Genetic mapping of the mouse ferritin light chain gene and 11 pseudogenes on 11 mouse chromosomes.

We typed the progeny of two sets of genetic crosses to determine the map locations for loci containing sequences related to the ferritin light chain (Ft11) gene. Twelve loci were positioned on 11 different chromosomes. One of these genes mapped to a position on Chr 7 predicted to contain the expressed gene on the basis of the previously determined position of the human homolog on 19q13.3-q13.4.

An array of murine leukemia virus-related elements is transmitted and expressed in a primate recipient of retroviral gene transfer.

Direct RNA-PCR analyses of T-cell lymphomas that developed in rhesus macaques during a gene transfer experiment revealed the presence of several different recombinant murine leukemia viruses (MuLV). Most prominent was the expected MuLV recombinant, designated MoLTRAmphoenv in which the amphotropic env of the helper packaging virus was joined to the long terminal repeat (LTR) of the Moloney MuLV-derived vector. This retrovirus does not exist in nature. An additional copy of the core enhancer acquired from the vector LTR may have augmented the replicative properties of MoLTRAmphoenv MuLV in several different rhesus cell types compared with the prototype amphotropic MuLV4070A. Unexpectedly, at least two types of mink cell focus-forming MuLV elements, arising from endogenous retroviral sequences expressed in the murine packaging cell line, were also transmitted and highly expressed in one of the macaques. Furthermore, murine virus-like VL-30 sequences were detected in the rhesus lymphomas, but these were not transcribed into RNA. The unanticipated presence of an array of MuLV-related structures in a primate gene transfer recipient demands ever-vigilant scrutiny for the existence of transmissible retroviral elements and replication-competent viruses possessing altered tropic or growth properties in packaging cells producing retroviral vectors.

Genomic cloning of mouse MIF (macrophage inhibitory factor) and genetic mapping of the human and mouse expressed gene and nine mouse pseudogenes.

The single functional mouse gene for MIF (macrophage migration inhibitory factor) has been cloned from a P1 library, and its exon/intron structure determined and shown to resemble that of the human gene. The gene was mapped to chromosome 10 using two multilocus crosses between laboratory strains and either Mus musculus musculus or Mus spretus. Nine additional loci containing related sequences, apparently all processed pseudogenes, were also mapped to chromosomes 1, 2, 3, 7, 8, 9, 12, 17, and 19. While most of these pseudogenes were found in inbred mice and M. spretus, some are species specific. This suggests that there have been active phases of pseudogene formation in Mus both before and after the separation of musculus and spretus. The human genome contains no pseudogenes; we assigned the human gene to chromosome 19, consistent with the location of mouse and human functional genes for MIF in a region of conserved linkage.

A highly divergent simian immunodeficiency virus (SIVstm) recovered from stored stump-tailed macaque tissues.

We report here the results of molecular analysis of a simian immunodeficiency virus (designated SIVstm) which was isolated from a rhesus monkey inoculated with stored lymph node tissue of an Asian stump-tailed macaque. The latter monkey had died in 1977 during an epidemic of acquired immunodeficiency and lymphoma at the California Regional Primate Research Center (L. J. Lowenstine, N. W. Lerche, P. A. Marx, M. B. Gardner, and N. C. Pedersen, p. 174-176, in M. Girard and L. Valette, ed., Retroviruses of Human AIDS and Related Animal Viruses, 1988). Nucleotide sequence analysis of the gag and env regions indicates that SIVstm is an ancient member of the SIV/human immunodeficiency virus type 2 group; it is quite divergent from known SIVs isolated from African sooty mangabeys as well as from Asian macaques. Furthermore, of all SIV strains described to date, SIVstm is the most closely related to human immunodeficiency virus type 2.

Genetic mapping of the mouse c-fms proto-oncogene to chromosome 18.

Chinese hamster X mouse somatic cell hybrids were analyzed by Southern blot hybridization with a probe specific for the cellular c-fms proto-oncogene. Results demonstrate that Fms, the genetic locus containing this sequence, maps to mouse chromosome 18. Mouse Fms is thus not linked to the same set of genes involved in growth regulation that human FMS is linked to.

