The role of RNA molecules in transduction of the proto-oncogene c-fps

The retroviral oncogene v-myb arose by transduction of the chicken proto-oncogene c-myb. We isolated and sequenced cDNA that represents the entire coding domain of chicken c-myb. By transcribing the cDNA into mRNA in vitro and then translating the RNA, we were able to document the integrity of the cDNA and to identify the codon responsible for initiation of translation from c-myb. Two different alleles of v-myb are extant, one in the genome of avian myeloblastosis virus (AMV) and the other in the genome of erythroblastosis virus 26 (E26V). The proteins encoded by the AMV and E26V alleles of v-myb differ from the product of c-myb in three ways: at their amino termini, they lack 71 and 80 amino acids respectively; at their carboxy termini, they are deficient in 199 and 278 residues; and 11 substitutions of amino acids are scattered throughout the product of AMV allele, whereas the product of the E26V allele contains only a single substitution. The structural origins of tumorigenicity by v-myb and the biological functions of c-myb remain enigmatic. The findings and molecular clones described here should now permit a systematic exploration of these enigmas.The retroviral oncogene v-myb arose by transduction of a chicken proto-oncogene known as c-myb (12). Two versions of v-myb have been identified (4), one in avian myeloblastosis virus (AMV) and the other in erythroblastosis virus 26 (E26V). The allele of v-myb found in AMV is expressed by means of a spliced mRNA that gives rise to a 45-kilodalton nuclear protein (p45v-myb) (13). By contrast, the E26V allele of v-myb is part of a tripartite gene that includes a portion of the viral structural gene gag, v-myb, and v-ets (another locus transduced from normal cells; see reference 20). The product of this chimera arises from translation of the viral genome itself rather than from a spliced mRNA (28).The polypeptides encoded by the AMV and E26V alleles of v-myb are appreciably smaller than the 75-kilodalton protein encoded by chicken c-myb (p75c-mYb) (13). Previous studies have attributed these differences to truncation of c-myb during the course of transduction into retroviruses (12,13 Approximately 5 x 105 independent XgtlO cDNA clones were then screened by plaque hybridization (2), using a nick-translated NcoI-BalI fragment (Fig. 1) from the cloned AMV genome that spans the majority of v-myb (12).Sequencing. Two independent c-myb cDNA clones that together span 3.5 kilobases (kb) were subcloned and sequenced by dideoxynucleotide chain termination (32), using phage vectors mpl8 and mpl9 (22) according to the strategy outlined in Fig. 1.Synthesis of RNA and translation in vitro. Molecular clones encompassing v-myb and c-myb coding regions were inserted into the SP65 expression vector as summarized below (see Fig. 3

Section: Resultsmentioning

confidence: 99%

Structure of the protein encoded by the chicken proto-oncogene c-myb.

Gerondakis¹,

Bishop²

1986

“…Therefore, deletion of this region is required for efficient transduction of a protooncogene into a retrovirus. In the case of PRCII or feline sarcoma virus, the 3' RNA processing site of c-fps is present in the viral genome, but it is apparently not functional because of the absence of other sequences thought to be essential for RNA processing (18).…”

Section: Resultsmentioning

confidence: 99%

Activation of oncogenicity of the c-rel proto-oncogene.

Sylla¹,

Temin²

1986

Reticuloendotheliosis virus strain T (Rev-T) induces a lethal lymphoma in young birds and transforms avian lymphoid cells in vitro. The transforming gene of Rev-T, v-rel, was derived from the turkey proto-oncogene c-rel. Comparison of the nucleotide sequences of v-rel and c-rel indicates that in addition to several internal amino acid changes relative to c-rel, p59V'-has amino acid sequences at both ends derived from the reticuloendotheliosis virus strain A-related virus env gene (K. C. Wilhelmsen, K. Eggleton, and H. M. Temin, J. Virol. 52:172-182, 1984). In this report, the v-rel sequences important for transformation were defined by constructing recombinant retroviruses in which c-rel sequences replaced the analogous v-rel sequences. These recombinant viruses expressing chimeric proteins were tested for their ability to transform spleen cells in vitro and to induce tumors in young chickens. Activation of the oncogenicity of c-rel in Rev-T required alteration of the amino terminus and the central region of the protein. Deletion of the noncoding sequences 3' to c-rel and of most of the helper virus-related env sequences was necessary for the formation of Rev-T.The transforming genes (viral oncogenes or v-onc) present in the genomes of highly oncogenic retroviruses are activated forms of similar genes present in the chromosomes of normal cells. These cellular genes, called proto-oncogenes or c-onc, are highly conserved and are thought to play an important role in cellular growth and differentiation (3). The mechanisms of activation of proto-oncogenes to transforming genes are of central interest in understanding the neoplastic process. A proto-oncogene can be converted into an activated viral oncogene by a variety of mutational events which affect either the primary structure of the gene or its expression, or both (4,5,9,11,20,22,27,36,39).Reticuloendotheliosis virus strain T (Rev-T), isolated from a turkey tumor, is a highly oncogenic retrovirus which causes rapidly lethal lymphomas in newly hatched chickens and turkeys (34). In cell culture, Rev-T transforms and immortalizes very early lymphoid cells from spleen or bone marrow (2). However, Rev-T does not transform chicken embryo fibroblasts (CEF) (13).In Rev-T-infected CEF, the v-rel protein, phosphoprotein p59v-rel (13), is expressed from spliced mRNA (8,24). The relative amounts of v-rel protein in infected CEF and in Rev-T-transformed chicken spleen cells are the same (13). This similarity indicates that the inability of Rev-T to transform CEF is not at the level of rel gene expression.Rev-T is defective for replication and requires a helper virus, reticuloendotheliosis virus strain A (Rev-A), for its propagation (6). The genome of Rev-T is derived from that of a virus like Rev-A by substitution of rel sequences for most of the envelope sequences and by deletion of most of the gag and pol genes (6, 28). However, the cis-acting sequences necessary for propagation of the virus are retained in Rev-T (6; Fig. 1).The cellular counterpart of v-rel, the turkey p...

