antigen of 35,000 molecular weight was detected in the KiMSV-infected cells, and was also observed induced by anaerobic stress of uninfected rat cells. This antigen is not present in cells infected with transforming gene mutants of KiMSV, or in cells transformed by a variety of agents other than KiMSV. The 35,000 molecular weight polypeptide is found as a native complex of molecular weight about 135,000 and copurifies with lactate dehydrogenase activity.

molecular weight polypeptide with protein kinase activity has been identified as a src Type-C sarcoma viruses represent gene product (Collett and Erikson, 1978; normal cellular genetic information which Levinson et al., 1978). In other avian syshas acquired the ability to transmit and tems alternate transforming mechanisms replicate as a virus, while simultaneously ef- apparently exist (Duesberg et al., 1977; fecting neoplastic transformation. Both in Stehlin and Graf, 1978). In the murine sysmurine and avian systems these sarcoma tem, in vitro translation of Harvey sarcoma viruses have been shown to be molecular virus RNA has revealed a polypeptide of recombinants of type-C leukemia viruses, around 20,000 molecular weight whose funcwhich in themselves are present in latent tion and relevance to a src gene product are form in normal cell DNA and can be induced not clear (Parks and Scolnick, 1977). as typical replicating viruses (Bishop, 1978). The Kirsten murine sarcoma virus Additional specific cell genes have recom- (KiMSV) offers distinct advantages as a bined with the leukemia viruses to create mammalian model system in that its transthe sarcoma viruses. These specific cell forming genes come from a different species genes (the src genes; MSV-rat sequences) (rat) than its nontransforming genes (of are believed to code for the transforming ac- mouse origin). This virus was first isolated tivity of the sarcoma viruses (Stehlin et al., from tumors which arose during passage of 1976; Anderson and Robbins, 1976; Scolnick the Kirsten murine leukemia virus (KiMuLV, and Parks, 1974; Andersson et al., 1979). a mouse virus) in rats (Kirsten et al., 1967). Identification of the transforming gene The genome of this sarcoma virus is comproducts of type-C sarcoma viruses and posed of two approximately equal parts: characterization of their functions should murine leukemia virus sequences, in turn provide valuable insights into the primary representing about half the KiMuLV events of neoplastic transformation. In the genome, and specific rat cellular sequences avian Rous sarcoma virus system, a 55,000 (MSV-rat sequences) (Anderson and Robbins, 1976; Scolnick et al., 1973). The mouse 1 To whom reprint requests should be addressed. INTRODUCTION

31

00426822!‘79/150031-18$02.00/O Copyright All rights

Q 1979 by Academic F’ress, Inc. of reproduction in any form reserved.

ANDERSON,

32

MAROTTI,

AND WHITAKER-DOWLING

leukemia virus sequences are not present in rat DNA, nor are the rat cell sequences present in mouse DNA. It has been proposed that the rat cell sequences of KiMSV represent those of a rat leukemia virus (Scolnick et al., 1973; Young et al., 1978), but there is also evidence that this rat genetic information represents other cellular genes (Anderson and Robbins, 1976). We have described how normal reproductive tissues, as well as a majority of spontaneous rat tumors, express elevated quantities of RNA corresponding to the rat sequences of the KiMSV genome (Anderson and Robbins, 1976). Young et al. (1978) have recently confirmed that many primary carcinogen induced tumors also express elevated quantities of the MSV-rat RNA. We have reported how anaerobic stress induces normal, uninfected rat fibroblasts in culture to express the MSV-rat RNA sequences at approximately 70-fold greater concentrations than seen with nonstressed cells (Anderson and Matovcik, 1977). In a manner similar to that reported in the case of drosophila tissues subjected to similar stress (heat shock or temporary anaerobiosis) (McKenzie and Meselson, 1977; Lewis et al., 1975), the anaerobic induction response of rat cells is also exceptionally specific, with fewer than four mRNA species induced to detectable levels (J. E. Richards, unpublished observation). We report here results of studies utilizing anaerobic stress of uninfected Fischer rat cells to induce synthesis of a class of specific polypeptides. Through use of techniques of immune precipitation, we have identified a polypeptide which is induced in uninfected rat cells subjected to anaerobic stress and which also appears to be coded for by a gene of the Kirsten murine sarcoma virus. Transforming gene mutants fail to produce this polypeptide, suggesting it may be a transforming gene product. Characterization of this polypeptide is described. MATERIALS

AND METHODS

Cell lines. The Fischer rat embryo line and BALB/c mouse lines were obtained

from the laboratory of Stuart Aaronson. The IMR-31 bison lung line was obtained from the American Type Culture Collection. Vero monkey cells were supplied by J. S. Youngner. BALB 3T3 cells infected with the Kirsten sarcoma virus point mutants R-20 and R-24 (Greenberger et al., 1974) were supplied by Joel Greenberger. Nucleic acid hybtidixution. [3H]cDNA product of Kirsten sarcoma virus was prepared by the endogenous reverse transcription reaction of virus purified by isopycnic centrifugation. Single-stranded [3H]cDNA labeled with [3H]TTP was synthesized during a 6-hr incubation at 37” in the presence of 20 pg/ml actinomycin D, and deoxynucleoside triphosphate concentrations were low4 M. The cDNA was fragmented, with an average size of 7-10 S. The representative character of this cDNA product has been reported in detail (Anderson and Robbins, 1976). Hybridization with uninfected rat cell RNA was only to the rat specific fraction of KiMSV cDNA, as verified by analysis of hybridization with cDNA prepared from KiMuLV alone. Cell RNA was prepared by the hot phenol method described previously (Anderson and Robbins, 1976). Hybridization was carried out in a 50-~1 reaction mixture containing [3H]cDNA (2 x lo3 cpm; 0.3 ng) and 0.1-200 pg cell RNA in a solution composed of 38% formamide, 600 m&l NaCl, 50 mM Tes pH 7.5, and 1 m&ZEDTA. After incubation for 7 days at 43”, hybridization was monitored with nuclease Sl. Specific RNA concentrations were quantitated by procedures described elsewhere (Anderson and Robbins, 1976). Polyribosome preparation. Polyribosomes were prepared by the procedure of Craig (1973). Cells were removed from plates by trypsinization and poured over crushed ice made from phosphate buffered saline. Cells were harvested by centrifugation and washed once with ice-cold PBS. The cell pellet was resuspended in 2 ml/12 plates 0.01 M Tris pH 8.9, 0.15 M KCI, 2 n-&f MgC12, 0.05% (w/w) Triton X-100. After 20 min incubation on ice, 0.3 ml of (6.7% Tween 40 + 3.3% deoxycholate) was added to each 2 ml of cell suspension. This mixture was vortexed briefly. After 10 min

