Avian Myelocytomatosis Virus Research Articles

Nucleotide sequence analysis of the proviral genome of avian myelocytomatosis virus (MC29).

The nucleotide sequence of the integrated proviral genome of avian myelocytomatosis virus (MC29) coding for gag-myc protein has been determined. By comparison of this nucleotide sequence with the helper virus as well as the c-myc region, it was possible to localize the junction points between helper viral and v-myc sequences. These studies demonstrate that (i) the large terminal repeat sequence of MC29 is very similar to that of Rous sarcoma virus, (ii) the viral genome has suffered extensive deletions in the gag, pol, and env genes, (iii) the gag region can code for p19, p10, and part of p27, (iv) the recombination between viral and cellular sequences occurred in the coding region of p27 such that the open reading frame extends for an additional stretch of 1,266 base pairs, resulting in a gag-myc hybrid protein, (v) the open reading frame terminated within the v-myc region 300 bases upstream of v-myc-helper viral junction, and (vi) the v-myc helper-viral junction at the 3' end occurred in the middle of env gene, rendering it defective.

Human and mouse cellular myc protooncogenes reside on chromosomes involved in numerical and structural aberrations in cancer.

A molecular clone of viral myc (v-myc), the oncogene of avian myelocytomatosis virus, MC29, detected homologous human, mouse, and Chinese hamster cellular myc (c-myc) sequences by Southern filter hybridization. A v-myc probe, containing sequences from the 3' domain of the gene, hybridized to single human HindIII and mouse EcoRI genomic DNA fragments of the cellular myc genes whose segregation could be followed in interspecies somatic cell hybrids. Human c-myc segregated concordantly with the enzyme marker glutathione reductase and with a karyotypically normal chromosome 8. A rearrangement of human c-myc was observed in Burkitt's lymphoma cells possessing the t(8;14) translocation. These results suggest that human c-myc is located close to the breakpoint on chromosome 8 (q24) involved in the t(8;14) translocation. The mouse c-myc gene segregated concordantly with chromosome 15 in mouse-Chinese hamster cell hybrids. These gene assignments are noteworthy, as structural and numerical abnormalities of human chromosome 8 and mouse chromosome 15 are associated frequently with B-cell neoplasms.

Nucleotide sequence analysis of the chicken c-myc gene reveals homologous and unique coding regions by comparison with the transforming gene of avian myelocytomatosis virus MC29, delta gag-myc.

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.

Altered pathogenicity of avian myelocytomatosis (MC29) viruses with mutations in the v-myc gene

Avian myelocytomatosis virus MC29 inoculated into the wing web of 1-day-old Brown Leghorn chickens causes a high incidence of tumors, predominantly endotheliomas that are apparently induced directly by the action of the viral myc gene. Mutants of MC29, in which portions of the v-myc gene have been deleted and which have reduced ability to transform macrophages in vitro, induce few tumors, among which lymphomas and osteopetrosis predominate. Analysis of lymphomas from birds infected with mutant MC29 suggested that they were a result of helper virus action rather than the mutant MC29.

Nucleotide sequence to the v-myc oncogene of avian retrovirus MC29.

Avian myelocytomatosis viruses are retroviruses whose oncogene (v-myc) induces an unusually wide variety of tumors, including carcinomas, endotheliomas, sarcomas, and myelocytomatoses. The viral gene v-myc arose by transduction of an undetermined portion of a cellular gene known as c-myc. In order to facilitate further studies of the functions of v-myc and c-myc and to permit detailed comparisons between the two genes, we have determined the nucleotide sequence of v-myc in the genome of the MC29 strain of myelocytomatosis virus. The v-myc domain in MC29 virus encodes a hydrophilic polypeptide with a molecular weight of 47,000, fused to a portion of the polyprotein encoded by the viral structural gene gag. The carboxyl-terminal half of the v-myc polypeptide is rich in basic amino acid residues. This feature may account for the DNA-binding properties of the hybrid gag-myc-encoded protein which would have a molecular weight of approximately 100,000, in accord with results from previous studies of the protein encoded by v-myc. The junctions between v-myc and the genome of the transducing virus are apparent but reveal no clues to the mechanism by which transduction might occur.

The human onc gene c-myc: structure, expression, and amplification in the human promyelocytic leukemia cell line HL-60.

