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A chimeric construct (VCD) consisting of parts from viral jun, chicken c-jun, and chicken junD was cloned into the replication-competent retroviral RCAS vector. This construct, RCAS-VCD, was found to have a higher focus forming potential in quail fibroblasts than the equivalent construct RCAS-VJ-1, expressing viral jun. DNAs from RCAS-VCD and RCAS-VJ-1 were transfected into primary quail embryo fibroblasts. Cells derived from one RCAS-VCD-induced focus survived cell crisis, which became manifest after 15 passages, and could be expanded into a long-term culture. This cell line, termed VCD, has been passaged for over 100 times so far. The cells grow to very high densities and then pile up into clumps of rounded cells. The culture releases a transforming virus with a titer of 105 FFU/ml, as tested on primary quail embryo fibroblasts. The transformed phenotype of VCD cells was verified by agar colony formation. VCD cells are capable of anchorage-independent growth with a cloning efficiency of 10%. Southern blot analysis of genomic DNA from VCD cells showed proviral integration of the RCAS construct without detectable rearrangements. Northern and Western blot analyses confirmed correct expression from integrated RCAS-VCD of predicted RNAs and of the chimeric Jun(VCD) protein. jun (VCD)-transformed cells provide a constant source of homogeneous cellular material for the investigation of the molecular mechanisms of jun -induced cell transformation and for the identification of direct and indirect targets of Jun protein function.

To investigate the molecular basis of oncogenesis induced by the v-jun oncogene of avian sarcoma virus 17 (ASV17), we developed a conditional cell transformation system in which transcription of the ASV17 v-jun allele is controlled by a doxycycline-sensitive transactivator (tTA) or a reverse (doxycycline-dependent) transactivator (rtTA), respectively. Permanent cell lines of quail embryo fibroblasts conditionally transformed by a doxycycline-controlled v-jun allele revert to the normal phenotype within 3 days and lose their ability to grow in soft agar, strictly dependent on the addition or removal of the drug, respectively. The reverted cells are rapidly retransformed on conditional activation of v-jun. While full-level synthesis of v-jun mRNA and v-Jun protein in these cells is established within 2 and 14 h, respectively, after switching to the permissive conditions, the first morphological alterations are observed after 24 h, and as early as 2 days later the morphology has changed entirely from flat cells resembling normal fibroblasts to spindle-shaped fusiform cells showing a typical jun-transformed phenotype. Kinetic expression analysis revealed that transcriptional activation of the direct jun target gene BKJ precisely coincides with the establishment of full-level v-Jun protein synthesis. Furthermore, we have analyzed the expression of a novel candidate v-jun target gene, termed JAC, which shows no sequence homology to known genes. Similar to BKJ, JAC is specifically activated in jun-transformed fibroblasts, and induction of JAC is tightly linked to the conditional expression of oncogenic v-Jun. These results demonstrate the high stringency of the doxycycline-controlled v-jun expression system, and they also indicate that expression of v-jun in these cells is indispensable for enhanced proliferation, cell transformation, and the induction of specific expression patterns of downstream target genes.

To investigate the molecular basis of oncogenesis induced by the v- myc oncogene of avian myelocytomatosis virus MC29, we developed a conditional cell transformation system in which expression of the MC29 v- myc allele is dependent on a doxycycline-sensitive transactivator (tTA). Clonal lines of quail embryo fibroblasts transformed by doxycycline-controlled v- myc revert to the normal phenotype and lose their ability to grow in soft agar after the addition of doxycycline. Repression of v- myc causes the cells to withdraw from the cell cycle, and long-term survival in culture requires reexpression of v- myc . Although complete repression of v- myc mRNA and v-Myc protein in these cells occurs within 14 h after the addition of doxycycline, the first morphological alterations are observed after 24 h, and after 3 days, the morphology changed entirely from small rounded cells showing a typical myc -transformed phenotype to large flat cells resembling normal fibroblasts. Cells exposed to doxycycline for 3 days reexpressed v- myc within 24 h after withdrawal of the drug from the culture medium, partial retransformation occurred after 2 days, and complete morphological transformation was reestablished after 6 days. Analogous results were obtained with a cell line in which expression of the v- myc allele is dependent on a reverse transactivator (rtTA) that is activated by doxycycline. The striking differential expression of known transformation-sensitive genes and of new candidate v- myc target genes revealed the tightness of the doxycycline-controlled v- myc expression system. The data also indicate that expression of v- myc in these cells is indispensable for enhanced proliferation, transformation, and immortalization.