A RAT GENE

PRODUCT

OF KiMSV

33

centrifugation at 10,000 g, 1 ml of this experiments. For direct analysis, cells supernatant was layered on a cold 34 ml were then lysed with 0.1% SDS and run lo-40% sucrose gradient containing 0.02 M on 12% SDS-polyacrylamide gels by the Tris pH 7.6, 0.05 M KCl, and 2 m&f MgCl,. procedure of Laemmli (1970). Parallel gels The gradients were spun in a Spinco SW 27 were run with the following molecular rotor for 150 min at 25,000 g at 4”. The weight standards: p-galaetosidase (116,000), gradients were then upwardly displaced phosphorylase (94,000), bovine serum althrough an Isco absorbance monitor and bumin (68,000), catalase (60,000), glutamate fractions collected. dehydrogenase (53,900), ovalbumin (43,000), RNA sedimentation analysis. Sedi- aldolase (40,000), lactate dehydrogenase mentation analysis was by the method of (35,000), carbonic anhydrase (29,000), and Travnicek and Riman (1973), as we have /3-lactoglobulin (18,000). Gels were sliced, used in this system previously (Anderson solubilized in NCS, and counted in toluene/ and Robbins, 1976). Cell RNA was dena- liquifluor in a Searle Mark III scintillation tured by heating 10 min at 37” in a solu- counter. tion containing 20% formamide, then cenLactate dehydrogenase assays and gels. trifuged 3 hr at 40,000 rpm in a 12.5 to 25% Total LDH assay was by the procedure of sucrose gradient in 0.1 M NaCl, 0.01 M Schwartz and Bodansky (1966). NondenaTris pH 7.5, and 0.001 M EDTA (TNE). turing gel electrophoresis was by the Fifty micrograms yeast tRNA was added procedure of Deitz and Lubrano (1967). to each fraction, followed by precipitation RESULTS with 2 vol of ethanol. Nucleic acid was resuspended in 100 ~1 TNE and assayed by Complete, Functional MSV-rat RNA nucleic acid hybridization. Induced by Anaerobic Stress Ribosomal RNA within the sample was Anaerobic stress induces uninfected, detected by monitoring the gradient with an Isco uv absorbance monitor as it was col- normal rat cells to massively express (ea. 1% of total cell RNA) rat cell RNA selected. quences indistinguishable by nucleic acid Cell culture and radiolabelingfor peptide analysis. Cells were grown on Falcon hybridization from those also present in the plastic culture dishes in the Dulbecco Kirsten (KiMSV) and Harvey (HaMSV) modification of Eagle’s medium (Gibco). murine sarcoma virus genomes. If this inSubconfluent cultures were prepared by a duced MSV-rat RNA is functioning as sixfold splitting of confluent cultures 24 hr mRNA, it might prove possible in view of its great quantity to identify its gene before labeling. Anaerobic culture was in a GasPak products, and in turn identify these same (or anaerobic chamber as described (Anderson very similar) gene products in KiMSV-inand Matovcik, 1977). Completely anaerobic fected cells. We thus examined if complete, conditions required approximately 6 hr to functional MSV-rat mRNA is induced in unachieve, as determined by a methylene blue infected rat cells subjected to anaerobic indicator. At this point, 1 ml of media con- stress. Although we have previously shown taining 100 $X/ml [3H]leucine or [35S]- such anaerobically induced RNA commethionine, or 10 $Zi/ml [14C]leucine was pletely hybridizes [3H]cDNA prepared added (via a catheter penetrating the cham- from the rat sequences of KiMSV, such ber wall) to the 10 ml of media already in induced RNA might be heavily degraded. each dish. Parallel, aerobically cultured Thus it was important to determine if intact, complete RNA molecules are actually cells were labeled simultaneously with rH]methionine or rH]- or [14C]leucine as in- transcribed. Accordingly, we compared RNA isolated from anaerobically stressed dicated. Cells were harvested 18 hr later. Preparation and polypeptide analysis of Fischer rat cells in culture with RNA cell extracts. Cells were washed with phos- isolated from the same cells cultured phate buffered saline, scraped with a rub- aerobically. As shown in Fig. 1, the MSVber policeman, and pooled for double label rat RNA sequencesin anaerobically stressed

34

ANDERSON,

MAROTTI,

AND WHITAKER-DOWLING

FRACTION

FIG. 1. Size distribution of MSV-rat RNA in uninfected Fischer rat cells. Total cell RNA was sedimented on a sucrose gradient, as described under Materials and Methods. Ribosomal RNA, 18 and 28 S, was detected by monitoring the A,,, as fractions were collected. MSV-rat RNA was detected by hybridization to [3H]cDNA prepared from Kirsten sarcoma virus, at a cDNA: homologous RNA ratio of less than 1:20. (0) RNA from cells cultured aerobically. (0) RNA from cells subjected to 24 hr of anaerobic culture.

cells were observed sedimenting in large part as intact 30 S molecules, along with some smaller material. In clear contrast, aerobically cultured cells showed no 30 S material, with only smaller, apparently degraded material seen. The 30 S RNA seen in the anaerobic cells is the same size as seen for the MSV-rat sequences spontaneously expressed by NRK cells (Tsuchida et al., 1974) or as packaged by typeC viruses grown in rat cells (Anderson and Robbins, 1976), and similar to that seen for analogous sequences in mouse (Duesberg and Scolnick, 1977) and chicken cells (Wang et al., 1977). This result confirms that anaerobic stress leads uninfected rat cells to transcribe complete copies of the MSVrat RNA sequences. While complete transcription is seen to occur, this still does not establish that such RNA molecules can function in an informational role. One criterion for functional mRNA is the presence of associated poly(A) sequences. Retention on oligo(T)cellulose provides a measure for attached poly(A). We thus isolated RNA from anaerobically stressed Fischer rat cells, and

found 45% of the MSV-rat RNA retained on the oligo(T)-cellulose column. This indicated the anaerobically induced MSVrat RNA has associated poly(A), and could function as mRNA. If the induced MSV-rat RNA sequences are indeed functioning as mRNA, they would have to become ribosome associated. We thus isolated RNA from cell fractions including polysomes prepared from anaerobically stressed Fischer rat cells and from aerobically cultured controls. As described in Table 1, over half of the MSV-rat RNA isolated from anaerobically induced cells was found in the cytoplasm, and a large majority of this in turn was polysome associated. This fraction of MSV-rat RNA associated with polysomes in the anaerobically cultured cells was substantially greater than that observed for the small total quantities of MSV-rat RNA seen in aerobically cultured cells. All together these results show that anaerobic stress induces uninfected rat cells in culture to transcribe functional genetic information indistinguishable from that believed to be the source of transforming activity of the K&ten and Harvey sarcoma viruses. TABLE

