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Rat thymic lymphomas induced by Moloney murine leukemia virus carry DNA rearrangements due to provirus integration in at least five independent cellular DNA domains (Mlvi-1, Mlvi-2, Mlvi-3, RMoInt-1, and c-myc). We had previously shown that rearrangements in more than one of these domains could occur in the same tumor. In this report we extend these findings by showing that, with one exception, tumors containing provirus insertions in Mlvi-1 always contained provirus insertions in a second locus, Mlvi-2. To determine whether both events occurred in the same population of tumor cells, we examined the clonal nature of these tumors by taking advantage of allelic polymorphisms that occur naturally in both Mlvi-1 and Mlvi-2. Tumors with provirus insertions in both Mlvi-1 and Mlvi-2 arising in rats heterozygous at one of these loci were identified. DNA from these tumors was analyzed by restriction endonuclease digestion and hybridization to DNA probes derived from both Mlvi-1 and Mlvi-2. Thus, we determined the clonal nature of three thymomas and showed that in these tumors both insertion events occurred in the same population of tumor cells. The concomitant appearance of provirus insertions in Mlvi-1 and Mlvi-2 suggests a synergism of these two events that may be important in tumor induction and progression.

The induction of thymic lymphomas by Moloney murine leukemia virus in the rat is linked to provirus integration in at least four independent cellular DNA regions (Mlvi-1, Mlvi-2, Mlvi-3, and c-myc). Because sequences homologous to at least three of these regions (Mlvi-1, Mlvi-2, and c-myc) map to chromosome 15 in the mouse, the question was raised whether they are closely linked in the rat genome and whether provirus integration in any one of these regions affects the same functional domain in rat DNA. In this study, we identified the chromosomal map location of Mlvi-1, Mlvi-2, and Mlvi-3 in the rat by using mouse-rat somatic cell hybrids that lose the rat chromosomes. The results showed that Mlvi-1 maps similarly to c-myc to chromosome 7, and Mlvi-2 maps to chromosome 2. Mlvi-3 probably maps to chromosome 15. We conclude that Mlvi-1, Mlvi-2, and Mlvi-3 are separate and independent genetic loci. Although Mlvi-1 and c-myc map to the same chromosome, they are not related, as determined by hybridization and restriction endonuclease mapping. The chromosomal map location of Mlvi-1 to chromosome 7 and Mlvi-2 to chromosome 2 is interesting, since chromosomal aberrations involving these two chromosomes are reproducibly observed in rat neoplasias induced by a variety of agents.

Avian leukosis viruses (ALVs) of endogenous origin do not appear to cause disease. These viruses have similar genetic compositions to ALVs of exogenous origin which cause non-acute disease. The major genetic differences between endogenous and exogenous ALVs are found in their envelope genes and c regions. Envelope genes of endogenous origin code for subgroup E antigens ( env E) whereas envelope genes of exogenous origin code for subgroups A, B, C, or D antigens ( env A), etc.). Endogenous viruses have characteristic constant regions c n, that are distinct from those of exogenous viruses, c x. We are using exogenous viruses, endogenous viruses and recombinants of exogenous and endogenous viruses to test for the role of envelope antigens and c regions in disease. Four cloned env E c x viruses (RAV-60s) have been compared for their oncogenic activity with a env A c x virus (RAV-1) and a env E c n virus (RAV-0). The RAV-60s caused the same incidence of disease as RAV-1. Individual RAV-1 and RAV-60 infected chickens developed different diseases. The most frequent disease was B cell lymphomas. Anemias, a fibrosarcoma, and osteopetrosis also occurred. These results suggest that c regions, not envelope antigens, are critical for the development of disease. They also demonstrate that cloned avian leukosis viruses cause more than one disease.

In this repot we review the evidence that synthesis and processing of Pr76 gag , the precursor to the core proteins (products of the gag gene) of avian retroviruses, is both necessary and sufficient to produce a virus particle. In addition, we describe the results of two-dimensional polyacrylamide gel electrophoretic analysis of Pr76 gag and its cleavage products. When the two-dimensional gel system is used in conjunction with protein blotting and antibody detection on nitrocellulose sheets, we observe multiply-charged species of both precursor and product proteins. Comparison with 32 PO 4 and 35 S-methionine labelled proteins indicates that much of the heterogeneity is probably due to phosphorylation. We also report results of an investigation into the site of synthesis of Pr76 gag . Immunoprecipitation across sucrose gradients containing polyribosomes derived from cytoplasmic and membrane fractions of infected cells indicates that Pr76 gag is found exclusively on cytoplasmic polyribosomes. Both Pr76 gag and, what we presume to be related incomplete nascent polypeptide chains, are localized in the 450S region of the gradient and are released by treatment with EDTA. Finally, we describe studies on a deletion mutant which, while failing to produce either reverse transcriptase or envelope glycoprotein, synthesizes pp60 src and a 63,000 dalton protein (P63) related to retrovirus core proteins. P63 contains an approximately 13,000 dalton internal deletion (in p27) as determined by tryptic mapping and analysis of products of in vitro cleavage by the gag-specific virion protease. The deletion does not appear to extend into the region of the p15 cleavage protease. P63 is resistant to normal processing and no product proteins are detectable even in cells superinfected with non-defective viruses. We conclude that the internal deletion in P63 has rendered this protein defective for processing either by inducing a conformational change, removal of a cleavage site, or both.

