Molecular analysis of myc gene mutants

We describe the generation and characterization of a series of deletion mutants of the avian acute leukaemia virus MC29 which allow the study of the function of the myc in transformation of quail embryo fibroblasts in vitro and tumour induction in vivo. These mutants, which are deleted in the 3' portion of the myc gene, fail to transform macrophages in vitro or induce tumours in vivo but are still able to transform morphologically fibroblasts. From one of these mutants a 'recovered' MC29 virus was generated which, like wild type MC29, transformed fibroblasts and macrophages in vitro. When tested in vivo this virus induced lymphomas of T and B cells rather that the endotheliomas induced by wild type MC29. This system allows us to investigate another question which is the mechanism by which the virus (or oncogene it contains) preferentially transforms one cell type.     

An avian retrovirus expressing chicken pp59c-myc possesses weak transforming activity distinct from v-myc that may be modulated by adjacent normal cell neighbors.

We demonstrate that EF168, an avian retrovirus that expresses the chicken pp59c-myc proto-oncogene, transforms quail embryo fibroblasts in vitro. An EF168-transformed quail clone, EF168-28, containing a single provirus, synthesizes several hundred copies of c-myc RNA and expresses elevated levels of the pp59c-myc gene product. The EF168 provirus in EF168-28 was isolated as a molecular clone, and the nucleotide sequence of its c-myc allele was confirmed as identical to that of exons 2 and 3 of the chicken c-myc proto-oncogene. Extended infection of quail embryo fibroblast cultures with EF168 induced a number of in vitro transformation-associated parameters similar to those elicited by the oncogenic v-myc-encoding retrovirus MC29, including alteration of cellular morphology, anchorage-independent growth, and induction of immortalized cell lines. Despite the fact that EF168 and MC29 shared these biological activities, further analysis revealed that EF168 initiated transformation in quail embryo fibroblasts, bone marrow, or adherent peripheral blood cultures 100- to 1,000-fold less efficiently than did MC29. Further, in contrast to MC29-induced foci, EF168 foci were smaller, morphologically diffuse, and less prominent. Analysis of newly infected cells demonstrated efficient expression of EF168 viral RNA in the absence of transformation. These differences suggest that while the pp59v-myc gene product can exert dominant transforming activity on quail embryo fibroblasts, its ability to initiate transformation is distinct from that of the pp110gag-v-myc gene product encoded by MC29 and may be suppressed by adjacent nontransformed cell neighbors.     

Nucleotide sequence of HBI, a novel recombinant MC29 derivative with altered pathogenic properties.

HBI is a recombinant avian retrovirus with novel pathogenic properties that was derived from the myc-containing virus MC29. In contrast to MC29, which causes endotheliomas in chickens, HBI induces lymphoid tumors. The results of molecular cloning and nucleotide sequencing of HBI reported here show that the virus contains sequences derived from both c-myc and ring-neck pheasant virus, in addition to MC29. The 3' half of the myc gene was largely replaced by c-myc sequences, and most of the long terminal repeat and gag regions were replaced by ring-neck pheasant virus sequences. The long terminal repeat contained a triplicate sequence which was homologous to the core enhancer sequence of the simian virus 40 72-base-pair repeat. The significance of these changes in relation to the unusual biological properties of the virus are discussed.     

Nuclear location of the putative transforming protein of avian myelocytomatosis virus

The putative transforming protein of avian myelocytomatosis virus MC29 is a 110,000 dalton (P110gag-myc) polyprotein comprised of sequences derived from both the gag region and the MC29-specific myc region. Two approaches have been taken to determine the location of the MC29 gag-related proteins in transformed cells: subcellular fractionation and immunofluorescence. Analysis of subcellular fractions of MC29-transformed cells by immunoprecipitation indicates that the majority of the gag-myc polyprotein is found in the nuclear fractions of Q8 cells (a nonproducer line of MC29-transformed quail embryo fibroblasts) and nonproducer cells derived from a liver tumor of MC20-infected quail. This is in contrast to the distribution of gag-related helper virus proteins lacking myc, which are found only in nonnuclear fractions of superinfected Q8 cells. The purity of unlabeled nuclei was assessed by electron microscopy and enzyme assays, revealing little contaminating material from other subcellular fractions. Immunofluorescence experiments using monospecific anti-gag serum showed specific, intense immunofluorescence in the nuclei of fixed Q8 cells. In contrast, the majority of P75gag-erb, a candidate transforming protein produced by avian erythroblastosis virus (AEV), is absent from the nuclei of nonproducer AEV-transformed chick embryo fibroblasts. The nuclear association of the MC29 transforming protein may be related to some of the unique properties of MC29-transformed cells.     

