JOURNAL OF VIROLOGY, Nov. 1977, p. 609-617
Vol. 24, No. 2 Printed in U.S.A.
Copyright © 1977 American Society for Microbiology
RNase Ti-Resistant Oligonucleotides of an N- and a B-Tropic Murine Leukemia Virus of BALB/c: Evidence for Recombination Between These Viruses DOUGLAS V. FALLER AND NANCY HOPKINS* Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication 8 July 1977
We used two-dimensional gel electrophoresis to obtain fingerprints of 32Plabeled RNase Tl-resistant oligonucleotides derived from the genomes of an Nand a B-tropic murine leukemia virus of BALB/c. These viruses share approximately 30 large Ti-resistant oligonucleotides. In addition, there are eight large oligonucleotides unique to the N-tropic virus, and there are six B-tropic virusspecific oligonucleotides. Viruses, designated XLP-N, which appear by biological criteria and analysis of virion proteins to be recombinants between these N- and B-tropic viruses, possess some but not all of the N or B virus-specific oligonucleotides. Both N- and B-tropic type C RNA viruses have been isolated from low-leukemic BALB/c mice (14, 15). N-tropic virus has been obtained by bromodeoxyuridine induction of BALB/c cells in culture (1) and from extracts of spleens of nondiseased mice (6, 7). B-tropic virus has been obtained from spontaneous or chemically induced tumors of BALB/c cells (13) and from spleen extracts of nondiseased mice (7). Hybridization studies, using N- and B-tropic isolates of BALB/c, showed that these viruses are >95%
homologous (2, 11). Two spleen isolates obtained by Hartley et al. (7) from a single nondiseased adult BALB/c mouse were cloned and studied extensively in several laboratories as prototype N-tropic and B-tropic viruses. These isolates are designated WN1802N and WN1802B. Hopkins and Jolicoeur (8) found that three biologically distinguishable N-tropic viruses could be isolated from WN1802N: one type, designated NP-N, was unable to form XC plaques; one, SP-N, made small XC plaques; and one, LP-N, made large XC plaques and was characteristic of uncloned WN18Q2N. The existence of viruses with unusual XC-plaque morphology provided a genetic marker that was used by Hopkins et al. (10) to isolate viruses that appeared, by biological criteria, to be recombinants: coinfection of SC-1 cells (5) (a cell line that does not show Fv-1 restriction) with SP-N and a large XC-plaqueforming B-tropic virus, derived from WN1802B and designated LP-B, yielded progeny with the N-tropism of SP-N and the large XC-plaque morphology of LP-B. The viruses with recombinant phenotype were designated XLP-N. Fur-
ther evidence that XLP-N viruses are recombinants was obtained from analysis of the antigenicity of infected cells and determination of the electrophoretic mobility on sodium dodecyl sulfate-gels of virion proteins of SP-N, LP-B, and 21 cloned XLP-N viruses obtained from seven independent crosses (9, 16). We were interested in obtaining additional evidence that XLP-N viruses are recombinants and also in attempting to map regions of the genome responsible for N- or B-tropism and large XC-plaque morphology. A method which has proved highly successful for mapping genes of avian type C viruses is analysis of the large oligonucleotides generated by RNase T1 digestion of parental and recombinant viral RNAs (3, 18). Thus, we analyzed the genomes of SPN, LP-B, and XLP-N viruses by this method. Consistent with hybridization studies showing extensive homology between N- and B-tropic viruses of BALB/c, we found that SP-N and LP-B share approximately 30 out of 36 to 38 of their large RNase Ti-resistant oligonucleotides. However, SP-N possesses eight and LP-B possesses six distinctive oligonucleotides. XLP-N viruses possessed some SP-N- and some LP-Bcharacteristic oligonucleotides, a result which provides substantial evidence that these viruses are genetic recombinants.
609
MATERIALS AND METHODS Cells and viruses. The origin and methods of propagation of SP-N (8, 10), LP-B (10), and XLP-N (10) viruses have been described. NIH/3T3 cells chronically infected with SP-N or XLP-N viruses and BALB/3T3 cells chronically infected with LP-B virus
610
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were used in the experiments reported here. Finger- are probably eight N-specific oligonucleotides. prints prepared from these viruses grown in SC-1 (5) Six of these were readily detected, and the prodcells yielded the same pattern of oligonucleotide spots ucts obtained after pancreatic RNase digestion as when the viruses were grown in NIH/3T3 or of these oligonucleotides are shown in Table 1. BALB/3T3 cells (unpublished data). migrated close RNA fingerprinting and characterization of A seventh oligonucleotide, N-37, oligonucleotides. These procedures have been de- to a mass of unresolved oligonucleotides and scribed in detail (4). The identity of apparently SP- was, therefore, difficult to analyze. Preliminary N- or LP-B-characteristic oligonucleotides inherited results indicated that this spot consists of U, by XLP-N viruses was confirmed by qualitative anal- 3C, (AC), (A3U), (A5C), (A2G). The existence of ysis of the products of pancreatic RNase digestion of an eighth N-specific oligonucleotide at position these spots. This means that autoradiograms of the 12 was inferred from the following observations: separated digestion products of these 3P-labeled T1 spot 12 of SP-N appeared "darker" relative to oligonucleotides were compared visually with autora- spots with similar electrophoretic mobility in diograms of the digestion products of the correspond- the second dimension than the corresponding ing oligonucleotides derived from SP-N or LP-B viruses. If the electrophoretic mobility and relative in- oligonucleotide of LP-B. Since mobility in the tensities of the products were the same, by eye, then second dimension of electrophoresis is based the oligonucleotides were considered to be the same. primarily on size, that spot 12 of SP-N is darker The secondary digestion products of the Ti oligonu- than oligonucleotides at a similar position imcleotides of SP-N and LP-B were analyzed quantita- plied that this spot is a doublet, consisting of tively as described previously (4). two (not necessarily identical) oligonucleotides
RESULTS