Substantial evidence indicates that retroviral transforming (v-onc) genes originated by means of recombination between a present nontransforming virus and normal cellular sequences (Duesberg et al., this volume). These sequences, called cellular onc gene, are highly conserved during evolution, suggesting that they may code for protein products which are essential for cell growth or tissue differentiation. As these normal cellular genes are homologous to viral- transforming genes, their potential role in tumorigenesis is of great interest. As an alternative to direct transformation by a viral onc gene, abnormal activation of a cellular onc gene may cause transformation. Two models have been proposed for such a mechanism. First, high levels of expression of a cellular onc gene may be caused by the insertion nearby of a viral promoter [12, 15, 16, 17] or by alteration of the physiological promoter by a mutagenic agent such as a chemical carcinogen. Secondly, a cellular onc gene may be relocated in a transcriptionally active region of the genome as a consequence of chromosomal rearrangements [2, 5, 6, 13]. In this chapter we review the evidence for a possible third mechanism for onc gene activation in neoplastic cells, that of gene amplification. The human homologue, c-myc, of the transforming gene of avian myelocytomatosis virus (MC29), which is expressed at relatively high levels in the human promyelocytic leukemia cell line HL-60, is stably amplified in the genome of these cells [7]. Amplification was also detected in primary, uncultured leukemic cells from the same individual, suggesting that the c-myc amplification may have been involved in the leukemic transformation in this case.

Characterization of the expression of cellular retrovirus genes and oncogenes in feline cells.

Expression of endogenous retrovirus genes and two different cellular oncogenes (c-onc genes) was examined at the transcriptional level in a variety of normal and lymphoma/leukemia tissues of the domestic cat. The two oncogenes, c-myb(related to avian myeloblastosis virus) and c-myc(related to avian myelocytomatosis virus) were selected for their association with the induction of hematopoietic malignancies, when present in the transforming retroviruses. Tissue-specific expression of endogenous feline leukemia virus (FeLV)-related genes was detected in cellular subpopulations of the cat placenta by in situ method of hybridization. Gel blotting analysis of placental poly(A)-selected RNA revealed that the FeLV-related RNA species were primarily subgenomic, representing the env gene region of the endogenous provirus elements. Like the endogenous retrovirus genes, c-myb and c-myc loci of the cat genomic DNA were also transcribed at differential levels in normal tissues of the cat. Dot-blot hybridization analysis showed that the expression of these two oncogenes was linked to growth and development as it varied with the gestational age of the fetus and from fetal to adult tissues. Among the major hematopoietic organs, spleen and bone marrow contained both c-myb and c-myc transcripts, while thymus preferentially expressed the c-myb gene. In contrast to the low level of c-myc expression in fetal thymus tissues, enhanced c-myc expression was detected in all five thymomas tested and also in several other neoplasms including two granulocytic leukemias. The feline c-myb gene was not very active in granulocytic leukemias or in three of the five thymomas. RNA gel blotting analysis of poly(A)-selected RNA of a thymoma and its lymph node metastasis showed identical pattern of c-myc transcripts.

Transcriptionally active c-myc oncogene is contained within NIARD, a DNA sequence associated with chromosome translocations in B-cell neoplasia.

NIARD (non-immunoglobulin-associated rearranging DNA) is located on mouse chromosome 15 at the break point of a commonly observed translocation event involving chromosomes 15 and 12 in murine plasmacytomas. The human cellular analogue of the v-myc oncogene of avian myelocytomatosis virus, strain MC-29, is known to reside on the distal end of human chromosome 8 and has been observed to translocate to chromosome 14 in Burkitt lymphomas. Using a cDNA clone specific for the transcript of the human c-myc gene (H c-myc), we show that the mouse c-myc (M c-myc) gene is contained within NIARD. NIARD-associated chromosome translocations occurred 1.3-2 kilobases (kb) 5' of the mouse c-myc gene where NIARD recombines with the switch region of the C(alpha) immunoglobulin gene in various murine plasmacytomas. The mouse c-myc encoding region within NIARD spanned <2.4 kb of DNA and expressed a low level of a 2.3-kb polyadenylylated RNA in BALB/c spleen. Increased (10- to 20-fold) levels of rearranged mouse c-myc transcripts (i.e., approximately 1.8-2.1 kb) were observed in plasmacytomas that have NIARD-associated chromosome translocations. Human c-myc and NIARD probes detected DNA rearrangements of human c-myc in four of seven Burkitt lymphomas. DNA sequences adjacent to the human c-myc gene recombined with the C(mu) immunoglobulin gene locus on chromosome 14 in several Burkitt lymphomas. The activation of the c-myc oncogene by chromosome translocation implicates its involvement in B-cell oncogenesis.

