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Rearrangement in the c-myb locus of each of four independently derived BALB/c plasmacytoid lymphosarcoma (ABPL9s) is due to the insertion of a defective Moloney murine leukemia virus (M-MuLV) into a 1.5-kilobase-pair stretch of cellular DNA at the 59 end of the v-myb-related sequences. This retroviral insertion is associated with abnormal transcription of myb sequences and probably represents a step in the neoplastic transformation of ABPL cells.

The nucleotide sequence of the integrated avian myeloblastosis virus (AMV) long terminal repeat (LTR) has been determined. The sequence is 385 bp long and is present at both ends of the viral DNA. The cell-virus junctions at each end consist of a six base pair direct repeat of cell DNA next to the inverted repeat of viral DNA, The LTR also contains promoter-like sequences, a mRNA capping site, and polyadenylation signals. Several features of this LTR suggest a structural and functional similarity with sequences of transposable and other genetic elements. Comparison of these sequences with LTRs of other avian retroviruses indicates that there is a great variation in the 3′ unique sequence (U3) while the 5′ specific sequences are highly conserved.

The sequence of the transforming region of the Moloney murine sarcoma virus genome has been determined by using molecularly cloned viral DNA. This region, 3.6 to 5.8 kilobase pairs from the left end of the molecule, contains the entire cellular insertion (src) sequence as well as helper viral sequences including the large terminal repeat (LTR). On the viral RNA strand, a long (1224 bases) open reading frame commenced to the left of the src-helper virus junction and terminated at a point 58 nucleotides into helper viral sequences to the right of src. Possible promoter and acceptor splice signals were detected in helper viral sequences upstream from this open reading frame. On the antiviral RNA strand, several promoter-like sequences, including one within the src region itself, were identified. However, no open reading frame downstream from these promoters was detected in the antiviral RNA strand. The LTR was found to contain promoter-like sequences as well as LTR was found to contain promoter-like sequences as well as mRNA capping and polyadenylylation signals. In addition, it possessed an 11-base inverted terminal repeat at each end. Thus, the structure of the Moloney murine sarcoma virus genome with an LTR at each end resembles that of prokaryotic transposable elements.

The genome of the avian myeloblastosis virus (AMV) has undergone a sequence substitution in which a portion of the region normally coding for the env protein has been replaced by cellular sequences. We have determined the complete nucleotide sequence of this region. Examination of the AMV oncogenic sequence revealed an open reading frame starting with the initiation codon ATG and terminating with the triplet TAG within the acquired cellular sequences and terminating with the triplet TAG at a point thirty-three nucleotides into helper viral sequences to the right of the helper-viral-cellular junction. The stretch of 795 nucleotides would code for a protein of 265 amino acids with a molecular weight of 30,000 daltons. The eleven amino acids at the carboxy terminus of such a protein would be derived from the env gene of helper virus. Antibodies were prepared against synthetic peptides derived from the predicted amino acid sequences. One such antibody precipitated two magnesium proteins of apparent nucleotide weight of 30,000 daltons and 51,000 daltons.

The integrated Abelson murine leukemia virus (A-MuLV) genome cloned in bacteriophage lambda gtWES.lambda B was used to localize viral genetic sequences required for transformation. Comparison of the biological activity of cloned A-MuLV genomic and subgenomic fragments showed that subgenomic clones that lacked the 5' long terminal repeat and adjoining sequences (300 base pairs downstream of the repeat) were not biologically active. In contrast, subgenomic clones that lacked the 3' long terminal repeat and as much as 1.3 kilobase pairs of the A-MuLV cell-derived abl gene were as efficient as wild-type viral DNA in transformation. The A-MuLV-encoded polyprotein P120 and its associated protein kinase activity were detected in transformants obtained by transfection with Cla I, BamHI, and HindIII subgenomic clones. In contrast, individual transformants obtained with subgenomic Sal I clones expressed A-MuLV proteins ranging in size from 82,000 to 95,000 daltons. Each demonstrated an associated protein kinase activity. These results provide direct genetic evidence that only the proximal 40% of abl with its associated 5' helper viral sequences is required for fibroblast transformation.

