Proc. Natl. Acad. Sci. USA Vol. 76, No. 9, pp. 4455-4459, September 1979 Cell Biology Presence and expression of Friend erythroleukemia -related sequences in normal and leukemic mouse tissues (RNA tumor /Friend spleen focus-forming virus/mechanism of transformation/differentiation/ expression) ALAN BERNSTEIN, CATHERINE GAMBLE, DONNA PENROSE, AND TAK W. MAK The Ontario Cancer Institute, and Department of Medical Biophysics, University of Toronto, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K9 Communicated by J. Tuzo Wilson, May 22, 1979

ABSTRACT The nature and distribution of sequences re- Although the origin and nature of sarcoma virus-specific lated to the murine erythroleukemia virus, Friend spleen sequences have been well characterized, no comparable studies focus-forming virus (SFFV), have been analyzed by using a ra- have been reported on the rapidly transforming mammalian dioactive cDNA probe specific for the SFFV genome (cDNAsff). From the proportion of high molecular weight viral [32PJRNA leukemia viruses. These viruses, which include the murine which hybridized to cDNAsff, it was estimated that these se- Abelson (9) and Friend (10-12) leukemia viruses, are replica- quences represent about 50% of the SFFV genome, indicating tion-defective and hence require helper virus to produce in- a genetic complexity of about 3300 nucleotides. cDNAff hy- fectious progeny. Immunological and molecular hybridization bridized extensively (80-95%) to SFFV virion RNA and to cel- analyses of cell clones nonproductively infected with the de- lular RNA from murine and rat cells productively or nonpro- fective Friend focus- ductively infected with SFFV. Only background homology was erythroleukemia-inducing virus, spleen detected between cDNAff and viral RNA from a number of forming virus (SFFV), have indicated that the SFFV genome murine [Friend (MuLV), Moloney-MuLV, contains some, but not all, of the sequences found on its helper and Kirsten sarcoma virus] and nonmurine (, lymphoid leukemia virus (13, 14). In addition, the SFFV ge- , baboon endogenous virus, and Mason- nome, like rapidly transforming sarcoma viruses, contains Pfizer mammary tumor virus) . Limited homology unique sequences that are not found on its helper lymphoid was also detected to a number of murine xenotropic and mink leukemia virus The that these cell focus-inducing viruses (20-35%) as well as Rauscher leu- (13-16). possibility SFFV-specific kemia virus (50%). Nucleotide sequences homologous to sequences code for a functional polypeptide is suggested by the cDNAsff were also detected in the DNA of normal cells of sev- observations that SFFV nonproducer cells express a high mo- eral mouse strains as single or a few copies per cell. Thermal lecular weight polyprotein that contains both virion structural denaturation analysis indicated that duplexes formed between and nonstructural components (17) and that the infection of cDNAsff and normal DBA/2J cellular DNA have a reduction in homologous or heterologous cells with SFFV results in the ap- melting temperature of 2VC when compared with the disso- of a new cell surface that is not found on ciation of hybrids between cDNAsff and homologous sequences pearance antigen in SFFV-infected mouse spleen cell DNA. Examination of cel- helper virus-infected cells (18). lular RNA from uninfected mouse cells indicated that SFFV- In this study, we have used a single-stranded DNA probe related RNA sequences were also expressed in varying degrees complementary to a portion of the replication-defective SFFV in different tissues of adult DBA/2J mice. The highest amounts genome that is not shared with its helper virus (cDNAff) (15) were observed in cells from bone marrow and spleen, whereas to demonstrate that nucleotide sequences that are highly related considerably lower amounts were found in cells from the thy- mus and kidney. No SFFV-related sequences could be detected to these SFFV-specific sequences are present in normal mouse in RNA extracted from liver, muscle, or fibroblasts. The presence cellular