Deletions Within the Transformation-Specific RNA Sequences of Acute Leukemia Virus MC29 Give Rise to Partially Transformation-Defective Mutants

The viral RNAs of three nonconditional mutants of avian myelocytomatosis virus MC29 were analyzed. These mutants, which were originally isolated from the quail producer line Q10 and were designated 10A, 10C, and 10H, have lost most of the ability to transform hematopoietic cells in vitro and to induce tumors in vivo, but they still transform cultured fibroblasts with the same efficiency as wild-type (wt) MC29. Electrophoretic analyses showed that the mutant genomic RNAs were smaller than the 5.7-kilobase genome of wt MC29; the genomes of mutants 10A, 10C, and 10H were about 5.5, 5.3, and 5.1 kilobases long, respectively. Analyses of the transformation-specific sequences of these mutant RNAs by a combination of T(1) oligonucleotide fingerprinting and hybridization with cDNA from the transformation-specific sequences myc of wt MC29 or competition hybridization including wt MC29 RNA revealed that deletions of myc-specific sequences had occurred. The deletions in all three mutants overlapped, since they all had lost one particular myc-specific oligonucleotide. In agreement with the size of the genomic RNAs, mutants 10C and 10H had lost two additional myc oligonucleotides, and mutant 10A contained a modified myc oligonucleotide. The locations of the deletions were deduced from comparisons with previously established oligonucleotide maps of several members of the MC29 subgroup of acute leukemia viruses and by hybridization of wt and mutant RNAs to molecularly cloned subgenomic fragments of wt MC29 proviral DNA, representing the 5' and 3' domains of the myc sequence. We found that the deleted sequences represented overlapping internal segments of the myc sequence and that the borders of myc with the partial complements of the virion genes gag and env appeared to be conserved in mutant and wt MC29 RNAs. The correlation between the altered transforming potential for hematopoietic cells and the partial deletion of myc in the mutant RNAs provided direct genetic evidence for the involvement of myc in oncogenesis. However, the unaffected efficiency of these mutants in fibroblast transformation suggested that the deleted sequences are not essential for the fibroblast-transforming potential of the onc gene of MC29.     

MC29 deletion mutants which fail to transform chicken macrophages are competent for transformation of quail macrophages.

A number of MC29 mutants with deleted myc genes have been previously characterized. Many of these mutants have been found to be defective for transformation of chicken macrophages in vitro and for tumor induction in chickens. Such mutants are capable of transforming Japanese quail macrophages in vitro and inducing a high incidence of tumors in Japanese quail. Thus, Japanese quail may contain a factor(s) capable of complementing the defective transforming proteins encoded by some deleted v-myc genes.     

Genome structure of HBI, a variant of acute leukemia virus MC29 with unique oncogenic properties.

We have analyzed the viral RNA of a variant of avian acute leukemia virus MC29, termed HBI. This virus was isolated during in vitro passage of a partially transformation-defective (td) mutant of MC29 (td10H-MC29) in chicken macrophages. While td10H-MC29 has a reduced ability to transform macrophages in vitro or to induce tumors in vivo, HBI-MC29 transforms macrophages efficiently and induces in vivo a high incidence of lymphoid tumors. Electrophoretic analysis of HBI-MC29 genomic RNA revealed that it has a complexity of 5.7 kilobases, like the RNA of wild-type (wt) MC29, and that it is 0.6 kilobases longer than the 5.1-kilobase RNA of the deletion mutant td10H-MC29. Analysis of the viral RNAs of two clonal isolates of HBI-MC29 by T1 oligonucleotide fingerprinting showed that sequences from the viral transformation-specific region, v-myc, which are deleted in td10H RNA, are present in HBI RNA. Moreover, hybridization of HBI RNA to molecularly cloned subgenomic fragments of wtMC29 proviral DNA, followed by fingerprint analysis of hybridized RNA, showed that the entire v-myc-specific RNA sequences defined previously are present. Hybridization to cloned DNA of the normal chicken locus c-myc shows a close relationship between HBI v-myc RNA and c-myc DNA, especially in the sequences which were deleted from td10H-MC29. T1 oligonucleotide maps of HBI and td10H RNAs were prepared and compared. Total conservation of the oligonucleotide pattern is observed in the overlapping v-myc regions, while the partial structural genes gag and env show some variations, most of which can be directly proven to be due to point mutations or recombination with helper viral RNAs that were analyzed in parallel. Recombination of td10H-MC29 with c-myc, followed by recombinational and mutational changes in the structural genes during passage with helper virus, could be a possible explanation for the origin of HBI.     

