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

Abstract

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 1OH, 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 1OH 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 T1 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 lOC and 1OH had lost two additional myc oligonucleotides, and mutant 1OA 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. 754 on July 7, 2020 by guest http://jvi.asm.org/ Downloaded from DELETION MUTANTS OF MC29 755 Recently, the chicken locus c-myc, which is related VOL. 41, 1982 on July 7, 2020 by guest

Inhibition of DNA binding of purified p55v-myc in vitro by antibodies against bacterially expressed myc protein and a synthetic peptide.

“…markers (from top to bottom): 97 K, 68 K, 53 K, 44 K, 30 K. acids specific for the carboxy-terminal half of the viral myc gene (corresponding to amino acids 228 -425) (Alitalo et al, 1983a). The cloned region comprises those sequences which presumably are deleted from the three non-transforming deletion mutants (Bister et al, 1982). The MS2-myc fusion protein was eluted from gels and used for immunization.…”

Phosphorylation of specific sites in the gag-myc polyproteins encoded by MC29-type viruses correlates with their transforming ability.

“…It has been proposed that these similarities in both pathogenicity and in vitro transformation potential are due to the presence of a MC29 subgroup-specific sequence (v-myc) derived from a cellular gene, c-myc (Bister and Duesberg, 1980;Bishop et al, 1980;Stehelin et al, 1980;Graf et al, 1980). Since then studies using transformation-defective (td) mutants of MC29 have defined essential and non-essential regions of v-myc, the oncogene of MC29 Bister et al, 1982;. In MC29, CM1 1, and MH2 transformed non-producer cells the only viral gene products detected were polyproteins of 110 000 (p110), 90 000 (p90), and 100 000 (p100) daltons, respectively (Bister et al, 1977(Bister et al, , 1980aHayman et al, 1979;Hu et al, 1978).…”

“…In addition, a subgenomic mRNA was detected in OK10 transformed cells which could code for a v-myc (Chiswell et al, 1981). That these proteins have a role in transformation rests on three lines of evidence: (i) they are transformation specific, (ii) they are the only gene products so far detected in transformed cells, and (iii) td mutants of MC29 have genetic lesions that map to the v-myc domain of p110 Bister et al, 1982;.…”

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

“…Conventional genetic analysis of the v-myc gene has yielded little information regarding the structurally and functionally important regions of the v-myc gene product. Ramsay et al (41) isolated three spontaneous MC29 mutants containing deletions ranging from 200 to 600 nucleotides in the myc gene (10,42). These mutants transform chicken and quail fibroblasts but exhibit significantly reduced efficiencies for transformation of chicken cells of hematopoietic origin (41).…”