A preferred region for integration of Friend murine leukemia virus in hematopoietic neoplasms is closely linked to the Int-2 oncogene.

Gene mapping experiments show that Fis-1, a preferred integration region for Friend murine leukemia virus in hematopoietic neoplasms, is extremely closely linked to Int-2, a preferred integration region for mouse mammary tumor virus in mammary carcinomas. Studies at the RNA and DNA level prove that these loci are distinct.

Identification of conserved and divergent domains within the envelope gene of the acquired immunodeficiency syndrome retrovirus.

The nucleotide sequences of the envelope genes of an African and a North American acquired immunodeficiency syndrome (AIDS) viral isolate have been determined. When their deduced amino acid sequences were aligned with the envelopes of the lymphoadenopathy and AIDS-associated retrovirus isolates, conserved and divergent regions were readily identified. Hypervariable stretches of 28-74 amino acids, exhibiting 20-30% amino acid identity at each position and characterized by reciprocal insertions and deletions, were confined to the gp120 external envelope protein. The origin and clinical implications of the AIDS viral env gene diversity are discussed.

Statistical considerations for linkage analysis using recombinant inbred strains and backcrosses.

Recombinant inbred (RI) mouse strains are extremely useful for gene mapping, especially for establishing preliminary map positions for new loci. However, the usual statistical analysis applied to such experiments may lead to erroneous conclusions about linkage unless unusually stringent criteria are adopted for rejecting the null hypothesis. We describe a Bayesian statistical approach for determining the probability of linkage when no prior information is available about the location of the gene to be mapped (the test locus). We present a table that gives the probability of linkage, the most likely position of the test locus with respect to a marker locus, and the interval around the marker locus that has a 95% chance of containing the test locus, for all possible experimental results suggesting linkage in sets of up to 40 RI strains. These results show that for the probability of linkage to be greater than 95%, the number of RI strains inheriting chromosomes recombinant for the test and marker loci must be smaller than previously assumed. The formulas derived for RI strains can be applied, with only minor modifications, to the analysis of Mendelian backcrosses. Differences between the Bayesian approach advocated here and the more traditional analysis of linkage are discussed in detail.

Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus.

An infectious NZB xenotropic murine leukemia virus (MuLV) provirus (NZB was molecularly cloned from the Hirt supernatant of NZB-IU-6-infected mink cells, and the nucleotide sequence of its env gene and long terminal repeat (LTR) was determined. The partial nucleotide sequence previously reported for the env gene of NFS-Th-1 xenotropic proviral DNA (Repaske, et al., J. Virol. 46:204-211, 1983) is identical to that of the infectious NZB xenotropic MuLV DNA reported here. Alignment of nucleotide or deduced amino acid sequences, or both, of xenotropic, mink cell focus-forming, and ecotropic MuLV proviral DNAs in the env region identified sequence differences among the three host range classes of C-type MuLVs. Major differences were confined to the 5' half of env; a high degree of homology was found among the three classes of MuLVs in the 3' half of env. Alignment of the nucleotide sequence of the LTR of NZB xenotropic MuLV with those of the LTRs of NFS-Th-1 xenotropic, mink cell focus-forming, and ecotropic MuLVs revealed extensive homology between the LTRs of xenotropic and MCF247 MuLVs. An inserted 6-base-pair repeat 5' to the TATA box was a unique feature of both NZB and NFS-Th-1 xenotropic LTRs.

A unique sequence related to the ecotropic murine leukemia virus is associated with the Fv-4 resistance gene.

