Vol. 24, No. 2 Printed in U.S.A.
JOURNAL OF VIROLOGY, Nov. 1977, p. 690-694 Copyright © 1977 American Society for Microbiology
RNase Ti-Resistant Oligonucleotides of Akv-1 and Akv-2 Type C Viruses of AKR Mice JEAN ROMMELAERE, DOUGLAS V. FALLER, AND NANCY HOPKINS* Cancer Center for Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication 8 June 1977
We used two-dimensional gel electrophoresis to obtain fingerprints of RNase Ti-resistant oligonucleotides derived from the genomes of Akv-1 and Akv-2 type C viruses of AKR mice. The fingerprints of these two viruses look identical. The products of pancreatic RNase digestion of corresponding oligonucleotides of the two viruses were indistinguishable. These observations are consistent with, but not proof of, the possible identity of the genomes of the Akv-i and Akv-2 viruses and, thus, of the viral genetic material believed to comprise the Akv-1 and Akv2 loci of AKR mice. AKR is a high-leukemic mouse strain characterized by the presence throughout life of significant titers of N-tropic ecotropic type C virus in its tissues. Genetic studies combined with hybridization studies have shown that two independently segregating chromosomal loci determine the high-virus phenotype of AKR mice and that these loci are probably integrated proviral DNA of the N-tropic virus(es) (2, 7). These loci are designated Akv-1 and Akv-2. By appropriate matings, the two loci were separated and isolated on the ecotropic virus-negative National Institutes of Health (NIH) Swiss mouse background (8). The presence of Akv-1 or Akv-2 in the NIH Swiss genome confers the high-N-tropic virus phenotype and allows one to obtain virus resulting from the presence of either locus alone. N-tropic virus obtained from mice carrying the Akv-1 or Akv-2 locus is called Akv-1 and Akv-2 virus, respectively. Akv-1 and Akv-2 viruses are indistinguishable by biological criteria; their genomes are indistinguishable by hybridization analysis (R. Gilden, unpublished data); and their glycoproteins (gp7O) are indistinguishable by tryptic peptide mapping (J. Elder, F. Jensen, and R. Lerner, personal communication). To further examine the degree of similarity between the genomes of Akv-i and Akv-2 viruses, we analyzed the large RNase Ti-resistant oligonucleotides generated by enzymatic cleavage of 32P-labeled viral RNA (1) and separated by two-dimensional gel electrophoresis (4). Autoradiograms of the second dimension of gel electrophoresis are termed fingerprints. RNase T1 cleaves RNA to yield free guanylic acid or oligonucleotides ending in guanylic acid. Long guanine-free tracts of RNA are rare; thus, cleavage of a large RNA molecule with RNase T1 yields primarily small, nonunique oligonucleo-
tides. However, unusual guanine-free sequences of approximately 10 to 40 bases in length can be found in large RNAs, and these give rise to a set of unique RNase Tl-resistant oligonucleotides. These unique oligonucleotides, in general, represent approximately 3 to 5% of the viral genome and are expected to be scattered along its length (3). The fingerprint of a particular RNA species is highly characteristic of that RNA. Procedures for preparing 32P-labeled viral RNA and for RNase T1 digestion and two-dimensional gel electrophoresis of the resulting oligonucleotides have been described (5). Ntropic viruses isolated from young (4- to 5-weekold) NIH Swiss mice inheriting either the Akv1 or the Akv-2 locus of AKR mice were obtained from W. Rowe and J. Hartley. NIH/3T3 cells chronically infected with these N-tropic viruses were used to prepare 32P-labeled viral RNA. Fingerprints of the 70S RNA of the Akv-i and Akv-2 viruses are shown in Fig. 1A and B, respectively. The two fingerprints look identical. A diagram of the fingerprints is shown in Fig. 2. That the oligonucleotides revealed as spots in Fig. 1 are derived from the viral genome is based on the observation that the same fingerprint is obtained after digestion of 35S viral RNA prepared from 70S RNA obtained from virions purified by isopycnic banding (data not shown). In addition, numbered oligonucleotides (Fig. 2) are present in equimolar amount (±15%). Molarity was determined by quantitation of the radioactivity present in each numbered spot combined with determination of the length of each oligonucleotide, as described below. Although the fingerprints of the Akv-i and Akv-2 viruses look identical, since oligonucleotides of different sequence and even different
690
VOL. 24, 1977
NOTES
691
. e
A
B
~~~~~~~AKV-1I
AKV-2
FIG. 1. Ti fingerprints of 70S RNA of Akv-1I and Akv-2 viruses. Autoradiographs of the second dimension of gel electrophoresis of 32P-labeled RNase Ti-resistant oligonucleotides of (A) Akv-1 and (B) Akv-2 viruses.