DNA. Furthermore, these cellular SFFV-related se- of these SFFV-related sequences in normal, uninfected mouse quences appear to be differentially expressed into RNA in cells cell DNA and their differential expression in hematopoietic from hematopoietic tissues of adult mice. tissues suggest that these sequences may be an integral part of the program of both normal and leukemic hematopoietic cell MATERIALS AND METHODS differentiation. Cells and Viruses. Cells were grown in alpha medium (19) Molecular analysis of the genomes of a number of avian and supplemented with 10% fetal bovine serum. Cell lines included murine sarcoma viruses has advanced considerably our un- NIH/3T3 and NRK cells nonproductively infected with SFFV, derstanding of viral carcinogenesis. Such studies have indicated NIH/3T3 cells productively infected with both the polycyth- that the genomes of these viruses contain unique sequences that emic strain of SFFV and its NB-tropic helper Friend murine have either been implicated or shown to be involved in the rapid leukemia virus (F-MuLV), and NIH/3T3 cells productively malignant transformation of fibroblasts (1-3). The observation infected with the cloned helper F-MuLV. These cell lines have that nucleotide sequences highly related to these sarcoma all been described (13). A fetal lung mink cell line CCL64 was virus-specific sequences (src) have been found in the DNA of obtained from J. Stephenson (National Institutes of Health, normal avian (4) and murine (5) cells has led to the hypothesis Bethesda, MD 20205). SFFV and its helper F-MuLV were that these rapidly transforming viruses may have arisen as the harvested from the above NIH/3T3 cell lines. Xenotropic result of a recombination event between a type C leukemia murine type C viruses, including BALB/virus-2 [BALB/c- virus and host src sequences. In addition, sequences related to murine xenotropic virus (XLV)], NZB-XLV, and a mink cell the src gene of Rous sarcoma virus appear to be transcribed (6) focus-inducing virus isolated from AKR mice (MCF-274) were and translated (7, 8) in normal chicken embryo fibroblasts, al- a role for these sequences in normal cell functon has not Abbreviations: SFFV, Friend spleen focus-forming virus; F-MuLV, though Friend murine leukemia virus; XLV, murine xenotropic virus; MCF, yet been determined. mink cell focus-inducing virus; cDNAsff, purified single-stranded DNA complementary to specific sequences on the SFFV genome; Crt, initial The publication costs of this article were defrayed in part by page concentration of RNA in moles of nucleotide per liter X time in sec- charge payment. This article must therefore be hereby marked "ad- onds; Cot, initial concentration of DNA in moles of nucleotide per liter vertisement" in accordance with 18 U. S. C. §1734 solely to indicate X time in seconds; Cot112, Cot necessary for 50% hybridization; Crtl/2, this fact. Crt necessary for 50% hybridization. 4455 Downloaded by guest on September 24, 2021 4456 Cell Biology: Bernstein et al. Proc. Natl. Acad. Sci. USA 76 (1979) obtained from J. Hartley (National Institutes of Health). These after incubation, the tubes were broken and the extent of hy- xenotropic viruses were propagated on CCL64 cells by using bridization was determined by hydrolysis with the single- described procedures (13). Moloney leukemia virus and strand-specific nuclease, S1. Rauscher leukemia virus were obtained from A. Wu (Depart- DNA-cDNA Hybridization. DNA was extracted from tissue ment of Anatomy, University of Toronto, Toronto, Canada). by a described method (22). For hybridization, cellular DNA Kirsten sarcoma virus, Rous sarcoma virus, feline leukemia sonicated to 6 S20,w was mixed with about 1000 cpm of cDNA virus, baboon endogenous virus, and Mason-Pfizer mammary in a mixture containing 0.75 M NaCl, 10 mM EDTA, 25 mM virus were all obtained from J. Gruber (National Cancer In- Tris-HCl (pH 7.4), and 50% formamide (crystallized five times). stitute). The reaction mixture containing about 15 mg of DNA per ml Mice. All mice used in these experiments were