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Isolation and Chromosomal Localizationof the Human Fgr Proc. Natl. Acad. Sci. USA Vol. 82, pp. 6595-6599, October 1985 Genetics Isolation and chromosomal localization of the human fgr protooncogene, a distinct member of the tyrosine kinase gene family (acute transforming retrovirus/heteroduplex analysis/in situ hybridization) STEVEN R. TRONICK*, NICHOLAS C. POPESCUt, MARC S. C. CHEAH*, DAVID C. SWAN*, SUZANNE C. AMSBAUGHt, CAROLE R. LENGEL*, JOSEPH A. DIPAOLOt, AND KEITH C. ROBBINS* *Laboratory of Cellular and Molecular Biology and tLaboratory of Biology, National Cancer Institute, Bethesda, MD 20205 Communicated by Robert J. Huebner, May 24, 1985 ABSTRACT The cell-derived domain of Gardner-Ra- growth factor receptors share functional and structural rela- sheed feline sarcoma virus (GR-FeSV) consists ofa y-actin- and tionships with retrovirus-encoded tyrosine kinases. a tyrosine-specific protein kinase-encoding sequence designat- Recently we have described the molecular organization of ed v-fgr. By utilizing a v-fgr probe, it was possible to detect Gardner-Rasheed feline sarcoma virus (GR-FeSV) (21), an related sequences present at low copy number in DNAs of a acutely transforming retrovirus (22) that encodes a tyrosine- variety of mammalian species and to isolate a human fgr specific protein kinase (23). Nucleotide sequence analysis homologue. Comparative studies revealed that this human has revealed that the GR-FeSV kinase is related to other DNA clone represented all but 200 base pairs of v-fgr. Analysis retrovirus-encoded protein kinases, in particular, the prod- of human genomic DNA demonstrated that thefgr protoonco- ucts of avian v-src and v-yes onc genes (24). In the present gene was distinct from the cellular homologues of other study, isolation and structural characterization of the human retrovirus onc genes. In addition, the fgr protooncogene was fgr protooncogene has made it possible to determine its localized to the distal portion of the short arm of human chromosomal localization and relationship to other members chromosome 1 at p36.1-36.2 by in situ hybridization. Taken of the tyrosine kinase gene family. together, our findings establish that thefgr protooncogene is a unique member of the tyrosine kinase gene family. MATERIALS AND METHODS Acutely transforming retroviruses have arisen in nature as Molecular Clones. Two DNA fragments representing nu- the result of apparently rare recombinational events involv- cleotides 915-1756 or 711-915 of the gene encoding the ing replication-competent type C retroviruses and a small set primary GR-FeSV translational product (24) were cloned into of evolutionarily conserved cellular genes termed protoon- pBR322. The former clone, designated pv-fgr-1 contained cogenes. Such transduced cellular (onc) sequences confer only tyrosine kinase-encoding sequences, whereas the latter upon the recombinant virus the ability to rapidly induce clone, designated pv-fgr-2, contained 3' y-actin- and 5' malignancies. Recent studies have indicated that protoonco- tyrosine kinase-encoding sequences. A pBR322 clone of genes can be activated as oncogenes within human tumor integrated GR-FeSV has been described (21). A DNA clone cells in the absence of retrovirus involvement and that such containing v-src sequences (pEcoRIB) (25) was obtained oncogenes may very well play a role in the development of from the American Type Culture Collection (no. 41005). malignancy. Thus, understanding the functions of protoon- Unintegrated Y73 avian sarcoma virus DNA provided by K. cogenes as well as the mechanisms by which they acquire Toyoshima was used to prepare a 1.15-kilobase-pair (kbp) transforming properties is likely to be critical for deciphering v-yes-specific probe that encompassed nucleotides 1652- the neoplastic process. 2801 (26). Recent findings regarding the relationship ofretrovirus onc Isolation of v-fgr-Related Human Sequences. A bacterio- genes to proteins of known function have strongly implicated phage library of human fetal liver DNA was provided by T. growth factor-mediated regulatory pathways in the steps Maniatis (27). The bacteriophage were plated at a density of leading to malignant transformation. The simian sarcoma 2 x 104 plaque-forming units per dish with Escherichia coli virus transforming gene, v-sis, encodes a protein closely strain BNN45, which was a gift from K. Shimizu and M. related in amino acid sequence (1, 2), secondary structure (3), Wigler. Nitrocellulose filter replicas of the infected bacterial and antigenic properties (3, 4) to human platelet-derived lawns, prepared by the method of Benton and Davis (28), growth factor (PDGF) (5, 6), a potent mitogen for connective were hybridized at 62°C for 24 hr in a solution containing tissue cells (7, 8). In addition, the amino acid sequences of 32P-labeled pv-fgr-1 probe (specific