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Human Urokinase Gene Is Located on the Long Arm Of Proc. Nati. Acad. Sci. USA Vol. 82, pp. 4448-4452, July 1985 Genetics Human urokinase gene is located on the long arm of chromosome 10 (plasminogen activator/somatic cell hybrids/in situ hybridization) PASQUALE TRIPPUTI*, FRANCESCO BLASIt, PASQUALE VERDEt, LINDA A. CANNIZZAROt§, BEVERLY S. EMANUEL0, AND CARLO M. CROCE* *The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, PA 19104; tliternational Institute of Genftics and Biophysics, Consiglio Nazionale delle Ricerche, via Marconi 10, 80123 Naples, Italy; and tDepartment of Pathology and Laboratory Medicine and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 Communicated by DeWitt Stetten, Jr., January 17, 1985 ABSTRACT Urokinase is one of the two plasminogen phenol, and precipitation with ethanol as described (23). activators that catalyze the conversion of inactive plasminogen Fifteen micrograms of DNA were digested with 30 units of to plasmin. By combining somatic cell genetics, in situ hybridi- the appropriate restriction endonuclease in standard condi- zation, and Southern hybridization, we localized the human tions recommended by the supplier (New England Biolabs). urokinase gene on the distal third of the long arm (q24-qter) of Fragments were separated by electrophoresis on a 1% chromosome 10. agarose gel. DNA was denatured and transferred to nitrocel- lulose as described by Southern (24). DNA on nitrocellulose Plasminogen activators catalyze plasminogen-dependent fi- sheets was hybridized to 32P-labeled probe DNA at 370C in brinolysis, a key reaction controlling extracellular 4x NaCl/Cit (lx NaCl/Cit = 0.15 M NaCl/0.015 M sodium proteolysis (1). Two forms of plasminogen activator have citrate, pH 7) containing 50% (vol/vol) formamide as de- been recognized: urokinase, isolated from human urine, and scribed (25). After hybridization, the filters were washed tissue plasminogen activator. Urokinase is a Mr 54,000 twice with 2x NaCl/Cit at room temperature and twice in two-chain protein (2) derived from a single-chain precursor O.1x NaCl/Cit containing 0.2% sodium dodecyl sulfate at (3-5). Tissue plasminogen activator is a single-chain protein 650C, air-dried, and exposed to Kodak XRA-5 film. of Mr 65,000 (6). Both tissue plasminogen activator (7) and Preparation of Labeled DNA Probe. The urokinase probe is urokinase (8) have been cloned. Increased production of a 637-base-pair EcoRI fragment from human cDNA clones plasminogen activator is associated with spontaneous human identified from a cDNA library prepared from total RNA of tumors (reviewed in ref. 9) and experimental tumors (10). human fibroblasts transformed by simian virus 40 (8). This Plasminogen activator production in malignant tumors is fragment contains the sequences encoding amino acids believed to be part of the biosynthetic program induced by 162-302 of human prourokinase and includes a 221-base-pair hormones that stimulate growth in vivo (10). Increases in intervening sequence (8). The DNA probes were labeled with plasminogen activator mRNA are an early and direct effect of 32Pby nick-translation (26) to specific activities of0.5-2 x 108 hormones (11) and of the potent tumor promoter phorbol cpm/0.2 ,ug ofDNA. DNA polymerase I was purchased from ester (refs. 12 and 13; Stoppelli et al., unpublished results). Boehringer Mannheim; [32P]NTP was from Amersham. These data are consistent with and may explain early findings Chromosome Preparation. Metaphase chromosome that plasminogen activator production is an early oncogene- spreads were prepared from peripheral blood lymphocytes of dependent effect of transformation with tumor viruses a normal (46, XY) male donor by using standard techniques. (14-17). Finally, recent data indicate that urokinase is re- Air-dried slides were kept in the cold (40C) for at least 1 week quired for the metastatic activity of human tumors (18). prior to their use in mapping studies. Because of the possible role of the genes encoding human In Situ Hybridization. In situ hybridization studies were plasminogen activator in tumor biology, it is important to performed by using modifications of the protocols of Can- determine their chromosomal location. We have combined nizzaro and Emanuel (27) and Yunis et al. (28). Air-dried somatic genetics, in situ hybridization, and Southern blot metaphase chromosome preparations on glass slides were hybridization to localize the human urokinase gene on the used 1-3 weeks after preparation. Slides were treated with distal third of the long arm (q24-qter) of chromosome 10. RNase to remove any chromosomally bound RNA. The slides were washed free of RNase and then dehydrated MATERIALS AND METHODS through an alcohol series. Chromosomal DNA was denatured by immersing the slides in 2x NaCl/Cit containing 70% Somatic Cell Hybrids. The cell lines and hybrid clones used formamide at 70'C, followed by rapid transfer through