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Characterization of a murine myeloma (MOPC-315) C-type virus by nucleic acid hybridization
VIROLOGY
93, 256-259
(1979)
Characterization
of a Murine Myeloma (MOPC-315) Nucleic Acid Hybridization ABRAHAM
Department
of Human Microbiology,
YANIV
AND
ARNONA
Sackler School of Medicine,
C-Type Virus by
GAZIT
Tel-Aviv
University,
Tel-Aviv,
Israel
Accepted August 10, 1978 Employing DNA:RNA hybridization, the murine myeloma MOPC-315 C-type virus was found to be related to the murine leukemia-sarcoma group, sharing the highest degree of homology with AKR leukemia virus. DNA:DNA hybridization experiments with cellular DNAs of various animal species revealed that MOPC-315 viral genome is related extensively to mouse DNA, pointing to the mouse as its species of origin. MOPC-315 viral transcript showed identical C&a values, final extents of hybridization, as well as T,,, values when hybridized either to MOPC-315 cellular DNA or to normal BALB/c DNA, thus indicating that the investigated particle is an endogenous virus of the mouse.
C-type particles have been isolated from several murine myelomas (1-7). According to immunological criteria, these viral particles were found to be related to the murine leukemia-sarcoma group. They contain the leukemia group-specific antigen (l-5), the G (Gross) viral envelope antigen (GVEA), and the xVEA, a viral envelope antigen distinct from GVEA (8). The measurements of the nucleic acid sequence homology among RNA tumor viruses distinguish between antigenically closely related viruses, thus providing a highly sensitive approach for classification. The extensive examination of the homology between the viral genomes of the known murine oncornaviruses ended up in a welldefined classification of these viruses (9-12). However, the status of the murine myeloma C-type viruses according to this classification has not yet been ascertained, except in one instance (5), where MOPC460 extracellular particles were found to share partial homology with Moloney leukemia virus RNA. The myeloma MOPC-315 cells, derived from BALB/c mice injected with Bayol F (13), were found to release C-type particles both in uivo (6) and in vitro (14). The present study was undertaken to classify this myeloma virus, to reveal its phylogenetic origin, and to establish whether it is 0042-6822/79/030256-04$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
an endogenous virus of the mouse. To establish the status of the MOPC-315 myeloma C-type particle within the murine leukemia-sarcoma group, the nucleotide sequence homology between these viral particles and some other known oncornaviruses was assayed. This was accomplished by measuring the extent of hybridization as well as the thermal stability of the hybrids formed between the myeloma virus [3H]cDNA and 70 S RNAs extracted from various oncornaviruses (Table 1). Consistant with its isolation from mouse cells, the MOPC-315 virus is more homologous to the murine oncornaviruses than to SSV and AMV. Among the murine oncornaviruses, the MOPC viral genome shares extensive nucleotide sequences with AKR and KiMSV (90 and 77% hybridization values, respectively), and only moderate sequence relationships with RLV (46%) and MoLV (45%). The relatedness inferred from the final extents of hybridization is in accord with the thermal stability of the formed hybrids as the highest thermal dissociation temperature (Z’,,J value (88’) was obtained for the homologous hybrid, followed by AKR and Ki-MSV with 85”, MoLV with 82”, and RLV with 81’. The considerable relatedness found between nucleotide sequences of the myeloma virus and the other murine oncornaviruses 256
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TABLE 1 NUCLEIC ACID HOMOLOGY OF MYELOMA MOPC-315 VIRUS WITH RNA TUMOR VIRUSES 70 S viral RNA”