Quail embryo fibroblasts transformed by four v-myc-containing virus isolates show enhanced proliferation but are non tumorigenic.

Quail embryo fibroblasts infected with any of the four natural avian myc gene-containing virus strains (MC29, CMII, OK10 and MH2) or with the myb, ets-containing E26 acute leukemia virus, were examined for their expression of several transformation-associated parameters. All myc-containing viruses, but not E26 or Rous sarcoma virus (used as a control) induced a dramatic stimulation of cell proliferation. In addition, the myc virus-transformed cells exhibited prominent nucleoli, possibly as a consequence of their increased proliferation. Cells transformed by MC29, OK10, MH2 and E26 were capable of growing in semi-solid medium and showed a loss of actin cables and, in most cases, of an ordered fibronectin distribution. All of the myc virus-transformed fibroblasts, as well as the E26-transformed cells, were unable to form tumors in nude mice, indicating that the myc gene (and the myb/ets genes) are not sufficient for the induction of a fully malignant phenotype in avian fibroblasts.     

Generation and characterization of a recombinant Moloney murine leukemia virus containing the v-myc oncogene of avian MC29 virus: in vitro transformation and in vivo pathogenesis.

A new retrovirus consisting of the v-myc oncogene sequences of avian MC29 virus inserted into the genome of Moloney murine leukemia virus (M-MuLV) was generated. This was accomplished by constructing a recombinant DNA clone containing the desired organization, introducing the recombinant DNA into mouse NIH 3T3 cells, and superinfecting the cells with replication-competent M-MuLV. The construction was designed so that an M-MuLV gag-myc fusion protein would be produced. The resulting virus, M-MuLV(myc), morphologically transformed uninfected NIH 3T3 cells. Stocks of M-MuLV(myc)-M-MuLV were infected into secondary mouse embryo cultures. M-MuLV(myc) induced striking growth and proliferation of hematopoietic cells. These cells were of the myeloid lineage by morphology, phagocytic properties, and surface staining with Mac-1 and Mac-2 monoclonal antibodies. They resembled mature macrophages, although they displayed minor properties of immaturity. The myeloid cells were transformed in comparison with uninfected myeloid cells since they were less adherent and had unlimited proliferative capacity and reduced growth factor requirements. The transformed myeloid cells with proliferative potential were actually myeloid progenitors which apparently underwent terminal differentiation to macrophages. It was possible to derive a permanent line of factor-independent macrophages from M-MuLV(myc)-transformed myeloid cells. M-MuLV(myc) also immortalized and morphologically transformed mouse embryo fibroblasts. These in vitro properties closely resembled the biological activity of MC29 virus in avian cells and suggested that the nature of the v-myc oncogene was an important determinant in transformation specificity. Neonatal NIH Swiss mice inoculated intraperitoneally with M-MuLV(myc)-M-MuLV only developed lymphoblastic lymphoma characteristic of the M-MuLV helper alone, and no acute fibrosarcomas or myeloid tumors resulted. In light of the strong myeloid transformation observed in vitro, the absence of acute in vivo myeloid disease was noteworthy. Interestingly, when a derivative of M-MuLV(myc) carried by a nonpathogenic amphotropic MuLV helper was inoculated, T lymphomas developed with long latency. Molecular hybridization confirmed that these tumors contained M-MuLV(myc).     

Two autonomous myc oncogenes in avian carcinoma virus OK10.

