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The first constant region of the Tcrb gene was completely deleted from the DNA of 8/10 mouse cell lines established from 3-methylcholanthrene-induced RF/J thymic lymphomas, but 6/7 primary lymphomas contained the first constant region sequences. DNA from RF/J thymic lymphomas induced by N-methyl-N-nitrosourea was then examined serially as the tumors were passaged in vivo and adapted to growth in culture as uncloned and, in some cases, cloned lines. Patterns of Tcrb-specific restriction fragments from most tumors changed extensively during continued propagation. Analysis of the patterns often suggested that initial DNA rearrangements within the Tcrb complexes of monoclonal tumors had been followed by further rearrangements within the same genes. However, these different patterns may alternatively have represented successive outgrowth of separate lineages from lymphomas that were polyclonal in origin.

The leukemogenic membrane glycoprotein (gp55) encoded by Friend spleen focus-forming virus appears to bind to erythropoietin receptors (EpoR) sto stimulate erythroblastosis [Li, J.-P., D'Andrea, A.D., Lodish, H.F. & Baltimore, D. (1990) Nature (London) 343, 762-764]. To directly compare the effects of gp55 with erythropoietin (Epo), we produced retrovirions that encode either gp55, Epo, or EpoR. After infection with EpoR virus, interleukin 3-dependent DA-3 cells bound 125I-labeled Epo and grew without interleukin 3 in the presence of Epo. These latter cells, but not parental DA-3 cells, became factor-independent after superinfection either with Epo virus or with Friend spleen focus-forming virus. In addition, Epo virus caused a disease in mice that mimicked Friend erythroleukemia. Although Fv-2r homozygotes are susceptible to all other retroviral diseases, they are resistant to both Epo viral and Friend viral erythroleukemias. These results indicate that both gp55 and Epo stimulate EpoR and that the Fv-2 gene encodes a protein that controls response to these ligands. However, the Fv-2 protein is not EpoR because the corresponding genes map to opposite ends of mouse chromosome 9. These results have important implications for understanding signal transduction by EpoR and the role of host genetic variation in controlling susceptibility to an oncogenic protein.

In an initial attempt to test the ability of replication-defective retroviruses to immunize against immunologically related pathogenic viruses, we have worked with the erythroleukemogenic Friend retrovirus complex (FV), which consists of a replication-competent helper component, Friend murine leukemia virus (FMuLV), and a related defective pathogenic component, spleen focus-forming virus (SFFV). An 81-base-pair deletion was introduced into the p15E-encoding region of the env gene of an otherwise replication-competent molecular clone of the FMuLV provirus. After transfection of this clone into cells that package the viral RNA in MuLV coats, infectious virus was released into the culture medium. Mouse fibroblasts infected with this virus, here called delta FMuLV, expressed the truncated viral env gene products in their cytoplasm but not on cell surfaces, and culture fluids from these cells did not transmit the infection to fresh mouse fibroblasts. In preliminary experiments, immunization of mice of H-2-congenic BALB/c strains with delta FMuLV conferred levels of immunity to FV disease ranging from weak to relatively strong. Immunized mice developed anti-FV IgM and IgG antibodies and cytotoxic T cells. Mice observed for 15 weeks after the first of two immunizations showed no detectable pathology, but delta FMuLV DNA was detectable in livers of some immunized mice for at least 3-6 weeks. These results suggest that our approach to development of retrovirus vaccines may be a useful one.

Mice homozygous for the H-2d haplotype at the major histocompatibility complex are markedly more susceptible to erythroleukemia induction by the Friend isolate of murine leukemia retrovirus (FV) than are congenic mice homozygous for the H-2b haplotype. The resistance conferred by the H-2b haplotype is recessive in this cross, since heterozygous F1 mice are as susceptible as parental strain H-2d homozygotes. However, H-2b-associated resistance is not an intrinsically recessive trait, since H-2b/H-2dm1 heterozygotes resemble H-2b homozygotes in their relative resistance to FV; the mutant H-2dm1 haplotype lacks the entire D region of the parental haplotype except for a single class I gene formed by the fusion of its terminal D-region genes to produce a class I gene differing from both parental genes, and thus this finding indicates that one or more D-region genes of the H-2d haplotype can actively suppress H-2b-associated resistance. Unlike H-2dm1, the mutant H-2dm2 haplotype, which retains only the class IDd gene in the D region of the H-2d haplotype, strongly suppresses resistance in H-2b/H-2dm2 heterozygotes, and the presence of Dd as a transgene significantly reduces the resistance of H-2b homozygotes. Since H-2b-associated resistance to FV appears to be due mainly to the capacity of Lb (also called Db), the only class I molecule encoded in the D region of the H-2b haplotype, to present viral epitopes for recognition by FV-specific cytotoxic T lymphocytes, suppression of resistance to FV by the Dd molecule implies that the presence of one class I molecule of the major histocompatibility complex can interfere with either the presentation of viral epitopes by another class I molecule or the generation of T cells that recognize viral epitopes so presented.