MOPC-315 AKR Ki-MSV MoLV RLV ssv AMV
Maximum hybridization values” VW
Tfll’
100 90 77 45 46 17 3
843 85 85 82 81 79.5 ND”
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o MOPC-315 60 to 70 S viral RNA was prepared from the myeloma viral particles obtained from culture fluids of MOPC-315 cells (14). The purification of viral particles and the isolation of viral RNA were described previously (6). 70 S RNAs from avian myeloblastosis virus (AMV), Rauscher leukemia virus (RLV), Moloney leukemia virus (MoLV), Kirstein murine sarcoma virus (Ki-MSV), AKR virus, and simian sarcoma virus 1 (SSV-1) were kindly supplied by Dr. R. W. Sweet of the Institute of Cancer Research, Columbia University, New York. “The myeloma virus ‘H-labeled cDNA was prepared in an endogenous reverse transcriptase reaction from purified virus particles. The cDNA made was 4 to 6 S in size, its sensitivity to S1 nuclease was 95%, and it protected about 90% of iodinated homologous 70 S RNA from RNase digestion at a 3:l to 4:l molar excess. A total of 1000 cpm of MOPC-315 [“H]cDNA was annealed to 0.2 ag 70 S RNA of the listed viruses in lo-al reaction volumes containing 0.06 M NaCl, 0.04 M Tris-HCl, pH 7.2, 0.002 M EDTA, and 0.1% SDS. The reaction mixtures were sealed in siliconized capillaries, denatured at 100” for 1 min, chilled on ice, and then incubated at 68” to reach a C,t of 10 mol/sec liter-‘. Hybrid formation was assayed by resistance to S, nuclease (14). The percentage of hybridization was normalized to that obtained at saturation with homologous viral RNA (actual value of 92%). ‘Samples were diluted lo-fold in 0.4 M NaCl, heated for 5 min at 5’ increments from 65’ to lOO”, and then digested with S1 nuclease. The temperature at which 50% of hybridized [3H]cDNA become dissociated ( T,) was determined from the melting profiles. ’ ND, Not determined.
suggests that the MOPC-315 virus is of mouse origin. This conclusion is based on studies (9) which showed that the murine oncornaviruses, whether endogenous or exogenous, are all closely related, in contrast to their lower degree of nucleic acid homology with type C viruses of other species. The close homology of MOPC cDNA to Ki-
TABLE 2 NUCLEIC ACID HOMOLOGY OF MYELOMA MOPC-315 VIRUS WITH CELLULAR DNAs Cellular
DNA”
BALB/c AKR C57 black NIH Swiss Rat Hamster Guinea pig Cat E. coli
Maximum hybridization values (%) b 60”
80”
93 91 92 85 60 60 20 20 5
76 64 61 57 2 2 2 2 0
T,’
82 81.5 81.5 81.5 65 63.5 63.5 63.5 <60
n Cellular DNAs from AKR, BALB/c, NIH Swiss, C57 black strain of mice, as well as DNA from rat (Charles River derived), Syrian golden hamster, and guinea pig were obtained from l-day-old embryos. Spleens were used as the source of cellular DNA of cat origin. DNAs were extracted as described elsewhere (20). ’ A total of 1000 cpm of MOPC-315 [3H]cDNA was annealed to a C,t of 10,000 with the various cellular DNAs in lo-al reaction mixtures containing 0.4 M NaCl, 0.05 M EDTA, and 0.1% SDS. Hybridization was initiated by heating the reaction mixtures to 105” for 2 min in an ethylene glycol water bath, followed by incubation at 66”. Following hybridization, reaction mixtures were diluted in 2 ml of 0.12 M sodium phosphate buffer (NaPB), pH 6.8, containing 0.4% SDS, and applied to hydroxylapatite (Bio-Rad) column at 60”. Unhybridized DNA was eluted with 20 ml of the same buffer and the hybrid was then melted at 5’ intervals and eluted with 8 ml buffer. After the 100” elution, the column was washed with 0.4 M NaPB to remove any remaining hybrids. The percentage of hybridization at 60’ or 80” refers to fraction of [3H]cDNA remaining hybridized when the column temperature was 60’ or was raised to 80”, respectively. ’ The T,,, values were calculated from the thermal elution profiles.
