alpha 1-Antichymotrypsin mRNA was isolated by specific polysome immunoprecipitation from turpentine-treated baboon liver. The highly enriched mRNA was used for synthesis and cloning of the corresponding cDNA. Baboon alpha 1-antichymotrypsin cDNA clones were identified by hybrid-selected translation, and the insert DNA fragment from one of the putative clones was used as a probe to screen a human liver cDNA library comprised of 40 000 independent transformants. One of the human cDNA clones was unambiguously identified to contain alpha 1-antichymotrypsin DNA sequences by comparison of its 5'-terminal nucleotide sequence with the N-terminal amino acid sequence of the protein. This cDNA clone, designated phACT235, contains 1524 base pairs of human DNA, which was sequenced in its entirety. The inserted DNA codes for a 25 amino acid signal peptide sequence and the entire mature alpha 1-antichymotrypsin of 408 amino acid residues. Comparison of the amino acid sequence of alpha 1-antichymotrypsin with that of the human alpha 1-antitrypsin has revealed a homology level similar to that between chymotrypsin and trypsin.
A human liver cDNA library was constructed by using poly(A)-containing RNA isolated from a human liver biopsy specimen. This library is comprised of 40,000 independent transformants with an average inserted DNA length of 1,200 base pairs. By using the previously cloned baboon antithrombin III cDNA as a specific hybridization probe, >30 human antithrombin mII cDNA clones were identified from this library. The Antithrombin III is a plasma protease inhibitor synthesized in the liver. The glycoprotein has a molecular weight of 55,000 and its entire amino acid sequence has almost been completed (1). It is a natural anticoagulant in that it specifically inhibits a number of serine proteases that participate in the blood coagulation cascade, including thrombin, factors IXa, Xa, XIa, and XIIa (2-5). The mechanism of inhibition involves the stoichiometric formation of protease-antiprotease complexes and the rate of complex formation is greatly enhanced in the presence of heparin, which is a well-known anticoagulant used clinically in myocardial infarction and surgery (6, 7). Deficiency of antithrombin III is a hereditary disorder that is associated with recurrent thrombophlebitis, acute aortic thrombosis, and thromboembolism (8-10). Heterogeneity of the classical antithrombin III deficiency has been observed (11). Abnormal antithrombin III also has been isolated from deficient patients and partially characterized (12), suggesting that the deficiency could be the result of mutations in the antithrombin III gene itself. Therefore, the genetic deficiency can be analyzed in molecular detail if the human antithrombin III gene can be isolated and characterized. We recently have reported the purification of antithrombin III mRNA from a baboon liver by polysome immunoprecipitation and the cloning of its cDNA (13). In this paper, we report the construction of a human liver cDNA library and the identification and sequence of a full-length human antithrombin III cDNA clone. MATERIALS AND METHODSConstruction of a Human Liver cDNA Library. A human liver biopsy specimen was kindly provided by Bill Williams (University of Texas Medical School in Houston). Total nucleic acid was extracted from the frozen tissue with phenol (14), and poly(A)-containing RNA was prepared by oligo(dT)-cellulose column chromatography (15). The RNA preparation was used for cDNA synthesis under conditions that favored full-length cDNA production (16). The cDNA preparation was sedimented through an alkaline sucrose gradient (16) and only fractions containing cDNA species of 1,000 nucleotides or more were pooled. The single-stranded cDNA was subsequently made double-stranded with reverse transcriptase (17). After nuclease S1 treatment, tracts of poly(dC) were added to the 3' termini of the DNA molecules by using terminal transferase (18). The enzymatically synthesized cDNA was rehybridized with Pst I-linearized pBR322 DNA that had been tailed with poly(dG) and the DNA was used for transformation of Escherichia coli RR1 (18). Bacterial transformants we...
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