The Mlvi-1/mis-1/pvt-1 locus, located approximately 270 kilobase pairs 3' of the c-myc protooncogene, was originally discovered as a common region of provirus integration in Moloney murine leukemia virus-induced rat T-cell lymphomas. The same locus was shown subsequently to be coamplified with c-myc and to be involved in chromosomal translocations in a variety of human and animal neoplasms. Provirus integration in Mlvi-1 in Moloney murine leukemia virus-induced rat T-cell lymphomas activates the c-myc protooncogene. The studies reported here were aimed to determine whether, in addition to the activation of c-myc, provirus integration affected the expression of other neighboring genes. Provirus integration was shown to occur in three clusters separated by regions of uninterrupted DNA. The proviruses in all three clusters had integrated in a single-transcriptional orientation, and they appeared intact. Systematic hybridization of Mlvi-1 clones to rat, mouse, and human genomic DNA revealed three patches of evolutionarily conserved sequences. Two of them were mapped in regions targeted by the provirus, and the third was mapped immediately 5' to the provirus clusters. A probe derived from the conserved sequences 5' of the integrated proviruses detected a tumor-specific RNA transcript in tumors carrying a provirus in Mlvi-1 or in the neighboring Mlvi-4 and c-myc loci. The highest level of RNA transcript expression, however, was seen in a CD4+ CD8+ tumor cell line that was not carrying a provirus in this region. We conclude that provirus insertion in this region activates both c-myc and another gene that is located in the immediate vicinity of the integrated Mlvi-1 proviruses and may be developmentally regulated in T cells.

Similarly to other mammalian and avian retroviruses that lack a transforming gene1, moloney murine leukaemia virus (MoMuLV) causes no morphological transformation in infected tissue culture cells. However, following injection in an appropriate animal host, MoMuLV induces mainly thymic lymphomas after a long latency period2,3. A common characteristic of neoplasms induced by retroviruses lacking transforming genes is their clonal origin4–9. Here we have generated MoMuLV-induced rat thymic lymphomas and confirmed their clonal nature. Furthermore, we took advantage of the clonality of these tumours to investigate the specificity of provirus integration in the tumour DNA. We reasoned that if several independently derived thymic lymphomas would contain the provirus integrated in the same region of cellular DNA, this would be a strong indication that this integration event is a contributing factor in oncogenesis. The results indicate that there is indeed a cellular DNA region (termed the ML VI-1 locus) that serves as the substrate for proviral DNA integration in 5 out of 16 tumours we examined.

Avian myelocytomatosis virus (MC29), a defective acute leukemia virus, has a broad oncogenic spectrum in vivo, and transforms fibroblasts and hematopoietic target cells in vitro. We have used recombinant DNA technology to isolate and characterize the sequences that are essential in the transformation process. Integrated MC29 proviral DNA was isolated from a library of recombinant phage containing DNA from the MC29-transformed nonproducer quail cell line Q5. The cloned DNA was analyzed by Southern blotting of restriction endonuclease digests and by electron microscopic visualization of R-loops formed between the cloned DNA and MC29 or helper virus RNA. It was found that the 9.2 kb cloned DNA insert contains approximately 4 kb of viral sequences and 5.2 kb of quail cellular sequences. The viral sequences contain all of the MC29-specific sequences and 5′ helper related sequences as well as part of the envelope region. The size of the cloned EcoRI fragment is the same as that of the major band in EcoRI-cleaved Q5 DNA that hybridizes to viral sequences. Transfection of the cloned DNA into NIH 3T3 cells revealed that the MC29-specific sequences are functional in that they induce foci of trans-formed cells with high efficiency.

