Leukemia (1997) 11, 619–623 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 REVIEW Fre2, a proviral integration site of Friend murine leukemia virus that is closely linked to Fv2 RW Friedrich1, M Veit1, D Eisel1, U Friedrich1, M Pass1,2 and CA Kozak3 1Institute of Medical Virology, Justus Liebig University, Giessen, Germany; and 3Laboratory ot Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Friend murine leukemia virus (F-MuLV) induces leukemia by Hoatlin et al8 suggested that the Fv2 gene product can interact integration into the cellular genome, thereby changing the with the erythropoietin receptor which plays an important role structure or expression of cellular oncogenes. In this report we describe a new F-MuLV integration site Fre2 isolated from in SFFV-induced erythroleukemia, thereby inducing F-SFFV splenic DNA of an erythroleukemic animal. This site was found resistance. to be rearranged in six out of 64 tumors tested; however, in F-MuLV proviruses have been found integrated in Fli1,a five out of these six cases no F-MuLV proviruses could be member of the ets gene family, in at least 90% of all primary detected in the vicinity of the rearrangement sites. The spleen tumors of erythroleukemic animals9 and in erythroid rearrangements represented closely clustered chromosomal cell lines.10 Several additional integration sites in erythroid or breakpoints, presumably chromosomal translocations. Exons myeloid leukemias have been described, including Fli2, Fim1, transcribed into differentially spliced mRNAs of 1.9 and 3.7 kb 11–16 have been found near the breakpoint. Fre2 is closely linked to Fim2/c-fms, Fim3/CB1/Evi1, Fis1, Pim1, c-myc and p53. Fv2, a locus on mouse chromosome 9 involved in erythropoi- In our own studies, it became clear that additional inte- esis. Sequences homologous to Fre2 could not be found in the gration sites for F-MuLV exist in spleen tumors of erythroleu- gene databases. kemic animals. In this article, we describe a new site, Fre2, Keywords: Friend murine leukemia virus; Fre2; Fv2; proviral closely linked to Fv2, with characteristics not seen in other integration site; erythroleukemia retroviral integration sites. Introduction Results and discussion Friend murine leukemia virus (F-MuLV) is a nondefective A genomic library in phage l was derived from DNA of a (ecotropic) retrovirus which can induce a variety of hemato- spleen tumor (tumor No. 25) in a BALB/c mouse. The F-MuLV logic neoplasms, including erythro- and myeloblastic leuke- which had been used for infection contained a bacterial supF mia when injected into newborn mice of appropriate strains. gene as a selectable marker.17 From positive l clones, Virally induced erythroleukemia is a multistage disease, sequences flanking supF-containing proviruses were isolated beginning with an early hemolytic anemia, partially compen- and used as probes for DNA rearrangements in Southern blots sated for by reactive erythropoiesis. Later, the animals become of splenic DNA from erythroleukemic mice. Sites that were severely anemic and show grossly enlarged spleens containing found rearranged in more than one of the tumors were called large numbers of transformed cells that do not differentiate Fre (Friend erythroleukemia; to be published elsewhere). Sev- into erythrocytes. This condition leads to the death of the ani- 1 eral such clones were mapped on mouse chromosomes using mals at an age of about 2–3 months. single copy genomic DNA flanking the viral inserts to identify F-MuLV can also act as a helper virus for the replication- RFLPs (M Pass, FT Sels, A Schulz, D Eisel, U Friedrich, C defective Friend spleen focus-forming virus, which causes Kozak and RW Friedrich, manuscript in preparation). Fre2 was either acute anemia (strain SFFVA) or polycythemia (strain initially mapped to distal chromosome 9 by analysis of an SFFVP) in susceptible newborn or adult mice 3 to 4 weeks intersubspecies cross. Because results suggested close proxim- 2 after infection. Some mouse strains, however, are resistant to ity of Fre2 and the Friend virus resistance gene, Fv2, we ana- SFFV-induced disease. Several genes have been held respon- lyzed a second interspecies cross: (NFS/N × M. spretus) × M. sible for Friend virus resistance, including Fv1 to Fv6 and Rfv1 spretus or C58/J.18 This cross could be typed for Fv2 since to Rfv3 (for reviews, see Refs 3–5). Fv2 is of special interest both C58/J and M. spretus carry the recessive resistance allele. in studies of hematopoiesis since it seems to be involved in In this cross, no recombinants were identified in 91 mice the regulation of early erythropoiesis. It was discovered by 6 typed for Fre2 and Fv2 indicating that, at the upper limit of Lilly and described in more detail by Axelrad and coworkers the 95% confidence level, these two genes are no