A correlative study was done to determine possible relationships between nonrandom aberrations in chromosomes 1, 6, and 7 occurring in human cutaneous malignant melanoma and the structure of oncogenes as well as specific genes encoding growth factors and growth factor receptors. Thirty cell lines derived from primary or metastatic melanomas of 28 patients were analyzed by Southern blotting with nick-translated probes for 28 different genes, some of which map near frequent chromosomal breakpoints observed in melanoma. An alteration in the MYB protooncogene was observed in a cell line derived from a primary melanoma in the vertical growth phase, which correlated with a 6q22 chromosomal abnormality. Another primary melanoma cell line had a cytogenetically undetected tumor-specific deletion within the gene for alpha-type protein kinase C. Polymorphic alleles for the genes encoding the epidermal growth factor receptor and alpha-type protein kinase C were also observed.

The retroviral long terminal repeat (LTR) contains transcriptional control elements that affect viral gene expression. By deletion mutagenesis of the genome of the cloned Abelson murine leukemia virus, regulatory signals could be mapped to at least three domains within the LTR. A defective 5' LTR that did not sustain transforming gene function was complemented by an intact LTR positioned at the 3' end of the genome. This versatility of the retroviral genome with respect to its transcriptional control elements appears to provide a strong selective advantage for viral gene expression.

The c-myb gene is the normal cellular homolog of v-myb, the oncogenic component of Avian Myeloblastosis Virus (AMV). The c-myb gene has previously been shown to code for a single protein species of about 75 kd. However, accumulating evidence indicates that this gene could code for multiple mRNAs as a result of differential splicing. This was first detected in the ABPL-2 tumor line and was later shown to occur in normal cells. To test if indeed these differentially spliced mRNAs code for a new protein species that went hitherto undetected, a series of antibodies directed against specific domains of the myb protein, including the putative sequences that could be generated from the alternatively spliced mRNAs were generated. Immunoprecipitation analysis using these antibodies show that both normal and tumor cell lines synthesize at least two myb proteins, one of 75 kd and the other of 89 kd.

Type-C RNA viruses may be horizontally transmitted as infectious cancer-inducing viruses or vertically transmitted from one generation to the next, often in an unexpressed form, within the host genome (for review see Aaronson and Stephenson 1976). To date the translational products of three leukemia viral genes have been identified (Baltimore 1975). The gag gene product is a polyprotein precursor that undergoes cleavage to form the major nonglycosylated viral structural proteins, which for mammalian type-C viruses are p30, p15, p12, and p10. It has been possible to determine that the information within the murine viral gag gene is arranged 5′-p15-p12-p30-p10-3′ (Barbacid et al. 1976). The products of the pol and env genes are, respectively, the viral reverse transcriptase and a precursor protein containing the major envelope glycoproteins, gp70 and p15E. Evidence primarily from the avian system indicates that the type-C viral genes are ordered 5′-gag-pol-env-3′ (for review see Wang et al. 1976).

Three types of tumors termed plasmacytomas (ABPC's), lymphosarcomas (ABLS's), and plasmacytoid lymphosarcomas (ABPL's) arise in BALB/c mice treated with pristane and Abelson murine leukemia virus (A-MuLV). While most ABPC's and BLS's contain integrated A-MuLV proviral genome and synthesize the v-abl RNA, most ABPL's do not. The ABPL tumors were examined for the expression of other oncogenes that may be associated with their transformed state, in the absence of transforming virus. These tumors expressed abundant c-myb RNA of unusually large size and showed DNA rearrangements of the c-myb locus.