RNase Tl-resistant oligonucleotides of parental viruses, SP-N and LP-B. 32P-labeled 70S viral RNA prepared from culture fluids of cells chronically infected with SP-N or LP-B was digested with RNase T1, and the resulting oligonucleotides were separated by two-dimensional gel electrophoresis. "Fingerprints" obtained by autoradiography of the second-dimension gels are shown in Fig. 1A and B, and diagrams of these fingerprints are shown in Fig. 1C and D. The fingerprints of SP-N and LP-B were strikingly similar. The two viruses appeared to share approximately 30 out of 36 to 38 oligonucleotide "spots." In Fig. 1C and D, oligonucleotides that appeared common to SP-N and LPB are shown as open circles. Since oligonucleotides of different sequence or even base composition can possess similar electrophoretic mobilities in this system, further characterization of apparently common oligonucleotides was necessary before identity could be reasonably assumed. Oligonucleotides were removed from the gels and digested with pancreatic RNase, and the digestion products were separated and quantitated. The results of this analysis for the numbered oligonucleotides in Fig. 1C and D are shown in Table 1. Oligonucleotides represented by open circles yielded the same digestion products whether they were derived from SP-N or LP-B, and therefore the products for these oligonucleotides are given only once in Table 1. It is likely that these oligonucleotides are the same in the two viruses. Oligonucleotides that are unique to SP-N, represented by solid black circles in Fig. 1C, are designated by the prefix "N" in Table 1. There
that comigrate, and quantitation of the radioactivity at this position corroborated this visual observation. Analysis of the secondary digestion products of spot 12 of SP-N revealed the presence of 2U, 7C, 6 (AC), (AU), (A2U), (A3G), and (A4G). The presence of two G-containing products confirmed the occurrence of two oligonucleotides at this position. Since the secondary digestion products of spot 12 of LP-B [U, 4C, 3(AC), (A2U), (A4G)] are a subset of the products of the SP-N spot 12, it seemed likely that an oligonucleotide, 12, was present in both SP-N and LP-B and that SP-N contained, in addition, an oligonucleotide, N-12, with the (inferred) composition U, 3C, 3(AC), (AU), (A3G). There are probably six LP-B-specific oligonucleotides. These are represented as hatched circles in Fig. 1D, and they are designated by the prefix "B" in Table 1. The products of pancreatic RNase digestion of five readily detected LP-B-specific oligonucleotides are shown in Table 1. The presence of a sixth LP-B-specific T1 oligonucleotide at position 35 was, like N-12, inferred from the relatively greater intensity and the presence of approximately twice the amount of radioactivity in this spot relative to the flanking oligonucleotides. However, as reported previously (4), determinations of the relative molarity and secondary digestion products of this spot were hampered by contamination from oligonucleotides migrating slightly ahead of it. Preliminary analysis of secondary digestion products of spot 35 of LP-B suggested that it contained all the products present in the oligonucleotide at position 35 of SP-N plus additional products (see reference 4). This result suggested that spot 35 of LP-B may consist of two oligonucleotides (35 and B-35) that comigrate and
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TABLE 1. Products of pancreatic RNase digestion of large RNase Tl-resistant oligonucleotides of SP-N and LP-B viruses Oligonucleotide no.a
Oligonucleotides common to SP-N and LP-B: 2 3
4 5 6 7 9
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 33 35
Composition
7U, 15C, (A2C), (A3C), G 2U, 7C, (AU), (A4C), (A2G) 1U, 5C, (AC), (AU), (A2U), (AsC), G 1U, 6C, (AC), 2(AU), (A2U), (A4C), G 2U, 5C, 2(A2C), (A2U), (AG) 5U, 10C, 2(AC), G 6U, 5C, 3(AC), (AU), (A2C), G 1U, 4C, 3(AC), (A2U), (A4G) 2U, 8C, 2(AC), (A2C), G 1U, 2C, 2(A2C), (AsC), (AG) 5U, 6C, 2(A2U), (AsU), G 7U, 8C, 2(AC), (AU), G 8U, 11C, G 4U, 3C, (AC), 2(AU), (A4G) 4U, 5C, (AC), (AU), (A2U), (AG) 4U, 6C, 2(AC), 2(AU), G 3U, 3C, 3(AU), (A2C), G 5U, 2C, 2(AC), (A2U), G 6U, 3C, (AC), (A2C), G 5U, 4C, (AC), 3(AU), G 4U, 3C, (AU), (A2U), G 5U, 1C, 2(AU), (AG) 2U, 8C, (AU), (AG) 1U, 3C, 3(AU), (A4C), G 2U, 8C, 2(AC), (A2C), (AG) 2U, 9C, 3(AC), (AG) 3U, 5C, 3(AC), (AsC), G 8U, 1C, (AC), (A2U), G 3U, 6C,-2(A2C), (AG)
Chain length
(nucleotidesr 30 ± 15% 19 20 22 18 20
23 19 18 17
24 22 20 18 18 19 16 15
15 18
13 12
14 16 19 19 21 15 17
Oligonucleotides present in SPN but not LP-B: N-1 N-10 N-11 N-32 N-34 N-36
5U, 6C, 2(AC), 3(AU), (AG) 2U, 10C, 3(AC), G 3U, 9C, (AC), (A2C), (A3C), G 4U, 2C, (AC), 3(AU), (AG) 3U, 3C, (AU), (A7C), (AG) 5U, 3C, G
22 19 22
16 17 10
Oligonucleotides present in LPB but not SP-N: B-1 32 5U, 8C, 4(AC), 2(AU), 2(A2C), G 23 4U, 7C, 2(AC), 3(AU), (AG) B-8 2U, 13C, 3(AC), (A3C), G B-10 26 3U, 12C, 2(AC), (A3C), (AG) B-11 25 1U, 7C, (AC), (A2C), (A3C), G B-32 18 a Numbers refer to oligonucleotide spots diagrammed in Fig. 1C and D. Homologous spots of SP-N and LPB are listed only once: the products of pancreatic RNase digestion of these spots were the same whether they were derived from SP-N or LP-B, except in the case of spots 12 and 35. These spots are doublets in one virus but contain only a single oligonucleotide in the other virus. The composition given for spot 12 is that of the single oligonucleotide (called 12) in LP-B. The products obtained from this oligonucleotide are contained among the products (not shown in table) of the doublet spot 12 of SP-N. The same analysis applies to spot 35. Oligonucleotides that are present in SP-N but not LP-B are designated by the prefix N-; those present in LPB but not SP-N are designated by the prefix B-. b Compositions are determined by quantitation of the products of pancreatic RNase digestion. Five determinations for the products of all the oligonucleotides of SP-N and five for the oligonucleotides of LP-B were made, averaged, and rounded to the nearest integer. The products of apparently common oligonucleotides were the same for the two viruses. Chain length was computed by addition of the nearest integers of the molar yields. c
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0
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FIG. 1. RNA fingerprints of SP-N and LP-B viruses. Autoradiograms of the second-dimension gel electrophoresis of 32P-labeled RNase Tl-resistant oligonucleotides of (A) SP-N and (B) LP-B viruses. Diagrams of the fingerprints of (C) SP-N and (D) LP-B viruses. 0, Oligonucleotides common to SP-N and LP-B. 0, Oligonucleotides present in SP-N but not LP-B. @, Oligonucleotides present in LP-B but not SP-N. O, in
