The endogenous ecotropic murine retroviruses Emv-16 and Emv-17 are both capable of producing new proviral insertions in the mouse genome.

New germ line proviral insertions are acquired at a high frequency by the progeny of SWR/J-RF/J hybrid female mice that carry the endogenous ecotropic murine leukemia proviruses Emv-16 and Emv-17. The tight linkage of these RF/J strain proviral loci has prevented genetic segregation of the retroviral genomes. Hence, it is not known whether both of these proviruses are capable of giving rise to new proviral insertions. We have molecularly cloned Emv-16 and Emv-17 and have characterized them in vitro and in vivo. Restriction enzyme analysis of the recombinant clones revealed that the proviral genomes are very similar to each other and closely resemble the wild-type AKR virus. A comparison of the flanking cellular DNA suggests that the Emv-16 and Emv-17 loci did not arise by simple duplication of a viral insertion site within the RF/J genome but most likely are independent integration events. Both proviruses produce infectious virus when transfected into NIH 3T3 cells, indicating that they are nondefective retroviruses. Exogenous infection of SWR/J mice with either Emv-16 or Emv-17 leads to viremia in the host animals, and in both cases, progeny of viremic females acquire new proviral insertions. The ability of these retroviruses to generate novel retroviral integration sites in the mouse genome provides a simple method for inducing insertional mutations in mice.     

Emv-13 (Akv-3): a noninducible endogenous ecotropic provirus of AKR/J mice

All AKR/J mice carry at least three endogenous ecotropic viral loci which have been designated Emv-11 (Akv-1), Emv-13 (Akv-3), and Emv-14 (Akv-4) (Jenkins et al., J. Virol. 43:26-36, 1982.) Using two independent AKR/J-derived sets of recombinant inbred mouse strains, AKXL (AKR/J x C57L/J) and AKXD (AKR/J x DBA/2J), as well as the HP/EiTy strain (an Emv-13-carrying inbred strain partially related to AKR/J mice) (Taylor et al., J. Virol. 23:106-109, 1977), we have examined the association of these endogenous viral loci with virus expression. Strains which transmit Emv-11 or Emv-14 or both were found to produce virus spontaneously, whereas strains that transmit Emv-13 alone were negative for virus expression. Restriction endonuclease digestion and hybridization with an ecotropic virus-specific hybridization probe of DNAs from strains which transmit only Emv-13 yielded enzyme cleavage patterns identical to those observed with DNAs from strains transmitting Emv-11 or Emv-14 or both. These findings indicate the absence of any gross rearrangement of Emv-13 proviral sequences. Cell cultures derived from recombinant inbred strains that carry only Emv-13 failed to express detectable infectious virus, viral proteins, or cytoplasmic ecotropic virus-specific RNA even after treatment with 5-iodo-2-deoxyuridine or 5-azacytidine, an inhibitor of DNA methylation. Our results indicate that a mechanism(s) other than methylation of Emv-13 proviral DNA is responsible for inhibition of Emv-13 expression.     

Genetic localization of Hao-1, blind-sterile (bs), and Emv-13 on mouse chromosome 2.

The blind-sterile (bs) mutation in the mouse was localized on Chromosome 2 between Hao-1 and Emv-13. N2 progeny from a backcross between congenic female 129.AKR-bs Emv-13 mice and (129.AKR-bs/bs x Mus musculus molossinus) F1 male mice were typed by analysis of isozyme variants for Hao-1, visible inspection for bs, and restriction fragment length polymorphism for Emv-13 and Emv-15. Comparison between markers on mouse Chromosome 2 and corresponding markers on human chromosomes suggest that the human homolog of bs will be located on 20q11-q13.     

A replication-competent, endogenous retrovirus from an aged DBA/2 mouse contains the complete env from Emv-3 and a novel gag partially related to AKT-8.