Several strains of laboratory and wild-derived mice from Japan carry the dominant allele at the Fv-4 locus (Fv-4r) that is responsible for resistance to infection by exogenous ecotropic murine leukemia virus. We have used blot hybridization with a probe specific for the ecotropic viral envelope to show that a unique envelope-reactive sequence is present in the Japanese mouse Mus musculus molossinus and in four independently derived partially congeneic strains carrying Fv-4r. Analysis of 31 backcross mice shows complete concordance between inheritance of this fragment and resistance to Friend virus complex-induced erythroblastosis. Inheritance of this sequence also suppresses spontaneous expression of the endogenous ecotropic viruses carried by M. m. molossinus. Restriction enzyme analysis shows that the Fv-4r-associated sequence is different from the full-length ecotropic proviruses of laboratory mice. Infectious virus cannot be induced from mice carrying only the Fv-4r-associated sequence. Examination of other wild-derived mice resistant to Friend virus complex shows that Mus cervicolor cervicolor also contains the Fv-4r sequence. Our data indicate that a unique or incomplete provirus containing ecotropic envelope-related sequences is responsible for Fv-4-mediated resistance to both exogenous and endogenous ecotropic virus in various Asian mice.

Relationship between a retroviral germ line reintegration and a new mutation at the ashen locus in B10.F mice. Retroviral integration and an ashen mutation.

In a litter of B10.F mice a single mouse was found with a "dilute-like" coat-color mutation. F2 matings were then carried out to establish sufficient numbers of mice carrying this mutation in a homozygous form. The coat-color mutation was found to be allelic to the ashen locus which is closely linked to the dilute locus on chromosome 9. Further analysis of the ashen mice showed they contained an additional ecotropic provirus integrated into their chromosomal DNA. This result initially suggested that retroviral integration was responsible for the mutation. However, backcross studies showed the mutation and provirus were genetically unlinked. The reintegrated provirus was molecularly cloned and transfection studies showed the virus was B tropic in host range. The restriction map of the cloned provirus was found to be similar to previously described B-tropic viruses. The results are discussed in view of the low probability of two rare and unlinked events occurring in a single mouse.

Cas spleen focus-forming virus. II. Further biological and biochemical characterization.

A new isolate of a murine erythroblastosis-inducing spleen focus-forming virus (Cas SFFV), derived from the wild mouse ecotropic murine leukemia virus Cas-Br-M, was further characterized after the production of a nonproducer cell line. When rescued from the nonproducer cells with a helper murine leukemia virus, the Cas SFFV induced rapid splenic enlargement and focus formation when inoculated into adult NFS/N mice. The Cas SFFV nonproducer cell line was also utilized to compare the envelope-related glycoprotein of Cas SFFV with gp52s from three strains of Friend SFFV. Cas SFFV was found to encode a 50,500-dalton glycoprotein (gp50) distinct in size to the envelope-related glycoproteins of the Friend SFFVs. The Cas SFFV was also compared in RNA blot hybridization studies. The genomic viral RNA of Cas SFFV was found to be slightly larger than two polycythemia-inducing strains of Friend SFFV and markedly larger than the anemia-inducing strain. Further comparisons between the SFFVs were made by examining their transforming capabilities in an in vitro erythroid burst assay. The erythroid bursts induced by Cas SFFV and the anemia-inducing strain of Friend SFFV showed similarities in their erythropoietin requirements. This study supports our recent observations that Cas SFFV is biologically similar to the anemia-inducing strain of Friend SFFV yet biochemically distinct from all Friend SFFVs.

Identification of a spleen focus-forming virus in erythroleukemic mice infected with a wild-mouse ecotropic murine leukemia virus.

An NFS/N mouse inoculated at birth with an ecotropic murine leukemia virus (MuLV) obtained from wild mice (Cas-Br-M MuLV) developed a lymphoma after 18 weeks. An extract prepared from the lymphomatous spleen was inoculated into newborn NFS/N mice, and these mice developed erythroleukemia within 9 weeks. Spleens from the erythroleukemic mice contained ecotropic and mink cell focus-inducing (MCF) MuLVs; however, when these viruses were biologically cloned and reinoculated into newborn NFS/N mice, no erythroleukemia was induced. In contrast, cell-free extracts prepared from the erythroleukemic spleens induced erythroleukemia within 5 weeks. Analysis of cell-free extracts prepared from the erythroleukemic spleens showed that they contained a viral species that induced splenomegaly and spleen focus formation in adult mice, with susceptibility controlled by alleles at the Fv-2 locus. The spleen focus-forming virus coded for a 50,000-dalton protein precipitated by antibodies specific to MCF virus gp70. RNA blot hybridization studies showed the genomic viral RNA to be 7.5 kilobases and to hybridize strongly to a xenotropic or MCF envelope-specific probe but not to hybridize with an ecotropic virus envelope-specific probe. The virus described here appears to be the fourth independent isolate of a MuLV with spleen focus-forming activity.