that one of these oligonucleotides (35) was present in SP-N. Only one G-containing product (AG) was found in spot 35 of LP-B; thus, the presence of two oligonucleotides at this position is less certain than in the case of N-12. (It is conceivable that spot 35 of LP-B might be a single oligonucleotide with abnormal electrophoretic mobility.) Attempts to separate the presumed doublets at position 35 of LP-B or 12 of SP-N into their component oligonucleotides by other chromatographic procedures were unsuc-
nucleotides. Consideration of the products of pancreatic RNase digestion (Table 1) of B-8 and N-i, of B-10 and N-10, and of B-32 and N-li suggested the possibility that each of these pairs may be related to one another. For example, N1 differs from B-8 by the absence of 1-2Cs, and a single base change might convert oligonucleotide N-1 to B-8 or vice versa. RNase Tl-resistant oligonucleotides of XLP-N viruses. The fingerprints of two cloned XLP-N viruses obtained from different coinfections (see reference 16) are shown in Fig. 2A cessful. It is interesting to note a possible relationship and B, and diagrams of these fingerprints are between certain SP-N- and LP-B-specific oligo- shown in Fig. 2C and D. Both viruses appeared
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, (C), Two oligonucleotides that comigrate: one (called 12) is also found in LP-B; one (N-12) is unique to SPN. O, in (D), Two oligonucleotides that comigrate: one (35) is also present in SP-N; the other (B-35) is unique to LP-B. XC and B, Positions of dye markers xylene cyanol FF and bromophenol blue, respectively. Arrows indicate direction of migration in first and second dimensions of the gel electrophoresis.
these XLP-N viruses with the corresponding SP-N and LP-B (Fig. 2C and D), although the parental oligonucleotides was substantiated by products of pancreatic RNase digestion of these qualitative analysis of pancreatic RNase digesoligonucleotides were not examined. In addition, tion products of these spots from recombinants both viruses yielded some SP-N- and some LP- 3-1 and 5-4. Note that both recombinants, like B-characteristic oligonucleotides. Isolate 3-1 SP-N, contained a doublet at position 12 and contains two of the six LP-B-specific oligonucle- apparently a single oligonucleotide at position otides (B-1 and B-8) and six of the eight SP-N- 35. The products of pancreatic RNase digestion specific oligonucleotides (N-10, N-11, N-12, N- of these spots were analyzed qualitatively and 32, N-34, and N-36). Isolate 5-4 contains LP-B- were indistinguishable from those of SP-N spots specific oligonucleotides B-8, B-10, B-11, and B- 12 and 35. Quantitation of the radioactivity present in 32 and SP-N oligonucleotides N-32, N-36, and N-12. The probable identity of apparently SP- the SP-N- or LP-B-specific oligonucleotides of N- or LP-B-characteristic oligonucleotides in XLP-N viruses indicated that they were present
to possess all the oligonucleotides common to
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FIG. 2. RNA fingerprints of two XLP-N recombinants, 3-1 and 5-4. Fingerprints of (A) 3-1 and (B) 5-4. Diagrams of the fingerprints of (C) 3-1 and (D) 5-4. 0, oligonucleotides also found in fingerprints of both SPN and LP-B. @, Oligonucleotides that are also found in SP-N but not LP-B and were presumably inherited
in molar amount (±15%), relative to other oligonucleotides (with the exception of spot 12, which, as noted above, was present in approximately twice molar amount). Neither 3-1 nor 54 contained all of the LP-B- or SP-N-specific oligonucleotides, and regions of the gel where the missing N- or B-specific oligonucleotides would be expected to migrate contained only background levels of radiation. These two obser-
vations provided strong evidence that 3-1 and 5-4 inherited some but not all of the genes of SP-N and LP-B and, thus, are genetic recombinants. Contamination of XLP-N viruses by either parental virus would have been detected if it were as high as 1%. A recurring problem in these studies was the presence of oligonucleotide spots which were present in nonmolar amounts (see, for example,
VIRUS RECOMBINATION
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*
615
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Oligonucleotides presumably inherited from the LP-B parent. (O, As in in Fig. 2B. XC, B, and arrows in (C) and (D) as in Fig. C
of three arrows
in Fig. 2B). Tentatively designated "contaminants," the origin of these spots remains unknown. They might be derived from contaminating RNA species, or they might be the result of aberrant cleavages of viral RNA. The intensity of these spots relative to viral spots (those invariably present in molar amount relative to one another) as well as their intensity relative to one another varied from experiment to experarrows
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iment and varied when different cell lines were used to propagate the viruses. They were observed in 25 cloned N-, B-, and NB-tropic viruses. The amount of radioactivity in these spots was usually too low to be quantitated, but, on some occasions, it was as great as the amount of radioactivity in viral spots. Whereas these putative "contaminants" are usually biochemically insignificant, the possibility of significant
