Identification and primary sequence of an unspliced human urokinase poly(A)+ RNA.

Abstract

Human urokinase cDNA clones have been identified from a cDNA library prepared from total RNA of human fibroblasts transformed by simian virus 40 [Okayama, H. & Berg, P. (1983) Mol. Cell. Biol. 3,[280][281][282][283][284][285][286][287][288][289]. Synthetic oligonucleotides, corresponding to urokinase protein sequence, were used as probes. The cloned cDNA covers most of the coding sequence and the entire 3' untranslated region. The nucleotide sequence of one of the clones identifies this as a copy of a partially spliced polyadenylylated precursor to urokinase mRNA. The introns separate functionally different domains of the enzyme. Human urokinase mRNA has been identified by RNA blot and its size was estimated at 2500 nucleotides.Urokinase is a plasminogen activator isolated from human urine (1) and used in therapy as a physiological thrombolytic agent in deep-vein thromboses, coronary obstructions, etc. (2). Plasminogen activators convert the inactive zymogen plasminogen to the active serine protease, plasmin, thus initiating and finely controlling extracellular proteolysis (for review, see ref.3). Besides and beyond its interest as a therapeutic tool, urokinase appears to play an important role in tissue formation and degradation (3) and in the metastatic activity of human tumors (4). The level of urokinase mRNA appears to be controlled by a variety of physiological and experimental effectors, including hormones (5), neoplastic transformation (6, 7), growth factors (8, 9), and tumor promoters (10).Two different active forms of urinary urokinase have been isolated: a high-and a low-molecular-weight form (Mr, 54,000 and 33,000, respectively). High-molecular-weight urokinase is split by reduction into the A and B chains of Mr 20,000 and Mr 30,000, respectively (11). The low-molecularweight form is made up by the same B chain and by a 21 amino acid long Al chain (12). The A, Al, and B chains have been sequenced (11-13) and the Al chain has been shown to be the COOH terminus of the A chain. The sequence data suggest that the A chain is the NH2-and the B chain is the COOH-terminal portion of a single chain pro-urokinase precursor (12 Fig. 1) was synthesized according to a published technique (15) and was purified through a Partisyl Sax 25 x 0.8 cm HPLC column, with a linear gradient (1-300 mM) of potassium phosphate (pH 6.5) in 30% ethanol. Oligonucleotides 1, 2, and 4 (see Fig. 1) (22); 4 x 106 clones were obtained and screened by the high-density screening procedure (24). Duplicate filters (20,000-30,000 colonies each) were hybridized to 32P end-labeled oligonucleotides in 5x standard saline citrate (NaCl/Cit)/lOx Denhardt's solution/250 ,ug of E. coli transfer RNA carrier per ml/0.5% sodium dodecyl sulfate (lx NaCl/Cit = 0.15 M NaCl/0.015 M Na citrate; lx Denhardt's solution = 0.02% bovine serum albumin/0.02% Ficoll/0.02% polyvinylpyrrolidone) (25). Filters were incubated at 550C and the temperature was allowed to drop to 320C (oligonucleotides 1 and 2) or to 370C (oligonucleotides 3 and 4) in 2 hr. The hy...

Serine phosphorylation of biosynthetic pro‐urokinase from human tumor cells

“…One or more such breaks can occur on the same pro-uPA molecule. The first type of cleavage (Lys-158) generates the active two-chain uPA [28,29]. The second type, at Lys-135, liberates at low Mr uPA or pro-uPA (the carboxy terminus) and an amino terminal fragment (residues 1-135).…”

Functions of the Fibrinolytic System in Human Ito Cells and Its Control by Basic Fibroblast and Platelet–Derived Growth Factor

“…The levels of u-PA, u-PAR, and PAI-1 mRNA were determined in total RNA isolated from cultured human HSC by the RNase protection assay, as previously detailed. 16 Antisense (complementary to mRNA) RNA probes were obtained by run-off transcription of pGEM1 plasmids pGEM1 (Promega, Madison, WI) containing the following cDNA fragments: a BamHi-BamHI 585-bp fragment of plasmid pQ2, encoding for part of the human u-PAR gene, 30 and a BamHi-BamHI 1,214-bp cDNA fragment of plasmid pHUK-8 encoding for part of human urokinase plasminogen activator 32 ; the PAI-1 probe was generated by oligo-(dT)-primed reverse transcription of total cell RNA prepared from human placenta, with subsequent amplification using specific oligodeoxyribonucleotide primers. 33 The HindIIIHindIII 110-bp cDNA fragment encoding for human glyceraldehyde phosphate dehydrogenase (GAPDH) was used as a ''housekeeping'' gene.…”

Enhanced expression of the urokinase-type plasminogen activator gene and reduced colony formation in soft agar by ectopic expression of PU.1 in HT1080 human fibrosarcoma cells

“…A PstI fragment of human urokinase cDNA (Veder et al, 1984) was derived from the American Type Culture Collection (ATCC; Rockville, MD, USA).…”