MSV does not contradict the notion of MOPC-virus being of mouse origin, although Ki-MSV is known to contain rat leukemia virus sequences (25). The KiMSV stock used here contained an excess of Ki-MoLV (R.W. Sweet, personal communication), which can be responsible for the high extent of hybridization observed between the myeloma virus and Ki-MSV. The partial homology of the myeloma virus to SSV is consistent with the relatedness of this simian virus with some murine RNA
258
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tumor viruses (9, 12, 16-18) as well as with normal mouse cellular DNA (17). The relatedness of MOPC-315 virus to other oncornaviruses, although suggesting its mouse origin, does not prove it unequivocally, since even horizontally transmitted viruses might exhibit a homology that would depend on the length of time since the species was infected (9). Therefore we adopted the approach (19) of measuring the homology between the viral genome and the cellular DNA of various animal species. This method is based on the observation that oncornaviruses exhibit homology with normal cellular DNA of their natural host. Viral cDNA was annealed to a C,t of 10,000 mol/sec liter-’ with a vast excess of cellular DNAs obtained from various strains of mice (BALB/c, AKR, C57 black, and NIH Swiss), as well as from other rodent species (rat, hamster, and guinea pig) and cat that represents a more remote mammalian speties (Table 2). Regarding the overall extent of hybridization, as well as the thermal stability of the hybrids, the myeloma virus was found to possess the greatest degree of
homology with cellular DNAs of the various species of mice. This fact was accentuated when attention was focused on perfeet duplexes of comparatively high thermal stability (80’ and above), where few, if any, stable duplexes were formed with DNAs extracted from rodent species other than mouse. All these hybridization results assign a mouse origin to the MOPC-315 virus. The presence of all the genetic information required for the production of an oncornavirus in the normal cellular DNA, provides a relevant parameter for establishing its endogenous nature. In association kinetits assays performed between MOPC viral probe and cellular DNA from myeloma cells and from normal uninfected BALB/c cells, identical extents of hybridization (-86%) and reannealing kinetics ( Cotl,z - 10 mol/sec liter-‘) were obtained, thus indieating quantitative similarity in MOPC315-viral information present in both cellular DNAs. Moreover, the fact that the hybrid formed with MOPC cellular DNA did not differ, and in fact was indistinguish-
SO-
TEMPERATURE 'C FIG. 1. Thermal stability of hybrids formed between MOPC-315 viral [3H]DNA probe and MOPC-315 (O), or BALB/c (W) cellular DNA. Hybridization carried out to a C,t of 10,000 mol/sec liter-’ at 66”, and thermal elution from hydroxylapatite columns were performed as described in Table 2. DNA:DNA dissociation profiles for MOPC-315 (0) BALB/c (0) cellular DNA, were monitored by absorbance at 260 nm.
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able in its melting profile, from the hybrid formed with BALB/c cellular DNA (Fig. l), shows that the viral information present in both cellular DNAs is also qualitatively similar. Thus, the same number of copies of MOPC viral-specific DNA exist both in MOPC-315 cellular DNA and the normal BALB/c DNA. Hence, the myeloma MOPC-315 virus can be established as an endogenous virus of the mouse. ACKNOWLEDGMENTS We wish to thank Drs. S. Spiegelman and SW. Sweet for valuable discussion and advice during this work and for the kind gift of viral RNAs. The skillful technical assistance of Mrs. M. Dvir is gratefully acknowledged. REFERENCES 1. WATSON, J., RALPH, P., SARKAR, S., and COHN, M., Proc. Nat. Acad. Sci. USA 66, 344-351 (1970). 2. KARPAS, A., and CAWLEY, J., Eur. J. Cancer, 8, 363-364 (1972a). 3. KARPAS, A., and CAWLEY, J., Eur. J. Cancer 6, 601-604 (1972b). 4. VOLKMAN, L. E., and KRUEGER, R. G., J. Viral. 12, 1589-1597 (1973). 5. ROBERTSON, D. L., YAU, P., DOBBERTIN, D. C., SWEENEY, T. K., THACH, S. S., BRENDLER, T., and THACH, R. E., J. Viral. 18.344-355 (1976). 6. YANIV, A., KLEINMAN, R., and EYLAN, E., J. Gen. Viral. 32,301-309 (1976).
