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

Ramsay, Gary; Hayman, Michael J.

doi:10.1128/jvi.41.3.745-753.1982

Cited by 52 publications

(15 citation statements)

References 35 publications

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“…However, deletion of as few as 11 amino acids within this same region (CH201) greatly reduced the efficiency of bone marrow cell transformations. Our results agree with and extend the previously reported results of Ramsey et al (41,42), who reported that large deletions within the 3' portion of the myc gene sequences greatly reduced the efficiency of hematopoietic cell transfor-mation. The deletion of 11 amino acids in the mutant CH201 resulted in the removal of residues within a particularly acidic region of the miyc protein (16 glutamic and aspartic acid residues within a sequence of 35 amino acids; Fig.…”

Section: Discussionsupporting

confidence: 93%

“…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).…”

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confidence: 99%

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

Heaney¹,

Pierce²,

Parsons³

1986

J Virol

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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, pAMst, resulted in morphological transformation, the synthesis of P11gag-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.

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Section: Discussionsupporting

confidence: 93%

mentioning

confidence: 99%

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

Heaney¹,

Pierce²,

Parsons³

1986

J Virol

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“…The loss of sequences from the viral RNAs was in good agreement with the observation that these mutant RNAs code for smaller gag-related proteins (Fig. 7) (24) which have lost myc-specific tryptic peptides (26).…”

Section: Resultssupporting

confidence: 87%

“…In this work we found that the altered oncogenic properties of these td mutants can be correlated with deletions within the transformation-specific (myc) sequences of their viral RNAs. The smaller genome sizes and the complete or partial loss of one to three myc-specific oligonucleotides are in excellent agreement with the analysis of the smaller gag-related polyproteins by peptide mapping techniques described in the accompanying paper (26). From these studies and from the data presented here, it clearly follows that all three of these mutants contain overlapping deletions of myc-specific sequences, increasing in size from mutant 1OA to lOC to 1OH.…”

Section: Discussionsupporting

confidence: 86%

“…forming (onc) gene of MC29; the viral RNA contains a specific sequence (myc) which is not related to known virion genes or to the src gene of Rous sarcoma virus (RSV) and is flanked by partial (A) sequence elements of gag and env (3,11,22). (Because of a recent proposal for a uniform nomenclature of transformation-specific RNA sequences of retroviruses [9a], the term myc is used throughout this paper and the accompanying paper [26]; this term is synonymous with mcv and mac, which we used previously [7,24].) The only viral protein product which is found in cells transformed by MC29 in the absence of helper virus (nonproducer cells) is a 110-kilodalton phosphorylated polyprotein (p110) that is antigenically related to the gag gene (4,5).…”

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confidence: 99%

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

Bister

Ramsay

Hayman

1982

J Virol

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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

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

Bunte¹,

Greiser‐Wilke²,

Donner³

et al. 1982

The EMBO Journal

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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.

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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

Cited by 52 publications

References 35 publications

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

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

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

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

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