We previously described an endogenous murine retrovirus, rv-DBA/2aged, isolated from an aged DBA/2 mouse. The previous report showed that a recombination which resulted in the replacement of Emv-3 gag sequences with gag sequences homologous to those found in the AKT-8 virus had taken place. This recombination allowed production of a competent virus from the defective Emv-3 locus. However, the extent of replacement of Emv-3 gag was not known. We report here the entire sequence for the gag gene of rv-DBA/2aged as well as the previously unsequenced 3' end of the Emv-3 gag gene. These data demonstrate that while sequences homologous to the entire gag gene fragment found in AKT-8 are represented in rv-DBA/2aged, the remainder of rv-DBA/2aged gag is not derived from Emv-3 but is a unique gag sequence. Furthermore, a complete comparison of env sequences shows that the env of rv-DBA/2aged is derived entirely from Emv-3. Additional data suggest that the recombination which led to production of the rv-DBA/2aged virus may be a common event in aging DBA/2 mice. Finally, comparison of the new sequences of Emv-3 with those of the Akv virus (also designated AKR-623 and Emv-11) and Emv-1 shows that this endogenous virus locus is very closely related to the other Emv loci at the nucleotide sequence level.     

Allelic Variation within the Emv-15 Locus Defines Genomic Sequences Closely Linked to the agouti Locus on Mouse Chromosome 2

Gene(s) at the agouti locus act within the microenvironment of the hair follicle to switch pigment synthesis in the melanocyte between eumelanin (black or brown pigment) and phaeomelanin (yellow pigment). Many phenotypic variants of this locus have been described. The mechanism(s) of gene action causing such variation in coat-color phenotype is not known. The close linkage of an endogenous ecotropic murine leukemia provirus, Emv-15, to the lethal yellow mutation of the agouti locus provides a means to molecularly access genes at or near the agouti locus. We have identified and used a unique mouse sequence flanking the Emv-15 provirus to define three alleles of the Emv-15 locus. We found a correlation between the presence of specific Emv-15 alleles and the origins of specific agouti locus mutations, confirming close linkage. However, we found some exceptions which suggest that the Emv-15 locus is closely linked to, but genetically separable from, the agouti locus.     

Mechanism of chemical activation of expression of the endogenous ecotropic murine leukemia provirus Emv-3.

DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus, Emv-3. This provirus is defective; it is very poorly expressed in young DBA/2 mice. The defect in Emv-3 is caused by a single base substitution in codon 3 of p15gag. The resulting amino acid substitution inhibits myristylation of the gag precursor and subsequent virus assembly. Despite this defect, percutaneous treatment of DBA/2 mice with the carcinogen and mutagen 7,12-dimethylbenz[a]anthracene (DMBA) induces ecotropic murine leukemia virus replication in virtually all treated mice. We hypothesized that this induction is the result of a DMBA-induced reverse mutation in codon 3 of p15gag which allows for efficient myristylation. We tested this hypothesis by isolating ecotropic viruses from DMBA-treated mice and determining the DNA sequences of selected regions of p15gag, including codon 3. In support of the above-described model, all of the viruses examined contained single nucleotide substitutions in codon 3. In addition, most of the replication-competent viruses that were sequenced appeared to result from simple mutation of Emv-3 rather than recombination with other endogenous murine leukemia viruses. These studies may provide a basis for development of a sensitive assay for the mutagenic activity of a variety of chemical carcinogens in vivo.     

Molecular cloning and mapping of the ecotropic leukemia provirus Emv-23 provides molecular access to the albino-deletion complex in mouse chromosome 7

Genetic analysis of radiation-induced deletion mutations involving the chromosome 7 albino (c) locus has expanded the functional map of this 6 to 11-cM region of the mouse genome. To generate one of many points of molecular access necessary for intensifying the analysis of the genes and phenotypes associated with this particular complex of deletions, we have cloned an endogenous ecotropic leukemia provirus (Emv-23), known to be closely linked to c, along with its flanking chromosome 7 sequences. A unique-sequence probe (23.3), derived from a region immediately 5' to the proviral integration site, was found to map less than 0.5 cM from c in a standard backcross analysis. Southern blot analysis of DNAs from animals carrying homozygous or overlapping albino deletions demonstrated that the 23.3 probe was deleted in several relatively small c-region deletions. The deletion mapping of the 23.3 probe places the Emv-23 locus between c and Mod-2, just proximal to a region important for male fertility and juvenile fitness. Mapping of this locus also provides a refinement of the genetic/deletion map for several mutations within this deletion complex.     