purchased was incubated at 400C. At various times the sealed glass tubes from the Jackson Laboratory. were broken and the SI-nuclease-resistant counts were deter- Preparation of High Molecular Weight Viral [32P]RNA. mined. NIH-3T3 cells chronically infected with SFFV (F-MuLV) were Thermal Denaturation Analysis. Hybrids between cellular used to prepare viral [32P]RNA. These cultures released virus DNA and viral cDNA were formed as described above [final stocks containing a SFFV-to-F-MuLV ratio of approximately Cot of >20,000 (mol/liter) X sec]. After hybridization, the re- 10:1 (15). The chronically infected NIH-3T3 cells were incu- action mixture was diluted 200-fold into 0.14 M sodium phos- bated in phosphate-free a-medium containing 10% dialyzed phate buffer (pH 7.4). The mixture was then loaded into a fetal calf serum and 2 mCi of 32P-phosphate (Amersham Searle) hydroxylapatite column and a temperature gradient ranging per ml for 18 hr (1 Ci = 3.7 X 1010 becquerels). The medium from 50°C to 95°C in 5°C intervals of prewarmed 0.14 M was then replaced with phosphate-free medium without iso- phosphate buffer plus 0.4% sodium dodecyl sulfate was used tope. The medium was harvested at intervals of 2 hr and virus to elute single-stranded DNA. Fractions (5 ml) were collected was purified by centrifugation through a 20-60% sucrose gra- and analyzed for acid-insoluble radioactivity. dient as described (13, 15). The 32P-labeled virus, which banded at densities of 1.14-1.16 gm/ml, was then pelleted at 100,000 RESULTS X g for 2 hr and resuspended in TNE (0.02 M Tris-HCl, pH Genetic Complexity of the Nucleotide Sequences in 7.4/0.14 M NaCI/10 mM EDTA) containing 1% sodium do- cDNAsff. Molecular hybridization was used to determine decyl sulfate. High molecular weight viral RNA (50-70s20,,) whether the cDNA to Friend virus complex was representative was purified by centrifugation through a 15-30% sodium do- of both SFFV and helper F-MuLV genomes, and also to de- decyl sulfate/sucrose gradient as described (15). High molecular termine what proportion of the SFFV genome was represented weight viral RNA was further purified by chromatography on on cDNAsff. Three viral cDNA probes were utilized: unfrac- oligo(dT) cellulose (grade T3; Collaborative Research, Wal- tionated cDNA complementary to SFFV and its helper F- tham, MA) as described (20). The bound viral [32P]RNA was MuLV (cDNASFFvF-MuLv)), SFFV-specific cDNA (cDNAsff), then precipitated with 3 vol of ethanol. and cDNA complementary to those sequences on the SFFV Preparation of Viral-Specific cDNAs. (i) CDNASFFV,MuLV. genome shared with F-MuLV (cDNAF-MuLV) Polyadenylated [3H]dGMP-labeled cDNA was synthesized from virus stocks 70S viral [3P]RNA, containing a ratio of approximately 10 SFFV containing SFFV to F-MuLV of approximately 10 to 1 (13) by genomes to 1 F-MuLV genome, was hybridized to varying a modified endogenous reverse transcriptase reaction as de- amounts of the three cDNAs listed above to a constant Crt scribed (15). The endogenous cDNA reaction was primed by value of 5 X 10-'. As shown in Fig. 1, the unfrac- 700 ,ug of calf thymus-activated primer per ml prepared as tionated cDNASFFVF-MULV hybridized to 80-90% of viral described (21). The cDNA synthesized consisted mainly of [32P]RNA at a cDNA-to-RNA ratio of 2:1, indicating that fragments with size of 6 s2ou and had a specific activity of 3 cDNASFFvF-MULV represented a uniform and fairly complete X 107 cpm/,ug. This cDNA protected about 90% of 70S viral transcript of the viral genomes. The fractionated cDNAsff, [32P]RNA at a cDNA-to-RNA ratio of 2:1 (see Fig. 1). specific for sequences on the SFFV genome, hybridized to cDNASFFVF-MuLV contains both sequences shared with helper approximately 50% of the viral [32P]RNA at a cDNA-to-RNA F-MuLV (cDNAF-MuLv) and cDNA unique to the SFFV ge- ratio of 2:1 (Fig. 1). Similarly, cDNAF-MULV hybridized to 50% nome (cDNAsff). of the viral [32P]RNA at a cDNA-to-RNA ratio of 2:1 (Fig. 1). (ii) cDNAsff. cDNAsff homologous to sequences specific for These results indicate that