The primate cell-derived transforming gene (v-sis) of simian sarcoma virus (SSV) is represented as a single copy marker within cellular DNAs of mammalian species including human. The human analogue of v-sis can be distinguished from its rodent counterparts by Southern blotting analysis of EcoRI-restricted DNAs. By testing for the presence of the human v-sis-related fragment, c-sis (human), in somatic cell hybrids possessing varying numbers of human chromosome, as well as in segregants of such hybrids, it was possible to assign c-sis to human chromosome 22.

Myelocytomatosis virus MC29 is a defective avian retrovirus with a hybrid transforming gene (delta gag-myc) consisting of a 1,358-base pair (bp) sequence from the retroviral gag gene and a 1,568-bp sequence (v-myc) shared with a cellular locus, termed c-myc. We have subjected to sequence analysis 2,735 bp of the cloned c-myc gene, which includes the v-myc-related region of 1,568 bp, an intervening sequence of 971 bp, and unique flanking sequences of 45 bp and 195 bp at the 5' and 3' ends, respectively. Analysis of the genetic information and alignment of the c-myc sequence with the known sequence of MC29 indicates that: (i) the two myc sequences share the same reading frame, including the translational termination signal; (ii) there are nine nucleotide changes between c-myc and v-myc that correspond to seven amino acid changes; (iii) the 971-bp intervening sequence of c-myc can be defined as an intron by consensus splice signals; (iv) the unique 5' sequence of c-myc could either extend its reading frame beyond the homology with v-myc or could be an intron because its junction with the myc region of the locus is a canonical 3' splice-acceptor site; (v) the v-myc contains 10 nucleotides at its 5' end not shared with the c-myc analyzed here and also not with known gag genes, probably derived from an upstream exon; and (vi) the c-myc locus can generate a mRNA whose termination signals have been identified to be located 83 bp and 119 bp from the point of divergence between the v-myc and c-myc. We conclude that the gene of the c-myc locus of the chicken and the onc gene of MC29 share homologous myc regions and differ in unique 5' coding regions and we speculate, on this basis, that their protein products may have different functions. The hybrid onc gene of MC29 must have been generated from the c-myc gene by deletion of the 5' cellular coding sequence, followed by substitution with the 5' region of the viral gag gene.

The murine c-myb gene has been recently shown to code for two protein products of 75kd and 89kd. The 89kd protein appears to be generated from an alternatively spliced mRNA which contains an additional stretch of 363 bases between exons 9 and 10. In this communication, we have examined whether similar alternatively spliced mRNAs of c-myb occur in human cells. Human c-myb exon 9A has been identified and sequenced in a cDNA clone (ML5) generated from the acute myeloid leukemic cell line ML-2. This alternatively spliced exon of c-myb has been found to contain the same number of nucleotides (363bp) as the corresponding mouse exon. Between murine and human exon 9A sequences, 81% sequence homology was found at the DNA level, while the homology at the predicted amino acid level was found to be 73%. A stretch of 14 amino acid residues at the junction of exons 9A and 10 have been found to be conserved between Drosophila and human sequences indicating that this region might perform an essential biological function which was deemed necessary through evolution.

Abelson murine leukemia virus (A-MuLV) is a replication-defective transforming retrovirus that arose by recombination of nondefective helper virus (M-MuLV) and cellular sequences present within the normal mouse genome. The latter sequences, termed abl, appear to code for the transforming properties of the virus (for review see Rosenberg and Baltimore, 1980). This virus induces in adult BALB/c mice a variety of lymphoid neoplasms predominantly of the pre-B cell series (ABLS tumors). However, when the mice are previously injected with pristane, which induces intraperitonal granulomatous tissue, this virus also rapidly induces plasmacytomas (ABPC tumors) and occasionally, a morphological subset of lymphosarcomas characterized by plasmacytoid cytoplasm but with very little immunoglobulin production (ABPL tumors) (Potter et al.,1978). Our preliminary experiments indicated that ABPC and ABLS tumors produced abundant amounts of infectious A-MuLV particles while most ABPL tumors, in striking contrast, did not. In an effort to understand the molecular mechanisms involved in the genesis of these tumors, a detailed study of the expression of abl, myc and myb oncogenes was undertaken (Mushinski et al., 1983).