base composition can possess similar electrophoretic mobilities in this system, further analysis of the oligonucleotides was necessary before identity between the fingerprints can be reasonably assumed. Oligonucleotides were removed from the second-dimension gel for digestion with pancreatic RNase, an enzyme that is specific for pyrimidine nucleoside linkages. Analysis and quantitation of the pancreatic RNase digestion products by electrophoresis on diethylamino-
ethyl paper followed by complete digestion of these secondary products to mononucleotides provided further characterization of each oligonucleotide (1) and allowed a determination of the oligonucleotide length (+15%), as previously described (5). The results of this analysis for numbered oligonucleotides of Akv-1 and Akv-2 viruses are shown in Table 1. The products of pancreatic RNase digestion of corresponding oligonucleotides of the two viruses appeared to
692
NOTES
J. VIROL.
FIG. 2. Schematic representation of fingerprints of Akv-1 and Akv-2 viruses. Oligonucleotides that are present in molar amount relative to one another have been arbitrarily numbered. "XC" and "B" indicate positions of dye marker xylene cyanol FF and bromophenol blue, respectively. Arrows indicate direction of migration in first and second dimensions of the gel electrophoresis.
be the same. (The quantitative discrepancies in molar yields of some products of pancreatic RNase digestion of corresponding oligonucleotides from Akv-1 and Akv-2 fall within the range of error of the method (standard deviation, ca. 15%). This result suggests that the large RNase Ti-resistant oligonucleotides of the two viruses are probably identical. RNA fingerprinting is a highly sensitive method of genome analysis: even single base changes can cause an oligonucleotide spot to appear or disappear from a fingerprint. Thus, the apparent identity of the fingerprints of Akv1 and Akv-2 viruses is noteworthy and suggests that the genomes of these two viruses may be identical. On the other hand, it should be kept in mind that this method of genome analysis displays only a small percentage of the viral RNA, and considerable differences in sequence between Akv-1 and Akv-2 viruses could go undetected. Assuming that the Akv-1 and Akv-2 virus genomes are identical, it seems possible that the Akv-1 and Akv-2 loci may have resulted from two integrations of the same viral genetic material, perhaps as a result of infection of germline cells by an Akv virus (6). Another possibility that can not be ruled out is that the Akv-1 and Akv-2 loci resulted from independent exogenous infections by two closely related viruses. In speculating on these alternatives, it is relevant to consider the following question: how similar to
Akv-1 and Akv-2 virus RNA fingerprints are fingerprints of the genomes of other endogenous ecotropic viruses? For example, if endogenous N-tropic ecotropic viruses from different inbred strains of mice had identical genomes, then the Akv-1 and Akv-2 loci could specify viruses that have indistinguishable fingerprints yet have independent origins. We have analyzed the large RNase Ti-resistant oligonucleotides of endogenous N-tropic type C virus of BALB/c, both an in vivo isolate and virus induced from cultured cells by bromodeoxyuridine (D. Faller and N. Hopkins, manuscript in preparation). We have also analyzed B-tropic viruses of BALB/c (5), C56BL/6, and B1O.BR mice (J. Rommelaere, unpublished data). The RNA fingerprints of all of these endogenous ecotropic viruses are strikingly similar to one another and to the fingerprints of the Akv-1 and Akv-2 viruses. However, they all differ from one another: the viruses from different strains of mice share between 70 and 90% of their large RNase Ti-resistant oligonucleotides. Oligonucleotides of Akv-1 and Akv-2 viruses that also appear to be present in an N-tropic virus of BALB/c are denoted with an asterisk in Table 1. That the fingerprint of endogenous N-tropic virus of BALB/c differs from the fingerprint of Akv-1 and -2 viruses and that the fingerprints of three endogenous B-tropic viruses differ from one another indicate that Akv-1 and -2 viruses are more closely related to each other than to
VOL. 24, 1977
NOTES
693
TABLE 1. Products ofpancreatic RNase digestion of Tl-resistant oligonucleotides of Akv-1 and Akv-2 virusesa Oligonucleotide no.'
