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

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 10H, 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 10H 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 T(1) 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 10C and 10H had lost two additional myc oligonucleotides, and mutant 10A 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.     

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.     

Genome structure of HBI, a variant of acute leukemia virus MC29 with unique oncogenic properties.

We have analyzed the viral RNA of a variant of avian acute leukemia virus MC29, termed HBI. This virus was isolated during in vitro passage of a partially transformation-defective (td) mutant of MC29 (td10H-MC29) in chicken macrophages. While td10H-MC29 has a reduced ability to transform macrophages in vitro or to induce tumors in vivo, HBI-MC29 transforms macrophages efficiently and induces in vivo a high incidence of lymphoid tumors. Electrophoretic analysis of HBI-MC29 genomic RNA revealed that it has a complexity of 5.7 kilobases, like the RNA of wild-type (wt) MC29, and that it is 0.6 kilobases longer than the 5.1-kilobase RNA of the deletion mutant td10H-MC29. Analysis of the viral RNAs of two clonal isolates of HBI-MC29 by T1 oligonucleotide fingerprinting showed that sequences from the viral transformation-specific region, v-myc, which are deleted in td10H RNA, are present in HBI RNA. Moreover, hybridization of HBI RNA to molecularly cloned subgenomic fragments of wtMC29 proviral DNA, followed by fingerprint analysis of hybridized RNA, showed that the entire v-myc-specific RNA sequences defined previously are present. Hybridization to cloned DNA of the normal chicken locus c-myc shows a close relationship between HBI v-myc RNA and c-myc DNA, especially in the sequences which were deleted from td10H-MC29. T1 oligonucleotide maps of HBI and td10H RNAs were prepared and compared. Total conservation of the oligonucleotide pattern is observed in the overlapping v-myc regions, while the partial structural genes gag and env show some variations, most of which can be directly proven to be due to point mutations or recombination with helper viral RNAs that were analyzed in parallel. Recombination of td10H-MC29 with c-myc, followed by recombinational and mutational changes in the structural genes during passage with helper virus, could be a possible explanation for the origin of HBI.     

DNA sequence analysis of X-ray-induced deletions at the white locus of Drosophila melanogaster

We have determined the nucleotide sequence of 5 X-ray-induced white mutants containing small rearrangements. Comparison with wild-type sequences showed deletions in the coding region ranging in size between 6 bp and 29 bp. These small deletions are distributed non-randomly over the white locus. Two mutants contain the same 29-bp deletion, while the other 3 deletions are clustered. All 5 deletions have occurred between 2 and 3 bp repeats. One of the repeats is preserved in the novel junction formed by the deletion. Our results suggest that recombinational processes may be involved in the generation of X-ray-induced deletions.     

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.     

Induction of tumors and generation of recovered sarcoma viruses by, and mapping of deletions in, two molecularly cloned src deletion mutants.

td108 , a transformation-defective (td) deletion mutant of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup A (SR-A), was molecularly cloned. Two isolates of td viruses, td108 -3b and td108 -4a, obtained by transfection of the molecularly cloned td108 DNAs into chicken embryo fibroblasts, were tested for their ability to induce tumors and generate recovered avian sarcoma viruses ( rASVs ) in chickens. Both td viruses were able to induce tumors with a latency and frequency similar to those observed previously with biologically purified td mutants of SR-A. rASVs were isolated from most of the tumors examined. The genomic RNAs of those newly obtained rASVs were analyzed by RNase T1 oligonucleotide fingerprinting. The results showed that they had regained the deleted src sequences and contained the same set of marker src oligonucleotides as those of rASVs analyzed previously. The src oligonucleotides of rASVs are distinguishable from those present in SR-A. We conclude that those rASVs must have been generated by recombination between the molecularly cloned td mutants and the c-src sequence. The deletions in the td mutants were mapped by restriction enzyme analysis and nucleotide sequencing. td108 -3b was found to contain an internal src deletion of 1,416 nucleotides and to retain 57 and 105 nucleotides of the 5' and 3' src coding sequences, respectively. td108 -4a contained a src deletion of 1,174 nucleotides and retained 180 and 225 nucleotides of the 5' and 3' src sequences, respectively. Comparison of sequences in the 5' src and its upstream region of td108 -3b with those of SR-A, rASV1441 (a td108 -derived rASV analyzed previously), and c-src suggested that the 5' recombination between td108 and c-src occurred from 7 to 20 nucleotides upstream from the beginning of the src coding sequence.     