1

INTRACELLULAR LOCATION OF MSV-RAT RNA SEQUENCES Percentage hybridizing Cell fraction Nuclei Cytoplasm Free RNA” Polysome associated’ l-4 Ribosomes 5- 10 Ribosomes Greater than 10 ribosomes

Anaerobic

of total RNAn Aerobic

26 74 13

33 67 41

32 18 11

26 0 0

4 RNA was isolated from Fischer rat cells cultured aerobically or subjected to 24 hr of anaerobic culture. Hybridizing RNA was quantitated as described (Anderson and Matovcik, 1977). b Free RNA was that detected in fractions corresponding to S values less than 60 S. c The actual association with polysomes was verified through specific release by puromycin or EDTA.

A RAT GENE PRODUCT OF KiMSV

Three Major Polypeptides Anaerobiosis

Induced

by

Since the large amounts of apparently functional MSV-rat RNA are induced by anaerobic stress of uninfected rat cells, one might expect their polypeptide gene products to also be induced. We thus compared the polypeptides synthesized by Fischer rat embryo cells sub-

35

ject to anaerobic stress with those synthesized by the same cells cultured in a normal atmosphere. As shown in Fig. ZA, substantially elevated quantities of three polypeptides are seen in anaerobically stressed confluent cells. Based on their mobilities in SDS-polyacrylamide gels, it can be estimated that these three major polypeptides have molecular weights of 95,000, 74,000,

FRACTION

FIG. 2. Polypeptides inducible by anaerobic stress of uninfected rat cells. Fischer rat embryo line cells

at (A) 30% confluency or (B) at confluency were labeled in parallel with [3H]leucine (10 @/ml) while under anaerobic conditions (O), or with [“Clleucine (1 #J/ml) while under aerobic conditions (0). Labeling was from 6 to 24 hr following initiation of anaerobic conditions, and extracts were analyzed on SDS-gels as described under Materials and Methods.

ANDERSON, MAROTTI, AND WHITAKER-DOWLING

36

and 35,000. Each band represents between 2 and 3% of the newly synthesized protein. Additional polypeptides may also be induced by anaerobiosis, but not in sufficient quantities to be detected here. We have observed that the induction of MSV-rat RNA in confluent cell cultures is greatly reduced compared with that shown by subconfluent cultures (Anderson and Matovcik, 1977). A comparison of those major polypeptides induced by anaerobic stress of subconfluent cultures as compared to confluent cultures may thus provide insights into those polypeptides more likely to be coded for by the MSV-rat RNA. As shown in Fig. 2B, anaerobic stress of confluent Fischer rat cell cultures still led to increased expression of the 95,000 and 74,000 molecular weight polypeptides, but expression of the 35,000 molecular weight material was not visibly elevated. For purposes of comparison, the MSV-rat RNA levels of subconfluent cells (Fig. 2A) were determined by nucleic acid hybridization to have increased 60-fold, while this RNA was increased only g-fold in the confluent cultures (Fig. 2B). This result is consistent with the 35,000 molecular weight polypeptide being encoded in the MSV-rat RNA sequences, but inconsistent with the 95,000 and 74,000 molecular weight polypeptides being coded by these same sequences. A Gene Product of K&ten

Sarcoma

Virus

If any anaerobically induced polypeptides (and particularly the more likely possibility, FISCHERPATCELLS f-4 24 HR)

RABBIT+

iwII(R+) SEwJl

KIFSWKIMULV) ,NFECTED+ % LEUZINE BISW CEILS

FIG. 3. An outline of the immune precipitation scheme used to identify a rat-specific antigen in cells infected with KiMSV.

of 35,000 molecular weight) are also encoded by the MSV-rat RNA as it occurs in the Kirsten sarcoma virus genome, it might prove possible to detect them in heterologous cells infected with KiMSV. We thus employed immune precipitation techniques outlined in Fig. 3 to determine if an antigen present in anaerobically stressed rat fibroblasts and absent in normal rabbit cells is also present in heterologous cells infected with KiMSV. Bison lung cells were selected as a test system, since these cells are evolutionarily distant from rat and would be less likely to exhibit immune cross-reactivity with rat proteins. Furthermore, those polypeptides which are antigenically similar in the bison and rat might also be expected to have their counterparts in the rabbit, and thus would be unlikely to elicit an immune response. Rabbit antisera was prepared against total protein from uninfected Fischer rat cells expressing the MSV-rat genetic information (and the p-35 polypeptide) as a result of anaerobic stress. If the KiMSV genome codes for the same or a very similar 35,000 molecular weight polypeptide, then bison cells transformed by KiMSV should contain a p-35 polypeptide that specifically reacts with this rabbit antirat sera. Other anaerobically inducible rat polypeptides with counterparts encoded by the KiMSV genome should also be detectable by these procedures. Bison lung cells were infected with Kirsten sarcoma virus and a Kirsten leukemia virus helper. Parallel bison cell cultures were infected with Kirsten leukemia virus alone. The KiMSV (KiMuLV)-infected cells were labeled with [3H]leucine and the KiMuLV-infected cells were labeled with [14C]leucine. The cells were pooled, homogenates prepared, and then were reacted with the rabbit antianaerobic rat sera. Goat anti-rabbit sera was used in a double antibody procedure to precipitate the immune complexes, which were then analyzed by SDS-polyacrylamide gel electrophoresis. A single major polypeptide, with a molecular weight of 35,000, was precipitated by this procedure from the KiMSVtransformed bison cell extracts (Fig. 4). When the anti-rat sera was preabsorbed