The entire nucleotide sequence of the transduced v-myc allele in the genome of avian oncogenic retrovirus CMII was determined. The CMII v-myc and the chicken c-myc alleles differ in their shared coding sequences by a single nucleotide substitution causing a glutamic acid/alanine exchange in the predicted sequences of the corresponding protein products. This mutation has not been found in the transduced v-myc alleles of avian oncogenic retroviruses MC29, MH2, and OK10. We conclude that no specific, if any, missense mutation of the c-myc coding sequence is necessary for oncogenic activation upon transduction of the cellular gene.

Nonproducer (NP) clones of chicken and quail cells transformed by avian myelocytomatosis virus MC29-A were isolated in focus and agar colony assays. Several quail MC29 NP clones were developed into long-term cultures. They have been kept in continuous culture for 6 months and about 35 passages. They do not release virus particles detectable by [ 3 H]uridine incorporation or reverse transcriptase assay. Rescue of transforming virus is possible at any time by superinfection of the NP cell clones with avian leukosis viruses such as Rous associated virus type 1 or ring-necked pheasant virus (RPV). [ 35 S]Methionine pulse-labeled protein extracts of NP and of superinfected MC29 transformed cell cultures were analyzed by immune precipitation and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In NP cells the common precursor polypeptide for the structural proteins of avian RNA tumor viruses (pr76) is not synthesized. Instead, a major polypeptide with a molecular weight of 110,000–120,000 was observed (MC29–110K). In superinfected cells, both MC29–110K and pr76 are synthesized. The MC29–110K polypeptide was precipitated by an antiserum against whole virus (PR RSV-B) as well as by a monospecific anti-p27 serum. It was not precipitated by an anti-glycoprotein serum. Pulse-chase experiments showed that the MC29–110K polypeptide turned over at a rate comparable to that of pr76. However, none of the major structural proteins (p27, p19, p15) could be detected after the chase. Competition radioimmune assays demonstrated that protein extracts of NP MC29 cells contain inhibitory activity for precipitation of p19 and p27, but not for p15.

The nucleotide sequence of the chicken gene c- mil was determined within and around all regions homologous to the oncogene v- mil of avian retrovirus MH2. The regions of homology to the previously determined v- mil sequence, ranging in size from 28 to 177 base pairs (bp), are distributed over 14 kilobase pairs (kbp) of the chicken genome and are organized in 11 exons. All exon-intron boundaries of c- mil , except the 5′ boundary of exon 1 and the 3′ boundary of exon 11, were unambiguously defined by the identification of consensus splice donor and acceptor sites precisely at positions where homology to v- mil ceases or resumes. The homology to v- mil starts within the coding sequence of exon 1 and ends within the 3′ untranslated region of exon 11, 12 nucleotides downstream from the nonsense codon terminating the large open reading frame shared between c- mil and v- mil . The c- mil and v- mil sequences differ at only 7 out of 1153 nucleotide positions, and the predicted sequences of v- mil and c- mil proteins differ by one conservative and four nonconservative substitutions among 379 amino acid residues. Hence, the carboxy-terminal domains of the MH2 gag-mil hybrid protein and of the putative c- mil protein are very similar. However, the amino-terminal domain of the cellular protein is possibly encoded by additional 5′ c- mil sequences not present in the transduced v- mil oncogene, while that of the MH2 hybrid protein is encoded by viral gag sequences. The sequence analysis also revealed that c- mil and c- myc derived sequences are immediately adjacent on the MH2 genome carrying both the v- mil and the v- myc oncogene. Hence, transduction of c- mil into MH2 involved recombination, at the 3′ site, with either the c- myc locus or a previously transduced v- myc gene, and, at the 5′ site, with gag sequences of the transducing virus. At both sites, no significant homologies were found between the sequence elements involved in the recombination.

Avian oncogenic retrovirus MH2 carries two cell-derived oncogenes, v- mil and v- myc . From an infectious stock of MH2 a spontaneous deletion mutant, MH2D12, that has lost most of the v- mil gene but has retained a complete and functional v- myc gene, has been isolated. Nonproducer quail embryo cells transformed by MH2D12 in the absence of helper virus contain two virus-specific proteins: a gag -related protein of 53,000 Da (p53 gag ), and a v- myc gene product of 59,000/61,000 Da (p59/61 v- myc ) indistinguishable from the v- myc protein encoded by MH2. MH2D12 viral RNA contains all T 1 -oligonucleotides specific for the MH2 v- myc gene but none of those characteristic for the v- mil gene. The genetic structure of molecularly cloned proviral DNA of MH2D12 was revealed by restriction mapping, blot hybridization, heteroduplex analysis, and nucleotide sequencing. The MH2D12 provirus is homologous to the MH2 genome but has suffered a deletion of 1271 nucleotides from the central region encompassing the 3′ end of Δ gag and all of v- mil except the very 3′ 31 nucleotides directly adjacent to the v- myc gene. A nine-nucleotide overlap of homology to gag or mil at the Δ gag /Δ mil junction suggests that recombination between homologous sequence elements of the Δ gag and v- mil domains of MH2 was involved in the genesis of MH2D12. The nucleotide sequence analysis predicts that the carboxyterminal 17 amino acids of p539 gag are encoded by the residual v- mil sequences and by intron-derived v- myc sequences. Transformation of quail embryo cells by MH2D12 can be assayed by focus and colony formation of transformed cells. This indicates that the v- mil gene is not essential for these activities. However, size and morphology of foci and colonies, and cellular morphology of cultured MH2D12-transformed cell lines can easily be distinguished from those observed in cell transformation by MH2 and resemble more those seen in cell transformation by viruses containing the myc oncogene only.