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

The putative transforming proteins of the four acute leukaemia viruses belonging to the MC29 subgroup were shown to be phosphorylated in vivo. Comparison of the MC29 and CM11 encoded phosphoproteins revealed identical tryptic phosphopeptide maps, with both the gag and myc domains being phosphorylated. In contrast, the MH2 phosphoprotein was only phosphorylated on the gag domain. Analysis of partial transformation-defective MC29 deletion mutants revealed that the deletions had removed the v-myc specific phosphopeptides. Phosphoamino acid analysis showed that these deleted phosphopeptides were phosphorylated on threonine. Moreover, a back mutant that had regained transforming ability had regained these phosphopeptides. These studies correlate the phosphorylation of the gag-myc protein with the transformation capability of the virus.     

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

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, p delta Mst, resulted in morphological transformation, the synthesis of P110gag-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.     

DNA base sequence changes in spontaneous and ethyl methanesulfonate-induced mutations of a chromosomally-integrated gene in Chinese hamster ovary cells

A series of spontaneous and ethyl methanesulfonate-induced 6-thioguanine-resistant mutants were isolated in the CHO-10T5 cell line. This cell line was constructed by the introduction of a shuttle vector containing the Escherichia coli gpt gene into a hypoxanthine-guanine phosphoribosyltransferase deficient derivative of the Chinese hamster cell line CHO-K1. Shuttle vector sequences were recovered from many of the mutant cell lines by the COS cell fusion technique and the DNA base sequence of the gpt genes was determined whenever possible.

The base sequences were determined for gpt genes recovered from 29 spontaneous mutants. Of these 29 mutants, 9 have single base substitutions, 1 has a small duplication, 17 have simple deletions, 1 has a deletion with additional bases inserted at the deletion site, and 1 has no change in the gpt coding sequence. Many of the deletions were less than 20 basepairs in length and several occurred in a region previously observed to be a hotspot for spontaneous deletions. The generation of the deletion/insertion mutation may have involved a quasi-palindromic intermediate.

A total of 59 ethyl methansesulfonate-induced mutants were isolated and vector sequences were recovered from 50 mutants. All 50 mutants sequenced had single base substitutions and most (45) were G:C to A:T transitions. While there were no strong hotspots in this collection of mutations, the site distribution was obviously nonrandom. Many of the G:C to A:T transitions either produced a nonsense codon or occurred at glycine codons.     

Definition of the Escherichia coli MC4100 genome by use of a DNA array

We have used an Escherichia coli K-12 whole-genome array based on the DNA sequence of strain MG1655 as a tool to identify deletions in another E. coli K-12 strain, MC4100, by probing the array with labeled chromosomal DNA. Despite the continued widespread use of MC4100 as an experimental system, the specific genetic relationship of this strain to the sequenced K-12 derivative MG1655 has not been resolved. MC4100 was found to contain four deletions, ranging from 1 to 97 kb in size. The exact nature of three of the deletions was previously unresolved, and the fourth deletion was altogether unknown.     

Identification of the viral sequence required for the generation of recovered avian sarcoma viruses and characterization of a series of replication-defective recovered avian sarcoma viruses.

The ability of transformation-defective deletion mutants of Schmidt-Ruppin Rous sarcoma virus to induce tumors and generate recovered sarcoma viruses (rASVs) was correlated with the partial src sequences retained in the transformation-defective viral genomes. Since all the transformation-defective viruses that were capable of generating rASVs retained a portion of the 3' src sequence, regardless of the extent of the 5' src deletion, and those lacking the 3' src were unable to generate rASVs, it appears that the 3', but most likely not the 5', src sequence retained in the transformation-defective viral genome is essential for rASV formation. However, rASVs derived from a particular mutant, td109, which retained a portion of the 3' src sequence, but lacked most (if not all) of the 5' src sequence, were all found to be defective in replication. Analyses of the genomic sequences of 13 isolates of td109-derived rASVs revealed that they contained various deletions in viral envelope (env), polymerase (pol), and structural protein (gag) genes. Ten isolates of rASVs contained env deletions. One isolate (rASV3812) contained a deletion of env and the 3' half of pol, and one isolate (rASV398) contained a deletion of env and pol. The one with the most extensive deletion (rASV374) had a deletion from the p12-coding sequence through pol and env. In addition, the 5' src region of td109-derived rASVs were heterogeneous. Among the 7 isolates analyzed in detail, one isolate of rASV had a small deletion of the 5' src sequence, whereas three other isolates contained extra new sequences upstream from src. Both env- and env- pol- rASVs were capable of directing the synthesis of precursor and mature gag proteins in the infected nonproducer cells. We attribute the deletions in the replication-defective rASVs to the possibility that the 5' recombination site between the td109 and c-src sequence, involved regions of only partial homology due to lack of sufficient 5' src sequence in the td109 genome for homologous recombination. A model of recombination between the viral genome and the c-src sequence is proposed to account for the requirement of the 3' src sequence and the basis for the generation of deletions in td109-derived rASVs.     