A RAT GENE PRODUCT OF KiMSV

25

50

75

37

100

FRACTION

FIG. 4. Immune precipitation of a polypeptide from KiMSV-infected bison lung cells, using sera directed against rat proteins. Rabbit antiserum was prepared against Fischer rat cells subjected to anaerobic stress for 24 hr. IMR-31 bison lung cells were infected with Kirsten murine sarcoma virus and a K&ten murine leukemia virus helper, [KiMSV(KiMuLV)-Bu] or with Kirsten leukemia virus alone [KiMuLV-Bu]. The KiMSV(KiMuLV)-Bu were labeled for 48 hr with 10 @i/ml [3H]leu, and the KiMuLV-Bu with 1 &i/ml [‘*C]Leu. Cells were pooled, swollen in RSB, and Dounce homogenized. Of this homogenate 400,000 cpm was centrifuged for 1 hr at 100,000 g. The supernatant fraction was immunoprecipitated with the rabbit antiserum and then the immune complexes were precipitated with goat anti-rabbit immunoglobulin. This immunoprecipitate was then analyzed on 12% SDSpolyacrylamide gel electrophoresis. Preimmune serum or the precipitate from the 100,000 g centrifugation failed to show any immune precipitation. (0) KiMSV(KiMuLV)-Bu, labeled with rH]Leu, (0) KiMuLV-Bu, labeled with [W]Leu. 14Cdpm are multiplied by 2.5, the ratio of 3H dpm to % dpm in the crude homogenate.

with extracts prepared from KiMSV-transformed BALB/c mouse cells, no material was immune precipitated from either the KiMSV-infected bison cells or the KiMuLVinfected cells. In contrast, preabsorption with uninfected mouse cell extracts showed no effect. To eliminate the possibility that these immune precipitation results reflect artifacts peculiar to the bison cell line used, the above studies were repeated using vero monkey cells. Very similar results were obtained, again showing a specific p-35 polypeptide in KiMSV-infected cells. To eliminate the possibility of labeling artifacts, the above studies were repeated but with [35S]methionine labeling of KiMSV-infected cells and [3H]methionine labeling of uninfected cells. Again, specific immune precipitation of a p-35 polypeptide was seen. It can be seen in Fig. 4 that bison cells infected with Kirsten murine leukemia

virus alone also showed a small amount of immune precipitating p-35 polypeptide, which might reflect either (a) presence of antigen in cells infected with KiMuLV alone, or (b) subunit interchange of a multimerit protein which occurs during the course of the double label experiments. In double label experiments where extracts were pooled after immune precipitation, a significant reduction was seen in the amount of p-35 immune precipitated from the KiMuLV infected cells. This would suggest that at least some of the immune precipitated p-35 which originated in cells infected with leukemia virus alone was seen as the result of subunit exchange. However it remained possible that cells not infected with the Kirsten sarcoma virus contained small amounts of the p-35 antigen. It was also possible that the immunologically reactive p-35 polypeptide was induced merely as a secondary effect of transformation. In

38

ANDERSON, MAROTTI, AND WHITAKER-DOWLING

fact, a recent report indicates many transformed cells release extracellularly large quantities of a 35,000 molecular weight polypeptide (Gottesman, 1978). If transformation per se induces a host p-35 polypeptide, then other transforming agents should also induce this same material. To further test these possibilities, we developed an immune competition assay, where anti (anaerobically stressed rat cell)sera was reacted with [35S]methioninelabeled KiMSV-infected vero monkey cell extracts. Various transformed or leukemia virus-infected cell extracts were added prior to the antisera and then examined for their ability to compete for immune precipitation of the labeled 35,000 molecular weight polypeptide. Analysis of all immune precipitates was then made by SDS-gel electrophoresis. As illustrated in Fig. 5, the p-35 antigen is present in both mouse and

I 0

1 100

200

Competmg

Protein

300 IPg)

FIG. 5. Immune competition assay for the p-35 polypeptide. KiMSV(KiMuLV)-infected vero monkey cells were labeled with 10 &i/ml [“Slmethionine for 24 hr. An S-100 extract was prepared as described under Materials and Methods. One hundred micrograms of this S-100 protein (containing around 700 cpm of the p-35 polypeptide) was reacted with limiting amounts of rabbit anti-sera (rat-O&, in the presence of the indicated amounts of other cell extracts. Goat anti-rabbit sera was then added, and the precipitate analyzed for p-35 on SDS-gel electrophoresis. Competing cell extracts were: (0) IMR-31 bison infected with KiMSV(KiMuLV); (m) KiMSV-infected 3T3 BALB; (A) SVIO-transformed 3T3 BALB; (0) uninfected 3T3 BALB; (0) 3T3 BALB infected with the transformationdefective KiMSV mutant R-20 (this is an absolute mutant); (0) 3T3 BALB infected with the transformation defective KiMSV mutant R-24 (an absolute mutant).

bison cells transformed by the Kirsten sarcoma virus, but is not present in uninfected cells or cells transformed by SV-40. Cells infected with transforming gene point mutants of KiMSV were also analyzed in this competition immunoassay, to further determine if the p-35 antigen may be a transforming (STC)gene product. Two nonconditional point mutants of KiMSV, R-20 and R-24, were examined in infected BALB/ c 3T3 mouse cells. As seen in Fig. 5, cells infected with these src gene mutants did not contain any detectable competing p-35 antigen. This result ties the p-35 antigen to the KiMSV arc gene, either directly as a src gene product or as a viral coded, rat specific polypeptide whose expression is controlled by the src gene. Using the same assay system as described in Fig. 5, further immune competition experiments were performed to independently test the possibility that infection with the helper leukemia virus alone is associated with the presence of the p-35 antigen. We also wanted to determine if the p-35 antigen is expressed in cells transformed by a wider variety of other agents, which could indicate its presence is a secondary effect of transformation. In addition, we wanted to explore the possibility that there is a host polypeptide which is inducible by anaerobic stress and which shares antigenic cross-reactivity over a wide range of species. As shown in Table 2, the p-35 antigen was not detectable in cells infected with Kirsten leukemia virus alone. This antigen was also not detectable in mouse cells which were spontaneously transformed, or transformed by methylcholanthrene, polyoma virus, or the Moloney sarcoma virus. It was also not seen in anaerobically stressed vero monkey cells. In contrast, p-35 antigen was again readily detectable in mouse or monkey cells transformed by the Kirsten sarcoma virus. Competing p-35 antigen was also detected in mouse cells transformed by the Harvey murine sarcoma virus. This observation is consistent with the fact that the Harvey murine sarcoma virus contains the same rat genetic information as does the Kirsten sarcoma virus (Anderson and Robbins, 1976).