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biological contamination cannot be ruled out until their source is elucidated. DISCUSSION In order to provide evidence for recombination and in the hope of ultimately mapping genes and functions of murine leukemia viruses, we have begun to analyze the large RNase Ti-resistant oligonucleotides of an N- and a B-tropic virus of BALB/c cells and viruses, designated XLP-N, that appear by biological criteria and analysis of virion proteins to be recombinants between SP-N and LP-B (9,10,16). Our analysis indicates that XLP-N viruses inherited some but not all of the genetic material of their N- or B-tropic virus parent and, thus, are probably true genetic recombinants. In addition to providing evidence for recombination, these studies reveal several interesting features of an N- and a B-tropic virus of BALB/c mice. Fingerprints of SP-N and LP-B viruses were very similar. Approximately 30 of 36 to 38 large Tl-resistant oligonucleotides are probably present in both viruses. Because single base changes can alter the position of a spot or cause an oligonucleotide to appear or disappear from the fingerprint, since the particular isolates examined were derived from virus stocks that have been passaged extensively in vitro, and because even clonal isolates derived from a single stock of virus can differ by one or more oligonucleotides (4), it is perhaps noteworthy that SP-N and LP-B fingerprints are so similar. The extensive similarity between the fingerprints of these viruses is also interesting because SP-N and LPB differ in a number of phenotypic and biochemically defined properties. These differences include N- or B-tropism, XC-plaque morphology (10), electrophoretic mobility of three virion proteins (p15, p30, and gp70) (16; N. Famulari and E. Fleissner, personal communication), and the presence or absence of the antigen GIX on infected cells (9, 12). (This latter property seems correlated with differences in the electrophoretic mobility of the gp7O's of these viruses [9, 16, 17].) When making these considerations, however, it should be kept in mind that the method of genome analysis used here reveals only a small percentage (approximately 3 to 5%) (3) of the viral genome, and extensive differences in sequence between SP-N and LP-B could go undetected. It is possible that three of the eight SP-Nspecific oligonucleotides are related to three of the six LP-B-specific oligonucleotides. Analysis of the products of pancreatic RNase digestion of the N and B virus-specific spots indicates that three of the N-specific oligonucleotides (N-
1, N-10, and N-il) could conceivably give rise to three of the B-specific oligonucleotides (B-8, B-10, and B-32, respectively), or vice versa, by a single base change. If this were the case, then related oligonucleotides would be allelic; in other words, they would derive from corresponding regions of the genome, and their inheritance would be mutually exclusive in XLP-N recombinants. This prediction has been upheld in 15 XLP-N viruses that have been examined so far (unpublished data). It is also interesting to note
that three other N-specific oligonucleotides (N32, N-34, and N-36) were not present in N-tropic virus induced by bromodeoxyuridine from BALB/c cells in culture and propagated on NIH/3T3 cells (unpublished data) and, thus, may have arisen during tissue culture passage of SP-N. It remains to be determined whether any of the SP-N- or LP-B-specific oligonucleotides will prove useful as markers for the biological differences or differences in electrophoretic mobility of virion proteins that have been observed between SP-N and LP-B, allowing us to map genes and phenotypes of murine leukemia viruses. ACKNOWLEDGMENTS This work was supported by Public Health Service grants from the National Cancer Institute (CA 19308 to N.H. and CA 14051 to S.E. Luria).
LITERATURE CITED 1. Aaronson, S. A., G. J. Todaro, and E. M. Scolnick. 1971. Induction of murine C-type viruses from clonal lines of virus-free BALB/3T3 cells. Science 174:157-159. 2. Callahan, R., R. E. Benveniste, M. M. Lieber, and G. J. Todaro. 1974. Nucleic acid homology of murine type-C viral genes. J. Virol. 14:1394-1403. 3. Coffin, J. M., and M. A. Billeter. 1976. A physical map of the Rous sarcoma virus genome. J. Mol. Biol. 100:293-318. 4. Faller, D. V., and N. Hopkins. 1977. RNase Ti-resistant oligonucleotides of B-tropic murine leukemia virus from BALB/c and five of its NB-tropic derivatives. J. Virol.
23:188-195. 5. Hartley, J. W., and W. P. Rowe. 1975. Clonal cell lines from a feral mouse embryo which lack host-range re-
striction for murine leukemia viruses. Virology 65:128-134. 6. Hartley, J. W., W. P. Rowe, W. I. Capps, and R. J.
7. 8. 9. 10.
Huebner. 1969. Isolation of naturally occurring viruses of the murine leukemia virus group in tissue culture. J. Virol. 3:126-132. Hartley, J. W., W. P. Rowe, and R. J. Huebner. 1970. Host-range restriction of murine leukemia viruses in mouse embryo cell cultures. J. Virol. 5:221-225. Hopkins, N., and P. Jolicoeur. 1975. Variants of Ntropic leukemia virus derived from BALB/c mice. J. Virol. 16:991-999. Hopkins, N., J. Schindler, and P. D. Gottlieb. 1977. Evidence for recombination between N- and B-tropic murine leukemia viruses. J. Virol. 21:1074-1078. Hopkins, N., P. Traktman, and K. Whalen. 1976. Ntropic variants obtained after co-infection with N- and B-tropic murine leukemia viruses. J. Virol. 18:324-331.
VOL. 24, 1977 11. Jolicoeur, P., and D. Baltimore. 1976. Effect of Fv-1 gene product on synthesis of N-tropic and B-tropic murine leukemia viral RNA. Cell 7:33-39. 12. O'Donnell, P. V., and E. Stockert. 1976. Induction of GLx antigen and Gross cell surface antigen after infection by ecotropic and xenotropic murine leukemia viruses in vitro. J. Virol. 20:545-554. 13. Peters, R. L., G. J. Spahn, L. S. Rabstein, G. J. Kelloff, and R. J. Huebner. 1973. Murine C-type RNA virus from spontaneous neoplasms: in vitro host range and oncogenic potential. Science 181:665-667. 14. Pincus, T., J. W. Hartley, and W. P. Rowe. 1971. A major genetic locus affecting resistance to infection with murine leukemia viruses. I. Tissue culture studies of naturally occurring viruses. J. Exp. Med. 133:1229-1233. 15. Pincus, T., W. P. Rowe, and F. Lilly. 1971. A major genetic locus affecting resistance to infection with murine leukemia viruses. II. Apparent identity to a major
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locus described for resistance to Friend murine leukemia virus. J. Exp. Med. 133:1232-1241. 16. Schindler, J., R. Hynes, and N. Hopkins. 1977. Evidence for recombination between N- and B-tropic murine leukemia viruses: analysis of three virion proteins by SDS polyacrylamide gel electrophoresis. J. Virol. 23:700-707. 17. Tung, J. S., E. S. Vitetta, E. Fleissner, and E. A. Boyse. 1975. Biochemical evidence linking the G1x thymocyte surface antigen to the gp69/71 envelope glycoprotein of murine leukemia virus. J. Exp. Med. 141:198-205. 18. Wang, L, D. Galehouse, P. P. Mellon, P. Duesberg, W. S. Mason, and P. K. Vogt. 1976. Mapping oligonucleotides of Rous sarcoma virus RNA that segregate with polymerase and group-specific antigen markers in recombinants. Proc. Natl. Acad. Sci. U.S.A. 73: 3952-3956.
Vol. 24, No. 2 Printed in U.S.A.