Nucleotide sequence analysis of human hypoxanthine phosphoribosyltransferase (HPRT) gene deletions.

We have determined the nucleotide sequences of 10 intragenic human HPRT gene deletion junctions isolated from thioguanine-resistant PSV811 Werner syndrome fibroblasts or from HL60 myeloid leukemia cells. Deletion junctions were located by fine structure blot hybridization mapping and then amplified with flanking oligonucleotide primer pairs for DNA sequence analysis. The junction region sequences from these 10 HPRT mutants contained 13 deletions ranging in size from 57 bp to 19.3 kb. Three DNA inversions of 711, 368, and 20 bp were associated with tandem deletions in two mutants. Each mutant contained the deletion of one or more HPRT exon, thus explaining the thioguanine-resistant cellular phenotype. Deletion junction and donor nucleotide sequence alignments suggest that all of these HPRT gene rearrangements were generated by the nonhomologous recombination of donor DNA duplexes that share little nucleotide sequence identity. This result is surprising, given the potential for homologous recombination between copies of repeated DNA sequences that constitute approximately a third of the human HPRT locus. No difference in deletion structure or complexity was observed between deletions isolated from Werner syndrome or from HL60 mutants. This suggests that the Werner syndrome deletion mutator uses deletion mutagenesis pathway(s) that are similar or identical to those used in other human somatic cells.     

Structural relationship between a normal chicken DNA locus and the transforming gene of the avian acute leukemia virus MC29.

We screened a recombinant chicken DNA/lambda phage library for sequences homologous to the transformation-specific sequences of the avian acute leukemia virus MC29 by hybridization with molecularly cloned MC29 proviral DNA. Three cellular DNA clones were found and compared with each other and with the viral genome by physical mapping with restriction endonucleases and by heteroduplex analysis. These experiments indicated that the three cellular clones overlap and represent a single cellular locus. The RNA genome of MC29 and normal cell DNA share a homologous region of 1.6 kilobases which is interrupted in the cellular DNA by 1.0 kilobase of sequences not present in the viral genome. Hybridization of the cloned cellular DNA to viral RNA and analysis of the protected viral RNA by fingerprinting techniques indicated that there is extensive sequence homology between the helper virus-unrelated mcv sequences of the viral RNA and the cellular DNA, with only minor base differences. The cellular mcv locus, however, lacks all helper virus-related sequences of MC29, including those of the partial viral gag gene which, together with mcv, encodes the probable transforming protein of MC29. We conclude that although the mcv locus of the normal cell does not represent a complete structural homolog to the onc gene of MC29, it is probably the precursor to the onc-specific sequence in the virus.     

Functional characterization of mutations in melanocortin-4 receptor associated with human obesity

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor implicated in the regulation of body weight. Genetic studies in humans have identified two frameshift mutations of MC4R associated with a dominantly inherited form of obesity. We have generated and expressed the corresponding MC4R mutants in 293T cells and found that cells transfected with the truncation mutants failed to exhibit agonist binding or responsiveness despite retention of structural motifs potentially sufficient for binding and signaling. Immunofluorescence studies showed that the mutant proteins were expressed and localized in the intracellular compartment but absent from the plasma membrane, suggesting that these mutations disrupted the proper cellular transport of MC4R. Further studies identified a sequence in the cytoplasmic tail of MC4R necessary for the cell surface targeting. We further investigated a possible dominant-negative activity of the mutants on wild-type receptor function. Co-transfection studies showed that the mutants affected neither signaling nor cell surface expression of wild-type MC4R. We also characterized three human sequence variants of MC4R, but these exhibited identical affinities for peptide ligands and identical agonist responsiveness. Thus, unlike the obesity-associated MC4R truncation mutants, the polymorphisms of MC4R are unlikely to be contributors to human obesity.     

Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene.

A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein.     

Evidence for p15 cleavage site in myc-specific proteins of avian MC29 and OK10 viruses

Myc-related proteins were precipitated from MC29 virus-transformed cells (PR-2) and from OK10 virus-transformed cells (9C) by anti-gag and anti-myc sera. Immunoprecipitates were cleaved with the avian retroviral protease p15 and the cleavage products analyzed in SDS-PAGE. Cleavage fragments of p110gag-myc (product of MC29 virus) and p58myc (product of OK10 virus) showed the presence of a p15 cleavage site within the myc-specific region. The site is missing in deletion mutants of MC29 virus.     