Compositions
Chain length (nucleotides)
ld 31-33 (±15%) 6.9 , 157:63C (A3C), (AXC), G 2 5U, 11C, (AC), (AU), (AX0), (AG) 28 3* 3U, 8C, (AC), (AU), (A40), (A2G) 23 4* 2U, 7C, (AC), (AU), (A50), (A2U), G 23 5* U, 6C, (AC), 2(AU), (A5C), (A2U), G 23 6* 5U, 7C, 3(AC), 3(AU), (AG) 26 7* 6U, 12C, 2(AC), G 23 8 5U, 5C, 2(AC), 2(AU), (A2C), G 22 9* 6U, 6C, 3(AC), (AU), (A20), G 24 10* 2U, 1102C, 3(AC), G 19-20 11* 22 3U, 9C, (AC), (A20), (A30), G 12 3U, 13C, 2(AU), G 21 13 U, 6C, 3(AC), (A20), G 17 14* 17-20 U2:9C,8:(A0), 2(A20), (AXC), (AG) 6U, 7C, 2(A2U), (A5U), G 15* 26 16* 22-23 78U, 8C, 2(AC), (AU), G 17* 9U, 12C, G 22 18* 20-23 5.6U 5C, 1- (AC), 2(AU), (A3G) 19* 5U, 6C, (AC), (AU), (A2U), (AG) 20 20* 19-20 3:9U, 6C, 2(AC), 2(AU), G 21* 4U, 3C, (AC), 2(AU), (A20), G 17 22* 6U, 3C, 2(AC), (A2U), G 17 9U, 40, (AC), (A20), G 23* 17-18 24* 5U, 4C, (AC), 3(AU), GG 18 (AC), (A2U), 13-15 25* 5U, 0, 2(AU), (AG) 12 26* 17-18 27* 5U, 1C, (AU), (AG) 19-20 3(AU), 28* -4IU, 4C, (AC), 2A) (AU), (AG (A40), G 42.866C 16-18 29* 8U, (AC), (A2U), G 14 30 6U, 6C, G 9-10 31* 13 32* 4U, 4C, 2(AU), G 17 lOU, C, (AC), (A2U), G 33* 15 3U, 3C, (AC), (A20), (A2U), G 34* 19-23 4U, 7C, 80(AC), 8:5(AU), 2(A20), (AG) 35* a Numbered oligonucleotides (Fig. 2) were digested with pancreatic RNase and the products of digestion were separated and quantitated. Approximately 200 cpm/nucleotide were typically found when 20 x 106 cpm of 70S viral RNA were used for an experiment. Because the object of these experiments was to compare Akv1 and Akv-2 as extensively as possible, oligonucleotides that are not well-separated on the fingerprint were analyzed despite the problem of cross-contamination, and their products are listed above. Cross-contamination may well account for the appearance of products in less than molar amount in some oligonucleotides. See Fig. 2. cAverage values of two determinations for each virus. If both averages could be rounded to the same nearest integer, the latter value is listed; if not, averages found for Akv-1 and Akv-2 are indicated as upper and lower values, respectively. d Oligonucleotides also present in an N-tropic virus obtained from BALB/c mice indicated by an asterisk.
