Purification of Antithrombin I11 mRNA and Cloning of Its cDNA*

Antithrombin I11 mRNA was enriched from a baboon liver by specific polysome immunoprecipitation. The partially purified antithrombin III mRNA preparation was used for cDNA synthesis and cloning. Candidate antithrombin 111 cDNA clones were identified by differ- ential hybridization using as probes [32P]cDNAs synthesized from the polysome-enriched and -depleted RNA fractions, respectively. The candidate clones were further analyzed by hybrid-selected translation. The authenticity of a cDNA clone positive to both tests was unambiguously confirmed by matching its nucleotide sequence with the known amino acid sequence of hu- man antithrombin III. The baboon antithrombin 111 cDNA clone hybridized well with human antithrombin 111 mRNA and can be used as a probe to isolate the corresponding human gene.

Antithrombin I11 is a plasma protease inhibitor synthesized in the liver. The glycoprotein has a molecular weight of 55,000 and its entire amino acid sequence is known (1). It is a natural anti-coagulant in that it specifically inhibits a number of serine proteases in the coagulation cascade, including thrombin, factors VIIa, IXa, Xa, and XIIa (2)(3)(4)(5). Its activity is enhanced tremendously after binding to heparin which is a well known anti-coagulant used clinically in myocardial infarction and surgery (6)(7). Deficiency of antithrombin I11 is a hereditary disorder that is associated with recurrent thrombophlebitis, acute aortic thrombosis, and thromboembolism (8)(9)(10). Heterogeneity of the classical antithrombin I11 deficiency has been observed (11). Abnormal antithrombin I11 has also been isolated from deficient patients and partially characterized (12), suggesting that the deficiency could be the result of mutations in the antithrombin 111 gene itself. The genetic deficiency, therefore, can be analyzed in molecular detail if the antithrombin I11 gene can be isolated and characterized. As a fist step toward this goal, we report the purification of antihrornbin I11 mRNA and the cloning of its cDNA.

MATERIALS AND METHODS
Purification of Human Antithrombin Iff and Its Antibody-Antithrombin 111 was partially purified from human plasma by column * This work was supported in part by Grant HL-27509 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article with 18 U.S.C. Section 1734 solely to indicate this fact. must therefore be hereby marked "aduertisement" in accordance $ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed. chromatography using Bio-Rad Affi-Gel blue (13). Column fractions were analyzed for the presence of antithrombin I11 by immunodiffusion (14) using a rabbit anti-serum against human antithrombin I11 purchased from Sigma. The partially purified antithrombin I11 preparation was covalently linked to cyanogen bromide-activated Sepharose (Pharmacia). Specific immunoglobulin molecules against human antithrombin I11 were purified from the crude rabbit antiserum and rendered ribonuclease free by antigen affinity column chromatography (15).
Purification of Antithrombin 1 1 1 mRNA by Polysome Immunopreczpitution-Polysomes were extracted from a baboon liver according to a previously reported procedure (16). Polysomes engaged in antithrombin I11 synthesis were enriched from total liver polysomes by specific immunoprecipitation using affinity purified antibody against antithrombin I11 and Staphylococcus aureus cells (15). RNA was released from the bound polysome by treatment with sodium dodecyl sulfate/EDTA. Polyadenylate-containing RNA was isolated by oligo(dT)-cellulose column chromatography (17). Enrichment of specific antithrombin I11 mRNA by this procedure was accessed by cellfree translation in an mRNA-dependent rabbit reticulocyte lysate system (18) in the presence of [35S]methlonine, followed by immunoprecipitation from the total translation products (19). Total and immunoprecipitated translation products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (20) and radioactive protein bands were visualized by fluorography (21).
Synthesis and Cloning of Baboon Antithrombin III cDNA-Previously reported procedures were employed for the synthesis of cDNA from enriched antithrombin I11 mRNA, and its insertion into the PstI site of pBR322 (16). Identification of antithrombin I11 cDNA clones by differential hybridization and hybrid-selected translation were as described (22). The positive clone was further analyzed by DNA sequencing (23). These experiments were performed according to the guidelines for recombinant DNA research from the National Institutes of Health.