A RAT GENE PRODUCT OF KiMSV

39

TABLE 2 IMMUNE COMPETITIONASSAYFORTHE p-~~POLYPEPTIDE" Competing cell extracts Species

BALB 3T3 BALB 3T3 BALB 3T3 BALB 3T3 BALB 3T12 Swiss 3T3 NIH 3T3 Monkey Vero Vero Vero

Percentage competition of p-35 immune precipitation

Infected with* KiMSV SV-40 (MethylcholanthreneY’ Polyoma KiMSV KiMSV(KiMuLV) (Anaerobic stress)

+ + + + + + + -p

90 290 430 280 225 270 375 225 270 100

0 25 2 3 3 7 18 4 30 0

MoMSV(MoMuLV) (Methylcholanthrene) KiMuLV HaMSV(MoMuLV) (KiMSV(KiMuLV)

+ + + + +

140 640 800 1050 1120 720 980

16 10 14 12 48 12 48

Cell line

Experiment 1 Mouse

Competing protein’ (CLg)

Transformed

Experiment 2 Mouse

Monkey

BALB 3T3 BALB 3T3 BALB 3T12 NIH 3T3 NIH 3T3 Vero Vero

a The immune competition assay system was as described in the legend to Fig. 5, with the exception that partially purified (%)-p-35 polypeptide was used. [35S]Met-labeled p-35 polypeptide was obtained from KiMSV(KiMuLV)-infected vero monkey cells. This was partially purified by fractionating into a 100,000g supernatant and then chromatography on Sephadex G-200. Five micrograms of this partially purified material was used in each assay. This would correspond to approximately 300 pg of unpurified S-100. Approximately 400-600 cpm of p-35 was immune precipitated in the absence of competing antigen. Experiment 2 used half as much antibody per sample as experiment 1. b Abbreviations used: KiMSV, Kirsten murine sarcoma virus; KiMuLV, Kirsten murine leukemia virus; MoMSV, Moloney murine sarcoma virus; MoMuLV, Moloney murine leukemia virus; HaMSV, Harvey murine sarcoma virus. c Crude cell extracts were centrifuged 1 hr at 100,000g and the resulting supernatant was used as immune competitor. Protein was determined by the Lowry method. d Transformation of these cells was induced by methylcholanthrene, in the laboratory of Joel Greenberger. They are carried as a transformed cell line. e Anaerobically stressed uninfected fibroblasts are not transformed, but exhibit some characteristics of the transformed phenotype (Anderson et al., submitted for publication).

It should be noted that this competition assay probably would not detect competing antigen present at concentrations much less than one-fifth as great as in KiMSVinfected cells. Nonetheless, it can be used to rule out substantial expression of such antigen as a secondary effect of transformation or anaerobic stress. Copuri$Txation of p-35 Polypeptide Lactate Dehydrogenase Activity

with

The above data strongly suggest that one gene product of the MSV-rat RNA is a

35,000 molecular weight polypeptide. To allow characterization of this polypeptide, we purified it from four systems, with purification monitored through use of double label procedures and SDS-gel electrophoresis. The four double label systems employed were: (1) KiMSV-infected transformed nonproducer BALB 3T3 mouse cells compared to uninfected BALB 3T3 cells, (2) vero monkey cells infected with KiMSV and a KiMuLV helper compared to cells infected with KiMuLV alone, (3) uninfected Fischer rat cells labeled while cultured with or

40

ANDERSON,

MAROTTI, AND WHITAKER-DOWLING

without oxygen, and (4) Fischer rat cells productively transformed by KiMSV and labeled while cultured with or without oxygen. Purification in the vero cell system was also monitored immunologically for the p-35 antigen. All four systems gave very similar results, and these results were seen regardless of whether labeling was done with [3H]- and [14C]leucine or with [35S]-and [3H]methionine. As a first step in purification, we determined which subcellular fraction contained the p-35 polypeptide. As described in Table 3, the anaerobically inducible p-35 was recovered exclusively from the soluble fraction (100,000 g supernatant) of the cytoplasm. Similarly, when KiMSV-infected vero monkey or bison lung cells were analyzed immunologically for p-35 antigen, it too was found only in the soluble fraction of the cytoplasm. A second step in purification was fractionation under nondenaturing conditions on Sephadex G-200 (Fig. 6). In view of the logistical difficulties in running gel analyses of each fraction, and in view of the small amounts of double label material involved, a relatively small (1.4 x 25 cm) Sephadex column was used. While knowing the p-35

had a denatured molecular weight of 35,000, mobility on Sephadex G-200 should provide evidence for the native molecular weight of the protein. SDS-gel electrophoresis was performed on the Sephadex column fractions to locate which contained the p-35. A single peak containing the p-35 was found, but the exact position of this peak was found reproducibly in either of two locations. By comparison to known standards (hexokinase, eatalase, ovalbumin, and aldolase) run separately, the approximate molecular weight of the p-35 under nondenaturing conditions was estimated as either 135,000 daltons (Fig. 6A), or 160,000 daltons (Fig. 6B) suggesting a tetrameric structure and/or an aggregate with other proteins. Candidate p-35 proteins were selected by the criteria: (1) a subunit molecular weight of 35,000, (2) a tetrameric structure, (3) presence in the soluble portion of the cytoplasm, and (4) a reasonable expectation to be inducible by anaerobic stress. Lactate dehydrogenase (LDH), with a tetramer molecular weight of 140,000 (Everse and Kaplan, 1973), and a known ability to be anaerobically inducible (Goodfriend et al., 1966), was prominent on this list. We thus

TABLE 3 LOCATIONOF 35,000 MW PROTEIN IN CELL FRACTIONS”

FO: Initial homogenate Fl: 10 min x 1000 g pellet F2: 10 min x 10,000 g pellet F3: 60 min x 100,000 g pellet F4: 60 min x 100,000 g supt

35,000 MW

Total material: percentage of input

YH dpm

14Cdpm

3H,‘4Cb

Total ‘H excess at 35,000 MW’

100 23 11 8 22

2,900 1,150 2,350 1,940 9,500

650 395 630 670 1,590

4.5 2.9 3.7 2.9 6.0

10,620 -500 -230 -880 7,710

n Subconfluent cultures of Fischer rat embryo cells in culture were labeled with [3H]leucine anaerobically and [‘4C]leucine aerobically, as described under Materials and Methods. Cells were rinsed with phosphate buffered saline and swollen in RSB (RSB = 0.01 J4 Tris pH 7.5, 0.01 M KCI, 0.0015 M MgCl,). Homogenization was achieved with 20 strokes of a Dounce homogenizer. Cell fractions were prepared by differential centrifugation at 4”, as indicated. Fraction Fl was rich in nuclei and cell membranes, F2 in membranes and cytoplasmic organelles, F3 in ribosomes, and F4 was the soluble residue. Aliquots of each fraction were then analyzed by electrophoresis on 12% SDS-polyacrylamide gels. * The ratio of 3H/*4C in the total initial sample was 3.55. c 3H excess at 35,000 MW was computed by subtracting from the observed 3H dpm at 35,000 MW (‘*C dpm at 35,000 MW) x the input 3H/‘4C ratio (3.55). This number was then multiplied by the ratio of total fraction volume/fraction volume loaded on gel.