Copyright © 1977 American Society for Microbiology
RNase Ti-Resistant Oligonucleotides of an N- and a B-Tropic Murine Leukemia Virus of BALB/c: Evidence for Recombination Between These Viruses DOUGLAS V. FALLER AND NANCY HOPKINS* Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication 8 July 1977
We used two-dimensional gel electrophoresis to obtain fingerprints of 32Plabeled RNase Tl-resistant oligonucleotides derived from the genomes of an Nand a B-tropic murine leukemia virus of BALB/c. These viruses share approximately 30 large Ti-resistant oligonucleotides. In addition, there are eight large oligonucleotides unique to the N-tropic virus, and there are six B-tropic virusspecific oligonucleotides. Viruses, designated XLP-N, which appear by biological criteria and analysis of virion proteins to be recombinants between these N- and B-tropic viruses, possess some but not all of the N or B virus-specific oligonucleotides. Both N- and B-tropic type C RNA viruses have been isolated from low-leukemic BALB/c mice (14, 15). N-tropic virus has been obtained by bromodeoxyuridine induction of BALB/c cells in culture (1) and from extracts of spleens of nondiseased mice (6, 7). B-tropic virus has been obtained from spontaneous or chemically induced tumors of BALB/c cells (13) and from spleen extracts of nondiseased mice (7). Hybridization studies, using N- and B-tropic isolates of BALB/c, showed that these viruses are >95%
homologous (2, 11). Two spleen isolates obtained by Hartley et al. (7) from a single nondiseased adult BALB/c mouse were cloned and studied extensively in several laboratories as prototype N-tropic and B-tropic viruses. These isolates are designated WN1802N and WN1802B. Hopkins and Jolicoeur (8) found that three biologically distinguishable N-tropic viruses could be isolated from WN1802N: one type, designated NP-N, was unable to form XC plaques; one, SP-N, made small XC plaques; and one, LP-N, made large XC plaques and was characteristic of uncloned WN18Q2N. The existence of viruses with unusual XC-plaque morphology provided a genetic marker that was used by Hopkins et al. (10) to isolate viruses that appeared, by biological criteria, to be recombinants: coinfection of SC-1 cells (5) (a cell line that does not show Fv-1 restriction) with SP-N and a large XC-plaqueforming B-tropic virus, derived from WN1802B and designated LP-B, yielded progeny with the N-tropism of SP-N and the large XC-plaque morphology of LP-B. The viruses with recombinant phenotype were designated XLP-N. Fur-
ther evidence that XLP-N viruses are recombinants was obtained from analysis of the antigenicity of infected cells and determination of the electrophoretic mobility on sodium dodecyl sulfate-gels of virion proteins of SP-N, LP-B, and 21 cloned XLP-N viruses obtained from seven independent crosses (9, 16). We were interested in obtaining additional evidence that XLP-N viruses are recombinants and also in attempting to map regions of the genome responsible for N- or B-tropism and large XC-plaque morphology. A method which has proved highly successful for mapping genes of avian type C viruses is analysis of the large oligonucleotides generated by RNase T1 digestion of parental and recombinant viral RNAs (3, 18). Thus, we analyzed the genomes of SPN, LP-B, and XLP-N viruses by this method. Consistent with hybridization studies showing extensive homology between N- and B-tropic viruses of BALB/c, we found that SP-N and LP-B share approximately 30 out of 36 to 38 of their large RNase Ti-resistant oligonucleotides. However, SP-N possesses eight and LP-B possesses six distinctive oligonucleotides. XLP-N viruses possessed some SP-N- and some LP-Bcharacteristic oligonucleotides, a result which provides substantial evidence that these viruses are genetic recombinants.
609
MATERIALS AND METHODS Cells and viruses. The origin and methods of propagation of SP-N (8, 10), LP-B (10), and XLP-N (10) viruses have been described. NIH/3T3 cells chronically infected with SP-N or XLP-N viruses and BALB/3T3 cells chronically infected with LP-B virus
610
FALLER AND HOPKINS
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were used in the experiments reported here. Finger- are probably eight N-specific oligonucleotides. prints prepared from these viruses grown in SC-1 (5) Six of these were readily detected, and the prodcells yielded the same pattern of oligonucleotide spots ucts obtained after pancreatic RNase digestion as when the viruses were grown in NIH/3T3 or of these oligonucleotides are shown in Table 1. BALB/3T3 cells (unpublished data). migrated close RNA fingerprinting and characterization of A seventh oligonucleotide, N-37, oligonucleotides. These procedures have been de- to a mass of unresolved oligonucleotides and scribed in detail (4). The identity of apparently SP- was, therefore, difficult to analyze. Preliminary N- or LP-B-characteristic oligonucleotides inherited results indicated that this spot consists of U, by XLP-N viruses was confirmed by qualitative anal- 3C, (AC), (A3U), (A5C), (A2G). The existence of ysis of the products of pancreatic RNase digestion of an eighth N-specific oligonucleotide at position these spots. This means that autoradiograms of the 12 was inferred from the following observations: separated digestion products of these 3P-labeled T1 spot 12 of SP-N appeared "darker" relative to oligonucleotides were compared visually with autora- spots with similar electrophoretic mobility in diograms of the digestion products of the correspond- the second dimension than the corresponding ing oligonucleotides derived from SP-N or LP-B viruses. If the electrophoretic mobility and relative in- oligonucleotide of LP-B. Since mobility in the tensities of the products were the same, by eye, then second dimension of electrophoresis is based the oligonucleotides were considered to be the same. primarily on size, that spot 12 of SP-N is darker The secondary digestion products of the Ti oligonu- than oligonucleotides at a similar position imcleotides of SP-N and LP-B were analyzed quantita- plied that this spot is a doublet, consisting of tively as described previously (4). two (not necessarily identical) oligonucleotides
RESULTS