approximately 50% of the SFFV the Friend SFFV genome was fractionated and purified by hydroxylapatite chromatography as described (15). (iii) cDNAF-MuLV. cDNAF-MuLv homologous to helper F- MuLV was obtained by hybridizing cDNASFFvFMuLV to excess 1' F-MuLV RNA. The annealed cDNA was separated from the C nonhybridized cDNA by hydroxylapatite chromatography as 0 described (15). The hybridized portion (cDNA homologous to N F-MuLV) was then treated with 0.3 M NaOH at 37°C for 12 hr to digest RNA and then neutralized with hydrochloric .0 acid. I Globin cDNA. The isolation of a- and (- mouse globin mRNA and synthesis of globin cDNA has been described pre- 0 2 4 6 8 viously (20). Ratio of complementary nucleotide RNA*cDNA Hybridization. Viral and cellular RNA was sequences RNA/ extracted as described (13, 15). Hybridization of cDNA to either viral or cellular RNA was performed at 40°C in the presence FIG. 1. Genetic complexity of cDNAsff. (SFFV, F-MuLV) [32P]RNA (50-70 S) (specific activity, 5 X 106 cpm/gg) was hybridized of five-times-recrystallized formamide (50% vol/vol)/0.5 M to varying amounts of I3HjcDNASFFVjF-MuLV, [3H]cDNAF-MuLV, or sodium chloride/10 mM EDTA/0.025 Tris-HCI (pH 7.4) in 5-,ul [3H]cDNAsff (107 cpm/lg). Hybridization was carried out to a Crt sealed glass capillary tubes. Between 500 and 1000 cpm of value of 5 X 10-1. Results represent averages of duplicate points. 0, [3H]cDNA was added to each reaction mixture. At various times cDNASFFV,F-MULV; A, cDNAF-MULV; *, cDNAjff. Downloaded by guest on September 24, 2021 Cell Biology: Bernstein et al. Proc. Natl. Acad. Sci. USA 76 (1979) 4457 gendffie is shared with its helper F-MuLV and 50% is SFFV- specific. Hybridization of cDNAsff to Other RNA Tumor Viruses. The presence of SFFV-specific sequences in a number of mu- 1000 C

rine and nonmurine retroviruses was determined by hybrid- Z 80F ization of cDNAsff to RNA extracted from these viruses. The cDNASff hybridized extensively to RNA from Friend virus 60~ complex (SFFV, F-MuLV) (96%), and had extensive homology (54%) with RNA from another murine erythroleukemia virus, 40r Rauscher leukemia virus (Table 1). In agreement with previous 20-_ reports (14), hybridization (20-35%) was also observed to RNA from two murine xenotropic viruses isolated from BALB/c and 100 101 102 103 104 NZB mouse cells and to RNA from a mink cell focus-inducing Crt, (mol liter-') X sec (MCF) virus isolated from AKR mice (Table 1). However, the FIG. 2. SFFV-specific sequences in NIH/3T3 and NRK clones extent of hybridization appears to be limited, suggesting that productively and nonproductively infected with SFFV. Total cellular additional sequences not shared with these viruses. RNA was extracted from (SFFV,F-MuLV)-producing NIH/3T3 cells there are (-), SFFV nonproducer NIH/3T3 cell clones NP 9(0) and NP 13(m), The cDNAsff did not hybridize to either cloned helper F-MuLV SFFV nonproducer NRK cell clones NP 501 (A) and NP 502 (V), isolated from the polycythemia-inducing variant of SFFV F-MuLV-infected NIH/3T3 cells (0), and uninfected NIH/3T3 (0) (F-MuLV), Moloney murine leukemia virus, or Kirsten murine and NRK (W) cells. The extent of hybridization of the various cellular sarcoma virus. In addition, no hybridization was observed be- to cDNA was then determined. tween cDNAsff and RNA from a number of nonmurine retro- viruses, including feline leukemia virus, Rous sarcoma virus, baboon endogenous virus, and Mason-Pfizer mammary tumor with helper F-MuLV. These observations indicate that a high virus. Table 1 also shows that sequences represented by cDNA proportion of the sequences represented by cDNAsff are on the to Friend helper F-MuLV (CDNAF-MuLv) were presented on SFFV genome. all the murine viruses tested. Presence of SFFV-Related Sequences in Normal Cell Hybridization of cDNAsff to Cellular RNA from Murine DNA. Studies on the avian (4) and murine (5) sarcoma viruses and Rat SFFV Nonproducers. We have previously described have suggested that sarcoma virus-specific sequences have the isolation of murine and rat cell clones