A RAT GENE PRODUCT OF KiMSV (A) :

t

:

:

6-

.2 6 .I -

2

5 0

05

4

.2

2

.I

0

41

Analysis on SDS-gels of the material released from the column by 1 mM NADH, showed p-35 of over 90% purity had been isolated, as shown in Fig. 8. The copurification of both the double labels in all the diverse systems suggests subunit exchange of the multimeric protein has occurred. The overall purification is summarized in Table 4. This highly purified p-35 was further analyzed by nondenaturing gel electrophoresis, with LDH activity located by enzyme specific staining. These gels were then sliced and counted for radioactivity. As

0

IO Fraction

20

FIG. 6. Sephadex G-200column fractionation: copurification with LDH. Supernatant of a 100,000 g centrifugation was concentrated by lyophilization and chromatographed on a 1.4 x 25-cm Sephadex G-200 column. Included and excluded volumes were determined with blue dextran and bromophenol blue, and the column was calibrated with the following standards run separately: hexokinase (h), catalase (c), ovalbumin (o), and aldolase (a). Individual column fractions were also analyzed by 12% SDS-polyacrylamide gel electrophoresis. Excess 3H dpm migrating as a polypeptide of 35,000 molecular weight are indicated by (0). Endogenous lactate dehydrogenase activity (0) was determined by the procedure of Schwartz (1966) for each fraction, (A) Fischer rat cells were infected with KiMSV(KiMuLV) and radiolabeled anaerobically with rH]Leu or aerobically with [W]Leu. (B) BALB 3T3 cells infected with KiMSV were labeled with [3H]Leu, and uninfected cells were labeled with [W]Leu.

assayed each of the Sephadex G-200 column fractions for endogenous lactate dehydrogenase activity. LDH activity was observed to copurify with the p-35 polypeptide (Fig. 6). Like the p-35, LDH activity was also seen in two forms, at regions corresponding to either 135,000 or 160,000 molecular weight. Affinity chromatographic procedures specific for NADH binding enzymes allow the rapid purification of NADH utilizing enzymes, including lactate dehydrogenase (Nadal-Ginard and Markert, 19’75). We thus effected further purification of the p-35 through use of an Affi-gel blue (Bio-Rad) column, as illustrated in Fig. 7. Again, p-35 copurified with endogenous LDH activity.

Fraction

FIG. 7. Aflinity chromatography of the p-35 polypeptide. The double label p-35 material from KiMSVinfected ([3H]Leu) and uninfected ([W]Leu) BALB 3T3 cells which was purified by Sephadex G-200 chromatography as described in Fig. B was further purified on a 0.5-ml column of Affi-Gel blue (Bio-Rad). Fractions 1 and 2 contain unadsorbed material and fractions 3, 4, and 5 are 2-ml washes with 0.01 M Tris pH 8.5, 10m4M dithiothreitol (buffer A). Fractions 6, 7, and 8 are 2-ml washes with buffer A containing 1 mM NAD+ and 1 mJ4 sodium lactate. The p-35 was then eluted with buffer A containing 1 m&f NADH,, in 2-ml fractions 9, 10, 11. The remaining material was removed from the column in fraction 12 with 2 ml buffer A containing 1 n-J4 NADH, and 0.2 M NaCl. Each fraction was assayed for LDH activity, an aliquot counted for SH and W, and samples were analyzed on 12% SDS-gels to detect the p-35 polypeptide. W dpm are normalized to reflect the ratio of 3H to *‘C incorporated in the crude cell extracts. (0) 3H dpm; (0) “C dpm; (A) p-35 polypeptide; (0) LDH activity.

ANDERSON, MAROTTI, AND WHITAKER-DOWLING

42

0 15

30

45

FRACTION

FIG. 8. SDS-gel electrophoresis of double labeled p-35 at its maximum purification. The p-35 polypeptide from KiMSV-infected @H]leucine label) and uninfected ([14C]leucine) BALB 3T3 cells was purified by a combination of differential centrifugation, Sephadex column chromatography, and affinity chromatography as described in Figs. 6B and 7. The p-35 eluted from the Affi-Gel blue column of Fig. 7 with 1 mJ4 NADH, was analyzed on 12% SDS-polyacrylamide gels.

shown in Fig. 9, our most purified p-35 again corn&rated with lactate dehydrogenase activity. TABLE 4 PURIFICATIONOF p-35 FROM KiMSV-INFECTED BALB 3T3

Crude homogenate Soluble cytoplasm (S-100) Sephadex G-ZOO Affi-gel blue (NADH, eluate)

Protein

p-35”

LDH

1.00”

1.00’

1.00

0.23 0.09

0.80 0.48

0.72 0.57

0.003

0.33

0.47

In parallel, when the p-35 antigen originating in KiMSV-infected vero cells was assayed for immunologically in extracts passed through the Sephadex G-200 column, it too was found to conurifv with LDH activity and to possess-an approximate native molecular weight of 135,000. When

i 6

12

B x B

n Determined by double label SDS-gels comparing KiMSV-infected with uninfected BALB 3T3. b Values represent recoveries relative to the total input material. r p-35 concentrations in total cell extracts are insufficient in KiMSV-infected cells to allow accurate quantitation. However, in anaerobically stressed cells where larger amounts of p-35 are present around 80% of the material in the crude extract is recovered in the S-100. At the next three steps of purification, the p-35 peak was of sufficient magnitude to allow accurate, reproducible quantitation.

3

0

20

IO

30

Frocflon

FIG. 9. Analysis of the purified p-35 polypeptide by nondenaturing gel electrophoresis. The same material which was analyzed on SDS-gels in Fig. 8 was also run on nondenaturing gels according to the procedure of Dietz and Lubrano (1967). Lactate dehydrogenase was located by staining for this activity in situ. The gel was then sliced and counted in a liquid scintillation counter. The scales used for the 3H and 14Cdpm reflect the labeling ratio observed in the crude extract.