An N-ras transforming gene was detected in human rectal carcinoma-derived cells (7060) and molecularly cloned. The genetic lesion responsible for the transforming activity of the 7060 oncogene was localized to a single nucleotide transition from A to T in codon 61 of the predicted protein. This lesion in the second exon results in substitution of histidine for glutamine at this position. We also found an EcoRI restriction fragment length polymorphism, consisting of two alleles, of the human c-myb gene. The 7060-transformed epithelial cells showed the homozygous phenotype, while normal fibroblasts of the same patient showed the heterozygous phenotype. This suggests a relationship between the phenotypic change in the c-myb locus and the induction of the 7060 tumor.

32D C13(G) is an interleukin 3(IL3)-dependent non-tumorigenic murine hematopoietic cell line which undergoes terminal differentiation into granulocytes when exposed to granulocytic colony stimulating factor (G-CSF). Infections of 32D C13(G) cells with either Kirsten rat sarcoma virus or Balb murine sarcoma virus, both containing a v-ras oncogene, generates clones that can permanently grow in G-CSF without differentiation. 32D-Ki-ras cells show a heterogeneous morphology ranging from the promyelocytic to the myelocytic stage of differentiation, and express high levels of both myeloperoxidase (MPO) and lactoferrin (LF) mRNA. 32D-Ha-ras cells show a more immature phenotype and express MPO but no LF mRNA. The apparent differentiation block of both 32D Ki-ras and 32D Ha ras can be reversed by treatment with the chemical inducers retinoic acid, sodium butyrate or dimethylsulphoxide, which leads to terminal differentiation into granulocytes. When 32D-Ki-ras and 32D-Ha-ras cells are cultured in medium containing IL-3 they become adherent and express some monocyte-macrophage markers. Upon prolonged exposure to IL3, 32D-Ki-ras, but not 32D-Ha-ras, resume suspension growth. Both 32D-Ki-ras and 32D-Ha-ras rapidly die if grown in chemically defined medium in the absence of any growth factor and are non-tumorigenic in immunosuppressed mice. These findings indicate that ras activation may interfere with the normal response to growth and differentiation factors in cells of the granulocytic lineage. These alterations may represent a critical, although non-sufficient, step in leukemogenesis.

Nonrandom involvement of chromosome 6 in cutaneous malignant melanoma have been noted by several investigators. Recently an alteration in the c-myb locus (6q22-23) has been identified by Southern analysis in the WM983A cell line which was derived from a primary melanoma of the vertical growth phase. In the present study, the nature of this rearrangement in the WM983A cell line has been further characterized by molecular cloning and nucleotide sequence analysis of the break-point region in the c-myb locus. The results of this investigation demonstrate that the rearrangement in the 6q22-23 region results in deletion of the 3'-end of the c-myb locus with the concomitant translocation of a portion of chromosome 12 to chromosome 6.

Bacteriophage libraries of mouse DNA were screened for sequences homologous to the v-myb oncogene and two overlapping clones containing the v-myb related region were isolated. Restriction enzyme mapping, heteroduplex analysis and nucleotide sequence analysis revealed the presence of nine exons. Six of these exons are homologous to the v-myb region while the other three exons are derived from the 5' region which is deleted in the viral oncogene. The sequences downstream to the sixth v-myb exon are not included in the 17 kbp of DNA sequences analyzed in this study. Comparison of the structure of the normal c-myb clone with its rearranged couterpart present in plasmacytoid lymphosarcomas revealed that the rearrangements occur in this locus as a result of viral integration. Present studies demonstrate that such a viral insertion interrupts the c-myb coding region at a region identical to that observed in the generation of the v-myb gene of avian myeloblastosis virus and results in the synthesis of mRNAs that lack the same 5' coding region.