259
7. KRUEGER, R. G., J. Gen. Viral. 37,475-485 (1977). 8. AOKI, T., and TAKAHASHI, T., J. Exp. Med. 135, 443-457 (1972). 9. BENVENISTE, R. E., and TODARO, G. J., Proc. Nat. Acad. Sci. USA 70,3316-3320 (1973). 10. HAAPALA, D. K., and FISCHINGER, P. J., Science 180,972-974 (1973). 11. CALLAHAN, R., BENVENISTE, R. E., LIEBER, M. M., and TODARO, G. J., J. Virol. 14, 1394-1403 (1974). 12. EAST, J. L., KNESEK, J. E., CHAN, J. C., and DMOCHOWSKI, L., J. Viral. 14.1396-1408 (1975). 13. POTTER, M., and LIEBERMAN, R., Adv. Immunol. 7,91-145 (1967). 14. YANIV, A., GAZIT, A., DVIR, M., GUTHMAN, D., and EYLAN, E., Eur. J. Cancer 14, 771-779 (1978). 15. SCOLNICK, E. M., RANDS, E., WILLIAMS, D., and PARKS, W. P., J. Virol. 12,458-463 (1973). 16. BENVENISTE, R. E., HEINEMANN, R., WILSON, G. L., CALLAHAN, R., and TODARO, G. J., J. Viral. 14,56-57 (1974). 17. WONG-STAAL, F., GALLO, R. C., and GILLESPIE, D., Nature (London) 256,670-672 (1975). 18. LIEBER, M. M., SHERR, C. J., TODARO, G. J., BENVENISTE, R. E., CALLAHAN, R., and COON, H. G., Proc. Nut. Acad. Sci. USA 72,2315-2319 (1975). 19. RUPRECHT, M. R., GOODMAN, N. C., and SPIEGELMAN, S., Proc. Nat. Acad. Sci. USA 70, 1437-1441 (1973). 20. SWEET, R. W., GOODMAN, N. C., CHO, J. R., RuPRECHT, R. M., REDFIELD, R. R., and SPIEGELMAN, S., Proc. Nat. Acad. Sci. USA 71, 1705-170s (1974).
93, 256-259
(1979)
Characterization
of a Murine Myeloma (MOPC-315) Nucleic Acid Hybridization ABRAHAM
Department
of Human Microbiology,
YANIV
AND
ARNONA
Sackler School of Medicine,
C-Type Virus by
GAZIT
Tel-Aviv
University,
Tel-Aviv,
Israel
Accepted August 10, 1978 Employing DNA:RNA hybridization, the murine myeloma MOPC-315 C-type virus was found to be related to the murine leukemia-sarcoma group, sharing the highest degree of homology with AKR leukemia virus. DNA:DNA hybridization experiments with cellular DNAs of various animal species revealed that MOPC-315 viral genome is related extensively to mouse DNA, pointing to the mouse as its species of origin. MOPC-315 viral transcript showed identical C&a values, final extents of hybridization, as well as T,,, values when hybridized either to MOPC-315 cellular DNA or to normal BALB/c DNA, thus indicating that the investigated particle is an endogenous virus of the mouse.
