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
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
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