Avian myelocytomatosis virus (MC29), a defective acute leukemia virus, has a broad oncogenic spectrum in vivo and transforms fibroblasts and hematopoietic target cells in vitro. We have used recombinant DNA technology to isolate and to characterize the sequences that are essential in the transformation process. Integrated MC29 proviral DNA was isolated from a library of recombinant phage containing DNA from the MC29-transformed nonproducer quail cell line Q5. The cloned DNA was analyzed by Southern blotting of restriction endonuclease digests and by electron microscopic visualization of R loops formed between the cloned DNA and MC29 or helper virus RNA. It was found that the 9.2-kilobase cloned DNA insert contains approximately 4 kilobases of viral sequences and 5.2 kilobases of quail cellular sequences. The viral sequences contain all of the MC29-specific sequences and 5' helper-related sequences as well as part of the envelope region. The size of the cloned EcoRI fragment is the same as that of the major band in EcoRI-cleaved Q5 DNA that hybridizes to viral sequences. Transfection of the cloned DNA into NIH 3T3 cells revealed that the MC29-specific sequences are functional in that they induce foci of transformed cells with high efficiency.

Mlvi-1 defines a locus of proviral integration in rat thymomas induced by Moloney murine leukemia virus. pvt-1/Mis-1 represents an independently identified locus which becomes rearranged either by chromosomal translocation in murine plasmacytomas or by provirus insertion in retrovirus-induced murine and rat thymic lymphomas. Although it had been claimed that pvt-1/Mis-1 and Mlvi-1 represent two different loci, we present here evidence showing that they are identical. This finding demonstrates the need for rigorous characterization of any newly identified common regions of integration in retrovirus-induced neoplasms.

TheGfi-1proto-oncogeneencodesazincfingerproteinwithsixC2H2-type,C-terminalzincfingermotifsand isactivatedbyprovirusintegrationinT-celllymphomalinesselectedforinterleukin-2independenceinculture and in primary retrovirus-induced thymomas. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis and is enhanced in mitogen-stimulated splenocytes. In this report, we show that Gfi-1 is a 55-kDa nuclear protein that binds DNA in a sequence-specific manner. The Gfi-1 binding site, TAAATCAC(A/ T)GCA, was defined via random oligonucleotide selection utilizing a bacterially expressed glutathioneS-transferase‐Gfi-1 fusion protein. Binding to this site was confirmed by electrophoretic mobility shift assays and DNase I footprinting. Methylation interference analysis and electrophoretic mobility shift assays with mutant oligonucleotides defined the relative importance of specific bases at the consensus binding site. Deletion of individual zincfingers demonstrated that only zincfingers 3, 4, and 5 are required for sequence-specific DNA binding. Potential Gfi-1 binding sites were detected in a large number of eukaryotic promoter-enhancers, including the enhancers of several proto-oncogenes and cytokine genes and the enhancer of the human cytomegalovirus (HCMV) major immediate-early promoter, which contains two such sites. HCMV major immediate-early‐chloramphenicol acetyltransferase reporter constructs, transfected into NIH 3T3fibroblasts, were repressed by Gfi-1, and the repression was abrogated by mutation of critical residues in the two Gfi-1 binding sites. These results suggest that Gfi-1 may play a role in HCMV biology and may contribute to oncogenesis and T-cell activation by repressing the expression of genes that inhibit these processes. Stimulation of normal T cells by a variety of mitogenic signals induces the expression of interleukin-2 (IL-2) and IL-2 receptor alpha. Expression of these molecules takes place within 2 to5hofthestart of the stimulation and leads to the expression of additional cytokines which induce the complex interplay of growth and differentiation events characteristic of the process of T-cell activation (reviewed in reference 10). To study the genetic regulation of the early steps of this process, we adopted an insertional-mutagenesis-based genetic strategy which was designed to identify genes contributing to the progression of IL-2-dependent T-cell lymphoma lines to IL-2independent growth. We reasoned that genes involved in the phenotypic progression of these cell lines to IL-2 independence would play a role either in the induction of IL-2 expression or in IL-2 signaling (16). The growth factor independence 1 (Gfi-1) gene cloned by using this strategy encodes a zincfinger protein with six C2H2type C-terminal zinc finger motifs. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis. In mitogen-stimulated spleen cells, its expression begins to rise approximately 12 h after the start of the stimulation, suggesting that it is unlikely to be involved in the induction of IL-2 expression during T-cell activation. In agreement with this,Gfi-1 failed to induce the expression of IL-2 when introduced into the IL-2-dependent cell line 2780d, which gave rise to the IL-2-independent 2780i-5 cells from which it was cloned (16). Gfi-1 not only is involved in the selection for IL-2 independence but also contributes to the induction and progression of

We report the isolation and sequence of a cDNA clone containing part of the env message of the Moloney murine leukemia virus (MoMuLV). This clone was derived from a rat thymic lymphoma induced by MoMuLV. The AG acceptor site employed in this message is located at position 5490 in the MoMuLV genome. This splice site is detectable at the cDNA level by the creation of a novel SacI restriction site not present in the viral genome. In the -1 to -40 region, this AG acceptor site is preceded by four conserved heptanucleotides (PyXPyTPuAPy) that may function as acceptors for removal of the 5' end of the intron.