nonproductively in- originated from normal host cell DNA. To determine whether fected with SFFV (13). These clones express high levels of RNA host sequences homologous to SFFV-specific sequences were sequences that hybridize to unfractionated cDNASFFVF-MuLV, also present in normal murine cellular DNA, cDNAsff was hy- although they release no infectious SFFV unless they are su- bridized to DNA extracted from both uninfected and SFFV- perinfected with helper F-MuLV (13). To determine if the infected mouse DBA/2J spleen cells. As a control, normal un- sequences on cDNAsff were in the RNA of cell clones nonpro- infected spleen DNA was also hybridized to cDNA to globin ductively infected with SFFV, cDNAsff was hybridized to RNA messenger RNA. As expected, cellular DNA from the greatly extracted from mouse and rat cell clones productively and enlarged spleens of mice infected 9 days previously with SFFV nonproductively infected with SFFV. The results in Fig. 2 in- had extensive homology with cDNAsff and hybridized with a dicate that, in addition to hybridizing to RNA extracted from C0t1/2 of approximately 1000 (Fig. 3). DNA from normal un- cells productively infected with SFFV, cDNAsff also hybridized infected mouse spleen cells also hybridized extensively to extensively to cellular RNA from two independently isolated cDNAsff, although the COtl/2 value was slightly higher than that NIH-3T3 nonproducer cell clones (NP9 and NP13) as well as observed to infected spleens. cDNA to a and f globin mes- to different heterologous rat NRK cell clones nonproductively senger RNA used as a standard also hybridized to uninfected infected with SFFV (NP501 and NP502). Only background spleen DNA with a C0t1/2 of 1000-2000. These results suggest homology was observed between cDNAsff and RNA from un- that SFFV-related sequences are presented as a single copy or infected NIH-3T3 or NRK cells, or to RNA from cells infected a few copies in the DNA of normal mouse cells. DNA from several other strains of mice and other species Table 1. Hybridization of cDNAff and CDNAFMuLv to RNA were also examined for the presence of SFFV-specific se- from murine and nonmurine retroviruses quences. As shown in Table 2, DNA sequences homologous to Percent hybridization cDNAsff were present in all mouse strains tested, but were not Viral RNA cDNAff cDNAF-MuLV F-SFFV (F-MuLV) 96 92 F-MuLV 10 100 Moloney-MuLV 4 74 BALB/c-XLV 22 39 10 NZB-XLV 18 41 M MCF-274 35 87 0 o20- c 40 3 Rauscher-MuLV 54 100 co ~~~~~~~~~30 Kirsten murine sarcoma virus 11 - 6 60\: Rous sarcoma virus 0 0 o o40> Feline leukemia virus 6 0 80- 50 Baboon endogenous virus 3 _ . 60 Mason-Pfizer mammary virus 0 1L--100 02 103 104 No RNA 0 Cot, (mol liter-') X sec RNA was extracted from sucrose gradient-purified virus and hy- FIG. 3. Hybridization of cDNAff to the DNA from either normal bridized to either cDNAff or cDNAF-MULV to Crt values of >10. (0) or erythroleukemic (0) DBA/2J spleen cells 9 days after SFFV 13HlcDNA.ff (500 cpm) and cDNAF-MULV (1000 cpm) was used in infection. DNA from mock-infected spleen DNA was also hybridized these experiments. to a and globin cDNA6~~~~~~~~~(A ). Downloaded by guest on September 24, 2021 4458 Cell Biology: Bernstein et al. Proc. Natl. Acad. Sci. USA 76 (1979) Table 2. Hybridization of cDNA8ff sequences to various murine and nonmurine DNA

Percent 100 hybridization Source of DNA with cDNAff I 80 ° N DBA mouse spleen 78 DBA mouse liver 80 40 - DBA mouse spleen (SFFV infected) 100 DBA mouse spleen (F-MuLV infected) 70 (C57BL/6 X C3H)F1 mouse spleen 80 20* BALB/c mouse spleen 70 100 10l 102 103 104 NIH/3T3 mouse fibroblasts 85 l05 Rat liver (Wistar) 26 Crt, (mol liter') X sec Chicken liver 0 FIG. 5. Expression of SFFV-related RNA sequences in tissues Hamster liver 22 from infected and uninfected mice. cDNAsffwas hybridized to cellular Human peripheral blood 0 RNA extracted from various infected and uninfected mouse tissues. The RNA was obtained from the following sources: SFFV-infected Human leukemic blood 0 DBA/2J spleen cells (0); bone marrow from 