MOLECULAR-CLONING OF CDNA FOR THE B-BETA-SUBUNIT OF XENOPUS FIBRINOGEN, THE PRODUCT OF A COORDINATELY-REGULATED GENE FAMILY

Fibrinogen, the principal blood-clotting protein, is made up of three different subunits synthesized in the liver. In vitro administration of glucocorticoids to liver cells from the frog Xenopus laevis causes a dramatic increase in fibrinogen synthesis. Investigations of molecular mechanisms underlying this hormonal stimulation at the mRNA level require cDNA clones complementary to the mRNAs coding for the three fibrinogen subunits, called A-alpha, B-beta, and gamma. We describe here the isolation and characterization of cDNA clones for the B-beta subunit of Xenopus fibrinogen. cDNA libraries in both plasmid (pBR322) and phage (lambda-gt10) cloning vectors were constructed from frog liver mRNA and screened with a rat B-beta cDNA. Clones thus isolated hybridized to two Xenopus liver mRNAs 2500 and 1800 bases long, the previously-determined sizes for B-beta mRNAs. The identity of the plasmid clone B-beta-27 was confirmed by hybridization-selection of complementary mRNA which translated in vitro into the B-beta polypeptide, as determined by size and susceptibility to thrombin cleavage. lambda/B-beta-10, a clone representing nearly all of the 2500-base B-beta mRNA, was isolated from the phage cDNA library. The 3'-end of this clone includes a polyadenylation signal about 20 residues upstream of a stretch of 34 adenosine residues, which probably represents the 3'-poly(A) tail of the messenger RNA. lambda/B-beta-10 lacks only 20 nucleotides of full-length B-beta mRNA at the 5'-end and there is one major start site of transcription. The 2500-base B-beta mRNA has a 700-base extension at the 3'-end that is not present in the 1800-base mRNA. The Xenopus laevis genome contains two or three genes for the B-beta fibrinogen subunit. Using the mDNA clone as a probe, B-beta mRNA was shown to be induced at least 20-fold by glucocorticoid treatment of purified parenchymal cells of Xenopus liver maintained in primary culture.