RNase Tl-resistant oligonucleotides of parental viruses, SP-N and LP-B. 32P-labeled 70S viral RNA prepared from culture fluids of cells chronically infected with SP-N or LP-B was digested with RNase T1, and the resulting oligonucleotides were separated by two-dimensional gel electrophoresis. "Fingerprints" obtained by autoradiography of the second-dimension gels are shown in Fig. 1A and B, and diagrams of these fingerprints are shown in Fig. 1C and D. The fingerprints of SP-N and LP-B were strikingly similar. The two viruses appeared to share approximately 30 out of 36 to 38 oligonucleotide "spots." In Fig. 1C and D, oligonucleotides that appeared common to SP-N and LPB are shown as open circles. Since oligonucleotides of different sequence or even base composition can possess similar electrophoretic mobilities in this system, further characterization of apparently common oligonucleotides was necessary before identity could be reasonably assumed. Oligonucleotides were removed from the gels and digested with pancreatic RNase, and the digestion products were separated and quantitated. The results of this analysis for the numbered oligonucleotides in Fig. 1C and D are shown in Table 1. Oligonucleotides represented by open circles yielded the same digestion products whether they were derived from SP-N or LP-B, and therefore the products for these oligonucleotides are given only once in Table 1. It is likely that these oligonucleotides are the same in the two viruses. Oligonucleotides that are unique to SP-N, represented by solid black circles in Fig. 1C, are designated by the prefix "N" in Table 1. There
that comigrate, and quantitation of the radioactivity at this position corroborated this visual observation. Analysis of the secondary digestion products of spot 12 of SP-N revealed the presence of 2U, 7C, 6 (AC), (AU), (A2U), (A3G), and (A4G). The presence of two G-containing products confirmed the occurrence of two oligonucleotides at this position. Since the secondary digestion products of spot 12 of LP-B [U, 4C, 3(AC), (A2U), (A4G)] are a subset of the products of the SP-N spot 12, it seemed likely that an oligonucleotide, 12, was present in both SP-N and LP-B and that SP-N contained, in addition, an oligonucleotide, N-12, with the (inferred) composition U, 3C, 3(AC), (AU), (A3G). There are probably six LP-B-specific oligonucleotides. These are represented as hatched circles in Fig. 1D, and they are designated by the prefix "B" in Table 1. The products of pancreatic RNase digestion of five readily detected LP-B-specific oligonucleotides are shown in Table 1. The presence of a sixth LP-B-specific T1 oligonucleotide at position 35 was, like N-12, inferred from the relatively greater intensity and the presence of approximately twice the amount of radioactivity in this spot relative to the flanking oligonucleotides. However, as reported previously (4), determinations of the relative molarity and secondary digestion products of this spot were hampered by contamination from oligonucleotides migrating slightly ahead of it. Preliminary analysis of secondary digestion products of spot 35 of LP-B suggested that it contained all the products present in the oligonucleotide at position 35 of SP-N plus additional products (see reference 4). This result suggested that spot 35 of LP-B may consist of two oligonucleotides (35 and B-35) that comigrate and
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TABLE 1. Products of pancreatic RNase digestion of large RNase Tl-resistant oligonucleotides of SP-N and LP-B viruses Oligonucleotide no.a
Oligonucleotides common to SP-N and LP-B: 2 3
4 5 6 7 9
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 33 35
Composition
7U, 15C, (A2C), (A3C), G 2U, 7C, (AU), (A4C), (A2G) 1U, 5C, (AC), (AU), (A2U), (AsC), G 1U, 6C, (AC), 2(AU), (A2U), (A4C), G 2U, 5C, 2(A2C), (A2U), (AG) 5U, 10C, 2(AC), G 6U, 5C, 3(AC), (AU), (A2C), G 1U, 4C, 3(AC), (A2U), (A4G) 2U, 8C, 2(AC), (A2C), G 1U, 2C, 2(A2C), (AsC), (AG) 5U, 6C, 2(A2U), (AsU), G 7U, 8C, 2(AC), (AU), G 8U, 11C, G 4U, 3C, (AC), 2(AU), (A4G) 4U, 5C, (AC), (AU), (A2U), (AG) 4U, 6C, 2(AC), 2(AU), G 3U, 3C, 3(AU), (A2C), G 5U, 2C, 2(AC), (A2U), G 6U, 3C, (AC), (A2C), G 5U, 4C, (AC), 3(AU), G 4U, 3C, (AU), (A2U), G 5U, 1C, 2(AU), (AG) 2U, 8C, (AU), (AG) 1U, 3C, 3(AU), (A4C), G 2U, 8C, 2(AC), (A2C), (AG) 2U, 9C, 3(AC), (AG) 3U, 5C, 3(AC), (AsC), G 8U, 1C, (AC), (A2U), G 3U, 6C,-2(A2C), (AG)
Chain length
(nucleotidesr 30 ± 15% 19 20 22 18 20
23 19 18 17
24 22 20 18 18 19 16 15
15 18
13 12
14 16 19 19 21 15 17
Oligonucleotides present in SPN but not LP-B: N-1 N-10 N-11 N-32 N-34 N-36
5U, 6C, 2(AC), 3(AU), (AG) 2U, 10C, 3(AC), G 3U, 9C, (AC), (A2C), (A3C), G 4U, 2C, (AC), 3(AU), (AG) 3U, 3C, (AU), (A7C), (AG) 5U, 3C, G
22 19 22
16 17 10
Oligonucleotides present in LPB but not SP-N: B-1 32 5U, 8C, 4(AC), 2(AU), 2(A2C), G 23 4U, 7C, 2(AC), 3(AU), (AG) B-8 2U, 13C, 3(AC), (A3C), G B-10 26 3U, 12C, 2(AC), (A3C), (AG) B-11 25 1U, 7C, (AC), (A2C), (A3C), G B-32 18 a Numbers refer to oligonucleotide spots diagrammed in Fig. 1C and D. Homologous spots of SP-N and LPB are listed only once: the products of pancreatic RNase digestion of these spots were the same whether they were derived from SP-N or LP-B, except in the case of spots 12 and 35. These spots are doublets in one virus but contain only a single oligonucleotide in the other virus. The composition given for spot 12 is that of the single oligonucleotide (called 12) in LP-B. The products obtained from this oligonucleotide are contained among the products (not shown in table) of the doublet spot 12 of SP-N. The same analysis applies to spot 35. Oligonucleotides that are present in SP-N but not LP-B are designated by the prefix N-; those present in LPB but not SP-N are designated by the prefix B-. b Compositions are determined by quantitation of the products of pancreatic RNase digestion. Five determinations for the products of all the oligonucleotides of SP-N and five for the oligonucleotides of LP-B were made, averaged, and rounded to the nearest integer. The products of apparently common oligonucleotides were the same for the two viruses. Chain length was computed by addition of the nearest integers of the molar yields. c
612
J. VIROL.
FALLER AND HOPKINS
0
0
&
0
Ilmomp-
0
00
.
SP-N
A
N.
