Urokinase is one of the two plasminogen activators that catalyze the conversion of inactive plasminogen to plasmin. By combining somatic cell genetics, in situ hybridization, and Southern hybridization, we localized the human urokinase gene on the distal third of the long arm (q24-qter) of chromosome 10.Plasminogen activators catalyze plasminogen-dependent fibrinolysis, a key reaction controlling extracellular proteolysis (1). Two forms of plasminogen activator have been recognized: urokinase, isolated from human urine, and tissue plasminogen activator. Urokinase is a Mr 54,000 two-chain protein (2) derived from a single-chain precursor (3)(4)(5) (19,20). The chromosomal content of hybrids was determined by enzymatic analysis using starch gel electrophoresis according to standard methods (21) and by karyotypic analysis using a combination of trypsin Giemsa and G11 banding techniques as described (22).DNA Extraction, Southern Blot Analysis, and Hybridization. DNA from human, mouse, and hybrid cell lines was prepared by cell lysis, proteinase K digestion, extraction with phenol, and precipitation with ethanol as described (23). Fifteen micrograms of DNA were digested with 30 units of the appropriate restriction endonuclease in standard conditions recommended by the supplier (New England Biolabs). Fragments were separated by electrophoresis on a 1% agarose gel. DNA was denatured and transferred to nitrocellulose as described by Southern (24). DNA on nitrocellulose sheets was hybridized to 32P-labeled probe DNA at 370C in 4x NaCl/Cit (lx NaCl/Cit = 0.15 M NaCl/0.015 M sodium citrate, pH 7) containing 50% (vol/vol) formamide as described (25). After hybridization, the filters were washed twice with 2x NaCl/Cit at room temperature and twice in O.1x NaCl/Cit containing 0.2% sodium dodecyl sulfate at 650C, air-dried, and exposed to Kodak XRA-5 film.Preparation of Labeled DNA Probe. The urokinase probe is a 637-base-pair EcoRI fragment from human cDNA clonesidentified from a cDNA library prepared from total RNA of human fibroblasts transformed by simian virus 40 (8). This fragment contains the sequences encoding amino acids 162-302 of human prourokinase and includes a 221-base-pair intervening sequence (8). The DNA probes were labeled with 32Pby nick-translation (26) to specific activities of0.5-2 x 108 cpm/0.2 ,ug of DNA. DNA polymerase I was purchased from Boehringer Mannheim; [32P]NTP was from Amersham. Chromosome Preparation. Metaphase chromosome spreads were prepared from peripheral blood lymphocytes of a normal (46, XY) male donor by using standard techniques. Air-dried slides were kept in the cold (40C) for at least 1 week prior to their use in mapping studies.In Situ Hybridization. In situ hybridization studies were performed by using modifications of the protocols of Cannizzaro and Emanuel (27) and Yunis et al. (28). Air-dried metaphase chromosome preparations on glass slides were used 1-3 weeks after preparation. Slides were treated with RNase to remove any chromosomally bound RNA. The slides were wash...