A RAT GENE

PRODUCT

OF KiMSV

43

these extracts were then passed through the Aft&gel blue affinity column, the p-35 antigen was also found capable of binding to this matrix. All together, these results strongly suggest that the p-35 is either a lactate dehydrogenase itself or else is complexed with lactate dehydrogenase subunits. In view of its 35,000 molecular weight, which is the same as that of known rat lactate dehydrogenases, and copurification with LDH activity, it seems most likely that the p-35 polypeptide is in fact a lactate dehydrogenase. The behavior of this activity on Sephadex G-200 suggests an additional polypeptide of around 25,000 molecular weight may associate with it.

valerate. In all instances, activity with pyruvate was at least lo-fold greater than with the other substrates. As would be expected if an LDH activity were the WCgene product, transformation-defective mutants of KiMSV did not effect elevated expression of this activity. And as would be expected if the MSV-rat RNA encodes a lactate dehydrogenase, this activity was also significantly elevated in uninfected Fischer rat cells subjected to anaerobic stress. It can be seen, however, that other means of transformation effected increases in LDH activity similar to that seen with Kirsten sarcoma virus infection. Although the immune competition data of Fig. 5 and Table 2 suggested this might be a different LDH activity, direct evidence was needed. If the Kirsten sarcoma virus actually encodes an LDH Activity in KNSV-Transformed LDH activity, cells transformed by this Cells virus should also exhibit qualitative differAs would be anticipated from KiMSV en- ences in their lactate dehydrogenases when coding an LDH activity, the amount of this compared with cells transformed by other enzyme was found to be significantly ele- agents. We thus examined by nondenavated in KiMSV-infected cells (Table 5). turing gel electrophoresis (Dietz and Substrate specificity was examined by com- Lubrano, 1967) the LDH activities present paring enzyme activity with pyruvate, Q- in BALB 3T3 mouse cells infected with or ketobutyrate, a-ketoglutarate, and a-keto- transformed by a variety of agents. As TABLE

5

LACTATE DEHYDROGENASEACTIVITYOFCELLEXTRACTS

Cell line Mouse

BALB BALB BALB BALB BALB BALB

3T3 3T3 3T3 3T3 3T3 3T12

Infected

with

KiMSV td KiMSV (R-20) td KiMSV (R-24) sv-40 -

Transformed -

LDH activity” (IU/mg protein)

+ + +

0.68 2.29 0.39 0.56 2.12 1.44

Rat

Fischer Fischer Fischer (anaerobic)

KiMSV(KiMuLV) -

+ -0

1.07 3.64 2.51

Bison

IMR-31 IMR-31

KiMuLV KiMSV(KiMuLV)

+

0.54 3.10

n Cells were harvested while in exponential growth. LDH activity was assayed by the method of Schwartz and Bodansky (1966) in a 100,000 9 supernatant of cell extracts prepared in RSB + 10s4 M dithiothreitol). Assays were performed immediately after extract preparation, on fresh extracts. Results are the mean of duplicate experiments. b Anaerobically stressed uninfected Fischer rat cells exhibit hexose transport rates elevated to an extent equivalent to KiMSV-transformed cells, but are not fully transformed (Anderson et al., 1979).

44

ANDERSON,

MAROTTI,

AND WHITAKER-DOWLING

shown in Fig. 10, KiMSV-transformed BALB 3T3 cells contain a major peak of LDH activity (at relative mobility 0.2) which is absent in the same cells transformed by SV-40 or in spontaneously transformed (BALB 3T12) cells. Furthermore, cells infected with the transformationdefective mutant of KiMSV, R-24, also are missing the KiMSV-specific LDH peak. In experiments not shown here, transformation-defective mutant R-20-infected cells, or uninfected cells, both were also found not to contain this specific LDH activity. If the rat sequences of the KiMSV genome actually encode a lactate dehydrogenase, then this KiMSV-specific LDH peak might also be detectable in normal, uninfected rat cells. To test this possibility, we compared the LDH isozyme profiles of Fischer rat cells (cultured aerobically or anaerobically) with the isozyme profiles of KiMSV transformed or uninfected BALB

mouse cells. As shown in Fig. 11, a normal rat LDH isozyme migrates in the same position as a KiMSV specific peak present in the KiMSV-transformed mouse cells. This same peak is absent in uninfected or spontaneously transformed mouse cells. These results provide independent evidence supporting the model that the Kirsten sarcoma virus carries a rat gene encoding a lactate dehydrogenase. Other Characteristics

of p-35

Further experiments characterizing the p-35/LDH activity have revealed the following: (1) “LDH-elevating virus” is not a contaminant of our cultures, as shown by direct assay. (2) The p-35 polypeptide is not a leukemia virus p-30. (3) The p-35 is not a virion structural component of KiMSV, and LDH activity is not found secreted extracellularly in detectable levels. (4) Pulsechase experiments show the p-35 is not processed from a larger precursor (e.g., a p-55), unless such processing is totally complete within 10 min. Furthermore, the p-35 is not normally processed to any smaller material within 90 min. (5) Anaerobically induced Fischer rat p-35 comigrates on SDS-gels with p-35 antigen from KiMSVinfected vero monkey cells. (6) There is no detectable protein kinase activity associated with the p-35 polypeptide. DISCUSSION

--I

~.

02

05

-

._-..

L

I

FIG. 10. Qualitative differences in the LDH activities of various BALB mouse cell extracts. Nondenaturing gels as in Fig. 8 were run on fresh 100,000 g supernatants of cell extracts. The gels were stained for LDH activity at the completion of the run, and then were scanned with a recording densitometer. Mobility is relative to a marker dye, bromphenol blue. Protein, 8-10 pg, was loaded on each gel. (A) KiMSV-infected BALB 3T3; (B) KiMSV transformation-defective mutant R-24-infected BALB 3T3; (C) SV-40-transformed BALB 3T3; (D) BALB 3T12, uninfected.