Virus gene expression in rat cells transformed by avian myelocytomatosis virus strain MC29 and avian erythroblastosis virus.

Virus gene expression in rat cells transformed by either avian myelocytomatosis virus strain MC29 or avian erythroblastosis virus has been studied by biological and biochemical methods. In the clones examined, virus-specific sequences were found to be transcribed into RNA and, in most clones, the characteristic gag-related proteins could be identified. The transformed rat cells were fused to permissive chick cells and the rescued virus was shown to transform both chick embryo fibroblasts and the appropriate haemopoietic cell type in chick bone marrow cultures. These results clearly demonstrate that, as with the non-defective avian sarcoma viruses, the genetic information responsible for transformation by the defective avian leukaemia viruses can be expressed in non-permissive mammalian host cells as well as in permissive avian cells.

Mouse c-myc oncogene is located on chromosome 15 and translocated to chromosome 12 in plasmacytomas.

Hybridization studies with viral oncogene probes indicate that c-myc, the cellular gene homologous to the transforming gene of avian myelocytomatosis virus, resides on mouse chromosome 15 and in many plasmacytomas is translocated to the antibody heavy chain gene locus on chromosome 12. The transcriptional orientation of the translocated c-myc sequence is opposite the orientation of the adjacent C alpha gene that codes for the heavy chain of immunoglobulin A. The translocated c-myc sequence is not the same oncogene detected in urine plasmacytomas by the NIH-3T3 cell transformation assay.

Human c-myc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells.

Human sequences related to the transforming gene (v-myc) of avian myelocytomatosis virus (MC29) are represented by at least one gene and several related sequences that may represent pseudogenes. By using a DNA probe that is specific for the complete gene (c-myc), different somatic cell hybrids possessing varying numbers of human chromosomes were analyzed by the Southern blotting technique. The results indicate that the human c-myc gene is located on chromosome 8. The analysis of hybrids between rodent cells and human Burkitt lymphoma cells, which carry a reciprocal translocation between chromosomes 8 and 14, allowed the mapping of the human c-myc gene on region (q24 leads to qter) of chromosome 8. This chromosomal region is translocated to either human chromosome 2, 14, or 22 in Burkitt lymphoma cells.

Migrant oncogene-Burkitt's lymphoma link

Two teams of investigators have provided the first evidence that the translocation of a single piece of DNA from one chromosome to another involves and perhaps "turns on" an oncogene in human cells. This evidence was presented at a recent Bristol-Myers symposium, "Chromosomes and Cancer: From Molecules to Man," at the University of Chicago. The laboratories of Carlo M. Croce, PhD, associate director of the Wistar Institute in Philadelphia, and Philip Leder, MD, chair, Department of Genetics, Harvard Medical School, Boston, pursued different avenues of research to arrive at the same conclusion: The translocation between the terminal ends of chromosomes 8 and 14 in Burkitt's lymphoma cells involves an exchange of two genes (among others)— one that codes for a region of the antibody molecule and one that is an oncogene called the<i>myc</i>gene (the human homologue of the avian myelocytomatosis virus). The translocation, which occurs in 90% of

Migrant oncogene-Burkitt's lymphoma link

ABSTRACT Two teams of investigators have provided the first evidence that the translocation of a single piece of DNA from one chromosome to another involves and perhaps turns on an oncogene in human cells. This evidence was presented at a recent Bristol-Myers symposium, Chromosomes and Cancer: From Molecules to Man, at the University of Chicago.The laboratories of Carlo M. Croce, PhD, associate director of the Wistar Institute in Philadelphia, and Philip Leder, MD, chair, Department of Genetics, Harvard Medical School, Boston, pursued different avenues of research to arrive at the same conclusion: The translocation between the terminal ends of chromosomes 8 and 14 in Burkitt's lymphoma cells involves an exchange of two genes (among others)— one that codes for a region of the antibody molecule and one that is an oncogene called the myc gene (the human homologue of the avian myelocytomatosis virus). The translocation, which occurs in 90% of

Recovery of myc-specific sequences by a partially transformation-defective mutant of avian myelocytomatosis virus, MC29, correlates with the restoration of transforming activity.

