Chromosomal Localization of DBL' Oncogene Sequences
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GENOMICS 5,546-553 (1989) Chromosomal Localization of DBL’ Oncogene Sequences STEVEN R. TRONICK,* 0. WESLEY MCBRIDE,t NICHOLAS C. POPESCU,$ AND ALESSANDRA EVA* *Laboratory of Cellular and Molecular Biology, tLaboratory of Biochemistry, and *Laboratory of Biology, National Cancer Institute, Bethesda, Maryland 20892 Received December 28, 1988, revised June 13, 1989 rearrangement led to the generation of NPDL-CM In The DBL oncogene was generated by rearrange- NIH/3T3 transfectants we have shown that the DBL ments involving three discontinuous regions of the oncogene-specific transcript is derived from 5’ and human genome. Analyses of panels of human X rodent middle sets of sequences, but not from the 3’ set,. Ex- somatic cell hybrids demonstrated that the DBL proto- amination of the DNA of the tumor cells from which oncogene located on the X chromosome (just proximal DBL and NPDL-d61 oncogenes were derived did not or distal to bands q26-27.2) underwent recombination reveal alteration of DBL-related sequences and led to at its 5’ and 3’ ends with sequences derived from chro- the conclusion that transforming activity was acquired mosomes 3 (pl3q-ter) and 16 (p13-q22), respectively. as result of the transfection process (Eva et al., 1987). DBL was localized to chromosome Xq27-q28 by in Nucleotide sequence analyses of cDNAs representing situ hybridization. Another oncogene, MCFS, was DBL oncogene and proto-oncogene transcripts showed previously shown to contain sequences derived from that the rearrangement resulted in the loss of the Xq27 as well. Comparison of the restriction maps and amino-terminal 497 codons of DBL and acquisition of nucleotide sequences of genomic and cDNA clones new sequences including a promoter region and an representing the chromosome X-specific sequences of amino terminus of 50 amino acids derived from an un- the DBL oncogene and MCFB, taken together with identified gene (Eva et al., 1988; Ron et al., 1988). A their chromosomal localization, indicates that these oncogenes were derived from the same genetic locus. molecular clone of the NPDL-cl61 oncogene has not yet been isolated. Thus, we do not have more detailed knowledge of its structure. The DBL cDNA is capable of inducing transfor- INTRODUCTION mation of NIH/3T3 cells provided that it.s expression is driven by a strong promoter; however, its transform- The DBL oncogene was initially isolated from a hu- ing activity is 20-50 times lower than that of the DBL man diffuse poorly differentiated B-cell lymphoma by oncogene (Ron et al., 1988). Truncating either the DBL DNA transfection of mouse NIH/3T3 cells (Eva and oncogene or the DBL cDNAs at a point corresponding Aaronson, 1985). A subsequent independent isolate was to the recombination site in the oncogene (that is, co- obtained following transfection of NIH/3T3 cells with dons l-497 and l-50 in the DBL and DBL oncogene DNA of a human nodular, poorly differentiated lym- sequences, respectively) generates molecules with phoma (designated NPDL-dbl by the authors) (Eva et transforming activities equivalent to that of the pa- al., 1987). Comparative restriction enzyme analyses of rental oncogene when each of these molecules is placed the DBL and NPDL-dbl oncogenes in transfected under the transcriptional control of the same promoter NIH/3T3 cells and of related sequences in normal hu- (Ron et al., 1989, in press). man placenta DNA (Eva et al., 1987) revealed that the The predicted amino acid sequence of DBL is not generation of these oncogenes involved recombination significantly similar to that of any known oncogene. is between at least two sets of human sequences derived markedly hydrophilic, and does not possess features from noncontiguous loci. In the case of the DBL on- characteristic of integral membrane proteins. In trans- cogene, recombination occurred within the 5’ and 3’ fected mouse cells the 66-kDa DBL oncogene protein regions of the DBL proto-oncogene, whereas only a 5’ is distributed approximately equally between soluble and membrane fractions of the cytoplasm and is not Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. 503639. detected in the nucleus (Srivastava et al.. 1986). Thus, ’ HGM symbol. DBL is considered to represent a unique class of human 088%7543/89 $3.00 546 CHROMOSOMAL MAPPING OF DBL 547 transforming gene. In order to understand better its DNAs using all four 3H-labeled deoxynucleoside tri- possible role in human malignancies and the events phosphates and were hybridized to RNase-treated and leading to the activation of DBL, we have determined formamide-denatured chromosomes for 20 h at 40°C. the chromosomal location of DBL sequences in the Hybridized chromosome preparations were washed in human genome. 