Cloning and analysis of a cDNA coding for bovine prothrombin.

Abstract

Poly(A)RNA enriched for prothrombin was isolated by specific immunoprecipitation ofbvine liver polysomes. Prothrombin consisted of about 8% of the cell-free translation products of this RNA. A double-stranded cDNA was synthesized by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and made blunt-ended with nuclease SI. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved previously at the single Pst I site and similarly tailed with dGTP. The resulting plasmids were used to transform Escherichia coli strain RRI under P3-EKI conditions. Sixtythree tetracycline-resistant clones were obtained that hybridized to nP-labeled cDNA synthesized from prothrombin-enriched mRNA. Recombinants containing cDNA to prothrombin mRNA sequences were screened by a solution hybridization assay with a[cDNA synthesized from mRNA. This enriched mRNA was 50% prothrombin mRNA, as determined by a reticulocyte lysate translation assay. Three positive clones were identified by this assay; they contained bovine DNA inserts of 700,500, and 400 base pairs. The DNA sequence of the 700-base-pair insert was then determined. This recombinant plasmid contained DNA coding for the carboxyl-terminal 160 residues of bovine prothrombin followed by a noncoding region of 119 base pairs and a poly(A) tail of 60 base pairs.The coagulation of blood is the result of a series of stepwise reactions in which many of the coagulation proteins in plasma are converted from zymogens to highly specific serine proteases (see ref. 1 for a recent review). In the final stages of the blood coagulation cascade, prothrombin is converted to thrombin by Factor Xa in the presence of phospholipid, calcium ions, and Factor Va. Thrombin then cleaves fibrinogen to form an insoluble fibrin clot. Although prothrombin is one of the most abundant coagulation proteins present in plasma, its mRNA constitutes only about 1% of the total poly(A)-RNA in bovine liver (2). This level of RNA makes it difficult to purify prothrombin mRNA to the degree of homogeneity required for many biochemical investigations. Recent developments in recombinant DNA techniques, however, permit the isolation of large amounts of homogeneous DNA corresponding to the specific mRNAs. Such cloned DNAs can be used for many different studies, such as the quantitation of cellular mRNA levels in response to various stimuli, the purification of specific mRNA by hybridization to immobilized DNA, and as a hybridization probe to isolate and study genomic DNA.In this paper, we describe the construction, cloning in Escherichia colh, and sequence of a recombinant plasmid containing DNA coding for the carboxyl-terminal portion of bovine prothrombin. Enrichment of mRNA. Polysomes were prepared from bovine liver as described (2). Polysomes synthesizing prothrombin were enriched by specific immunoprecipitation with antiprothrombin prepared in rabbits and either goat anti-rabbit immunoglobulin coupled to p-aminobenzyl-cellulose (3) or ...

Characterization of a cDNA clone coding for the beta chain of bovine fibrinogen.

“…Reverse transcription of enriched bovine liver mRNA, synthesis of doublestranded cDNA, homopolymeric G/C tailing, construction of recombinant plasmids by using pBR322 as vector, and cloning into E. coli K-12, strain RRI, have been reported (19). The cloning was performed under P3-EK1 conditions in compliance with the revised (1978) guidelines for recombinant DNA research from the National Institutes of Health.…”

“…This was probably an underestimation of the mRNA purity, however, because the antibodies used in these studies were prepared against the intact fibrinogen molecule, and the efficiency of these antibodies in recognizing and binding to individual nascent chains may be low (29). The radiolabeled cDNA probe was then hybridized individually to a collection of recombinant plasmids containing bovine liver cDNA (19). Each hybrid was examined for protection against nuclease Si digestion.…”

“…In order to identify clones carrying inserted cDNA coding for fibrinogen, radiolabeled cDNA was synthesized from enriched fibrinogen mRNA and used in a solution hybridization assay (19). As estimated by cell-free translation and immunoprecipitation, about 14% of the translation products from the enriched mRNA were specifically precipitated by antifibrinogen antibodies.…”

“…mRNA for fibrinogen was enriched by immunoadsorption with Staphylococcus aureus cells and affinity-purified antibodies to bovine fibrinogen (21). Radiolabeled cDNA was prepared from the enriched mRNA and was used as a probe in a solution hybridization screening procedure as described (19).…”

The Prothrombin Gene

“…As it was not possible to determine the complete structure of preprothrombin by au tomated sequenator analysis, MacGillivray et al [38] cloned a cDNA copy of prothrombin mRNA into a bacterial plasmid. The amino acid sequence of the precursor was then pre dicted from the DNA sequence.…”