RESULTS
Enrichment of Baboon Antithrombin 111 mRNA by Polysome Immunoprecipitation-Antithrombin I11 mRNA constitutes about 0.1% of total liver mRNA. Cell-free translation of baboon liver polyadenylate-containing RNA showed an array of proteins of different molecular weights on a denaturing polyacrylamide gel (Fig. lA, lane 4 ) , and only minute quantities of antithrombin I11 were detectable by immunoprecipitation of the translation products with the specifk immunoglobulins against antithrombin 111 (Fig. lB, lane 4 ) . A preparation of baboon liver polysomes was used for enrichment of antithrombin I11 mRNA by specific polysome immunoprecipitation. Cell-free translation of this mRNA preparation has shown that there was a marked decrease in total translational activity (Fig. lA, lane Z), but immunoprecipitation of these products yielded a distinct antithrombin I11 band (50,000 daltons) plus 2 apparently truncated antithrombin I11 protein products (Fig. lB, lane 2). Polysomal RNA depleted of antithrombin 111 mRNA produced a pattern similar to that of total polysornal RNA from baboon liver (Fig.  lA, lane 3 ) , and no antithrombin I11 was immunoprecipitable from these translation products ( Fig. 1 B, lane 3 ) . The enrichment for antithrombin I11 mRNA from total liver polysomes achieved by specific immunoprecipitation is estimated to be at least 50-fold.
Construction and Identification of a Baboon Antithrombin I Z I Clone-Single-stranded cDNA was synthesized from 3 pg of polysome-enriched antithrombin I11 mRNA and centrifuged through an alkaline-sucrose gradient. Fractions containing DNA longer than 1000 nucleotides was made double- stranded and inserted into the PstI site of pBR322 by the dG/ dC homopolymer addition method. Candidate antithrombin I11 cDNA clones were identified among recombinants by differential hybridization. Recombinant plasmid DNAs were prepared by the mini-lysate procedure (24), digested with BamHI and electrophoresed in an agarose gel. The gel was bi-directionally transferred to 2 nitrocellulose fiters (25). The duplicate fiters were hybridized separately with ["PIcDNA probes synthesized from polysome-enriched ( Fig. 2A) and -depleted (Fig. 2B) mRNA preparations. DNAs in lanes 1 and 6 hybridized strongly with both probes, while DNA in lanes 2 and 5 hybridized moderately with both probes. DNAs in lanes 3 and 4, however, hybridized strongly with the antithrombin 111-enriched probe but only moderately with the antithrombin 111-depleted probe. These are, therefore, candidate clones to contain baboon antithrombin I11 DNA sequences. Candidate antithrombin I11 cDNA clones were further analyzed by hybrid-selected translation. Immunoprecipitation of translation products from total baboon liver mRNA again showed very little radioactive antithrombin I11 (Fig. 3, lane  6), while the polysome-enriched polysomal mRNA again yielded a 50,000-dalton band plus 2 truncated translation product (Fig. 3, lane 3 ) . As a positive control for hybrid selection, a previously cloned baboon a1-antitrypsin cDNA (26) was also immobilized on to DBM-cellulose and used for hybrid selection. Immunoprecipitation of the translation products with anti-al-antitrypsin immunoglobulins yielded a strong al-antitrypsin band as expected (Fig. 3, lane 2). While protein synthesized from RNA selected by the cloning vector pBR322 showed no immunoprecipitable product with immunoglobulins against antithrombin I11 (Fig. 3, lane I ) , RNA selected by one of the candidate recombinant clones (pbAT 111) was apparently enriched for antithrombin iil mRNA as indicated by the presence of an immunoprecipitable 50,000dalton antithrombin I11 band (Fig. 3, lane 4 ) .
Confirmation of the Baboon Antithrombin ZZZ cDNA Clone  Confirmation of a baboon antithrombin I11 cDNA clone by nucleotide sequence analysis. The amino acid sequence has been deduced from the nucleotide sequence at the 5' terminus of the pbAT I11 DNA insert and compared to that of human antithrombin 111. The asterisks represent differences in amino acid residues between the two sequences.
The two sequences were identical to each other for 37 out of 40 residues compared, and this cDNA clone apparently contained antithrombin I11 DNA sequences that coded for amino acid residues 71 to the COOH terminus of the protein. Discrepancies at positions 73 (Lys uersus Asn) and 100 (Lys uersus Glx) could represent genuine amino acid substitutions in the antithrombin I11 genes of the two species (Fig. 4).

DISCUSSION
Antithrombin 111 mRNA has been enriched from total polysomes of a baboon liver by specific immunoprecipitation and used for cloning of its cDNA. A genuine baboon AT I11 cDNA clone has been identified by differential hybridization, hybrid-selected translation, and DNA sequencing. Since antithrombin 111 mRNA constitutes only about 0.1% of total liver mRNAs, the cloning procedure described in this report should be of general application to the cDNA cloning of other mRNAs present at similarly low cellular concentrations, provided that specific antisera to the particular proteins are available. We have recently reported the use of this approach to clone the cDNA for phenylalanine hydroxylase which is a hepatic enzyme deficient in phenylketonuria (22).
Since the baboon antithrombin I11 cDNA clone hybridized well with human antithrombin 111 mRNA (data not shown), it could be used as a hybridization probe to isolate the corresponding human gene and analyze the familial antithrombin 111 deficiency by gene mapping. This type of analysis has led to the development of gene mapping methodologies for prenatal diagnosis of various thalassemias and sickle cell anemia by genetic polymorphism linkage to the hereditary disorders (27,28). More recently, methods for direct analysis of the point mutation in the sickle cell trait have also been developed, mainly by identification of restriction enzymes that could distinguish the mutated nucleotide in the , 5 globin gene (29, 30). The cloning, sequencing, and comparison of the normal and deficient antithrombin 111 cDNAs should permit the development of such methodologies for prenatal diagnosis of antithrombin I11 deficiency.
Antithrombin 111 shares significant amino acid sequence homology with human al-antitrypsin (1,26), which is another plasma protease inhibitor, and with chicken ovalbumin (31), which is the major egg white protein that apparently has no protease inhibitor activity. The 3 proteins had been classified as members of a super family that had diverged 500 million years ago (31). We have recently reported the cloning and characterization of the human chromosomal al-antitrypsin gene (32). Comparison of its molecular structure with that of the chicken ovalbumin gene showed that the number, size, and positioning of the two sequence-related genes are completely different, suggesting that intronic sequences could also be inserted into pre-existing exonic sequences if the two genes arose by divergent evolution (32). Since the extent of sequence homology between antithrombin I11 and a1-antitrypsin is greater than that between a1-antitrypsin and chicken ovalbumin, it would be interesting to examine the genomic organization of the antithrombin I11 gene and compare its molecular structure with those of the human al-antitrypsin and chicken ovalbumin genes. These studies could lead to a better understanding on the evolutionary origin of this interesting gene family.