The c-myb proto-oncogene is the cellular homolog of the avian myeloblastosis virus oncogene (v-myb) (Roussel et al., 1979; Souza et al., 1980a; Bergman et al., 1981). The boundaries of c-myb are not yet known; however, studies have shown that the v-myb sequence is transduced from a portion of the coding region of c-myb into the avian myeloblastosis virus (AMV) (Klempnauer and Bishop 1983). AMV is a retrovirus that causes myeloblastic or monocytic leukemia in chickens and transforms myelomonocytic hematopoietic cells in culture (Moscovici 1975). The v-myb sequence is thought to be essential for the oncogenic potential of AMV (Duesberg et al., 1980; Souza 1980a; Gonda et al., 1981) and it appears that only certain target cells are responsive to the v-myb gene product (Moscovici 1975).

The results summarized in this review show that the normal chicken myb gene codes for a protein of 77 kd which appears to play an important role in the control and/or differentiation of hematopoietic cells of myeloid and T lymphoid series. The activation of this gene has been observed in chicken and murine systems. In the avian system, this has been achieved by transduction of the myb oncogene into a retrovirus. Such a transduction resulted in the deletion of coding sequences from both the 5' and 3' ends of the gene. Initiation and terminator codons in helper viral sequences have been substituted for the analogous sequences in the proto-oncogene. Deletion of similar stretches of sequence in both the viruses suggested the possibility that these deletions may play an important role in the activation of this gene. The availability of the murine model system allowed us to examine this question further. In the ABPL tumor system, the activation of the myb locus occurred as a result of viral integration in a region immediately upstream to the v-myb related sequences. In NSF-60 cell line, the activation is due to the viral integration toward the 3' end of the gene. In both cases the viral integration results in the synthesis of aberrant mRNAs that have suffered deletions similar to those observed in the avian system. In all instances this results in the synthesis of truncated proteins which appear to mediate the transforming function. The availability of chicken and mouse c-myb cDNA clones makes it possible to test this hypothesis directly by construction of retroviruses containing various deletion mutations.

The nucleotide sequence of the integrated avian myeloblastosis virus long terminal repeat has been determined. The sequence is 385 base pairs long and is present at both ends of the viral DNA. The cell-virus junctions at each end consist of a 6-base-pair direct repeat of cell DNA next to the inverted repeat of viral DNA. The long terminal repeat also contains promoter-like sequences, an mRNA capping site, and polyadenylation signals. Several features of this long terminal repeat suggest a structural and functional similarity with sequences of transposable and other genetic elements. Comparison of these sequences with long terminal repeats of other avian retroviruses indicates that there is a great variation in the 3' unique sequence (U3), whereas the 5' specific sequences (U5) and the R region are highly conserved.

Using a panel of anti-myb antibodies, we have examined the c-myb proteins present in the NFS-60 and ABPL-1, ABPL-2, and ABPL-4 tumor cell lines, all of which have an altered myb locus due to viral insertional mutagenesis. As predicted from previous DNA and RNA analysis, NFS-60 cells produce myb protein with a truncated C-terminus and a normal N-terminus. The three ABPL tumor cell lines studied here were previously shown to have undergone similar rearrangements towards the 5' end of myb locus and were expected to synthesize identical myb RNAs and proteins. These cell lines were found to produce myb proteins with N-terminal modifications. Surprisingly, however, the three cell lines were found to synthesize a heterogenous array of myb proteins of different sizes. These observations suggest that oncogenic activation of myb in ABPL tumors may involve additional changes in the coding region in addition to those occurring at the N-terminus.