50% formamide, 2~ SSC at 39°C and then with an alcohol series and covered with a 50% solution of NTB- MATERIALS AND METHODS 2 nuclear track emulsion (Kodak, Rochester, NY). The slides were stored desiccated at 4°C for 14 days and Cell Hybrids, Molecular Clones, DNA Probes, and then developed. Grains situated on nonoverlapping Southern Analysis chromosomal regions were counted and their specific locations determined on G-banded chromosomes as Construction and karyotypic and biochemical anal- previously described (Popescu et al., 1985; Tronick et yses of human X mouse and human X hamster somatic al., 1985). cell hybrids retaining varying numbers of human chro- mosomes have been reported in detail (McBride et al., 1982a,b,c). Genomic (Eva and Aaronson, 1985) and RESULTS cDNA (Eva et al., 1988) clones of the DBL oncogene have been described and they are shown diagrammat- Somatic Cell Hybrids ically, along with the hybridization probes used in this We initially employed genomic probes (lacking re- study, in Fig. 1. The DBL oncogene was isolated by petitive sequences) representing transcribed regions of cloning DNA of an NIH/3T3 transfectant into a cosmid the DBL oncogene isolated by cloning DNA of a third- vector; this cosmid clone transforms NIH/3T3 cells. A cycle NIH/3T3 transfectant (Eva et al., 1987). One restriction map of this clone is shown in Fig. 1A along probe was derived from the 5’ region, 2.4-3.5 kb on the with the regions used as hybridization probes. This map of the cosmid clone (probe a in Fig. 1A). The sec- clone has been described in more detail previously (Eva ond probe corresponds to 13-14.1 kb on the map (probe et al., 1987). Two DBL oncogene cDNA clones were b in Fig. 1A). Each probe detected the 3.0-kb DBL- also used as probes. The cDNAs were derived from specific mRNA in NIH/3T3 transfectants (Eva et al., poly( A)+ RNA isolated from NIH/3T3 transfectants 1987). These probes were hybridized to several restric- (Eva et al., 1988). Sequences in clone lbl-1 were derived tion enzyme digests of DNAs isolated from a series of solely from the middle region of the DBL oncogene, human-rodent somatic cell hybrids containing differ- indicated by the solid line. ent human chromosomes (McBride et al., 1982a,b,c). Isolation and purification of molecularly cloned DNA Probe a detected a 6-kb EcoRI DNA fragment that fragments and high-molecular-weight eucaryotic cell could be localized to chromosome 3 (Table 1). Two of DNA were performed according to standard techniques. the hybrids examined contained spontaneous breaks The hybridization conditions used for analyzing involving this chromosome. Both of these cellular Southern blots of somatic cell hybrid DNA have been DNAs hybridized with the 5’ probe and contained the described (Jaiswal et al., 1988). Briefly, nylon mem- human a2-HS-glycoprotein sequence (3q27-q29), but branes to which DNA fragments had been transferred they did not contain the RAF1 oncogene sequence were hybridized with 32P-labeled probes at 42°C for (3~25) or express aminoacylase 1 (3~21). In addition, 24-48 h in a solution containing 50% formamide and one of these hybrids retained the functional glutathione 10% dextran sulfate and were washed at 55°C in 0.15 peroxidase 1 gene (3p13-q12), whereas the other did M NaCl, 0.0015 M sodium citrate, pH 7.0. In some not. This indicates that the probe a sequences repre- cases, after autoradiography, the bound probe was re- senting 5’ DBL oncogene-transcribed sequences were moved from the blot with 0.4 M NaOH at 42°C and located on the human chromosome 3 long arm or prox- the membrane was rehybridized with another probe. imal short arm (3p13-qter). In contrast, probe b (Fig. 1) detected a human-spe- In Situ H-vbridization and Autoradiography cific 3.3-kb EcoRI fragment in different hybrid cell lines Peripheral blood lymphocytes from a normal male of this same series and could be shown instead to be subject (46;XY) were cultured for 96 h in RPM1 me- present on the human X chromosome (Table 1). A dium supplemented with 15% fetal bovine serum, phy- cDNA clone isolated from a third-cycle NIH/3T3 cell tohemagglutinin, and antibiotics. Air-dried chromo- transfectant, designated lbl-1 (Eva et al., 1988), and some preparations were obtained from methotrexate- derived entirely from the set of DBL coding sequences synchronized cultures as previously described (Popescu that are not rearranged with respect to those found in et al., 1985). Radiolabeled probes (sp act, -l/5x107 normal human placental DNA (Eva et al., 1987) (that cpm/pg) were prepared by nick translation of plasmid is, only DNA fragments from within the region between 548 TRONICK ET AL E HP E E H E E E E E E E SE E 1 Ia I I 1, 1 I I , I , c-!I .,. ,,i I I I a b c B (E SS N A E X SC s ti (El ‘1 ..~.