5:9U,4:C,
b
the other viruses analyzed. Thus, with the reservation that only a limited sample of endogenous viruses were studied, our data are consistent with the possibility that the Akv-1 and Akv-2 loci may have arisen as a result of two integrations of Akv viral genetic material. We thank J. W. Hartley and W. P. Rowe, National Institutes of Health, for generously providing the Akv-1 and Akv2 viruses. This work was supported by Public Health Serivce grants from the National Cancer Institute (CA 19308 to N.H. and
CA 14051 to S. E. Luria). J.R. is a Charge de Recherches du Fonds National de la Recherche Scientifique de Belgique and Fellow of the Fondation Rose et Jean Hoguet.
LITERATURE CITED 1. Brownlee, G. G. 1972. Determination of sequences in RNA. North Holland/American Elsevier Publishing Co., Amsterdam, London, and New York. 2. Chattopadhyay, S. K., W. P. Rowe, N. M. Teich, and D. R. Lowy. 1975. Definitive evidence that the murine C-type virus-inducing locus Akv-1 is viral genetic ma-
694
NOTES
terial. Proc. Natl. Acad. Sci. U.S.A. 72:906-910. 3. Coffin, J. M., and N. A. Billeter. 1976. A physical map of the Rous sarcoma virus genome. J. Mol. Biol. 100: 293-318. 4. DeWachter, R., and W. Fiers. 1972. Preparative twodimensional polyacrylamide gel electrophoresis of 32p_
labelled RNA. Anal. Biochem. 49:184-197. 5. Faller, D. V., and N. Hopkins. 1977. RNase Tl-resistant oligonucleotides of B-tropic murine leukemia virus from BALB/c and five of its NB-tropic derivatives. J. Virol.
J. VIROL. 23:188-195. 6. Jaenisch, R. 1976. Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc. Natl. Acad. Sci. U.S.A. 73:1260-1264. 7. Rowe, W. P. 1972. Studies of genetic transmission of murine leukemia virus by AKR mice. I. Crosses with Fv-ln strains of mice. J. Exp. Med. 136:1272-1285. 8. Rowe, W. P., J. W. Hartley, and T. Bremner. 1972. Genetic mapping of a murine leukemia virus-inducing locus of AKR mice. Science 178:860-862.
JOURNAL OF VIROLOGY, Nov. 1977, p. 690-694 Copyright © 1977 American Society for Microbiology
RNase Ti-Resistant Oligonucleotides of Akv-1 and Akv-2 Type C Viruses of AKR Mice JEAN ROMMELAERE, DOUGLAS V. FALLER, AND NANCY HOPKINS* Cancer Center for Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication 8 June 1977
We used two-dimensional gel electrophoresis to obtain fingerprints of RNase Ti-resistant oligonucleotides derived from the genomes of Akv-1 and Akv-2 type C viruses of AKR mice. The fingerprints of these two viruses look identical. The products of pancreatic RNase digestion of corresponding oligonucleotides of the two viruses were indistinguishable. These observations are consistent with, but not proof of, the possible identity of the genomes of the Akv-i and Akv-2 viruses and, thus, of the viral genetic material believed to comprise the Akv-1 and Akv2 loci of AKR mice. AKR is a high-leukemic mouse strain characterized by the presence throughout life of significant titers of N-tropic ecotropic type C virus in its tissues. Genetic studies combined with hybridization studies have shown that two independently segregating chromosomal loci determine the high-virus phenotype of AKR mice and that these loci are probably integrated proviral DNA of the N-tropic virus(es) (2, 7). These loci are designated Akv-1 and Akv-2. By appropriate matings, the two loci were separated and isolated on the ecotropic virus-negative National Institutes of Health (NIH) Swiss mouse background (8). The presence of Akv-1 or Akv-2 in the NIH Swiss genome confers the high-N-tropic virus phenotype and allows one to obtain virus resulting from the presence of either locus alone. N-tropic virus obtained from mice carrying the Akv-1 or Akv-2 locus is called Akv-1 and Akv-2 virus, respectively. Akv-1 and Akv-2 viruses are indistinguishable by biological criteria; their genomes are indistinguishable by hybridization analysis (R. Gilden, unpublished data); and their glycoproteins (gp7O) are indistinguishable by tryptic peptide mapping (J. Elder, F. Jensen, and R. Lerner, personal communication). To further examine the degree of similarity between the genomes of Akv-i and Akv-2 viruses, we analyzed the large RNase Ti-resistant oligonucleotides generated by enzymatic cleavage of 32P-labeled viral RNA (1) and separated by two-dimensional gel electrophoresis (4). Autoradiograms of the second dimension of gel electrophoresis are termed fingerprints. RNase T1 cleaves RNA to yield free guanylic acid or oligonucleotides ending in guanylic acid. Long guanine-free tracts of RNA are rare; thus, cleavage of a large RNA molecule with RNase T1 yields primarily small, nonunique oligonucleo-
tides. However, unusual guanine-free sequences of approximately 10 to 40 bases in length can be found in large RNAs, and these give rise to a set of unique RNase Tl-resistant oligonucleotides. These unique oligonucleotides, in general, represent approximately 3 to 5% of the viral genome and are expected to be scattered along its length (3). The fingerprint of a particular RNA species is highly characteristic of that RNA. Procedures for preparing 32P-labeled viral RNA and for RNase T1 digestion and two-dimensional gel electrophoresis of the resulting oligonucleotides have been described (5). Ntropic viruses isolated from young (4- to 5-weekold) NIH Swiss mice inheriting either the Akv1 or the Akv-2 locus of AKR mice were obtained from W. Rowe and J. Hartley. NIH/3T3 cells chronically infected with these N-tropic viruses were used to prepare 32P-labeled viral RNA. Fingerprints of the 70S RNA of the Akv-i and Akv-2 viruses are shown in Fig. 1A and B, respectively. The two fingerprints look identical. A diagram of the fingerprints is shown in Fig. 2. That the oligonucleotides revealed as spots in Fig. 1 are derived from the viral genome is based on the observation that the same fingerprint is obtained after digestion of 35S viral RNA prepared from 70S RNA obtained from virions purified by isopycnic banding (data not shown). In addition, numbered oligonucleotides (Fig. 2) are present in equimolar amount (±15%). Molarity was determined by quantitation of the radioactivity present in each numbered spot combined with determination of the length of each oligonucleotide, as described below. Although the fingerprints of the Akv-i and Akv-2 viruses look identical, since oligonucleotides of different sequence and even different
690
VOL. 24, 1977
NOTES
691
. e
A
B
~~~~~~~AKV-1I
AKV-2
FIG. 1. Ti fingerprints of 70S RNA of Akv-1I and Akv-2 viruses. Autoradiographs of the second dimension of gel electrophoresis of 32P-labeled RNase Ti-resistant oligonucleotides of (A) Akv-1 and (B) Akv-2 viruses.
base composition can possess similar electrophoretic mobilities in this system, further analysis of the oligonucleotides was necessary before identity between the fingerprints can be reasonably assumed. Oligonucleotides were removed from the second-dimension gel for digestion with pancreatic RNase, an enzyme that is specific for pyrimidine nucleoside linkages. Analysis and quantitation of the pancreatic RNase digestion products by electrophoresis on diethylamino-
ethyl paper followed by complete digestion of these secondary products to mononucleotides provided further characterization of each oligonucleotide (1) and allowed a determination of the oligonucleotide length (+15%), as previously described (5). The results of this analysis for numbered oligonucleotides of Akv-1 and Akv-2 viruses are shown in Table 1. The products of pancreatic RNase digestion of corresponding oligonucleotides of the two viruses appeared to
692
NOTES
J. VIROL.
FIG. 2. Schematic representation of fingerprints of Akv-1 and Akv-2 viruses. Oligonucleotides that are present in molar amount relative to one another have been arbitrarily numbered. "XC" and "B" indicate positions of dye marker xylene cyanol FF and bromophenol blue, respectively. Arrows indicate direction of migration in first and second dimensions of the gel electrophoresis.
