Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe. Five overlapping lambda phages were identified that contained the gene for factor IX. The complete DNA sequence of about 38 kilobases for the gene and the adjacent 5' and 3' flanking regions was established by the dideoxy chain termination and chemical degradation methods. The gene contained about 33.5 kilobases of DNA, including seven introns and eight exons within the coding and 3' noncoding regions of the gene. The eight exons code for a prepro leader sequence and 415 amino acids that make up the mature protein circulating in plasma. The intervening sequences range in size from 188 to 9473 nucleotides and contain four Alu repetitive sequences, including one in intron A and three in intron F. A fifth Alu repetitive sequence was found immediately flanking the 3' end of the gene. A 50 base pair insert in intron A was found in a clone from one of the genomic libraries but was absent in clones from the other library. Intron A as well as the 3' noncoding region of the gene also contained alternating purine-pyrimidine sequences that provide potential left-handed helical DNA or Z-DNA structures for the gene. KpnI repetitive sequences were identified in intron D and the region flanking the 5' end of the gene. The 5' flanking region also contained a 1.9-kb HindIII subfamily repeat. The seven introns in the gene for factor IX were located in essentially the same position as the seven introns in the gene for human protein C, while the first three were found in positions identical with those in the gene for human prothrombin.

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A human genomic DNA library was screened for the gene for protein C by using a cDNA probe coding for the human protein. Three different overlapping A Charon 4A phage were isolated that contain inserts for the gene for protein C. The complete sequence of the gene was determined by the dideoxy method and shown to span about 11 kilobases of DNA. The coding and 3' noncoding portion of the gene consists of eight exons and seven introns. The eight exons code for a preproleader sequence of 42 amino acids, a light chain of 155 amino acids, a connecting dipeptide of Lys-Arg, and a heavy chain of 262 amino acids. The preproleader sequence and the connecting dipeptide are removed during processing, resulting in the mature protein composed of a heavy and a light chain held together by a disulfide bond. The heavy chain also contains the catalytic region for the serine protease. Two Alu sequences and two homologous repeats of about 160 nucleotides were found in intron E. The seven introns in the gene for protein C are located in essentially the same positions in the amino acid sequence as the seven introns in the gene for human factor IX, while the first three introns in protein C are located in the same positions as the first three in the gene for human prothrombin.Protein C is a precursor to a serine protease present in plasma that plays an important physiological role in the regulation of blood coagulation (1,2). Human protein C is a vitamin K-dependent glycoprotein containing nine residues ofcarboxyglutamic acid and one equivalent of p-hydroxyaspartic acid. Protein C shows considerable structural homology with the other vitamin K-dependent plasma proteins involved in blood coagulation, including prothrombin, factor VII, factor IX, and factor X. Protein C is synthesized as a single-chain polypeptide that undergoes considerable processing to give rise to a two-chain molecule held together by a disulfide bond. The two-chain form is converted to activated protein C by thrombin by the cleavage of a 12-residue peptide from the amino terminus of the heavy chain (2). This reaction is greatly accelerated by the presence of thrombomodulin (3). Activated protein C regulates the coagulation process by the inactivation of factor Va (4, 5) and factor VIIIa (4, 6) by minor proteolysis. Consequently, individuals lacking protein C often have a history of thrombotic disease (7,8).Studies from our laboratory (9) and that of others (10) have led to the isolation and characterization of the cDNA coding for human and bovine protein C. In the present investigation, the cDNA for human protein C has been used for the isolation of overlapping genomic clones from a X Charon 4A phage library. The nucleotide sequence of the gene was then determined and compared with the genes for human factor LX (11, 12) and prothrombin (13).MATERIALS AND METHODS Screening of the Genomic Library. A human genomic library in X Charon 4A phage (14) was screened for genomic clones of human protein C by the plaque hybridization procedure ofBenton and Davis as modi...

A lambda gt 11 library containing cDNA inserts prepared from human liver mRNA has been screened with an affinity-purified antibody to human histidine-rich glycoprotein (HRG) and then with a restriction fragment isolated from the 5' end of the largest cDNA insert obtained by antibody screening. A number of positive clones were identified and shown to code for HRG by DNA sequence analysis. A total of 2067 nucleotides were determined by sequencing 3 overlapping cDNA clones, which included 121 nucleotides of 5'-noncoding sequence, 54 nucleotides coding for a leader sequence of 18 amino acids, 1521 nucleotides coding for the mature protein of 507 amino acids, a stop codon of TAA, and 352 nucleotides of 3'-noncoding sequence followed by a poly(A) tail of 16 nucleotides. The length of the noncoding sequence of the 3' end differed in several clones, but each contained a polyadenylylation or processing sequence of AATAAA followed by a poly(A) tail. More than half of the amino acid sequence of HRG consisted of five different types of internal repeats. Within the last 3 internal repeats (type V), there were 12 tandem repetitions of a 5 amino acid segment with a consensus sequence of Gly-His-His-Pro-His. This repeated portion, referred to as a "histidine-rich region", contained 53% histidine and showed a high degree of similarity to a histidine-rich region of high molecular weight kininogen.

HCV infection has a significant impact on kidney transplant recipients over the long term and in particular affects them in the second decade. Our pilot study revealed only partial efficacy of IFN-alpha therapy for HCV-infected recipients, but with the high risk of acute rejection.