“…4a). A short match of this type would be sufficient for a template-switch, or copy-choice, recombination at this site (7,12,37,39). If this had occurred, the v-ski sequence upstream of the recombination site should be identical to the c-ski sequence, and the SKV sequence downstream of the site should be identical to the corresponding region of the ALV sequence.…”

Section: Resultsmentioning

confidence: 99%

“…At the 3' ALV/ski junction, the short stretch in which 6 of 7 bases are in common between c-ski and ALV provides a possible site for copy-choice recombination via a template switch during reverse transcription (7,12). An additional deletion event is required to account for the product of this recombination, since the v-ski sequence immediately upstream of the site is missing 2 bases of c-ski sequence.…”

Section: Discussionmentioning

confidence: 99%

The v-ski oncogene encodes a truncated set of c-ski coding exons with limited sequence and structural relatedness to v-myc.

Stavnezer¹,

Brodeur²,

Brennan³

1989

The nucleotide sequence of a biologically active v-ski gene from a cloned proviral segment shows that ski is a 1,312-base sequence embedded in the p19 region of the avian leukosis virus gag gene. The v-ski sequence contains a single open translational reading frame that encodes a polypeptide with a molecular mass of 49,000 daltons. The predicted amino acid sequence includes nuclear localization motifs that have been identified in other nuclear oncoproteins. It also contains a proline-rich region and a set of cysteine and histidine residues that could constitute a metal-binding domain. Two regions of the amino acid sequences of v-ski and v-myc are related, and the two proteins exhibit similar distributions of hydrophobic and hydrophilic amino acids. Cloned segments of the chicken c-ski proto-oncogene totaling 65 kilobases have been analyzed, and regions related to v-ski have been sequenced. The results indicate that v-ski is derived from at least five coding exons of c-ski, that it is correctly spliced, and that it is missing c-ski coding sequences at both its 5' and 3' ends. The c-ski and avian leukosis virus sequences that overlap the 5' virus/v-ski junction in Sloan-Kettering virus contain an 18-of-20-base sequence match that presumably played a role in the transduction of ski by facilitating virus/c-ski recombination.The Sloan-Kettering viruses (SKVs) are replication-defective, acute transforming retroviruses that contain the onco- We previously reported the molecular cloning and restriction endonuclease mapping of an internal fragment of a provirus corresponding to the 5.7-kilobase (kb) genome of the virus SKV5.7 (34). This fragment, containing the ski gene bounded by helper virus sequences, was used to construct an analog of the SKV5.7 genome by insertion at shared restriction sites in a cloned provirus of the avian leukosis virus (ALV) Rous-associated virus (RAV)-1. This constructed provirus, transfected into chicken embryo fibroblasts, yielded a biologically active virus (RAV-SKV) that was indistinguishable in transforming activity from the uncloned parental virus. Chicken embryo fibroblasts transformed by RAV-SKV were found to produce a P110 virus/ski fusion protein, as do cells infected by the parental virus, SKV5.7.We have determined the nucleotide sequence of this biologically active ski gene. This information has permitted a sequence comparison of v-ski and other previously sequenced genes, determination of the coding capacity of ski, and analysis of the predicted amino acid sequence of the gene. Since ski-encoded proteins have been localized to the nucleus of SKV-transformed cells (2), we paid particular attention to possible relationships between ski and other nuclear oncogenes and to the possible presence in ski of domains related to functional motifs that have been found in * Corresponding author. other nuclear proteins (4,8,10,13,17,18,27,28,41). We report here the sequence of v-ski, some potentially relevant structural features of its predicted amino acid sequence, and the results of our compar...