C-type particles have been isolated from several murine myelomas (1-7). According to immunological criteria, these viral particles were found to be related to the murine leukemia-sarcoma group. They contain the leukemia group-specific antigen (l-5), the G (Gross) viral envelope antigen (GVEA), and the xVEA, a viral envelope antigen distinct from GVEA (8). The measurements of the nucleic acid sequence homology among RNA tumor viruses distinguish between antigenically closely related viruses, thus providing a highly sensitive approach for classification. The extensive examination of the homology between the viral genomes of the known murine oncornaviruses ended up in a welldefined classification of these viruses (9-12). However, the status of the murine myeloma C-type viruses according to this classification has not yet been ascertained, except in one instance (5), where MOPC460 extracellular particles were found to share partial homology with Moloney leukemia virus RNA. The myeloma MOPC-315 cells, derived from BALB/c mice injected with Bayol F (13), were found to release C-type particles both in uivo (6) and in vitro (14). The present study was undertaken to classify this myeloma virus, to reveal its phylogenetic origin, and to establish whether it is 0042-6822/79/030256-04$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
an endogenous virus of the mouse. To establish the status of the MOPC-315 myeloma C-type particle within the murine leukemia-sarcoma group, the nucleotide sequence homology between these viral particles and some other known oncornaviruses was assayed. This was accomplished by measuring the extent of hybridization as well as the thermal stability of the hybrids formed between the myeloma virus [3H]cDNA and 70 S RNAs extracted from various oncornaviruses (Table 1). Consistant with its isolation from mouse cells, the MOPC-315 virus is more homologous to the murine oncornaviruses than to SSV and AMV. Among the murine oncornaviruses, the MOPC viral genome shares extensive nucleotide sequences with AKR and KiMSV (90 and 77% hybridization values, respectively), and only moderate sequence relationships with RLV (46%) and MoLV (45%). The relatedness inferred from the final extents of hybridization is in accord with the thermal stability of the formed hybrids as the highest thermal dissociation temperature (Z’,,J value (88’) was obtained for the homologous hybrid, followed by AKR and Ki-MSV with 85”, MoLV with 82”, and RLV with 81’. The considerable relatedness found between nucleotide sequences of the myeloma virus and the other murine oncornaviruses 256
SHORT
TABLE 1 NUCLEIC ACID HOMOLOGY OF MYELOMA MOPC-315 VIRUS WITH RNA TUMOR VIRUSES 70 S viral RNA”
MOPC-315 AKR Ki-MSV MoLV RLV ssv AMV
Maximum hybridization values” VW
Tfll’
100 90 77 45 46 17 3
843 85 85 82 81 79.5 ND”
257
COMMUNICATIONS
o MOPC-315 60 to 70 S viral RNA was prepared from the myeloma viral particles obtained from culture fluids of MOPC-315 cells (14). The purification of viral particles and the isolation of viral RNA were described previously (6). 70 S RNAs from avian myeloblastosis virus (AMV), Rauscher leukemia virus (RLV), Moloney leukemia virus (MoLV), Kirstein murine sarcoma virus (Ki-MSV), AKR virus, and simian sarcoma virus 1 (SSV-1) were kindly supplied by Dr. R. W. Sweet of the Institute of Cancer Research, Columbia University, New York. “The myeloma virus ‘H-labeled cDNA was prepared in an endogenous reverse transcriptase reaction from purified virus particles. The cDNA made was 4 to 6 S in size, its sensitivity to S1 nuclease was 95%, and it protected about 90% of iodinated homologous 70 S RNA from RNase digestion at a 3:l to 4:l molar excess. A total of 1000 cpm of MOPC-315 [“H]cDNA was annealed to 0.2 ag 70 S RNA of the listed viruses in lo-al reaction volumes containing 0.06 M NaCl, 0.04 M Tris-HCl, pH 7.2, 0.002 M EDTA, and 0.1% SDS. The reaction mixtures were sealed in siliconized capillaries, denatured at 100” for 1 min, chilled on ice, and then incubated at 68” to reach a C,t of 10 mol/sec liter-‘. Hybrid formation was assayed by resistance to S, nuclease (14). The percentage of hybridization was normalized to that obtained at saturation with homologous viral RNA (actual value of 92%). ‘Samples were diluted lo-fold in 0.4 M NaCl, heated for 5 min at 5’ increments from 65’ to lOO”, and then digested with S1 nuclease. The temperature at which 50% of hybridized [3H]cDNA become dissociated ( T,) was determined from the melting profiles. ’ ND, Not determined.