6-month-old uninfected mice (0); spleen cells from 6-month-old (A) or 6-week-old DNAs were extracted from murine and nonmurine tissue and hy- DBA/2J to to > 20,000. [3H]cDNA8ff (650 cpm) (4 uninfected DBA/2J mice; thymus (U), kidney (0), liver (v), and bridized cDNAsff Cot values (v) 6-month-old uninfected DBA/2J mice; unin- was to mg per ml. Maximum hybridization was muscle cells from hybridized 15 ofDNA cells from 2-month-old 62% of cDNAsff to DNA from DBA mouse spleen infected with fected NIH/3T3 cells (*); uninfected spleen input BALB/c mice (b); and uninfected 2-month-old RNC spleen cells SFFV. (x). detected in chicken or human DNA. A limited degree of ho- mology was observed between cDNAsff and rat or hamster marrow, spleen, thymus, kidney, liver, and muscle, was hy- DNA. bridized to cDNAsff. As shown in Fig. 5, cellular RNA from The relatedness of the DNA sequences homologous to leukemic spleens of mice infected with SFFV contained very cDNAsff in normal mouse DNA was determined by thermal high levels of SFFV-specific sequences (Crtl/2 z10). When denaturation analysis. Duplexes between cDNAsff and DNA RNA extracted from uninfected bone marrow and spleen cells from SFFV-infected spleen cells had a melting temperature from DBA/2J mice was examined, sequences highly related of 820C (Fig. 4), as expected for sequences that are totally to cDNAsff were also found to be expressed at a level approxi- complementary. Duplexes formed between cDNAsff and nor- mately 1/50th to 1/100th that of leukemic spleens (Crt1/2 of mal, uninfected DBA/2J mouse cell DNA had a melting tem- 500-1000). Lower levels of these RNA sequences were also perature of 800C. This 20C reduction in melting temperatures found in cells from the thymus and kidney (Crt1/2 -1-3 X 104) indicates that the viral SFFV-specific sequences, although they and sequences homologous to cDNAsff were not detected in have diverged somewhat from the homologous sequences found RNA extracted from liver or muscle cells or NIH/3T3 fibro- in normal host cells, are still highly related to these endogenous blasts. The presence of SFFV-related sequences could also be cellular sequences. detected in RNA from spleen cells of two other inbred mouse Expression of SFFV-Related Nucleotide Sequences in strains examined, BALB/c and RNC. Normal and Leukemia Cellular RNA. The presence of nu- cleotide sequences that were highly related to SFFV-specific sequences in the DNA of normal mouse cells raised the possi- DISCUSSION bility that these cellular sequences might be transcribed into RNA in normal cells. To examine this possibility, total RNA The results presented in this report, as well as previous obser- extracted from various uninfected mouse tissues, including bone vations from this laboratory (15), indicate that the replica- tion-defective SFFV genome contains unique sequences not present on either its helper virus or other nonerythroleu- kemia-inducing viruses. The genetic complexity of these se- 10( quences, as determined by hybridization with cDNAsff, was found to be approximately 50% of the SFFV genome. Because 3i the defective SFFV genome has a complexity of about 6600 NO nucleotides (23), the genetic complexity of these sequences as represented by cDNAsff would be about 3300 nucleotides. If they are contiguous, these sequences are sufficiently complex 0) to encode for one or more proteins with a total molecular weight z 4( of 100,000. 0 The present findings provide insight into the possible origin 24 of the SFFV genome. The presence of nucleotide sequences in normal mouse cell DNA that are highly related to SFFV-spe- ( cific sequences suggests that these viral sequences originated 50556065 70 7580 8590 95 in normal host cell DNA. This conclusion, together with the Temperature, 0C observations that SFFV shares sequences with other murine FIG. 4. Thermal denaturation of hybrids formed between DNA type C viruses (13-16), suggests that the SFFV genome arose from SFFV-infected and uninfected spleens with cDNAff. DNA ex- by a recombination event between a replication-competent