activity, 2 x 108 cpm/,ug), receptors for epidermal growth factor (EGF) and insulin have 6x NaCl/Cit (lx NaCl/Cit = 0.15 M NaCl/0.015 M sodium been shown to possess striking similarity with the tyrosine- citrate), 5 x Denhardt's solution (lx Denhardt's solution = specific protein kinases encoded by the v-erbB and v-ros 0.02% polyvinylpyrrolidone/0.02% bovine serum albumin/ transforming genes, respectively (9, 10). Several other acute- 0.02% Ficoll), and 100 pgg of sheared salmon testes DNA per ly transforming retroviruses encode protein kinases that are ml. Filters were washed at 45°C in 0.1 x NaCl/Cit containing related to one another (11, 12) and possess amino acid 0.1% NaDodSO4 and autoradiographed at -70°C for 24 hr sequence homology with EGF (9) and insulin (10, 59) recep- with Kodak XAR film and Dupont Cronex Lightning Plus tors. Moreover, receptors for EGF (13), insulin (14-16), and screens. PDGF (17-20) all respond to their respective growth factors Analysis of Cloned and Genomic DNAs. DNAs were digest- by autophosphorylation on tyrosine residues. Thus, certain ed with restriction enzymes, fractionated by electrophoresis through agarose gels, and blotted onto nitrocellulose filters as The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: FeSV, feline sarcoma virus; GR-FeSV, Gardner- in accordance with 18 U.S.C. §1734 solely to indicate this fact. Rasheed-FeSV; kbp, kilobase pair(s). 6595 Downloaded by guest on September 25, 2021 6596 Genetics: Tronick et al. Proc. Natl. Acad. Sci. USA 82 (1985) described (29). DNA fragments were labeled with [32P]dCTP a b c d by nick translation to a specific activity of 2 x 101 cpm/,ug kbp and incubated with nitrocellulose filters in a buffer containing 20 mM sodium phosphate (pH 6.4), 50% formamide, 10%o w dextran sulfate, 5 mM EDTA, 2x Denhardt's solution, 5x 23.0 NaCl/Cit, and 50 ,ug of sonicated single-stranded salmon 4 FIG. 1. Detection of v-fgr-related testes DNA per ml. After hybridization at 42°C for 24-48 hr, sequences in mammalian DNAs. 9.6 1 from normal cat (lane filters were washed sequentially at room temperature in 2 x DNAs isolated NaCl/Cit and then at 420C in 0.1 x NaCl/Cit containing 0.1% a), raccoon (22) (lane b), rhesus mon- o key (lane c), and human (lane d) cells were as 6.6 NaDodSO4. Filters autoradiographed described were digested with HindIII, fraction- above. ated by agarose gel electrophoresis, Heteroduplex Analysis ofCloned DNAs. Linearized plasmid and subjected to Southern analysis 4.3 b DNAs were hybridized in the presence of 50% formamide at with the pv-fgr-1 probe. HindIII di- 22°C for 4-6 hr. Samples were spread onto a distilled-water gests of bacteriophage X DNA were hypophase and prepared for electron microscopy as de- used as molecular size standards. scribed (30). Contour lengths were determined by compari- son to 4X174 single- and double-stranded DNAs with the aid of a Zeiss Videoplan 2 image analysis system. nick-translated pv-fgr-1 DNA as described by Wahl et al. In Situ Chromosome Hybridization. Cultured peripheral (36). blood lymphocytes from a normal male donor were stimu- lated with phytohemagglutinin and synchronized with RESULTS methotrexate (0.1 ,uM) prior to harvest (31). Chromosomes were prepared by a standard air-dried method after hypotonic v-fgr Is Derived from an Evolutionarily Conserved Cellular treatment in 0.075 M KCI and several fixations in methanol/ Gene. Previous studies have shown that the primary GR- glacial acetic acid, 3:1 (vol/vol). The method described by FeSV translational product possesses a tyrosine-specific Harper and Saunders (32) was used for in situ hybridization. protein kinase activity encoded by a sequence, v-fgr, which Chromosome preparations were hybridized with 1 ,ug of is not related to the genome of its parental helper virus (21). [3H]DNA probe (specific activity, 2.2 x 107 cpm/,ug) per ml, To examine the relationship of v-fgr to the mammalian washed, covered with nuclear track emulsion NTB2 (Ko- genome, a DNA fragment representing v-fgr was subcloned dak), and stored dessicated at 4°C for 14 days. Autoradio- into a plasmid vector for use as a probe. This probe, graphs were developed, rinsed briefly in water, and fixed; designated pv-fgr-1, detected related sequences in cellular air-dried slides were stained for 5 min with 0.25% DNAs from a variety of species (Fig. 1). Single bands were Wright stain in 60 mM phosphate, 1:3 (vol/vol) at pH 6.8 (32). observed in HindIII digests of cat, racoon, and human DNA, Slides were then destained through an alcohol series and and two bands were detected in rhesus monkey DNA. These restained with Wright stain to improve the quality of G- findings demonstrated that v-fgr was cell-derived and con- bands. Grains were counted directly on banded chromo- served within the mammalian genome at low- or single-copy somes.
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