Analysis of the primary translational product and integrated DNA of a new feline sarcoma virus, GR-FeSV

107

We have isolated cDNA molecules representing the complete coding sequence of a new human gene which is a member of the src family of oncogenes. Nucleotide sequence analysis revealed that this gene, termed slk, encoded a 537-residue protein which was 86% identical to the chicken proto-oncogene product, p60CSrc, over a stretch of 191 amino acids at its carboxy terminus. In contrast, only 6% amino acid homology was observed within the amino-terminal 82 amino acid residues of these two proteins. It was possible to activate slk as a transforming gene by substituting approximately two-thirds of the slk coding sequence for an analogous region of the v-fgr onc gene present in Gardner-Rasheed feline sarcoma virus. The resulting hybrid protein molecule expressed in transformed cells demonstrated protein kinase activity with specificity for tyrosine residues.Normal cellular genes which have given rise to retrovirus onc sequences represent the most abundant class of genes capable of acquiring oncogenic properties. In addition to these, dominant transforming genes present in certain tumor cells have been found by their ability to induce foci of transformation when transfected into susceptible assay cells (for a review, see reference 1). More recently, other genes implicated in the malignant process by virtue of their amplification in tumor cells have been identified because of their genetic relatedness to known oncogenes (15,17,32). Of the transforming genes identified to date, roughly half encode products which possess structural or enzymatic relatedness to protein-tyrosine kinases. This family includes altered versions of genes encoding cell surface receptors for certain growth factors (8,35,40), as well as a much larger number that have not yet been linked to normal cellular functions.The prototype protein-tyrosine kinase gene v-src (6) and its close relatives, v-yes (16) and v-fgr (23), were each identified initially within oncogenic retroviruses as components derived from distinct cellular genes (38). In an effort to search for new src-related genes within the human genome, we have taken a more general approach not reliant upon the identification of novel oncogenic viruses or transforming genes, but dependent only upon normal gene expression. In the present study, we report the isolation of cDNA molecules representing the complete coding sequence of a new human src-related gene and demonstrate that the gene can acquire transforming properties by substituting a portion of its coding sequence for a related onc gene in a retrovirus vector. New England Nuclear Corp.) per ml or with 4 ml methionine-free Dulbecco modified Eagle medium containing 125 ,uCi of [35S]methionine (New England Nuclear) per ml. Radiolabeled cells were lysed with 1 ml of lysing buffer (10 mM sodium phosphate [pH 7.5], 100 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1 mM phenylmethylsulfonyl fluoride) per petri dish, clarified by centrifugation at 100,000 x g for 30 min, and divided into five identical aliquots. Extracts were incubated wi...

Section: Methodsmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Isolation and oncogenic potential of a novel human src-like gene.

Kawakami¹,

Pennington²,

Robbins³

1986

107

“…The amino acid sequence of the v-fgr product, p70gag-fgr (25), was deduced from nucleotide sequence data. The carboxy-terminal half of the oncogene product showed highest homology (80%) with that of the v-yes product (26).…”

mentioning

confidence: 99%

Structure, expression, and chromosomal location of the human c-fgr gene.

Nishizawa¹,

Semba²,

Yoshida³

et al. 1986

The nucleotide sequence of seven exons of the human c-fgr gene, a cellular homolog of the oncogene of Gardner-Rasheed feline sarcoma virus, was determined. Twenty-six independent genomic clones were obtained from a human gene library with a DNA clone of Y73 avian sarcoma virus oncogene, v-yes, as a probe under relaxed hybridization conditions. Restriction mapping and partial sequence analyses revealed that two of these clones were derived from the c-fgr gene, distinct from the c-yes gene. Interestingly, the splicing points of the c-fgr gene were identical with those of the c-src gene throughout the seven exons, suggesting that the two proto-oncogenes were generated by gene duplication of an ancestral gene containing intervening sequences. On RNA blot hybridization the major transcript was found to be 2.6 kilobases long. Two additional transcripts of 3.5 and 4.7 kilobases were also detected. Furthermore, karyotype analysis of several human-mouse hybrid cells and Southern blot analyses of DNAs of the hybrids with a human c-fgr locus-specific probe showed that this gene is located on chromosome 1.It is widely accepted that an acutely oncogenic retrovirus contains a viral oncogene that is derived from a cellular counterpart and is responsible for the initiation and maintenance of cellular transformation. In animals, cellular oncogenes have been highly conserved throughout evolution, suggesting that they play important roles in normal cells. However, the normal functions of most cellular oncogenes are still unknown (2). More than 10 oncogenes were found to be structurally related to the most well-characterized oncogene, the src gene of Rous sarcoma virus, and so are called the "src family." Most members of this gene family have been found to encode products that are associated with tyrosine kinase activity, and it is generally assumed that kinase activity plays a key role in cell transformation (2).In the src family, the yes gene, identified as an oncogene of avian Y73 sarcoma virus (15) Chromosome mapping. To determine the chromosomal location of the human c-fgr gene, we needed a human c-fgr-specific probe. For this purpose, we used an

“…This group of oncogenes is known as the src gene family and it includes the src, fpslfes, yes, ros, fms, abl, erbB, and fgr genes (3). The proteins encoded by these genes have a high degree of amino acid sequence homology (9,18,19,25,34,35,39,40,47,50,55,61), and they are associated with tyrosine-specific protein kinase activities (1,2,8,14,15,17,21,24,26,33,37,41,43,57) that are believed to be essential to the mechanism of cell transformation. Viral oncogenes were derived from cellular oncogenes by a process of recombination between viral and cellular sequences.…”

mentioning

confidence: 99%

Antipeptide antiserum identifies a widely distributed cellular tyrosine kinase related to but distinct from the c-fps/fes-encoded protein.

Feldman¹,

Tam²,

Hanafusa³

1986

We raised antibodies directed against a synthetic peptide representing an amino acid sequence of the conserved kinase domain of the transforming protein of Fujinami sarcoma virus (FSV) (P140). The antiserum obtained specifically recognized FSV-P140 and its cellular homolog and in addition, it recognized a new cellular protein of 94,000 daltons (NCP94) in avian and mammalian cells. NCP94 was found to be associated with a cyclic nucleotide-independent protein kinase activity that was specific for tyrosine residues. Although NCP94 and FSV-P140 share antigenic determinants, NCP94 is not a cellular homolog of FSV-P140: NCP94 and the previously identified c-fps/fes product were different in their tryptic fingerprints and in their tissue specificities. Thus, the function of NCP94 in normal cells is probably different than that of the c-fps/fes product. NCP94 was expressed in every tissue and cell line that was examined. In chickens, NCP94 levels were highest during embryonic development and NCP94 expression was high in gizz4rd, brain, and spleen throughout embryonic and adult life. The universal expression of NCP94 suggests that this protein may be involved in an essential function of normal cells. NCP94 may be a new ceDlular tyrosine kinase of the src gene family.