Organization and stability of endogenous xenotropic murine leukemia virus proviral DNA in mouse genomes.

In a series of blot hybridization experiments, using a xenotropic envelope probe and restriction enzymes known to cut xenotropic proviral DNA a single time (EcoRI) or not at all (HindIII), we have studied the organization and relationship of endogenous xenotropic env-related sequences in various mouse strains. Multiple copies (18 to 28) of xenotropic env-reactive fragments were found in all mouse DNAs after digestion with either HindIII or EcoRI, and the majority of fragments were of sizes compatible with their origin from full-length proviral DNA. Five HindIII and five EcoRI restriction fragments were common to all inbred mouse DNAs tested. In addition, each strain exhibited unique characteristic xenotropic env-reactive bands; these bands were remarkably stable during many years of inbreeding. The cleavage patterns characteristic of each strain were also useful for showing genealogical relatedness among the various inbred mice.

Variation in the number of copies and in the genomic organization of ecotropic murine leukemia virus proviral sequences in sublines of AKR mice.

DNAs isolated from individual mice of four AKR sublines (AKR/J, AKR/N, AKR/Cum, and AKR/Boy) were examined by hybridization of electrophoretically separated restriction enzyme fragments to a 500-base pair, 32P-labeled probe specific for env sequences of ecotropic murine leukemia virus. Variation in the number of proviral DNA copies and in their genomic organization, as reflected by the location of restriction enzyme sites in flanking cellular sequences, was observed both between and within AKR sublines. Evidence is presented for the continual acquisition of new proviruses in the four sublines studied. The ecotropic proviral DNA copies present in the four AKR sublines can be related to their genealogy; each subline contains two or three copies of proviral DNA in common with other sublines and from one to six unique ecotropic proviruses. Overall, a new copy appears about every 12 generations of inbreeding. Some of the unique proviral DNA copies contain internal alterations, as reflected by restriction enzyme maps that differ from those of prototype ecotropic proviruses.

Internal organization of endogenous proviral DNAs of xenotropic murine leukemia viruses.

The internal organization of endogenous xenotropic murine leukemia virus proviruses was determined in a series of blot hybridization experiments in which DNA from several different inbred mouse strains, digested with restriction enzymes known to cleave xenotropic proviral DNAs at least twice, was annealed to generalized murine leukemia virus or xenotropic env-specific DNA probes. Comigrating bands of variable intensity which hybridized to the xenotropic env probe were identified in all inbred mouse DNA preparations. At least seven classes of endogenous xenotropic proviral DNA with respect to SacI cleavage maps were detected in mouse DNA. Two of the seven classes were indistinguishable from proviruses associated with known infectious xenotropic murine leukemia viruses. These results are consistent with the existence of related but organizationally distinct families of endogenous xenotropic proviral DNA that are present in different relative abundances in mouse genomic DNA.

Cloning and characterization of an envelope-specific probe from xenotropic murine leukemia proviral DNA.

An 8.9-kilobase EcoRI restriction fragment was cloned from mink cells chronically infected with NFS-Th-1 xenotropic murine leukemia virus by using a lambda phage host vector system. After its transfer into pBR322, the EcoRI DNA insert was characterized and found to contain 6.7 kilobases of proviral DNA sequences and 2.2 kilobases of mink cellular DNA flanking the 5' end of the viral genome. A 500-base pair fragment which was located at the 3' terminus of the cloned DNA insert and which mapped to the env region of xenotropic proviral DNA was subcloned into pBR322. This xenotropic envelope proviral DNA segment did not hybridize to ecotropic murine leukemia proviruses but did anneal to representative alpha and beta xenotropic and seven different mink cell focus-inducing proviral DNAs. The cloned xenotropic envelope-specific probe was also used in blot hybridization experiments to analyze the arrangement of related sequences in preparations of different mouse liver DNAs.