3-Methylcholanthrene (MCA) skin painting rapidly induced a 100% incidence of thymic lymphomas in RF/J mice. Tumors developed exclusively in cells of the T-lymphocyte lineage and displayed the surface phenotype: Thy-1+, Lyt-1+, Lyt-2+, Qa-1+, H-2K+, H-2D+, TL-, Ig-, Ia-. This phenotype resembled that of cortisone-resistant, medullary thymocytes, whereas the phenotype of spontaneous AKR thymomas resembles that of cortisone-sensitive, cortical thymocytes. The phenotype differed very little from one tumor to another and was maintained during syngeneic passage in vivo and adaptation to growth in culture. Infectious ecotropic murine leukemia virus (MuLV) was produced in a minority of primary tumors and cell lines, but no xenotropic or mink cell focus-inducing virus (MCF) MuLV production was reliably detected. MuLV-related antigen expression on normal thymocytes increased in an age-dependent manner, and levels on thymoma cells were similar to those on thymocytes from age-matched normal controls. MuLV nonproducer tumor cells expressed few or no epitopes of AKR ecotropic gp70, but they were positive when tested with an antiserum prepared against MCFenv glycoprotein.

FVB/N mice offer a system suitable for most transgenic experiments and subsequent genetic analyses. The inbred FVB/N strain is characterized by vigorous reproductive performance and consistently large litters. Moreover, fertilized FVB/N eggs contain large and prominent pronuclei, which facilitate microinjection of DNA. The phenotype of large pronuclei in the zygote is a dominant trait associated with the FVB/N oocyte but not the FVB/N sperm. In experiments to generate transgenic mice, the same DNA constructs were injected into three different types of zygotes: FVB/N, C57BL/6J, and (C57BL/6J x SJL/J)F1. FVB/N zygotes survived well after injection, and transgenic animals were obtained with efficiencies similar to the F1 zygotes and much better than the C57BL/6J zygotes. Genetic markers of the FVB/N strain have been analyzed for 44 loci that cover 15 chromosomes and were compared with those of commonly used inbred strains. In addition to the albino FVB/N strain, pigmented congenic strains of FVB/N are being constructed. These features make the FVB/N strain advantageous to use for research with transgenic mice.

Mice of the RF/J strain are highly susceptible to induction of thymic lymphoma by skin painting with 3-methylcholanthrene (MCA), whereas mice of the 129/J and I/LnJ strains are resistant. Resistance was the dominant trait in F1 mice of crosses of RF with each resistant strain. Analysis of the lymphoma incidence in MCA-painted backcross populations indicated segregation of a single dominant gene for resistance in both crosses. None of these strains show inducibility of the aryl hydrocarbon hydroxylase enzyme system, a phenotype attributed to the dominant Ahb gene which is also known to influence susceptibility to MCA-induced lymphoma. The occurrence of the disease in these backcrosses was independent of the hosts' phenotype at either the H-2 or Fv-1 locus, both of which have shown an influence on susceptibility to murine leukemia virus-associated lymphoma in other experimental systems.

Expression of 11 cellular oncogenes was determined in normal vs. lymphomatous thymic tissues of RF and AKR mice; only c-myc and c-myb transcripts were detected in an age-inappropriate pattern in thymomas. Normal thymocytes from young RF mice contained RNA transcripts of both genes, but the transcripts were no longer detected at 9 or more weeks of age. More than 90% of RF thymomas, occurring at 20-28 weeks of age after skin painting with 3-methylcholanthrene at 12 weeks, contained c-myc transcripts, and 70% of the tumors contained c-myb transcripts. Seven cell lines derived from these 3-methylcholanthrene thymomas expressed both cellular genes, as did 2 rare spontaneous thymomas of 12-month-old RF mice. No indication of rearrangement or amplification of either gene was seen in any of the RF tumors or cell lines. In AKR mice, transcripts of the 2 genes persisted longer in the normal thymus than in RF mice, but they were no longer detected at 26 weeks of age. Of 3 thymomas in 6-month-old 3-methylcholanthrene-treated AKR mice, all expressed c-myb and 2 expressed c-myc. Among 11 spontaneous AKR thymomas, however, only 2 showed detectable levels of both genes, and 2 more expressed c-myc or c-myb but not both.

BALB/c mice injected with seven 0.15-ml samples of whole rabbit serum over a 2-week period developed nonneoplastic proliferation of the extrahepatic bile duct epithelium and glandular components. Sera from other animals, including bovines, humans, pigs, goats, and chickens as well as non-serum-containing secretions such as human breast milk and bile also produced this effect. Partial purification utilizing gel filtration and affinity chromatography of the active 33-65% saturated ammonium sulfate precipitate of whole serum indicated that the distribution and characteristics of this glycoprotein showed some similarities with those of IgA. Chromatographically purified human IgA was administered to BALB/c mice and was found to induce bile duct proliferation identical to that seen with whole human serum. Purified human IgG and IgM had no activity. Since IgA-containing serum from BALB/c mice was inactive, it appears that heterologous IgA functions as a specific extrahepatic bile duct growth factor (BDGF) in BALB/c mice. Murine susceptibility to the growth-stimulating effect of serum was strain specific; genetic studies utilizing crosses of susceptible (BALB/c) and resistant (C57BL/10) strains of mice revealed that the ability to respond to the infusion of BDGF is inherited in a polygenic fashion.