Molecular cloning of the avian acute transforming retrovirus MH2 reveals a novel cell-derived sequence (v-mil ) in addition to the myc oncogene

Abstract

Mill‐Hill‐2 virus (MH2) proviral DNA was cloned from a transformed non‐producer cell culture (MH2QB2) through insertion of randomly cut high mol. wt. cellular DNA in the lambdoid vector L47.1. Restriction analysis of a suitable recombinant phage by Southern DNA blotting and hybridization with different probes allowed us to characterize the genetic organization of the provirus and to identify a novel MH2‐specific sequence of at least 1.1kbp. Such a sequence, for which we propose the name v‐mil, from MilI‐Hill‐2 virus, is not homologous to v‐myc, the previously described oncogene of MH2, nor to avian leukaemia virus‐related sequences. Evidence is presented here that v‐mil has a cellular counterpart (c‐mil) phylogenetically conserved in birds and mammals, including man, and expressed as a single RNA species at least in some tissues. MH2 virus might thus be regarded, like avian erythroblastosis virus or E26, as another example of retroviruses having recombined with more than one cellular gene.

Protein stabilization: a common consequence of mutations in independently derived v-Myc alleles

“…c-Myc (KK 54,54 RR 54,55 ) was generated by ligation of a PstI ± SacI linker containing the appropriate mutation into spt ± c-Myc (Crouch et al, 1993), and the subsequent HindIII fragment ligated into SFCV-sa 7 (La Rocca et al, 1994). For the construction of SFCV ± MH2 vMyc, a 285 bp amino terminal fragment and a carboxy terminal 621 bp fragment were generated by PCR from a pMH2 proviral cDNA template (Coll et al, 1983). The primers pairs used were 5'-gacaagcttatcgataccatgccgctcagcgccagc-3' and 5'-cgtcaccatctccagctggtcggcgg-3' for the amino terminal fragment, and 5'-gacaagcttctatgcacgagagttcttt-3' and 5'-gcggctctgctgggggtcgacacgccgccc-3' for the carboxy terminal fragment.…”

“…The primers pairs used were 5'-gacaagcttatcgataccatgccgctcagcgccagc-3' and 5'-cgtcaccatctccagctggtcggcgg-3' for the amino terminal fragment, and 5'-gacaagcttctatgcacgagagttcttt-3' and 5'-gcggctctgctgggggtcgacacgccgccc-3' for the carboxy terminal fragment. After HindIII/PstI digestion and HindIII/ClaI digestion of the amino terminal and carboxy terminal fragments respectively, the puri®ed fragments were ligated into the HindIII site of SFCV-sa + , together with a central 584 bp PstI/ClaI spanning exon 2/exon 3 boundary, which was isolated from pMH2 (Coll et al, 1983). All sequences generated by PCR or mutagenesis were con®rmed by doublestranded sequencing using T7-Sequenase v2.0 essentially as described by the manufacturers (Amersham).…”

Protein product of proto-oncogene c-mil.

“…The genome of the highly oncogenic avian retrovirus MH2 contains two cell-derived sequences, termed v-mil and vmyc (4,9,11,13). The v-myc oncogene of MH2 is expressed via a subgenomic mRNA (17) as a 59,000/61,000-molecularweight protein (5,18) and is closely related to v-myc alleles present in the genomes of three other independently isolated avian RNA tumor viruses (1; K. Bister and H. W. Jansen, Adv.…”

A revised and complete map of the chicken c-mil/raf-1 locus

“…B-raf and raf-1 orthologs have c-raf-1 been identified in bony fish, amphibians, birds, and mammals, suggesting that B-raf and c-raf-1 are the result of a gene duplication event that occurred before the divergence of the teleost and tetrapod lineages (T.B., unpublished data). The discovery of the c-raf-1 protooncogene dates back to 1927 when Begg isolated the Mill Hill 2 (MH 2) retrovirus, which causes endotheliomas and ovarian carcinomas in chicken (Vogt et al, 1979;Coll et al, 1983). MH2 contains two unrelated, retroviral oncogenes that were designated as v-myc and v-mil (Jansen et al, 1983a, b;Kan et al, 1983).…”

“…Subsequent analysis of v-mil revealed its high sequence homology to the v-raf oncogene of the murine sarcoma retrovirus 3611 (Kan et al, 1984;Sutrave et al, 1984). Further analyses demonstrated that v-mil and v-raf arose independently by retroviral transduction from cellular genes and led to the identification of the avian cmil and mammalian raf-1 genes, respectively (Coll et al, 1983;Jansen et al, 1984;Bonner et al, 1986;Koenen et al, 1988). Subsequently, the chicken c-mil/raf-1 locus was localized to the microchromosomal fraction, a hallmark of the chicken karyotype (Symonds et al, 1986).…”