,.2
90
0 21rQ v2) 95
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1' 2? 3
0 0
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160
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FIG. 1. RNA fingerprints of SP-N and LP-B viruses. Autoradiograms of the second-dimension gel electrophoresis of 32P-labeled RNase Tl-resistant oligonucleotides of (A) SP-N and (B) LP-B viruses. Diagrams of the fingerprints of (C) SP-N and (D) LP-B viruses. 0, Oligonucleotides common to SP-N and LP-B. 0, Oligonucleotides present in SP-N but not LP-B. @, Oligonucleotides present in LP-B but not SP-N. O, in
that one of these oligonucleotides (35) was present in SP-N. Only one G-containing product (AG) was found in spot 35 of LP-B; thus, the presence of two oligonucleotides at this position is less certain than in the case of N-12. (It is conceivable that spot 35 of LP-B might be a single oligonucleotide with abnormal electrophoretic mobility.) Attempts to separate the presumed doublets at position 35 of LP-B or 12 of SP-N into their component oligonucleotides by other chromatographic procedures were unsuc-
nucleotides. Consideration of the products of pancreatic RNase digestion (Table 1) of B-8 and N-i, of B-10 and N-10, and of B-32 and N-li suggested the possibility that each of these pairs may be related to one another. For example, N1 differs from B-8 by the absence of 1-2Cs, and a single base change might convert oligonucleotide N-1 to B-8 or vice versa. RNase Tl-resistant oligonucleotides of XLP-N viruses. The fingerprints of two cloned XLP-N viruses obtained from different coinfections (see reference 16) are shown in Fig. 2A cessful. It is interesting to note a possible relationship and B, and diagrams of these fingerprints are between certain SP-N- and LP-B-specific oligo- shown in Fig. 2C and D. Both viruses appeared
VOL. 24, 1977
VIRUS RECOMBINATION
613
.4i
qip
IS 4
9
* vqm
S.
0
0
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B
0 0
0 0
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23 24
e
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, (C), Two oligonucleotides that comigrate: one (called 12) is also found in LP-B; one (N-12) is unique to SPN. O, in (D), Two oligonucleotides that comigrate: one (35) is also present in SP-N; the other (B-35) is unique to LP-B. XC and B, Positions of dye markers xylene cyanol FF and bromophenol blue, respectively. Arrows indicate direction of migration in first and second dimensions of the gel electrophoresis.
these XLP-N viruses with the corresponding SP-N and LP-B (Fig. 2C and D), although the parental oligonucleotides was substantiated by products of pancreatic RNase digestion of these qualitative analysis of pancreatic RNase digesoligonucleotides were not examined. In addition, tion products of these spots from recombinants both viruses yielded some SP-N- and some LP- 3-1 and 5-4. Note that both recombinants, like B-characteristic oligonucleotides. Isolate 3-1 SP-N, contained a doublet at position 12 and contains two of the six LP-B-specific oligonucle- apparently a single oligonucleotide at position otides (B-1 and B-8) and six of the eight SP-N- 35. The products of pancreatic RNase digestion specific oligonucleotides (N-10, N-11, N-12, N- of these spots were analyzed qualitatively and 32, N-34, and N-36). Isolate 5-4 contains LP-B- were indistinguishable from those of SP-N spots specific oligonucleotides B-8, B-10, B-11, and B- 12 and 35. Quantitation of the radioactivity present in 32 and SP-N oligonucleotides N-32, N-36, and N-12. The probable identity of apparently SP- the SP-N- or LP-B-specific oligonucleotides of N- or LP-B-characteristic oligonucleotides in XLP-N viruses indicated that they were present
to possess all the oligonucleotides common to
614
J. VIROL.
FALLER AND HOPKINS
S
A
/rSADX
:;;,
."
--
7.
:7
#. }4 I< ) *_
3-1 T
0
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FIG. 2. RNA fingerprints of two XLP-N recombinants, 3-1 and 5-4. Fingerprints of (A) 3-1 and (B) 5-4. Diagrams of the fingerprints of (C) 3-1 and (D) 5-4. 0, oligonucleotides also found in fingerprints of both SPN and LP-B. @, Oligonucleotides that are also found in SP-N but not LP-B and were presumably inherited
in molar amount (±15%), relative to other oligonucleotides (with the exception of spot 12, which, as noted above, was present in approximately twice molar amount). Neither 3-1 nor 54 contained all of the LP-B- or SP-N-specific oligonucleotides, and regions of the gel where the missing N- or B-specific oligonucleotides would be expected to migrate contained only background levels of radiation. These two obser-
vations provided strong evidence that 3-1 and 5-4 inherited some but not all of the genes of SP-N and LP-B and, thus, are genetic recombinants. Contamination of XLP-N viruses by either parental virus would have been detected if it were as high as 1%. A recurring problem in these studies was the presence of oligonucleotide spots which were present in nonmolar amounts (see, for example,
VIRUS RECOMBINATION
VOL. 24, 1977
*
615
*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *
ri
I 0
0
0 0
0
0
0
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B 62
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from SP-N in the recombinants. Fig. I C. See text for explanation and D.
5-4 C,
Oligonucleotides presumably inherited from the LP-B parent. (O, As in in Fig. 2B. XC, B, and arrows in (C) and (D) as in Fig. C
of three arrows
in Fig. 2B). Tentatively designated "contaminants," the origin of these spots remains unknown. They might be derived from contaminating RNA species, or they might be the result of aberrant cleavages of viral RNA. The intensity of these spots relative to viral spots (those invariably present in molar amount relative to one another) as well as their intensity relative to one another varied from experiment to experarrows
0
iment and varied when different cell lines were used to propagate the viruses. They were observed in 25 cloned N-, B-, and NB-tropic viruses. The amount of radioactivity in these spots was usually too low to be quantitated, but, on some occasions, it was as great as the amount of radioactivity in viral spots. Whereas these putative "contaminants" are usually biochemically insignificant, the possibility of significant