be the same. (The quantitative discrepancies in molar yields of some products of pancreatic RNase digestion of corresponding oligonucleotides from Akv-1 and Akv-2 fall within the range of error of the method (standard deviation, ca. 15%). This result suggests that the large RNase Ti-resistant oligonucleotides of the two viruses are probably identical. RNA fingerprinting is a highly sensitive method of genome analysis: even single base changes can cause an oligonucleotide spot to appear or disappear from a fingerprint. Thus, the apparent identity of the fingerprints of Akv1 and Akv-2 viruses is noteworthy and suggests that the genomes of these two viruses may be identical. On the other hand, it should be kept in mind that this method of genome analysis displays only a small percentage of the viral RNA, and considerable differences in sequence between Akv-1 and Akv-2 viruses could go undetected. Assuming that the Akv-1 and Akv-2 virus genomes are identical, it seems possible that the Akv-1 and Akv-2 loci may have resulted from two integrations of the same viral genetic material, perhaps as a result of infection of germline cells by an Akv virus (6). Another possibility that can not be ruled out is that the Akv-1 and Akv-2 loci resulted from independent exogenous infections by two closely related viruses. In speculating on these alternatives, it is relevant to consider the following question: how similar to
Akv-1 and Akv-2 virus RNA fingerprints are fingerprints of the genomes of other endogenous ecotropic viruses? For example, if endogenous N-tropic ecotropic viruses from different inbred strains of mice had identical genomes, then the Akv-1 and Akv-2 loci could specify viruses that have indistinguishable fingerprints yet have independent origins. We have analyzed the large RNase Ti-resistant oligonucleotides of endogenous N-tropic type C virus of BALB/c, both an in vivo isolate and virus induced from cultured cells by bromodeoxyuridine (D. Faller and N. Hopkins, manuscript in preparation). We have also analyzed B-tropic viruses of BALB/c (5), C56BL/6, and B1O.BR mice (J. Rommelaere, unpublished data). The RNA fingerprints of all of these endogenous ecotropic viruses are strikingly similar to one another and to the fingerprints of the Akv-1 and Akv-2 viruses. However, they all differ from one another: the viruses from different strains of mice share between 70 and 90% of their large RNase Ti-resistant oligonucleotides. Oligonucleotides of Akv-1 and Akv-2 viruses that also appear to be present in an N-tropic virus of BALB/c are denoted with an asterisk in Table 1. That the fingerprint of endogenous N-tropic virus of BALB/c differs from the fingerprint of Akv-1 and -2 viruses and that the fingerprints of three endogenous B-tropic viruses differ from one another indicate that Akv-1 and -2 viruses are more closely related to each other than to
VOL. 24, 1977
NOTES
693
TABLE 1. Products ofpancreatic RNase digestion of Tl-resistant oligonucleotides of Akv-1 and Akv-2 virusesa Oligonucleotide no.'
Compositions
Chain length (nucleotides)