Transformation-defective mutants of Rous sarcoma virus with src gene deletions of varying length.

The RNAs of transformation-defective (td) deletion mutants of the Schmidt-Ruppin strain of Rous sarcoma virus were found to vary in size when compared by polyacrylamide gel electrophoresis. Three of seven td mutants appeared to recombine with a mutant of Rous sarcoma virus (Schmidt-Ruppin), which has a temperature-sensitive sarcoma (src) gene and is termed ts68, to give rise to recombinants with a reduced temperature sensitivity. The results suggested that different clones of td mutants exist: some in which the src gene appears to be deleted, and others in which the src gene is only partially deleted. A direct correlation between RNA size and the extent of src gene deletion measured by recombination was not obtained, possibly because the recombination assay could only detect src sequences homologous to the lesion(s) of ts68, whereas the electrophoretic analysis of the RNA measured src deletions as well as other possible alterations of the RNA.     

DNA and RNA from Uninfected Vertebrate Cells Contain Nucleotide Sequences Related to the Putative Transforming Gene of Avian Myelocytomatosis Virus

The avian carcinoma virus MC29 (MC29V) contains a sequence of approximately 1,500 nucleotides which may represent a gene responsible for tumorigenesis by MC29V. We present evidence that MC29V has acquired this nucleotide sequence from the DNA of its host. The host sequence which has been incorporated by MC29V is transcribed into RNA in uninfected chicken cells and thus probably encodes a cellular gene. We have prepared radioactive DNA complementary to the putative MC29V transforming gene (cDNA(mc) (29)) and have found that sequences homologous to cDNA(mc) (29) are present in the genomes of several uninfected vertebrate species. The DNA of chicken, the natural host for MC29V, contains at least 90% of the sequences represented by cDNA(mc) (29). DNAs from other animals show significant but decreasing amounts of complementarity to cDNA(mc) (29) in accordance with their evolutionary divergence from chickens; the thermal stabilities of duplexes formed between cDNA(mc) (29) and avian DNAs also reflect phylogenetic divergence. Sequences complementary to cDNA(mc) (29) are transcribed into approximately 10 copies per cell of polyadenylated RNA in uninfected chicken fibroblasts. Thus, the vertebrate homolog of cDNA(mc) (29) may be a gene which has been conserved throughout vertebrate evolution and which served as a progenitor for the putative transforming gene of MC29V. Recent experiments suggest that the putative transforming gene of avian erythroblastosis virus, like that of MC29V, may have arisen by incorporation of a host gene (Stehelin et al., personal communication). These findings for avian erythroblastosis virus and MC29V closely parallel previous results, suggesting a host origin for src (D. H. Spector, B. Baker, H. E. Varmus, and J. M. Bishop, Cell 13:381-386, 1978; D. H. Spector, K. Smith, T. Padgett, P. McCombe, D. Roulland-Dussoix, C. Moscovici, H. E. Varmus, and J. M. Bishop, Cell 13:371-379, 1978; D. H. Spector, H. E. Varmus, and J. M. Bishop, Proc. Natl. Acad. Sci. U.S.A. 75:4102-4106, 1978; D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature [London] 260:170-173, 1976), the gene responsible for tumorigenesis by avian sarcoma virus. Avian sarcoma virus, avian erythroblastosis virus, and MC29V, however, induce distinctly different spectra of tumors within their host. The putative transforming genes of these viruses share no detectable homology, although sequences homologous to all three types of putative transforming genes occur and are highly conserved in the genomes of several vertebrate species. These data suggest that evolution of oncogenic retroviruses has frequently involved a mechanism whereby incorporation and perhaps modification of different host genes provides each virus with the ability to induce its characteristic tumors.     

Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV

We have isolated three types of pBR322-AAV recombinant plasmids that contain deletions within the 145 bp AAV terminal repeats. When the plasmids were transfected into human cells, mutants that contained deletions within the left (type I) or right (type II) terminal repeat were viable. Of four mutants examined that contained deletions in both termini (type III), only one was viable. All of the viable mutants produced AAV virions that contained wild-type AAV DNA. Furthermore, the viable type III deletion could be converted to a nonviable mutant by deleting all copies of an 11 bp sequence from its termini. We conclude that there is an efficient mechanism for correcting deletions within the AAV termini. A model that could account for these observations is also discussed.