The above data provide evidence that a 35,000 molecular weight polypeptide is a gene product of the rat cell sequences of the Kirsten murine sarcoma virus genome. This polypeptide is seen specifically in cells transformed by the Kirsten sarcoma virus, evidently not being a host polypeptide induced or modified as a general secondary effect of neoplastic transformation. Since cells infected with transforming gene mutants of KiMSV do not contain this p-35 antigen it is most likely either a transforming gene product or else its expression is regulated by a transforming gene product. Antigenically closely related polypeptides are also present in two other systems: normal rat cells subjected to anaero-

A RAT GENE PRODUCT OF KiMSV

45

ABCDEFG

c

FIG. 11. Corn&ration of a normal rat LDH isozyme and a KiMSV specific LDH isozyme. Nondenaturing gels as in Figs. 8 and 10 were run on 100,000 g supernatants of cell extracts. Protein, 5- 10 pg, was loaded on each gel. The gels were stained (90 min at 20”) for LDH activity and then photographed. No isozyme bands were visible with a mobility greater than 0.35. Extracts analyzed were prepared from (A-C) Fischer rat cells and (D-F) BALB mouse cells. (A) Fischer rat cells cultured aerobically. (B) Fischer rat cells cultured anaerobically for 24 hr immediately prior to extract preparation. (C) Fischer rat cells productively infected with and transformed by the Kirsten sarcoma virus. (D) BALB 3T3 mouse cells infected with and transformed by the Kirsten sarcoma virus. (E) Uninfected BALB 3T3 cells. (F) Uninfected BALB 3T12 cells. (G) Assay blank.

bit stress, and heterologous cells transformed by the Harvey murine sarcoma virus, a virus which contains the same rat genetic information as KiMSV. It should be noted, however that differences may exist in the p-35 antigen as it is expressed in each of the three systems. The p-35 polypeptide encoded in the KiMSV genome may be a mutant version of a normal rat cell constituent. Other interpretations can be made for our results. As was initially argued with many other virus T antigens, it is still possible that transformation by KiMSV induces or modifies a specific host polypeptide (p-35), which in our case would be immunologically cross-reactive with a rat p-35. By this model

the shared antigenic determinants on this polypeptide would exist in rat, bison, monkey, and mouse, and yet would still be capable of eliciting an immune response in rabbits. Furthermore, the induction of this host antigen would have to be specific to transformation by KiMSV, and not other transforming agents. And finally, this induced or modified host polypeptide (with LDH activity) would now coincidentally comigrate with a rat lactate dehydrogenase. While these possibilities are conceivable, we believe it is far more likely that (1) the rat sequences of the KiMSV genome actually encode the p-35 polypeptide, (2) this polypeptide is antigenically similar to one produced by rat cells subjected to anaerobic

46

ANDERSON, MAROTTI, AND WHITAKER-DOWLING

lactate dehydrogenase activity could relate to neoplastic transformation. To begin with, high levels of LDH should directly result in high levels of aerobic glycolysis. This would explain the Warburg effect (Warburg, 1956) by which transformed cells consume great quantities of glucose and produce corresponding quantities of lactate, even though respiratory pathways themselves are not blocked. The many diverse phenomena associated with neoplastic transformation suggest a requirement for a pleiotropic effector; one such candidate is the protein kinase associated with the SYCfunction of the (avian) Rous sarcoma virus. It is not difficult to envision a model whereby lactate dehydrogenase could also indirectly act as a pleiotropic effector. Lactate dehydrogenase is believed to play a key metabolic regulatory role in many tissues (Guppy and Hochachka, 1978). Abnormal expression of lactate dehydrogenase activity should have additional indirect consequences, as outlined in Fig. 12. Basically, the interaction of LDH and pyruvate would expedite the conversion of a cell’s NADH, to NAD+, by circumventing the need for mitochondrial function (Gregg, 1972). In turn, this should lead to (1) increased synthesis of poly ADP-ribose, which has been associated with the activation of DNA synthesis, and (2) increased ADP-ribosylation of proteins. One enzyme subject to modulation via ADP-ribosylation is adenyl cyclase (Gill and Meren, 1978) which in turn is linked to expression of some A Model for Neoplastic Transformation characteristics of the transformed phenoIt is not unreasonable to ask at this point type. how uncontrolled or modified expression of This model is consistent with the observation by Schwartz et al. (1974) that the combined level of NADHz + NAD+ is greatly reduced in transformed cells, and that a prime mechanism for NADH,/NAD+ destruction is by nicotinamide removal from NAD+ (Reichsteiner et al., 1976). It is obvious, however, that with a multifunctional molecule like NAD+ numerous other interactions exist which in turn could be formulated into a corresponding number of models for neoplastic transformation. It FIG. 12. A model of how elevated, abnormally con- should also be clear that this system might represent only a part of the transforming trolled expression of lactate dehydrogenase might activity of the Kirsten sarcoma virus. lead to neoplastic transformation. stress (cells which are also induced to transcribe the MSV-rat RNA sequences), and (3) this p-35 polypeptide is an actual transforming gene product. It should be clear that additional polypeptides may be encoded by the rat sequences of the KiMSV genome. Since these sequences constitute at least half of the KiMSV genome, their approximate coding capacity can be estimated as more than 150,000 daltons of protein. Thus additional polypeptides, or multiple copies of the p-35 polypeptide, are quite probably encoded by this RNA. Since the p-35 polypeptide copurifies with lactate dehydrogenase activity, in a native complex of 135,000 or 160,000 molecular weight, and since lactate dehydrogenase is known to be a tetramer with a subunit molecular weight of 35,000, the p-35 polypeptide presumably represents lactate dehydrogenase itself. We do not at present have sufficient data to associate a specific rat tissue lactate dehydrogenase with either the anaerobically induced or KiMSVencoded p-35 polypeptides, although a very similarly migrating isozyme is seen in Fischer rat fibroblasts. Beyond the major M, H, and X tissue isozymes, there are other immunologically distinct minor isozymes, in both free and membrane-associated forms. Further characterization of the apparent KiMSV encoded LDH is currently in progress.

A RAT GENE PRODUCT OF KiMSV ACKNOWLEDGMENTS These studies were supported by grants from the National Cancer Institute and the National Science Foundation. We wish to acknowledge the excellent technical assistance of Lisa Matovcik and William Kovacik. We also wish to thank Victor Fried for his invaluable advice and encouragement, and those individuals too numerous to name whom have provided us with helpful criticisms and suggestions. REFERENCES ANDERSON,G. R., MAROTTI, K. R., and MATOVCIK, L. M. (1979). Anaerobic induction of src related RNA in uninfected rat fibroblasts: The state of the induced cells. Submitted. ANDERSON,G. R., and MATOVCIK, L. M. (1977). Expression of murine sarcoma virus genes in uninfected rat cells subjected to anaerobic stress. Science 197, 1371- 1374. ANDERSON, G. R., and ROBBINS, K. C. (1976). Rat sequences of the Kirsten and Harvey murine sarcoma virus genomes: Nature, origin and expression in rat tumor RNA. J. Viral. 17, 335-351. ANDERSSON,P., GOLDFARB,M. P., and WEINBERG, R. A. (1979). A defined subgenomic fragment of in vitro synthesized Moloney sarcoma virus DNA can induce cell transformation upon transfection. Cell 16, 63-75.

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