Chromosomal assignment of two endogenous ecotropic murine leukemia virus proviruses of the AKR/J mouse strain.

The AKR/J mouse strain is genetically fixed for three different ecotropic murine leukemia virus genomes, designated Akv-1, Akv-3, and Akv-4 (Emv-11, Emv-13, and Emv-14). With recombinant inbred strains and crosses with linkage-testing stocks, Akv-3 and Akv-4 were placed on the mouse chromosome map. Akv-3, which encodes a replication-defective provirus, maps near the agouti coat color locus, a, on chromosome 2. Akv-4, which is replication competent, maps near the neurological mutant gene locus trembler, Tr, on chromosome 11. Akv-1 and Akv-2 (Emv-12), an ecotropic provirus carried by AKR/N but not AKR/J, have previously been mapped to chromosome 7 and 16, respectively. Thus, the four Akv proviruses mapped to date are on four different chromosomes. Akv-3 is the second ecotropic murine leukemia virus provirus to be mapped near the agouti locus. The results are discussed in relation to possible nonrandomness of viral integration.     

Role of recombinant ecotropic and polytropic viruses in the development of spontaneous thymic lymphomas in HRS/J mice.

The biological and genetic characteristics of murine leukemia viruses (MuLV) derived from leukemic and normal HRS/J mice were studied. T1-oligonucleotide fingerprinting and mapping of viral RNAs from unpassaged isolates revealed the presence of complex mixtures of viral genomes. MuLV that were purified by endpoint dilution were genetically heterogeneous. Thus, endogenous retroviral sequences expressed in the tissues of HRS/J mice readily recombined with one another. Furthermore, the regular recovery of recombinant ecotropic MuLV suggested reciprocal in vivo complementation of a genetic defect(s) in each of the endogenous ecotropic proviruses Emv-1 and Emv-3. Some recombinant ecotropic viruses contained sequences in the p15E-U3 region that were not derived from Emv-1 and Emv-3 but were found in recombinant polytropic HRS/J viruses. Finally, comparison of the genetic structures of leukemogenic and nonleukemogenic MuLV of this strain implied that the oncogenic phenotype of these MuLV is encoded within env or the U3 region of the genome or both. Our results are consistent with a stepwise convergent evolution of recombinant MuLV in vivo in individual HRS/J mice. Ultimately, this process of selection results in formation of leukemogenic polytropic viruses.     

A nucleotide substitution in the gag N terminus of the endogenous ecotropic DBA/2 virus prevents Pr65gag myristylation and virus replication.

The endogenous ecotropic provirus Emv-3 present in DBA/2 mice is poorly expressed in the animal, as well as in cell cultures. Transfection of proviral DNA into NIH 3T3 cells localized the expression defect to the 5' region of the viral genome, spanning the untranslated region and the N-terminal part of the gag gene. Comparison of the nucleotide sequence of the Emv-3 provirus with the sequence of the highly infectious Akv murine leukemia virus revealed three nucleotide differences within the gag coding region. One of these differences was found in codon 3 of the gag polyprotein, where a Gln codon is seen in Akv and a Pro codon is differences was found in codon 3 of the gag polyprotein, where a Gln codon is seen in Akv and a Pro codon is seen in Emv-3. By site-directed mutagenesis, we showed that the defect of Emv-3 expression indeed is localized to codon 3 of the gag gene. The gag polyprotein of mammalian type C retrovirus contains myristic acid covalently linked to the N-terminal glycine. This myristylation is not seen in the Emv-3-coded gag polyprotein. We showed that the in vitro-mutagenized Emv-3 genome containing a Gln codon at position 3 of the gag gene yields a myristylated gag polyprotein. Thus, it seems most likely that the defect of expression of the Emv-3 provirus is due to the presence of a proline is position 3 of the gag polyprotein, preventing the myristylation.     