The oncogenic avian retrovirus OK10 has the genetic structure gag-delta pol-myc-delta-env. The myc sequence is transduced from a cellular gene, proto-myc, while gag, pol, and env are essential retrovirus genes. By analogy with other directly oncogenic retroviruses, the specific myc sequence of OK10 is thought to be essential for transforming function. However, unlike the specific sequences of all other transforming retroviruses that encode unique transforming proteins, the myc sequence of OK10 encodes two potential transforming proteins, p58 and p200. p200 is translated from the gag-delta pol-myc region of genomic RNA, while p58 is thought to be translated from the gag leader and the open reading frame of myc via a subgenomic mRNA. In this paper, we ask whether both myc genes of OK10 are autonomous transforming genes. By differentially inactivating the p200 myc gene of OK10 provirus in vitro and analyzing transforming function in quail embryo cells, it was found that mutants expressing only p58 transformed like wild-type OK10. Further, it was shown that p58 with and without the gag leader had transforming function and that p58 of wild-type OK10 is initiated from the gag leader. Mutants expressing only p200 were also transforming but less efficiently than mutants that express only p58. A mutant OK10 virus in which the native frameshift of retroviruses between gag and pol was deleted expressed a shortened p200 (delta p200). Although this virus expressed more delta p200 than wild-type OK10 did, it transformed cells less efficiently. It follows that OK10 expresses two autonomous transforming genes, which is unique among retroviruses with onc genes.     

Targets of v-myc tumorigenesis in the avian embryo depend on time and not on site of retroviral infection

The present study extends our previous data, showing that the v-myc oncogene induces heart tumors and skin anomalies in young avian embryos [Saule et al., Proc. Natl. Acad. Sci. USA 84, 7982–7986 (1987)]. We now report that the target cells which become transformed are the same, whether the MC29 retrovirus is injected at E3 in various sites of the embryo (coelom, heart, brain, lateral plate mesoderm) or deposited on the embryo. Furthermore we confirm, in the quail, the time-specific pattern previously observed in the chick. In the quail, the incidence of heart tumors falls from 100% to 28% when injection is delayed from E3 to E4. By contrast, the incidence of skin anomalies rises from 30% to 64% when injection is delayed from E3 to E4. The skin defect, which consists of the presence of bell-shaped cornified feathers, could be assigned to hyperkeratinization of the epidermis. Both the dermis and the epidermis displayed hyperproliferation, whereas skin muscle hypertrophy during the embryonic period could not be confirmed. The presence of myc gene products was investigated using an antibody that recognizes both the c- and v-myc proteins. In the skin of control embryos, nuclei were well stained at E12–E13. At E14 the signal had disappeared. In abnormal skin patches from infected embryos, the antibody still marked heavily epidermal and dermal nuclei at E18. Finally we injected MC29 through the chorioallantoic vein in E10 chickens. No tumors were found during embryonic life, but 81% of the chickens developed tumors of hemopoietic or endothelial origin from the 14th posthatching day onwards. Studies of MC29 integration sites demonstrated that these tumors were derived from only a few transformed cells. Thus, contrasting with in vitro experiments, in vivo this virus has a restricted number of targets varying with the time of injection.     

Association of gag-myc proteins from avian myelocytomatosis virus wild-type and mutants with chromatin.

The localization of the transformation-specific proteins was analyzed in quail embryo fibroblast cell lines transformed by wild-type avian myelocytomatosis virus MC29 and by three of its deletion mutants, Q10A , Q10C , and Q10H , with altered transforming capacities, and in a chicken fibroblast cell line transformed by the avian erythroblastosis virus (AEV). These viruses code for polyproteins consisting of part of the gag gene and of a transformation-specific region, myc for MC29 and erb A for AEV. Analysis by indirect immunofluorescence using monoclonal antibodies against p19, the N-terminal region of the polyprotein, showed that the gag-myc proteins in cells transformed by the wild-type MC29 as well as by the three deletion mutants are located in the nucleus. In contrast, cells transformed by AEV, which express the gag-erb A protein, give rise to cytoplasmic fluorescence. Fractionation of cells into nuclear and cytoplasmic fractions and analysis by immunoprecipitation and gel electrophoresis confirmed these results. About 60% of the gag-myc proteins of wild-type as well as of mutant origin were found in the nucleus, while 90% of the gag-erb A protein was present in the cytoplasm. Also, pulse-chase analysis indicated that the gag-myc protein rapidly accumulates in the nucleus in just 30 min. Further, it was shown that the wild-type and also mutant gag-myc proteins are associated with isolated chromatin. Association to chromatin was also observed for the gag-myc protein from MC29-transformed bone marrow cells, which are believed to be the target cells for MC29 virus in vivo.     

gag as well as myc sequences contribute to the transforming phenotype of the avian retrovirus FH3.