more than (see Ref. 3 for references) as a locus responsible for the resist- 3.2 centimorgans apart. ance of some mouse strains to Friend-SFFV induced disease The F-MuLV flanking sequence isolated from DNA of tumor (allele Fv2r is assumed to be involved in the control of DNA 7 No. 25 was used to screen a genomic library of normal replication associated with early erythropoiesis ). Recently, BALB/c DNA in phage l. One such clone (25.5-D) which con- tained a 16.4 kb insert was sequenced. A diagram of the gen- omic structure of this region is shown in Figure 1. The position Correspondence: R Friedrich, Institute of Medical Virology, Frank- of the proviral insert in the DNA of tumor 25 was determined furter Str. 107, D-35392 Giessen, Germany 2Present address: Dept. f. Innere Medizin, Universita¨tsspital Zu¨rich, by PCR and is indicated in this Figure. Ha¨lderliweg 4, CH-8044 Zu¨ rich The flanking sequence (designated probe A in Figure 1) and Received 25 September 1996; accepted 18 January 1997 a sequence derived from the l insert (probe B) were used to F-MuLV integration site Fre2 RW Friedrich et al 620 Figure 1 Genomic structure of Fre2. A genomic library of normal BALB/c spleen DNA in phage l was screened with probe A originally isolated from tumor 25. The restriction enzymes shown were derived after sequencing the insert of one such clone designated 25.5-D. The diagram shows the position of proviral DNA found in tumor 25 and the position of the rearrangements found in tumors 3, 24, 29, 32 and 39. The location of probes used for Southern blot analysis is shown. The lower part of the diagram represents the structure of two exons as derived from the analysis of cDNA clones. Potential initiation and termination codons as well as a potential polyadenylation signal are also given. screen 63 spleen tumors from mice infected with F-MuLV as only partially to these fragments. This led us to the tentative newborns for DNA rearrangements. By this procedure, five localization of the rearrangement site as shown in Figure 1. additional tumors were found which showed rearranged Fre2 We tried to localize proviral sequences in the vicinity of the DNAs. A Southern blot of tumor DNAs cut with either EcoRI rearrangement sites in tumors 3, 24, 29, 32 and 39 by or EcoRV and hybridized with probe B (see Figure 1) revealed hybridization of viral probes (derived from LTR or env that restriction enzyme fragments of DNAs from all tumors sequences) to Southern blots similar to those shown in Figure were of similar sizes (Figure 2). The amount of DNA 2 as well as by PCR with oligonucleotides derived from viral rearranged varies among the tumors. As can be seen from Fig- and cellular sequences. All attempts to find proviral sequences ure 2, only a small fraction of tumor 32 seems to contain in this region were unsuccessful. All tumor DNAs, however, rearranged DNA since most of the hybridizing DNA fragments contained F-MuLV proviruses integrated near Fli1 as expected migrate with the restriction fragments from the control DNA. from our earlier results9 (data not shown). These results sug- In contrast, the intensities of the wild-type EcoRI and EcoRV gest that the observed rearrangement found in these tumors restriction fragments obtained from tumors 29 and 39 (10.4 represents a chromosomal translocation, either a gross and 12.3 kb, respectively) are similar (tumor 39) or even lower rearrangement in the same chromosome or a recombination than the 6 kb EcoRI and EcoRV fragments indicative of the with another chromosome. Fre2 rearrangement. In tumor 3, only little DNA remains at The size of the restriction fragments corresponding to the the position of the wild-type fragment; this could, at least in rearranged DNA is surprisingly similar in all tumors that are part, be due to the degradation of this DNA. Since DNA of positive for this rearrangement. This suggests a rather precise tumor 29 shows only minor degradation, the stronger intensity recombination not only in Fre2 (chromosome 9) but also in of the 6 kb band suggests that either both alleles of Fre2 are the (still unknown) recombination partner. rearranged in most of the tumor cells or that one allele is To localize possible coding regions within the l insert, the deleted. whole insert was hybridized to Northern blots of poly(A+) RNA This was also obvious using restriction enzymes such as from normal mouse tissue. Two RNA species of 1.9 and 3.7 kb HindIII and PstI which generate smaller fragments than those could be found in all mammalian tissues and cell lines tested produced by EcoRI or EcoRV (Figure 3). Smaller fragments (Figure 4). Southern blot hybridizations with probes from vari- than expected of 480 and 560 bp were found in HindIII ous regions of the Fre2 locus did not show cross hybridization digests of tumors 3, 29 and 39.