suggests that the MOPC-315 virus is of mouse origin. This conclusion is based on studies (9) which showed that the murine oncornaviruses, whether endogenous or exogenous, are all closely related, in contrast to their lower degree of nucleic acid homology with type C viruses of other species. The close homology of MOPC cDNA to Ki-
TABLE 2 NUCLEIC ACID HOMOLOGY OF MYELOMA MOPC-315 VIRUS WITH CELLULAR DNAs Cellular
DNA”
BALB/c AKR C57 black NIH Swiss Rat Hamster Guinea pig Cat E. coli
Maximum hybridization values (%) b 60”
80”
93 91 92 85 60 60 20 20 5
76 64 61 57 2 2 2 2 0
T,’
82 81.5 81.5 81.5 65 63.5 63.5 63.5 <60
n Cellular DNAs from AKR, BALB/c, NIH Swiss, C57 black strain of mice, as well as DNA from rat (Charles River derived), Syrian golden hamster, and guinea pig were obtained from l-day-old embryos. Spleens were used as the source of cellular DNA of cat origin. DNAs were extracted as described elsewhere (20). ’ A total of 1000 cpm of MOPC-315 [3H]cDNA was annealed to a C,t of 10,000 with the various cellular DNAs in lo-al reaction mixtures containing 0.4 M NaCl, 0.05 M EDTA, and 0.1% SDS. Hybridization was initiated by heating the reaction mixtures to 105” for 2 min in an ethylene glycol water bath, followed by incubation at 66”. Following hybridization, reaction mixtures were diluted in 2 ml of 0.12 M sodium phosphate buffer (NaPB), pH 6.8, containing 0.4% SDS, and applied to hydroxylapatite (Bio-Rad) column at 60”. Unhybridized DNA was eluted with 20 ml of the same buffer and the hybrid was then melted at 5’ intervals and eluted with 8 ml buffer. After the 100” elution, the column was washed with 0.4 M NaPB to remove any remaining hybrids. The percentage of hybridization at 60’ or 80” refers to fraction of [3H]cDNA remaining hybridized when the column temperature was 60’ or was raised to 80”, respectively. ’ The T,,, values were calculated from the thermal elution profiles.
MSV does not contradict the notion of MOPC-virus being of mouse origin, although Ki-MSV is known to contain rat leukemia virus sequences (25). The KiMSV stock used here contained an excess of Ki-MoLV (R.W. Sweet, personal communication), which can be responsible for the high extent of hybridization observed between the myeloma virus and Ki-MSV. The partial homology of the myeloma virus to SSV is consistent with the relatedness of this simian virus with some murine RNA
258
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tumor viruses (9, 12, 16-18) as well as with normal mouse cellular DNA (17). The relatedness of MOPC-315 virus to other oncornaviruses, although suggesting its mouse origin, does not prove it unequivocally, since even horizontally transmitted viruses might exhibit a homology that would depend on the length of time since the species was infected (9). Therefore we adopted the approach (19) of measuring the homology between the viral genome and the cellular DNA of various animal species. This method is based on the observation that oncornaviruses exhibit homology with normal cellular DNA of their natural host. Viral cDNA was annealed to a C,t of 10,000 mol/sec liter-’ with a vast excess of cellular DNAs obtained from various strains of mice (BALB/c, AKR, C57 black, and NIH Swiss), as well as from other rodent species (rat, hamster, and guinea pig) and cat that represents a more remote mammalian speties (Table 2). Regarding the overall extent of hybridization, as well as the thermal stability of the hybrids, the myeloma virus was found to possess the greatest degree of
homology with cellular DNAs of the various species of mice. This fact was accentuated when attention was focused on perfeet duplexes of comparatively high thermal stability (80’ and above), where few, if any, stable duplexes were formed with DNAs extracted from rodent species other than mouse. All these hybridization results assign a mouse origin to the MOPC-315 virus. The presence of all the genetic information required for the production of an oncornavirus in the normal cellular DNA, provides a relevant parameter for establishing its endogenous nature. In association kinetits assays performed between MOPC viral probe and cellular DNA from myeloma cells and from normal uninfected BALB/c cells, identical extents of hybridization (-86%) and reannealing kinetics ( Cotl,z - 10 mol/sec liter-‘) were obtained, thus indieating quantitative similarity in MOPC315-viral information present in both cellular DNAs. Moreover, the fact that the hybrid formed with MOPC cellular DNA did not differ, and in fact was indistinguish-
SO-
TEMPERATURE 'C FIG. 1. Thermal stability of hybrids formed between MOPC-315 viral [3H]DNA probe and MOPC-315 (O), or BALB/c (W) cellular DNA. Hybridization carried out to a C,t of 10,000 mol/sec liter-’ at 66”, and thermal elution from hydroxylapatite columns were performed as described in Table 2. DNA:DNA dissociation profiles for MOPC-315 (0) BALB/c (0) cellular DNA, were monitored by absorbance at 260 nm.