The avian retrovirus FH3, like MC29 and CMII, encodes a Gag-Myc fusion protein. However, the FH3-encoded protein is larger, about 145 kDa, and contains almost the entire retroviral gag gene. In contrast to the other gag-myc avian retroviruses, FH3 fails to transform fibroblasts in vitro, although macrophages are transformed both in vitro and in vivo (C. Chen, B. J. Biegalke, R. N. Eisenman, and M. L. Linial, J. Virol. 63:5092-5100, 1989). We have used the polymerase chain reaction technique to obtain a molecular clone of FH3. Sequence analysis of the FH3 myc oncogene revealed a single proline----histidine change (position 223) relative to c-myc. However, substitution of the FH3 myc sequence with the chicken c-myc sequence did not alter the transformation potential of the virus. Hence, overexpression of the proto-oncogene as a Gag-Myc retroviral protein is sufficient for macrophage, but not fibroblast, transformation. After passage of FH3 in fibroblast cultures, a virus (FH3L) that is capable of rapidly transforming fibroblasts appears. The Gag-Myc protein encoded by FH3L is smaller (ca. 130 kDa) than that encoded by the original viral stock (FH3E). Sequencing of an FH3L molecular clone revealed a 212-amino-acid deletion within the Gag portion. Using FH3E/FH3L recombinants, we have demonstrated that the ability of encoded viruses to transform fibroblasts directly correlates with the presence of this deletion. Moreover, the addition of the Gag sequence deleted from FH3L to the MC29 oncoprotein significantly reduces its transforming activity as measured by focus assay. These data suggest that the C-terminal segment of Gag attenuates the oncogenic potential of Gag-Myc fusion proteins.     

Transfection by DNAs of avian erythroblastosis virus and avian myelocytomatosis virus strain MC29.

Chicken embryo fibroblasts and NIH 3T3 mouse cells were transformable by DNAs of chicken cells infected with avian myelocytomatosis virus strain MC29 or with avian erythroblastosis virus. Transfection of chicken cells appeared to require replication of MC29 or avian erythroblastosis virus in the presence of a nontransforming helper virus. In contrast, NIH 3T3 cells transformed by MC29 or avian erythroblastosis virus DNA contained only replication-defective transforming virus genomes.     

FH3, a v-myc avian retrovirus with limited transforming ability.

We have isolated a new acute avian transforming virus which contains the oncogene myc. This virus, designated FH3, was isolated after injection of a 10-day-old chick embryo with avian leukosis virus. While FH3 shares many properties with other v-myc-containing avian retroviruses, it also has several unique properties. The primary target for transformation in vitro is chicken macrophages; infection of chicken fibroblasts does not lead to complete morphological transformation. FH3 also exhibits a limited host range, in that Japanese quail macrophages and fibroblasts are infected but are not completely transformed. FH3 induces in vivo a limited tumor type if injected into 10-day-old chick embryos; only a cranial myelocytoma, which does not appear to be metastatic, can be detected. The v-myc gene of FH3 is expressed predominantly as a P145 Gag-Myc protein which is encoded by a ca. 8-kilobase genomic RNA. This FH3-encoded polyprotein is localized in the nucleus of all infected cells, whether or not they are transformed.     

Characterization of lymphoid tumors induced by a recombinant murine retrovirus carrying the avian v-myc oncogene. Identification of novel (B-lymphoid) tumors in the thymus

Lymphoid tumors induced by a recombinant murine retrovirus carrying the v-myc oncogene of avian MC29 virus were characterized. The Moloney murine leukemia virus myc oncogene (M-MuLV (myc], carried by an amphotropic MuLV helper, induced tumors in NIH Swiss and NFS/N mice after a relatively long latency (8 to 24 wk). Tumor masses appeared in the thymus, spleen, and lymph nodes. Flow cytometry of the tumor cells indicated that approximately 50% were positive for Thy 1.2. Most of these tumors also expressed one or more other cell surface markers of thymocytes and mature T cells (CD4, CD8). Southern blot hybridization revealed genomic rearrangements for the TCR beta genes. The TCR beta analysis suggested that the M-MuLV(myc)-induced Thy 1.2+ tumors were derived from somewhat less mature cells than tumors induced by M-MuLV, which is a classical non-acute retrovirus lacking an oncogene. The remainder of the M-MuLV(myc)-induced tumors were Thy 1.2-, but they were positive for Ly-5 (B220) and also for MAC-2. The Thy 1.2- tumors were characteristically located in the thymus. However, they were negative for TCR beta gene rearrangements. Some, but not all, of the Thy 1.2- tumors contained rearrangements for Ig genes. Additionally, they typically expressed mRNA specific for B but not for T cells. Thus, these thymic tumors had characteristics of the B cell lineage. Tumor transplantation experiments demonstrated that the Thy 1.2- tumor cells could reestablish in the thymus and spleen of irradiated hosts, and low level expression of the Thy 1 molecule was observed in the thymus but not the spleen on the first passage. After serial passage, one Thy 1- tumor altered its cell surface phenotype to Thy 1low B220-.     