Avian myelocytomatosis virus MC29 transforms fibroblasts and macrophages in vitro. Recently we isolated three deletion mutants of MC29 that have a decreased ability to transform macrophages while retaining their capacity to transform fibroblasts. One of these mutants, MC29 td10H, on passage through chicken embryo cultures gave rise to a recovered virus MC29 10H B1, which has regained the ability to transform macrophages efficiently. Immunoprecipitation analysis of MC29 10H B1-infected cells revealed a 108,000-dalton gag-myc polyprotein as opposed to the 90,000-dalton protein of MC29 td10H or the 110,000-dalton polyprotein of wtMC29. Tryptic peptide mapping studies demonstrated that the 108,000-dalton protein had acquired v-myc peptides that were lost from the td10H 90,000-dalton polyprotein and two novel peptides. Restriction enzyme analysis of the MC29 10H B1 proviral DNA also showed that myc sequences had been acquired. These results suggest that MC29 td10H has recombined with c-myc sequences to generate a recovered virus, MC29 10H B1.

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.

Isolation and characterization of c-myc, a cellular homolog of the oncogene (v-myc) of avian myelocytomatosis virus strain 29.

The chicken genome contains nucleotide sequences homologous to transforming genes (oncogenes) of a number of avian retroviruses. We have isolated chicken DNA (c-myc) that is homologous to the oncogene (v-myc) of the avian myelocytomatosis virus MC29 and have compared the structures of the cellular and viral genes. Results from restriction endonuclease mapping of c-myc and from analysis of heteroduplexes between the DNAs of the cellular and viral genes show that c-myc is homologous to 1,500 nucleotides in v-myc DNA. This homologous region is interrupted in c-myc by an intron-like sequence of 1,100 nucleotides which is absent from v-myc. Nuclear RNA from normal chicken cells contains at least five species of transcripts from c-myc ranging from 2.5 to 6.5 kilobases in length. By contrast, cytoplasm contains only the 2.5-kilobase c-myc RNA. These features of the c-myc gene and its nuclear transcripts are characteristic of normal cellular genes and suggest that the myc gene is of cellular rather than viral origin. The exons in c-myc may define two functional domains in the gene and may therefore facilitate the dissection of the different oncogenic potentials of the MC29 virus.

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.

Expression of cellular homologues of retroviral onc genes in human hematopoietic cells.

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.

Isolation and biochemical characterization of partially transformation-defective mutants of avian myelocytomatosis virus strain MC29: localization of the mutation to the myc domain of the 110,000-dalton gag-myc polyprotein.

Recently, we isolated three mutants of MC29 virus which, although able to transform fibroblasts with the same efficiency as wild-type MC29, were 100-fold less efficient at transforming macrophages. In this study we found that MC29-transformed quail producer cell line Q10 was able to generate these partially transformation defective mutants at a high frequency. Using tryptic peptide mapping, we determined that the smaller gag-myc polyproteins encoded by the transformation-defective viruses had lost myc-specific tryptic peptides. This suggested that the mutations which resulted in the transformation-defective viruses being inefficient at transforming macrophages were located in the v-myc sequence and thus directly implicated v-myc and the gag-myc polyprotein in transformation by MC29.

Nuclear localization and DNA binding of the transforming gene product of avian myelocytomatosis virus.

Oncornaviruses transform cells either directly through a virus-coded oncogene1 or, if they lack such a gene, indirectly by promoter insertion2–4 into the cellular genome and activation of a cellular gene. Both transformation mechanisms ultimately result in abnormally high expression of normal genes. Recently, the activated cellular gene in certain lymphomas4 has been shown to be homologous to the oncogene of an acute avian leukaemia virus, MC29. In MC29 the oncogene is fused to the viral structural gene, gag. The product of this fused gene is a protein of molecular weight 110,000 (110 K)5–7, designated p110gag–myc. We have characterized this protein by using monoclonal antibodies against p19, the N-terminal portion of the gag–myc fusion or 110 K protein and purified it 3,700-fold by immune affinity column chromatography. Immunofluorescence studies and cell fractionation of MC29-transformed fibroblasts indicate that the 110 K protein is predominantly located in the nucleus. Moreover, the purified protein binds to double-stranded DNA. These properties may be related to the role of the protein in transformation.