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COMMUNICATIONS
able in its melting profile, from the hybrid formed with BALB/c cellular DNA (Fig. l), shows that the viral information present in both cellular DNAs is also qualitatively similar. Thus, the same number of copies of MOPC viral-specific DNA exist both in MOPC-315 cellular DNA and the normal BALB/c DNA. Hence, the myeloma MOPC-315 virus can be established as an endogenous virus of the mouse. ACKNOWLEDGMENTS We wish to thank Drs. S. Spiegelman and SW. Sweet for valuable discussion and advice during this work and for the kind gift of viral RNAs. The skillful technical assistance of Mrs. M. Dvir is gratefully acknowledged. REFERENCES 1. WATSON, J., RALPH, P., SARKAR, S., and COHN, M., Proc. Nat. Acad. Sci. USA 66, 344-351 (1970). 2. KARPAS, A., and CAWLEY, J., Eur. J. Cancer, 8, 363-364 (1972a). 3. KARPAS, A., and CAWLEY, J., Eur. J. Cancer 6, 601-604 (1972b). 4. VOLKMAN, L. E., and KRUEGER, R. G., J. Viral. 12, 1589-1597 (1973). 5. ROBERTSON, D. L., YAU, P., DOBBERTIN, D. C., SWEENEY, T. K., THACH, S. S., BRENDLER, T., and THACH, R. E., J. Viral. 18.344-355 (1976). 6. YANIV, A., KLEINMAN, R., and EYLAN, E., J. Gen. Viral. 32,301-309 (1976).
259
7. KRUEGER, R. G., J. Gen. Viral. 37,475-485 (1977). 8. AOKI, T., and TAKAHASHI, T., J. Exp. Med. 135, 443-457 (1972). 9. BENVENISTE, R. E., and TODARO, G. J., Proc. Nat. Acad. Sci. USA 70,3316-3320 (1973). 10. HAAPALA, D. K., and FISCHINGER, P. J., Science 180,972-974 (1973). 11. CALLAHAN, R., BENVENISTE, R. E., LIEBER, M. M., and TODARO, G. J., J. Virol. 14, 1394-1403 (1974). 12. EAST, J. L., KNESEK, J. E., CHAN, J. C., and DMOCHOWSKI, L., J. Viral. 14.1396-1408 (1975). 13. POTTER, M., and LIEBERMAN, R., Adv. Immunol. 7,91-145 (1967). 14. YANIV, A., GAZIT, A., DVIR, M., GUTHMAN, D., and EYLAN, E., Eur. J. Cancer 14, 771-779 (1978). 15. SCOLNICK, E. M., RANDS, E., WILLIAMS, D., and PARKS, W. P., J. Virol. 12,458-463 (1973). 16. BENVENISTE, R. E., HEINEMANN, R., WILSON, G. L., CALLAHAN, R., and TODARO, G. J., J. Viral. 14,56-57 (1974). 17. WONG-STAAL, F., GALLO, R. C., and GILLESPIE, D., Nature (London) 256,670-672 (1975). 18. LIEBER, M. M., SHERR, C. J., TODARO, G. J., BENVENISTE, R. E., CALLAHAN, R., and COON, H. G., Proc. Nut. Acad. Sci. USA 72,2315-2319 (1975). 19. RUPRECHT, M. R., GOODMAN, N. C., and SPIEGELMAN, S., Proc. Nat. Acad. Sci. USA 70, 1437-1441 (1973). 20. SWEET, R. W., GOODMAN, N. C., CHO, J. R., RuPRECHT, R. M., REDFIELD, R. R., and SPIEGELMAN, S., Proc. Nat. Acad. Sci. USA 71, 1705-170s (1974).