Site-directed mutagenesis of the gag-myc gene of avian myelocytomatosis virus 29: biological activity and intracellular localization of structurally altered proteins.

Transfection of chicken embryo cells with pMC29, a plasmid vector containing the sequences for the acute transforming virus MC29, and a cloned transformation-defective helper virus, p delta Mst, resulted in morphological transformation, the synthesis of P110gag-myc (the product of the gag-myc oncogene), and the production of infectious virus. MC29 mutants bearing site-directed deletions within the gag-specific sequences or within the middle portion of the myc sequences efficiently induced transformation of chicken embryo cells in culture. However, variants containing deletions of sequences in the amino-terminal half or carboxy-terminal portion of the myc gene were defective for transformation. The gag-myc proteins encoded by these variants efficiently localized to the cell nucleus. Premature termination mutants were isolated which encoded gag-myc proteins lacking the carboxy-terminal 185 residues; these truncated proteins localized to both the nucleus and the cytoplasm. Deletion of as few as 11 residues within the middle of the myc-specific sequences (residues Ile-239 to Glu-249) significantly reduced the efficiency of chicken hematopoietic cell transformation.     

Rous sarcoma virus mutant dlPA105 induces different transformed phenotypes in quail embryonic fibroblasts and neuroretina cells.

dlPA105 is a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion in the N-terminal portion of the v-src gene coding sequence. This virus was isolated on the basis of its ability to induce proliferation of quiescent quail neuroretina cells. The altered v-src gene encodes a phosphoprotein of 45,000 daltons which possesses tyrosine kinase activity. DNA sequencing of the mutant v-src gene has shown that deletion extends from amino acid 33 to 126 of wild-type p60v-src. We investigated the tumorigenic and transforming properties of this mutant virus. dlPA105 induced fibrosarcomas in quails with an incidence identical to that induced by wild-type virus. Quail neuroretina cells infected with the mutant virus were morphologically transformed and formed colonies in soft agar. In contrast, dlPA105 induced only limited morphological alterations in quail fibroblasts and was defective in promoting anchorage-independent growth of these cells. Synthesis and tyrosine kinase activity of the mutant p45v-src were similar in both cell types. These data indicate that the portion of the v-src protein deleted in p45v-src is dispensable for the mitogenic and tumorigenic properties of wild-type p60v-src, whereas it is required for in vitro transformation of fibroblasts. The ability of dlPA105 to induce different transformation phenotypes in quail fibroblasts and quail neuroretina cells is a property unique to this Rous sarcoma virus mutant and provides evidence for the existence of cell-type-specific response to v-src proteins.     

Identification of nucleotide sequences which may encode the oncogenic capacity of avian retrovirus MC29.

The retrovirus strain MC29 induces a variety of tumors in chickens, including myelocytomatosis and carcinomas of the kidney and liver. In addition, the virus can transform cultures of embryonic avian macrophages and fibroblasts. We have characterized the genome of MC29 virus and have identified nucleotide sequences that may encode the oncogenic potential ofthe virus. MC29 virus can replicate only with the assistance of a related helper virus. The defect in replication is apparently a consequence of a deletion in one or more viral genes: the haploid genome of the MC29 virus has a molecular weight of ca. 1.7 X 10(6), whereas the genome of the helper virus MCAV has a molecular weight of ca. 3.1 X 10(6). Although MC29 virus transforms fibroblasts in culture, its genome has no detectable homology with the gene src that is responsible for transformation of fibroblasts by avian sarcoma viruses. We prepared radioactive single-stranded DNA complementary to nucleotide sequences present in the genome of MC29 virus but not in the genome of MCAV (cDNA(MC29)). If they are contiguous, these sequences (ca. 1,500 nucleotides) are sufficiently complex to encode at least one protein. Homologous sequences were not detectable in several strains of avian sarcoma viruses or in an endogenous virus of chickens. Our findings confirm and extend recent reports from other laboratories and lead to the conclusion that MC29 virus may contain a previously unidentified gene(s) that is capable of transforming several distinct target cells. The evolutionary origins of this putative gene and its location on the viral genome can be explored with cDNA(MC29).