Poorly expressed endogenous ecotropic provirus of DBA/2 mice encodes a mutant Pr65gag protein that is not myristylated.

DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus designated Emv-3. Although this provirus appears to be nondefective by genomic restriction enzyme mapping, weanling mice do not produce virus and only about one-third of adult mice ever express virus. 5-Iododeoxyuridine and 5-azacytidine, two potent inducers of ecotropic virus expression, are relatively ineffective at inducing Emv-3 expression. However, the chemical carcinogen 7,12-dimethylbenz(a)anthracene can induce ecotropic virus expression in approximately 95% of treated DBA/2 mice. Previous experiments involving DNA transfection and marker rescue analysis of molecularly cloned Emv-3 DNA suggested that Emv-3 carries a small defect(s) in the gag gene, not detectable by restriction enzyme mapping, that inhibits virus expression in vivo and in vitro. Using a combination of approaches, including DNA sequencing, peptide mapping, and metabolic labeling of cells with [3H]myristate, we have demonstrated that the defect in Emv-3 most likely results from a single nucleotide substitution within the gene for p15gag that inhibits myristylation of the Pr65gag N terminus. Myristylation of Pr65gag is thought to be required for this protein to associate with the plasma membrane and is essential for virus particle formation. These results provide a conceptual framework for understanding how Emv-3 expression is regulated during development and after chemical induction.     

Genetic Organization of the agouti Region of the Mouse

The agouti locus on mouse chromosome 2 acts via the hair follicle to control the melanic type and distribution of hair pigments. The diverse phenotypes associated with various agouti mutations have led to speculation about the organization of the agouti locus. Earlier studies indicated that two presumed agouti alleles, lethal yellow (Ay) and lethal light-bellied nonagouti (ax), are pseudoallelic. We present genetic data showing probable recombination between Ay and three agouti mutations (at, a, and ax), which suggest that Ay is a pseudoallele of the agouti locus. The close linkage of an endogenous ecotropic murine leukemia provirus, Emv-15, to Ay provides a molecular access to genes at or near the agouti locus. However, previous studies suggested that the Emv-15 locus can recombine with some agouti alleles and therefore we analyzed mice from recombinant inbred strains and backcrosses to measure the genetic distance between various agouti alleles and the Emv-15 locus. Our data indicate that the Emv-15 locus is less than 0.3 cM from the agouti locus. These experiments provide a conceptual framework for initiating chromosome walking experiments designed to retrieve sequences from the agouti locus and give new insight into the genetic organization of the agouti region.     

Dominant hemimelia and En-1 on mouse chromosome 1 are not allelic.

Previous studies have shown that En-1, a homeobox-containing gene, maps close to or at the Dh locus in the mouse. Since homeobox-containing genes are key genes in the control of development the close proximity of En-1 to the developmentally significant gene Dh raised the possibility that the Dh mutation represented a mutant allele of En-1. A genetic analysis involving En-1, Dh, and other chromosome 1 markers (Emv-17, ln and Pep-3) shows that although Dh and En-1 are closely linked they are separable by recombination (4/563). The likely gene order and recombination frequencies of these loci are: ln (5.2 +/- 0.9) Emv-17 (1.1 +/- 0.4) Dh (0.7 +/- 0.4) En-1 (3.0 +/- 0.7) Pep-3. This shows that Dh is not a mutant allele of En-1.     

