Xenopus fibrinogen: characterization of the mRNAs for the three subunits.

Abstract

We purified and characterized the mRNAs coding for each of the three subunits of Xenopus fibrinogen. Purification was accomplished by electrophoretic separation of liver polyadenylated RNA in a fully denaturing gel, followed by recovery of the RNA from the gel via transfer to an ion-exchange membrane. This procedure yielded fractions which were highly enriched for the mRNAs for each of the fibrinogen chains. The fibrinogen mRNAs were identified by two methods: (i) in vitro translation followed by subunit-specific cleavage with the proteases thrombin and batroxobin; and (ii) cross-hybridization with cDNA clones for individual subunits of rat fibrinogen. The results demonstrate that the Aca and -y chains of frog fibrinogen are each coded by a single mRNA species. The Aca mRNA is ca. 3,100 nucleotides in length, which is nearly twice the minimum size required to code for the Aca precursor polypeptide. The y chain mRNA comprises about 1,600 bases and includes only a small untranslated region. In contrast, the BP subunit is synthesized from two mRNAs, one of which is 2,500 and the other 1,800 nucleotides long. The 2,500-base mRNA includes a large noncoding region, whereas the smaller one is near the minimum required size. The larger BP8 mRNA is ca. fivefold more abundant that the smaller species.Fibrinogen, which is synthesized and secreted by the liver, is found in the plasma of all vertebrates. The intact molecule comprises two sets of three nonidentical subunits designated Aax, BP, and -y. During the process of blood coagulation, the proteolytic enzyme thrombin cleaves the NH2-terminal A and B fibrinopeptides from the Aa and BP chains, thus generating fibrin which forms the structural matrix of a blood clot.We have previously described the polypeptide chain composition of fibrinogen from the frog Xenopus laevis (14,33 inducing the acute-phase response in mammals (16,19), was used in an effort to increase fibrinogen production in frogs. Polyadenylated RNA was purified (13) from the liver offrogs that had received a single injection of pure spirits of turpentine (Parks) into the leg muscle at a dose of 83 ,Il/50 g of body weight 12 to 24 h before sacrifice. This mRNA preparation was slightly enriched for the fibrinogen mRNAs relative to albumin mRNA, based on the in vitro translation assay, and was therefore used for the experiments described here. The sizes of all of the fibrinogen translation products and mRNAs were identical whether the RNA had been isolated from normal or turpentine-treated animals.To isolate the mRNAs coding for the Aa, BP, and -y chains of fibrinogen, liver mRNA was fractionated in a methylmercuric hydroxide agarose gel and recovered from the gel by transfer to and elution from a DEAE membrane (13). The high resolution of the RNA achieved during electrophoresis was preserved by slicing the DEAE membrane into 1-mm sections. The RNA eluted from each membrane slice was translated in vitro, and the products were resolved in a sodium dodecyl sulfate (SDS)-polyacrylamide gel (Fig. 1). All translated...