type tracted from infected (-) or uninfected (0) DBA/2J spleen DNA was C virus and host cell SFFV-related sequences. hybridized to cDNA8ff to a Crt value of 2 X 104. The sample was then loaded on hydroxyapatite columns at 50'C and a 50-950C tempera- The demonstration that the SFFV genome contains both ture gradient was used to elute denatured DNA. unique sequences as well as sequences related to MCF raises Downloaded by guest on September 24, 2021 Cell Biology: Bernstein et al. Proc. Natl. Acad. Sci. USA 76 (1979) 4459 several possibilities for its mechanism of carcinogenesis. Hartley grants from the Medical Research Council and the National Cancer et al. (24) have reported that AKR thymic lymphoma may be Institute of Canada. C.G. is a Research Student of the National Cancer induced by MCF, whose RNA genorne structure (25) and gly- Institute of Canada. coprotein peptide map (26) indicate the presence of structural components similar to those found in XLVs. The presence of 1. Stehelin, D., Guntaka, R., Varmus, H. & Bishop, J. M. (1976) J. sequences homologous to XLV and MCF on the SFFV genome Mol. Biol. 101, 349-365. reported here and earlier by Troxler et al. (14) suggest that these 2. Scolnick, E. M., Howk, R. S., Anisowicz, A., Peebles, P. T., Scher, sequences may be playing a role in the transformation process C. D. & Parks, W. P. (1975) Proc. Natl. Acad. Sci. USA 72, by this virus. Alternatively, it is possible that carcinogenesis by 4650-4654. 3. Sheiness, D., Fanshier, L. & Bishop, J. M. (1978) J. Virol. 28, acute leukemia viruses such as SFFV involves the acquisition 600-610. of host endogenous genetic sequences that are not found on 4. Stehelin, D., Varmus, H. E., Bishop, J. M. & Vogt, P. K. (1976b) these xenotropic viral genomes. This latter model is consistent Nature (London) 260, 170-173. with the present observations that the SFFV genome contains 5. Frankel, A. E. & Fischinger, P. J. (1976) Proc. Natl. Acad. Sci. murine host cell sequences not found on xenotropic viruses and USA 73,3705-3709. with the observation that a number of different acute leukemia 6. Spector, D. H., Smith, K., Padgett, T., McCombe, P., Roulland- viruses, including Abelson (27-29) and Friend (17, 18) leukemia Dussoix, D., Moscovici, D., Varmus, H. E. & Bishop, J. M. (1978) viruses, code for polypeptides that are not antigenically related Cell 13, 371-379. to known virion structural proteins. 7. Spector, D. H., Baker, B., Varmus, H. E. & Bishop, J. M. (1978) As shown in this paper, these SFFV-related sequences are Cell 13, 381-386. 8. Collett, M. S., Brugge, J. S. & Erikson, R. L. (1978) Cell 15, present in normal mouse cell DNA, and furthermore appear 1363-1369. to be differentially expressed in tissues involved in hemato- 9. Abelson, H. T. & Rabstein, L. S. (1970) Cancger Res. 30, 2208- poietic function in these uninfected mice. The highest levels 2212. of these sequences were observed in RNA from bone marrow 10. Friend, C. (1957) J. Exp. Med. 105,307-318. and spleen cells and represented about 1-2% of the levels found 11. Axelrad, A. A. & Steeves, R. A. (1964) Virology 24,513-518. in the enlarged erythroleukemic spleens of mice infected with 12. Steeves, R. A. (1975) J. Natl. Cancer Inst. 54, 289-297. SFFV. The lower levels of these RNA sequences in normal 13. Bernstein, A., Mak, T. & Stephenson, J. (1977) Cell 12, 287- spleens, compared to the erythroleukemic spleens, may be due 294. either to a lower level of expression of these sequences in normal 14. Troxler, D. H., Boyars, J. K., Parks, W. P. & Scolnick, E. M. (1977) hematopoietic cells or to a minor population of cells in hema- J. Virol. 22,361-372. 15. Mak, T. W., Penrose, D., Gamble, C. L. & Bernstein, A. (1978) topoietic tissues that express these sequences at a level compa- Virology 87,73-80. rable to that found in leukemic spleen cells. Lower amounts of 16. Mak, T. W., Penrose, D., Gamble, C. & Bernstein, A. (1979) in these SFFV sequences were also found in the RNA of cells from Oncogenic Viruses and Host Cell , eds. Ikawa, Y. & the thymus and kidney, whereas no SFFV-related RNA se- Odaka, T. (Academic, New York), pp.183-193. quences were detectable in cells from liver, muscle, or fibro- 17. Barbacid, M., Troxler, D. H., Scolnick, E. M. & Aaronson, S. blasts in culture. It is of interest to note that these RNA se- (1978) J. Virol. 