an for the mapping of urokinase sequences have been exten- alcohol series for dehydration. sively described in previous studies (19, 20). The chromo- Probe DNA was labeled with 3H by nick-translation to a somal content of hybrids was determined by enzymatic specific activity of 4 x 107 cpm/tag according to the protocol analysis using starch gel electrophoresis according to stan- of Lai et al. (29), and the DNA was separated from dard methods (21) and by karyotypic analysis using a com- unincorporated labeled nucleotides by chromatography on a bination of trypsin Giemsa and G11 banding techniques as Sephadex column. Carrier DNA, salmon sperm, was added described (22). at a 100Ox excess and DNA was ethanol-precipitated and DNA Extraction, Southern Blot Analysis, and Hybridiza- resuspended in hybridization mixture [2x NaCl/Cit contain- tion. DNA from human, mouse, and hybrid cell lines was prepared by cell lysis, proteinase K digestion, extraction with Abbreviation: GOT, glutamic-oxaloacetic transaminase (aspartate aminotransferase, EC 2.6.1.1). The publication costs of this article were defrayed in part by page charge §Present address: Department of Pediatrics, The Milton S. Hershey payment. This article must therefore be hereby marked "advertisement" Medical Center, The Pennsylvania State University, Hershey, PA in accordance with 18 U.S.C. §1734 solely to indicate this fact. 17033. 4448 Downloaded by guest on October 2, 2021 Genetics: Tripputi et al. Proc. Natl. Acad. Sci. USA 82 (1985) 4449 ing 50% formamide and 10% dextran sulfate (pH 7.0)]. Probe Human Chromosomes DNA was denatured for 5 min at 70'C and quickly chilled on -1 Hybrids 1 2 3 4 5 6 7 8 9 10111213141516171819202122 x Human ice. Probe DNA was added to the slides at a concentration of urokinase DSK1B2A5 02 0.05 gg/ml. The slides were coverslipped, put in moist DSK1B2A5 C20_ chambers, and hybridized for 18 hr at 370C. After hybridiza- PT47 M3 tion, the slides were extensively washed at 390C to remove Nufi nonspecifically bound labeled DNA and were dehydrated PAFxBaWbI C5 through an alcohol series. Slides were dipped in liquid 57-37-IFI M21 nuclear track emulsion (Kodak NTB-2), stored in the dark, MUM2S5 771-C105 + and developed at appropriate intervals. Slides were banded 077-Bl0CIO_ ____ by using a modified Wright's Giemsa protocol (27) and 77-810 CU25 * 77-BIO Ct26 * + analyzed under the light microscope. 77-BIOCt2 _____ _ Metaphase spreads with good chromosome morphology 77-B10 C30 __ __ and few background grains were selected. The location of 77-Bl0 C31 * _ __* *+ specific grains was determined using an idiogram from Yunis GMxLM 013 et al. (28). GMxLM 0_ __ GMxLMI RESULTS CUC275 S ______ 57-77F7DC7 __ Mapping ofthe Human Urokinase Gene on Chromosome 10. D206 53 A 637-base-pair-long urokinase cDNA fragment was used as ipi+ probe in Southern blot experiments. Fig. 1 A and B shows the results ofhybridization ofthe probe with the Sst I and HindIII FIG. 2. Histogram ofhuman chromosome complement contained digests, respectively, ofDNA from (i) human-mouse somatic in urokinase-positive hybrids from Fig. 1 and additional hybrids. cell hybrids containing different complements of human Chromosome content was determined by isoenzyme and cytogenetic chromosomes and (ii) from the parental human and mouse analysis. cells. A single positive hybridization band was detected in control human DNA (Fig. 1 A and B, lane 1), but no bands 2 shows that the urokinase plasminogen activator gene were detected in the mouse DNA (Fig. 1 A and B, lane 16). segregates concordantly with human chromosome 10. As expected on the basis ofthe restriction map and sequence Subregional Mapping of the Urokinase Gene. To map the of genomic DNA clones (34), bands of -7 kb and 10 kb were precise region of chromosome 10 containing the urokinase obtained with Sst I and HindIII, respectively. The DNA of gene, we used somatic cell hybrids between mouse cells IT22 three of the 14 human-mouse hybrids was positive for and human fibroblasts carrying a translocation of the distal hybridization (Fig. 1, lanes 4, 7, and 8). Digestion with third ofthe long arm ofchromosome 10 to chromosome 17 (10 BamHI endonuclease gave a 10.3-kb positive band in these pter-- 10q24 :: 17 q13 -- 17 qter; 17 pter-* 17 q13 :: 10 q24 same hybrids (not shown). The histogram in Fig. 2 was -* 10 qter) (30). These hybrids have been used to assign the constructed from the analysis ofthese and other hybrids. The gene for cytoplasmic glutamic-oxaloacetic transaminase complement of human chromosomes of the different hybrids (GOT; aspartate aminotransferase, EC 2.6.1.1) to the region was established by isoenzyme and cytogenetic analyses. Fig. q24 -- qter of human chromosome 10(31). As shown in Fig. 3, three of the hybrids were positive (10.3-kb band) When the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 BamHI digests were hybridized with the urokinase probe.
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