616
J. VIROL.
FALLER AND HOPKINS
biological contamination cannot be ruled out until their source is elucidated. DISCUSSION In order to provide evidence for recombination and in the hope of ultimately mapping genes and functions of murine leukemia viruses, we have begun to analyze the large RNase Ti-resistant oligonucleotides of an N- and a B-tropic virus of BALB/c cells and viruses, designated XLP-N, that appear by biological criteria and analysis of virion proteins to be recombinants between SP-N and LP-B (9,10,16). Our analysis indicates that XLP-N viruses inherited some but not all of the genetic material of their N- or B-tropic virus parent and, thus, are probably true genetic recombinants. In addition to providing evidence for recombination, these studies reveal several interesting features of an N- and a B-tropic virus of BALB/c mice. Fingerprints of SP-N and LP-B viruses were very similar. Approximately 30 of 36 to 38 large Tl-resistant oligonucleotides are probably present in both viruses. Because single base changes can alter the position of a spot or cause an oligonucleotide to appear or disappear from the fingerprint, since the particular isolates examined were derived from virus stocks that have been passaged extensively in vitro, and because even clonal isolates derived from a single stock of virus can differ by one or more oligonucleotides (4), it is perhaps noteworthy that SP-N and LP-B fingerprints are so similar. The extensive similarity between the fingerprints of these viruses is also interesting because SP-N and LPB differ in a number of phenotypic and biochemically defined properties. These differences include N- or B-tropism, XC-plaque morphology (10), electrophoretic mobility of three virion proteins (p15, p30, and gp70) (16; N. Famulari and E. Fleissner, personal communication), and the presence or absence of the antigen GIX on infected cells (9, 12). (This latter property seems correlated with differences in the electrophoretic mobility of the gp7O's of these viruses [9, 16, 17].) When making these considerations, however, it should be kept in mind that the method of genome analysis used here reveals only a small percentage (approximately 3 to 5%) (3) of the viral genome, and extensive differences in sequence between SP-N and LP-B could go undetected. It is possible that three of the eight SP-Nspecific oligonucleotides are related to three of the six LP-B-specific oligonucleotides. Analysis of the products of pancreatic RNase digestion of the N and B virus-specific spots indicates that three of the N-specific oligonucleotides (N-
1, N-10, and N-il) could conceivably give rise to three of the B-specific oligonucleotides (B-8, B-10, and B-32, respectively), or vice versa, by a single base change. If this were the case, then related oligonucleotides would be allelic; in other words, they would derive from corresponding regions of the genome, and their inheritance would be mutually exclusive in XLP-N recombinants. This prediction has been upheld in 15 XLP-N viruses that have been examined so far (unpublished data). It is also interesting to note
that three other N-specific oligonucleotides (N32, N-34, and N-36) were not present in N-tropic virus induced by bromodeoxyuridine from BALB/c cells in culture and propagated on NIH/3T3 cells (unpublished data) and, thus, may have arisen during tissue culture passage of SP-N. It remains to be determined whether any of the SP-N- or LP-B-specific oligonucleotides will prove useful as markers for the biological differences or differences in electrophoretic mobility of virion proteins that have been observed between SP-N and LP-B, allowing us to map genes and phenotypes of murine leukemia viruses. ACKNOWLEDGMENTS This work was supported by Public Health Service grants from the National Cancer Institute (CA 19308 to N.H. and CA 14051 to S.E. Luria).
LITERATURE CITED 1. Aaronson, S. A., G. J. Todaro, and E. M. Scolnick. 1971. Induction of murine C-type viruses from clonal lines of virus-free BALB/3T3 cells. Science 174:157-159. 2. Callahan, R., R. E. Benveniste, M. M. Lieber, and G. J. Todaro. 1974. Nucleic acid homology of murine type-C viral genes. J. Virol. 14:1394-1403. 3. Coffin, J. M., and M. A. Billeter. 1976. A physical map of the Rous sarcoma virus genome. J. Mol. Biol. 100:293-318. 4. Faller, D. V., and N. Hopkins. 1977. RNase Ti-resistant oligonucleotides of B-tropic murine leukemia virus from BALB/c and five of its NB-tropic derivatives. J. Virol.
23:188-195. 5. Hartley, J. W., and W. P. Rowe. 1975. Clonal cell lines from a feral mouse embryo which lack host-range re-
striction for murine leukemia viruses. Virology 65:128-134. 6. Hartley, J. W., W. P. Rowe, W. I. Capps, and R. J.
7. 8. 9. 10.
Huebner. 1969. Isolation of naturally occurring viruses of the murine leukemia virus group in tissue culture. J. Virol. 3:126-132. Hartley, J. W., W. P. Rowe, and R. J. Huebner. 1970. Host-range restriction of murine leukemia viruses in mouse embryo cell cultures. J. Virol. 5:221-225. Hopkins, N., and P. Jolicoeur. 1975. Variants of Ntropic leukemia virus derived from BALB/c mice. J. Virol. 16:991-999. Hopkins, N., J. Schindler, and P. D. Gottlieb. 1977. Evidence for recombination between N- and B-tropic murine leukemia viruses. J. Virol. 21:1074-1078. Hopkins, N., P. Traktman, and K. Whalen. 1976. Ntropic variants obtained after co-infection with N- and B-tropic murine leukemia viruses. J. Virol. 18:324-331.
VOL. 24, 1977 11. Jolicoeur, P., and D. Baltimore. 1976. Effect of Fv-1 gene product on synthesis of N-tropic and B-tropic murine leukemia viral RNA. Cell 7:33-39. 12. O'Donnell, P. V., and E. Stockert. 1976. Induction of GLx antigen and Gross cell surface antigen after infection by ecotropic and xenotropic murine leukemia viruses in vitro. J. Virol. 20:545-554. 13. Peters, R. L., G. J. Spahn, L. S. Rabstein, G. J. Kelloff, and R. J. Huebner. 1973. Murine C-type RNA virus from spontaneous neoplasms: in vitro host range and oncogenic potential. Science 181:665-667. 14. Pincus, T., J. W. Hartley, and W. P. Rowe. 1971. A major genetic locus affecting resistance to infection with murine leukemia viruses. I. Tissue culture studies of naturally occurring viruses. J. Exp. Med. 133:1229-1233. 15. Pincus, T., W. P. Rowe, and F. Lilly. 1971. A major genetic locus affecting resistance to infection with murine leukemia viruses. II. Apparent identity to a major
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locus described for resistance to Friend murine leukemia virus. J. Exp. Med. 133:1232-1241. 16. Schindler, J., R. Hynes, and N. Hopkins. 1977. Evidence for recombination between N- and B-tropic murine leukemia viruses: analysis of three virion proteins by SDS polyacrylamide gel electrophoresis. J. Virol. 23:700-707. 17. Tung, J. S., E. S. Vitetta, E. Fleissner, and E. A. Boyse. 1975. Biochemical evidence linking the G1x thymocyte surface antigen to the gp69/71 envelope glycoprotein of murine leukemia virus. J. Exp. Med. 141:198-205. 18. Wang, L, D. Galehouse, P. P. Mellon, P. Duesberg, W. S. Mason, and P. K. Vogt. 1976. Mapping oligonucleotides of Rous sarcoma virus RNA that segregate with polymerase and group-specific antigen markers in recombinants. Proc. Natl. Acad. Sci. U.S.A. 73: 3952-3956.