27, 826-830. quences in adult mice show a tissue distribution similar to that 18. Risser, R. (1979) J. Exp. Med., 149, 1152-1167. observed for the cell surface antigen specified by SFFV (18), 19. Stanners, C. P., Eliceiri, G. L. & Green, H. (1971) Nature (Lon- indicating that they may be related gene products. don) New Biol. 230,52-54. 20. Bernstein, A., Hunt, D. M., Crichley, V. & Mak, T. W. (1976) Cell Although the function of these endogenous SFFV-related 9,375-381. sequences in normal mice is not known, the observation that 21. Taylor, J. M., Illmensee, R. & Summers, J. (1976) Biochim. Bio- these sequences are differentially expressed in cells from he- phys. Acta 442,324-330. matopoietic tissues suggests that they play a role in normal as 22. Varmus, H. E., Vogt, P. K. & Bishop, J. M. (1973) Proc. Natl. well as leukemic hematopoiesis. Consistent with this hypothesis Acad. Sci. USA 70,3067-3071. are several observations demonstrating similarities between 23. Duesberg, P., Bister, K., Vogt, P., Troxler, D. & Scolnick, E. (1979) SFFV-transformed cells and their normal counterparts. First, in Oncogenic Viruses and Host Cell Genes, eds. Ikawa, Y. & Johnson and Metcalf (30) have shown that normal mouse fetal Odaka, T. (Academic, New York), pp. 95-115. liver, like SFFV-infected spleens, contains cells that are also 24. Hartley, J. W., Wolford, N. K., Old, L. J. & Rowe, W. P. (1977) capable of erythroid Proc. Natl. Acad. Sci. USA 74,789-793. differentiation in the absence of added 25. Rommelaere, J., Faller, D. V. & Hopkins, N. (1978) Proc. Natl. normal regulator, erythropoietin. Second, genetically anemic Acad. Sci. USA 75,495-499. mice carrying mutations at loci (W and steel) that are known 26. Elder, J. H., Gantsch, J. W., Jensen, F. C., Lerner, R. A., Hartley, to affect the proliferation of normal hematopoietic stem cells J. W. & Rowe, W. P. (1977) Proc. Natl. Acad. Sci. USA 74, also exhibit altered susceptibility to erythroleukemic trans- 4676-4680. formation by SFFV (31-33). And third, recent observations 27. Witte, 0. N., Rosenberg, N., Paskind, M., Shields, A. & Baltimore, have indicated that the Fv-2 locus, which controls susceptibility D. (1978) Proc. Natl. Acad. Sci. USA 75,2488-2492. to exogenous infection by SFFV (34), also affects expression of 28. Reynolds, F. H., Jr., Sacks, T., Deobagkar, D. N. & Stephenson, both endogenous SFFV-related sequences (35) and a SFFV- J. R. (1978) Proc. Natl. Acad. Sci. USA 75,3974-3978. specified cell surface antigen (18) in normal uninfected mice. 29. Risser, R., Stockert, E. & Old, L. J. (1978) Proc. Natl. Acad. Sci. USA 75,3918-3922. Thus, if these sequences are involved in the regulation of normal 30. Johnson, G. R. & Metcalf, D. (1977) Proc. Natl. Acad. Sci. USA development, then it is not unlikely that the constitutive ex- 74,3879-3884. pression of highly related viral sequences in hematopoietic cells 31. Steeves, R. A., Bennet, M., Mirand, E. A. & Russell, L. B. (1968) as a result of exogenous viral infection could lead to alterations Nature (London) 218,372-374. in cell functions that are ultimately recognized at the cellular 32. Bennet, M., Steeves, R. A., Cudkowicz, G., Mirand, E. A. & level as leukemia. Russell, L. B. (1969) Science 162,564-565. 33. McCool, D., Mak, T. W. & Bernstein, A. (1979) J. Exp. Med., 149, The authors thank George Cheong for technical assistance, Dr. J. 837-846. Hartley for kindly providing various strains of murine xenotropic and 34. Lilly, F. & Pincus, T. (1973) Adv. Cancer Res. 17, 231-277. MCF viruses, and Dr. L. Siminovitch for his comments during the 35. Mak, T. W., Axelrad, A. A. & Bernstein, A. (1979) Proc. NatI. preparation of the manuscript. This study was supported in part by Acad. Sci. USA 76, in press. Downloaded by guest on September 24, 2021