The long-range restriction map surrounding the mouse agouti locus reveals a disparity between physical and genetic distances

Isolation of a gene based on its location, which depends on aligning physical landmarks with the genetic map, can yield basic information about genome structure and organization. As a first step toward isolating the mouse agouti (A) locus, we have begun to define the physical position of this gene relative to genetically linked DNA probes from the Psp, Emv-15, and Src loci. Using a combination of pulsed-field gel techniques that include partial digestion with rare-cutting restriction enzymes and analysis of polymorphic sites present in certain inbred strains, we have constructed long-range restriction maps for each of the probes that span a total of more than 3000 kb. The Src and Emv-15 probes are less than 600 kb apart, but are separated from the Psp probe by at least 1500 kb. By determining the position of a 75-kb deletion that inactivates agouti function, we have localized the A locus to within 500 kb of the Psp probe, but more than 600 kb away from the Emv-15 probe. These physical distances contrast with the known recombination frequencies, 3 +/- 3 cM for A-Psp and less than 0.3 cM for A-Emv-15, and suggest that recombination between A and Emv-15 may be suppressed.     

Synteny on mouse chromosome 5 of homologs for human DNA loci linked to the Huntington disease gene

Comparative mapping in man and mouse has revealed frequent conservation of chromosomal segments, offering a potential approach to human disease genes via their murine homologs. Using DNA markers near the Huntington disease gene on the short arm of chromosome 4, we defined a conserved linkage group on mouse chromosome 5. Linkage analyses using recombinant inbred strains, a standard outcross, and an interspecific backcross were used to assign homologs for five human loci, D4S43, D4S62, QDPR, D4S76, and D4S80, to chromosome 5 and to determine their relationships with previously mapped markers for this autosome. The relative order of the conserved loci was preserved in a linkage group that spanned 13% recombination in the interspecific backcross analysis. The most proximal of the conserved markers on the mouse map, D4S43h, showed no recombination with Emv-1, an endogenous ecotropic virus, in 84 outcross progeny and 19 recombinant inbred strains. Hx, a dominant mutation that causes deformities in limb development, maps approximately 2 cM proximal to Emv-1. Since the human D4S43 locus is less than 1 cM proximal to HD near the telomere of chromosome 4, the murine counterpart of the HD gene might lie between Hx and Emv-1 or D4S43h. Cloning of the region between these markers could generate new probes for conserved human sequences in the vicinity of the HD gene or possibly candidates for the murine counterpart of this human disease locus.     

Genetic background affects integration frequency of ecotropic proviral sequences into the mouse germ line.

Germ line acquisition of ecotropic proviruses occurs at a high frequency in the progeny of SWR/J-RF/J hybrid mice carrying two genetically linked RF/J ecotropic proviral loci, Emv-16 and Emv-17 (N. A. Jenkins and N. G. Copeland, Cell 43:811-819, 1985). To determine if genetic background affects proviral integration frequency, I analyzed a series of crosses in which the two RF/J proviral loci were transferred onto different provirus-negative background strains. Unlike SWR/J-RF/J hybrid progeny, few CBA/CaJ-RF/J hybrid mice were identified that carried new germ line proviral loci. These results indicate that genetic factors other than the linked RF/J proviral loci contribute to the increased frequency of germ line provirus integration seen in the SWR/J-RF/J hybrids. The frequency of proviral acquisition appeared to increase when females carrying Emv-16, Emv-17, and at least one new proviral locus were further backcrossed, suggesting that integration frequency can be increased by genetic manipulation. The breeding data are consistent with the hypothesis that virus from the mother infects the egg or the early embryo. Analysis of the transmission frequency and cosegregation patterns of new proviral loci indicated that viral integration occurs after the first round of DNA replication and before the germ line is set aside during embryogenesis, with a majority of viral integrations occurring at the two-cell stage of development, and independent viral integrations can occur in the same or in different cells of the embryo.     