ld 31-33 (±15%) 6.9 , 157:63C (A3C), (AXC), G 2 5U, 11C, (AC), (AU), (AX0), (AG) 28 3* 3U, 8C, (AC), (AU), (A40), (A2G) 23 4* 2U, 7C, (AC), (AU), (A50), (A2U), G 23 5* U, 6C, (AC), 2(AU), (A5C), (A2U), G 23 6* 5U, 7C, 3(AC), 3(AU), (AG) 26 7* 6U, 12C, 2(AC), G 23 8 5U, 5C, 2(AC), 2(AU), (A2C), G 22 9* 6U, 6C, 3(AC), (AU), (A20), G 24 10* 2U, 1102C, 3(AC), G 19-20 11* 22 3U, 9C, (AC), (A20), (A30), G 12 3U, 13C, 2(AU), G 21 13 U, 6C, 3(AC), (A20), G 17 14* 17-20 U2:9C,8:(A0), 2(A20), (AXC), (AG) 6U, 7C, 2(A2U), (A5U), G 15* 26 16* 22-23 78U, 8C, 2(AC), (AU), G 17* 9U, 12C, G 22 18* 20-23 5.6U 5C, 1- (AC), 2(AU), (A3G) 19* 5U, 6C, (AC), (AU), (A2U), (AG) 20 20* 19-20 3:9U, 6C, 2(AC), 2(AU), G 21* 4U, 3C, (AC), 2(AU), (A20), G 17 22* 6U, 3C, 2(AC), (A2U), G 17 9U, 40, (AC), (A20), G 23* 17-18 24* 5U, 4C, (AC), 3(AU), GG 18 (AC), (A2U), 13-15 25* 5U, 0, 2(AU), (AG) 12 26* 17-18 27* 5U, 1C, (AU), (AG) 19-20 3(AU), 28* -4IU, 4C, (AC), 2A) (AU), (AG (A40), G 42.866C 16-18 29* 8U, (AC), (A2U), G 14 30 6U, 6C, G 9-10 31* 13 32* 4U, 4C, 2(AU), G 17 lOU, C, (AC), (A2U), G 33* 15 3U, 3C, (AC), (A20), (A2U), G 34* 19-23 4U, 7C, 80(AC), 8:5(AU), 2(A20), (AG) 35* a Numbered oligonucleotides (Fig. 2) were digested with pancreatic RNase and the products of digestion were separated and quantitated. Approximately 200 cpm/nucleotide were typically found when 20 x 106 cpm of 70S viral RNA were used for an experiment. Because the object of these experiments was to compare Akv1 and Akv-2 as extensively as possible, oligonucleotides that are not well-separated on the fingerprint were analyzed despite the problem of cross-contamination, and their products are listed above. Cross-contamination may well account for the appearance of products in less than molar amount in some oligonucleotides. See Fig. 2. cAverage values of two determinations for each virus. If both averages could be rounded to the same nearest integer, the latter value is listed; if not, averages found for Akv-1 and Akv-2 are indicated as upper and lower values, respectively. d Oligonucleotides also present in an N-tropic virus obtained from BALB/c mice indicated by an asterisk.
5:9U,4:C,
b
the other viruses analyzed. Thus, with the reservation that only a limited sample of endogenous viruses were studied, our data are consistent with the possibility that the Akv-1 and Akv-2 loci may have arisen as a result of two integrations of Akv viral genetic material. We thank J. W. Hartley and W. P. Rowe, National Institutes of Health, for generously providing the Akv-1 and Akv2 viruses. This work was supported by Public Health Serivce grants from the National Cancer Institute (CA 19308 to N.H. and
CA 14051 to S. E. Luria). J.R. is a Charge de Recherches du Fonds National de la Recherche Scientifique de Belgique and Fellow of the Fondation Rose et Jean Hoguet.
LITERATURE CITED 1. Brownlee, G. G. 1972. Determination of sequences in RNA. North Holland/American Elsevier Publishing Co., Amsterdam, London, and New York. 2. Chattopadhyay, S. K., W. P. Rowe, N. M. Teich, and D. R. Lowy. 1975. Definitive evidence that the murine C-type virus-inducing locus Akv-1 is viral genetic ma-
694
NOTES
terial. Proc. Natl. Acad. Sci. U.S.A. 72:906-910. 3. Coffin, J. M., and N. A. Billeter. 1976. A physical map of the Rous sarcoma virus genome. J. Mol. Biol. 100: 293-318. 4. DeWachter, R., and W. Fiers. 1972. Preparative twodimensional polyacrylamide gel electrophoresis of 32p_
labelled RNA. Anal. Biochem. 49:184-197. 5. Faller, D. V., and N. Hopkins. 1977. RNase Tl-resistant oligonucleotides of B-tropic murine leukemia virus from BALB/c and five of its NB-tropic derivatives. J. Virol.
J. VIROL. 23:188-195. 6. Jaenisch, R. 1976. Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc. Natl. Acad. Sci. U.S.A. 73:1260-1264. 7. Rowe, W. P. 1972. Studies of genetic transmission of murine leukemia virus by AKR mice. I. Crosses with Fv-ln strains of mice. J. Exp. Med. 136:1272-1285. 8. Rowe, W. P., J. W. Hartley, and T. Bremner. 1972. Genetic mapping of a murine leukemia virus-inducing locus of AKR mice. Science 178:860-862.
