DNA from a transducing bacteriophage carrying a fusion of the tryptophan and lactose operons of E. coli (Xdtrp-lac) has been used to direct cell-free synthesis of j3-galactosidase (EC 3.2.1.23). Whereas normal lac operon (Xdlac) DNA requires adenosine-3': 5'-cyclic monophosphate (cAMP) for i3-galactosidase synthesis, trp-lac DNA is unaffected by cAMP. This difference in cAMP dependence verifies the presence of a cAMP-requiring promoter in the lac operon that has been removed from the trp-lac DNA. Synthesis with trp-lac DNA is controlled by the protein product of the tryptophan repressor gene (trpR). Synthesis in extracts of trpR-(repressor-negative) cells is progressively reduced by increased additions of extract from trpR+ cells. No trpR product repression is seen when P-galactosidase synthesis is programmed by normal lac DNA. This highly sensitive and specific assay has facilitated quantitation and partial purification of the trp repressor.
A variety of novel transducing lines of phage lambda can be obtained by induction of a mixed culture of abnormal lysogens. Such a culture is simply made by mass lysogenization of a host lacking the normal prophage attachment site.
We have isolated clones ofEscherichia coli strain K-12 that contain a hybrid pBR322 plasmid having a 4.2-kilobase insert of a DNA transcript of the mRNA of human plasminogen activator, urokinase. The bacterially produced enzyme has properties similar to those of urokinase from human fetal kidney cells. Both enzymes occur in discrete forms ranging from 32,000 to 150,000 daltons in size. They react with antibody to purified urokinase from human kidney cells, bind to a benzamidine-Sepharose column, and induce plasminogen-dependent lysis of a fibrin clot.For the dissolution of a blood clot, it is necessary to activate plasminogen to form the protease plasmin which then degrades the fibrin network of the clot. A plasminogen activator called "urokinase" was first observed in human urine in 1951 by Williams (1). Urokinase-like activity has been found in cultures of human kidney cells (2), and the material was subsequently shown to be immunologically indistinguishable from urokinase derived from urine. In urine, as well as in tissue culture, urokinases of two sizes are found; they are called "type S2" and "type Si,, and have molecular weights of 54,700 and 31,400, respectively (3, 4).Human urokinase can be used to treat acute thromboembolic events such as venous and arterial thrombosis, pulmonary embolism, intracardiac thrombosis, and systemic embolism. However, the high cost of isolation of urokinase from either tissue culture cells or urine limits the use of this enzyme as a therapeutic agent. If urokinase could be obtained from microorganisms by recombinant DNA technology, one might have a more economical method of production. This report describes the construction of hybrid bacterial plasmids that produce human urokinase in Escherichia coli.MATERIALS AND METHODS Bacterial Strain. E. coli K-12 strain x1776 (5) dapD8, minAl, minB2, supE42, A-, rfb-2, nalA25, oms-2, thyA57, metC65, oms-1, 29 (bioH-asd), cycB2, cycAl, hsdR) was provided by R. Curtiss III.DNA and Enzymes. Plasmid pBR322 was isolated as described by Ratzkin and Carbon (6). Reverse transcriptase was obtained from Life Sciences (St. Petersburg, FL). E. coli DNA polymerase I (the large fragment) and various restriction enzymes were purchased from New England BioLabs. Terminal transferase was obtained from P-L Biochemicals and nuclease S1 was purchased from Miles. Digestions with restriction enzymes were carried out under conditions recommended by the supplier.Antibody Against Urokinase. Anti-urokinase antibody synthesis was induced in New Zealand White rabbits by injection of purified urokinase (0.8 mg, type SI) emulsified in Freund's adjuvant. After three more injections of antigen over a period of2.5 months, the rabbits were bled. The antibody was purified by ammonium sulfate precipitation, followed by DEAE-cellulose chromatography and urokinase affinity chromatography (7). This antibody was checked for purity by electrophoresis (8) and for activity by the Ouchterlony test (9).Isolation ofmRNA from Human Fetal Kidney Cells. Human fetal kidney ce...
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