Sequence and Insertion Sites of Murine Melanoma-Associated Retrovirus

We previously showed that B16 melanoma cells produce ecotropic melanoma-associated retrovirus (MelARV) which encodes a melanoma-associated antigen recognized by MM2-9B6 monoclonal antibody. The biological significance of MelARV in melanoma formation remains unknown. We found that infection of normal melanocytes with MelARV resulted in malignant transformation. It is likely that MelARV emerged from the defective Emv-2 provirus, a single copy of ecotropic provirus existing in the genome of C57BL/6 mice. In the present study, we cloned and sequenced the full-length MelARV genome and its insertion sites and we completed sequencing of the Emv-2 provirus. Our data show that MelARV has a typical full-length retroviral genome with high homology (98.54%) to Emv-2, indicating a close relationship between both viruses. MelARV probably emerged as a result of recombination between Emv-2 and an endogenous nonecotropic provirus. Some observed differences in the gag and pol regions of MelARV might account for the restoration of productivity and infectivity of a novel retrovirus that somatically emerged during melanoma formation. MelARV does not contain any oncogene and therefore might induce transformation by insertional mutagenesis. We sequenced two insertion sites of MelARV. The first insertion site represents the 3' coding region of the c-maf proto-oncogene at 67.0 centimorgans (cM) on chromosome 8. The c-maf proto-oncogene encodes a basic leucine zipper protein homologous to c-fos and c-jun. Insertion of MelARV in BL6 melanoma cells resulted in the up-regulation of c-maf. It is noteworthy that the Emv-2 provirus is also inserted into a noncoding region at 61.0 cM on the same chromosome 8. The second insertion site is the 3' noncoding region of the DNA polymerase gamma (PolG) gene on chromosome 7. The expression of PolG was not affected by the MelARV insertion. Further investigation of the biological significance of MelARV in melanoma formation is being undertaken.     

Lack of ecotropic virus involvement in induction of lymphomas in DBA/2J mice by 7,12-dimethylbenz(a)anthracene.

DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus, Emv-3, that is replication defective because of a single nucleotide substitution in codon 3 of p15gag. However, when weanling DBA/2 mice are treated percutaneously with 7,12-dimethylbenz(a)anthracene (DMBA), ecotropic virus replication is induced in almost all of the treated mice. Previous studies have shown that this induction results from DMBA-induced reverse mutations in codon 3 that allow efficient virus replication. In addition to ecotropic virus replication, DMBA also induces lymphomas in 100% of the treated mice. These results have raised the possibility that ecotropic virus replication is causally associated with the development of lymphomas in DBA/2 mice, perhaps via the insertional activation or mutation of cellular proto-oncogenes. To test this possibility, we compared lymphoma incidence after percutaneous DMBA treatment in DBA/2J-dv/dv mice, which carry two copies of Emv-3, with lymphoma incidence in DBA/2J-d+18J/d+18J mice, which lost both copies of Emv-3 by homologous recombination involving the long terminal repeat sequences. The results of this study conclusively demonstrated that Emv-3 is not causally associated with the development of DMBA-induced lymphomas in DBA/2J mice. Interestingly, histopathological and molecular analyses of the lymphomas indicated that the majority of the lymphomas in both strains of mice were of the B-cell lineage. This was unanticipated, since the majority of chemically induced lymphomas in other inbred strains are thymic lymphomas, presumably of the T-cell lineage. Thus, DBA/2 mice appear to present a unique model system for the investigation of chemically induced B-cell lymphomas in mice.     

Recombinant Inbred Strain and Interspecific Backcross Analysis of Molecular Markers Flanking the Murine Agouti Coat Color Locus

Recombinant inbred strain and interspecific backcross mice were used to create a molecular genetic linkage map of the distal portion of mouse chromosome 2. The orientation and distance of the Ada, Emv-13, Emv-15, Hck-1, Il-1a, Pck-1, Psp, Src-1 and Svp-1 loci from the beta 2-microglobulin locus and the agouti locus were established. Our mapping results have provided the identification of molecular markers both proximal and distal to the agouti locus. The recombinants obtained provide valuable resources for determining the direction of chromosome walking experiments designed to clone sequences at the agouti locus. Comparisons between the mouse and human genome maps suggest that the human homolog of the agouti locus resides on human chromosome 20q. Three loci not present on mouse chromosome 2 were also identified and were provisionally named Psp-2, Hck-2 and Hck-3. The Psp-2 locus maps to mouse chromosome 14. The Hck-2 locus maps near the centromere of mouse chromosome 4 and may identify the Lyn locus. The Hck-3 locus maps near the distal end of mouse chromosome 4 and may identify the Lck locus.