Isolation and characterization of the human tissue-type plasminogen activator structural gene including its 5' flanking region.
mRNA specific for tissue type plasminogen activator (t-PA) is induced in HeLa cells by the tumor promoter phorbol myristate acetate (Waller, E.K., and Schleuning, W.D. (1985) J. Biol. Chem. 260, 6354-6360). To study the underlying mechanism, a cDNA library was constructed from phorbol myristate acetate stimulated HeLa cell mRNA and screened with two t-PA mRNA specific oligonucleotides (Edlund, T., Ny, T., Rånby, M., Heden, L.-O., Palm, G., Holmgren, E., and Josephson, S. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 349-352). Two nearly full length double-stranded cDNA clones were obtained. Suitable restriction fragments from the cDNA were employed as probes for the isolation of three recombinant bacteriophages, containing overlapping fragments of the t-PA gene. By restriction analysis, heteroduplex mapping, and DNA sequencing it was determined that the three overlapping fragments contain the complete t-PA structural gene and that the 2658 bases long t-PA mRNA is encoded by a gene of approximately 29 kilobases overall length, which is interrupted by 13 introns. To characterize the presumptive control region, a subcloned gene fragment, containing the 5' sequence of the cDNA, was sequenced, and the transcription initiation site was identified by nuclease S1 protection experiments. The putative transcription start site is located 24 base pairs (bp) downstream of a typical TATA consensus sequence. Two additional TATA motifs with hitherto unknown functions are found 93 and 226 bp upstream of the putative cap site. A recombinant plasmid was constructed, which accommodates the cap site including 475 bp of upstream sequences, fused to a double-stranded cDNA of t-PA mRNA which contains the complete translated and parts of the 5' and 3' untranslated regions. This plasmid directs t-PA biosynthesis in Xenopus laevis oocytes after microinjection into the germinal vesicle.
Fisher
R
R
Waller
E K
EK
Grossi
G
G
Thompson
D
D
Tizard
R
R
Schleuning
W D
WD
D000595:Amino Acid Sequence; D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004273:DNA, Neoplasm / Q000302:isolation & purification; D005260:Female; D005796:Genes / Q000187:drug effects; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D009865:Oocytes / Q000378:metabolism; D010957:Plasmids; D010960:Plasminogen Activators / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014981:Xenopus
D004273:DNA, Neoplasm; D012333:RNA, Messenger; D004247:DNA; D010960:Plasminogen Activators; D013755:Tetradecanoylphorbol Acetate