Total cellular poly(A)-enriched RNA from a variety of fresh human leukemic blood cells and hematopoietic cell lines was analyzed for homology with molecularly cloned DNA probes containing the onc sequence of Abelson murine leukemia virus (Ab-MuLV), Harvey murine sarcoma virus (Ha-MuSV), simian sarcoma virus (SSV), and avian myelocytomatosis virus strain MC29. Results with the fresh blood cells paralleled those obtained with the cell lines. With Ab-MuLV and Ha-MuSV, multiple RNA bands were visualized in all cell types examined without significant variation in the relative intensities of the bands. When SSV was used as the probe, expression of related onc sequences was absent in all of the hematopoietic cell types examined except for one neoplastic T-cell line (HUT 102), which produces the human T-cell leukemia (lymphoma) retrovirus HTLV. In this cell line, a single band (4.2 kilobases) was observed. With MC29 as the probe, a single band of 2.7 kilobases was visualized in all cell types examined with only a 10 to 2-fold variation in intensity of hybridization. An exception was the promyelocytic cell line, HL60, which expressed approximately 10-fold more MC29-related onc sequences. With induction of differentiation of HL60 with either dimethyl sulfoxide or retinoic acid, a marked diminution in the amount of the MC29-related, but not the Ab-MuLV-related, onc message was observed.

A single genetic alteration, a guanine-to-cytosine transversion, is responsible for the acquisition of malignant properties by K-ras genes of two human tumor cell lines established from carcinomas of the bladder (A1698) and lung (A2182). As a consequence, arginine instead of the normal glycine is incorporated into the K-ras-coded p21 proteins at amino acid position 12. This mutation creates a restriction enzyme polymorphism that can be used to screen human cells for transforming K-ras genes. This approach was used to identify the mutational event responsible for the malignant activation of a K-ras oncogene in a squamous cell lung carcinoma of a 66-year-old man; this point mutation was not present in either the normal bronchial or parenchymal tissue or in the blood lymphocytes. Hence, malignant activation of a ras oncogene appears to be specifically associated with the development of a human neoplasm.

The murine diploid hematopoietic cell line 32D Cl3 strictly requires interleukin-3 (IL-3) for proliferation. When 32D Cl3 cells are transferred to IL-3-free medium which contains recombinant human granulocyte colony stimulating factor (rhG-CSF), the cell number increases four- to five-fold, and after 14 days the whole cell population is differentiated into morphologically normal and myeloperoxidase- and lactoferrin-positive metamyelocytes and granulocytes. Infection with Abelson murine leukemia virus (A-MuLV) of 32D Cl3 cells growing in the presence of IL-3 induces, within 2 weeks, the appearance of cells that are IL-3-independent for growth. The latter cells lack myeloid, T and B cell markers, and are unable to differentiate, even in the presence of very high doses of rhG-CSF. However, once the 32D Cl3 cells have been exposed to G-CSF, they become resistant to the transforming effects of A-MuLV as judged by the appearance of the IL-3-independent clones. These findings suggest that the ability of Abelson virus to transform immature progenitor cells is due to interference of the v-abl gene product with the mechanisms that control the commitment of the cells to differentiate.

The genome of the avian myeloblastosis virus (AMV) has undergone a sequence substitution in which a portion of the region normally coding for the env protein has been replaced by cellular sequences. We have determined the complete nucleotide sequence of this region. Examination of the AMV oncogenic sequence revealed an open reading frame starting with the initiation codon ATG and terminating with the triplet TAG within the acquired cellular sequences and terminating with the triplet TAG at a point thirty-three nucleotides into helper viral sequences to the right of the helper-viral-cellular junction. The stretch of 795 nucleotides would code for a protein of 265 amino acids with a molecular weight of 30,000 daltons. The eleven amino acids at the carboxy terminus of such a protein would be derived from the env gene of helper virus. Antibodies were prepared against synthetic peptides derived from the predicted amino acid sequences. One such antibody precipitated two magnesium proteins of apparent nucleotide weight of 30,000 daltons and 51,000 daltons.