Rabbit antisera were prepared against a synthetic peptide corresponding to the carboxyterminal amino acid sequences of the transforming protein p110 gag-myc of avian oncovirus MC29. Analysis of immunoprecipitates formed with these sera (anti- myc C) demonstrated that the gag-myc hybrid proteins encoded by the avian leukemia viruses MC29, CMII, and OK10 were recognized by the anti-peptide sera, but not the gag or pot precursor proteins of helper viruses or the MH2-encoded p100 gag-mil protein. In cells transformed by OK10 or MH2, putative v- myc proteins of 60K (OK10) and 59/61K (MH2) were also precipitated by anti- myc C. In addition, the anti-peptide sera reacted specifically with the gag-myc proteins encoded by three partially transformation-defective mutants of MC29, td 10A, td 10C, and td10H, and by MC29 variant HBI.

The RNAs and proteins of five laboratory strains of defective avian acute leukemia virus MC29, differing in passage history and in some cases in their helper viruses and oncogenic spectra, were investigated for genetic variation. Earlier work based on homology with nondefective viruses of the avian tumor virus group has distinguished in 5.7-kb (kilobase) MC29 RNA a 5′ group-specific section, an internal MC29-specific section, and a 3′ group-specific section, and has determined that 5′ and internal sections together are translated into a nonstructural, gag gene-related 110K (kilodaltons) protein. The RNAs of the five MC29 strains analyzed here shared 20 conserved T 1 -oligonucleotides and differed in specific sets selected from 10 variable oligonucleotides. Since some, but not all, variable and some conserved MC29-oligonucleotides were shared with helper viruses, and since the 5′ cap-oligonucleotides of MC29 RNA rescued from the same nonproducer cell varied with that of the helper virus RNA, it is concluded that genetic variation of MC29 includes recombination with helper virus. Oligonucleotide maps of the five MC29 RNAs located 8 variable oligonucleotides in their 3′ group-specific sections, only conserved oligonucleotides in their internal-specific section, and mostly conserved oligonucleotides in their 5′ group-specific section (except for variable 5′ cap-oligonucleotides and the lack of an internal oligonucleotide in one strain). The 110K proteins of the MC29 strains were electrophoretically indistinguishable, which is in accord with the conserved nature of their templates. The ratio of MC29 RNA to helper virus RNA in virions was found to reflect, by a constant, the ratio of 110K MC29 to helper viral proteins in transformed cells. MC29 RNA is proposed to consist of two genetic units: one made of the highly conserved 5′ and internal-specific RNA sections, which codes for the 110K protein and appears to serve a primary function in oncogenicity; the other made of the variable 3′ group-specific RNA sections which may affect oncogenicity indirectly.

The avian leukemia and carcinoma inducing retrovirus MH2 contains the novel oncogene v- mil in addition to the cell-derived oncogene v- myc High-molecular-weight chicken DNA contains sequences closely related to v- mil and also sequences more distantly related to this gene. Several phage clones were isolated by screening of a chicken recombinant DNA library with a v- mil -specific probe in stringent conditions. These clones contain overlapping segments of v- mil -related chicken DNA. Hence, the sequences closely related to v- mil , termed c- mil , appear to represent a single-copy locus of the chicken genome. The close relationship between the cellular and the viral gene was demonstrated by hybridization between c- mil DNA and MH2 viral RNA, or between c- mil DNA and cloned v- mil DNA, and by a comparison of their restriction maps, which revealed total conservation in c- mil DNA of all restriction sites found in v- mil DNA. The c- mil locus spans at least 10 kb, with nine regions of homology to v- mil , ranging in size from about 0.07 to 0.17 kb, interrupted by eight intervening sequences with complexities of about 0.5 to 3.5 kb. Analyses of the cloned chicken c- mil and c- myc loci by nucleic acid hybridization employing specific probes from the mil-myc junction of MH2 proviral DNA revealed that c- mil - and c- myc -related sequences are directly adjacent in the viral genome and that MH2 contains additional 5′ c- myc -related sequences not present in the genomes of other leukemia viruses carrying the v- myc oncogene.