Prostate cancer is the most frequently diagnosed cancer in American men. Screening for prostate-specific antigen (PSA) has led to earlier detection of prostate cancer, but elevated serum PSA levels may be present in non-malignant conditions such as benign prostatic hyperlasia (BPH). Characterization of gene-expression profiles that molecularly distinguish prostatic neoplasms may identify genes involved in prostate carcinogenesis, elucidate clinical biomarkers, and lead to an improved classification of prostate cancer. Using microarrays of complementary DNA, we examined gene-expression profiles of more than 50 normal and neoplastic prostate specimens and three common prostate-cancer cell lines. Signature expression profiles of normal adjacent prostate (NAP), BPH, localized prostate cancer, and metastatic, hormone-refractory prostate cancer were determined. Here we establish many associations between genes and prostate cancer. We assessed two of these genes-hepsin, a transmembrane serine protease, and pim-1, a serine/threonine kinase-at the protein level using tissue microarrays consisting of over 700 clinically stratified prostate-cancer specimens. Expression of hepsin and pim-1 proteins was significantly correlated with measures of clinical outcome. Thus, the integration of cDNA microarray, high-density tissue microarray, and linked clinical and pathology data is a powerful approach to molecular profiling of human cancer.

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.

Factor VII is a precursor to a serine protease that is present in mammalian plasma. In its activated form, it participates in blood coagulation by activating factor X and/or factor IX in the presence of tissue factor and calcium. Clones coding for factor VII were obtained from two cDNA libraries prepared from poly(A) RNA from human liver and Hep G2 cells. The amino acid sequence deduced from the cDNAs indicates that factor VII is synthesized with a prepro-leader sequence of 60 or 38 amino acids. The mature protein that circulates in plasma is a single-chain polypeptide composed of 406 amino acids. The amino acid sequence analysis of the protein and the amino acid sequence deduced from the cDNAs indicate that factor VII is converted to factor VII8 by the cleavage of a single internal bond between arginine and isoleucine. This results in the formation of a light chain (152 amino acids) and a heavy chain (254 amino acids) that are held together by a disulfide bond. The light chain contains a -carboxyglutamic acid (Gla) domain and two potential epidermal growth factor domains, while the heavy chain contains the serine protease portion of the molecule. Factor VII shows a high degree of amino acid sequence homology with the other vitamin K-dependent plasma proteins.

A cDNA library prepared from human liver has been screened for factor IX (Christmas factor), a clotting factor that participates in the middle phase of blood coagulation. The library was screened with a single-stranded DNA prepared from enriched mRNA for baboon factor IX and a synthetic oligonucleotide mixture. A plasmid was identified that contained a cDNA insert of 1,466 base pairs coding for human factor IX. The insert is flanked by G-C tails of 11 and 18 base pairs at the 5' and 3 ' ends, respectively. It also included 138 base pairs that code for an aminoterminal leader sequence, 1,248 base pairs that code for the mature protein, a stop codon, and 48 base pairs of noncoding sequence at the 3' end. The leader sequence contains 46 amino acid residues, and it is proposed that this sequence includes both a signal sequence and a pro sequence for the mature protein that circulates in plasma. The 1,248 base pairs code for a polypeptide chain composed of 416 amino acids. The amino-terminal region for this protein contains 12 glutamic acid residues that are converted to y-carboxyglutamic acid in the mature protein. These glutamic acid residues are coded for by both GAA and GAG. The arginyl peptide bonds that are cleaved in the conversion ofhuman factor IX to factor IXa by factor XI. were identified as Arg145-Ala146 and Argl"NValls1. The cleavage of these two internal peptide bonds results in the formation of an activation peptide (35 amino acids) and factor IXa, a serine protease composed of a light chain (145 amino acids) and a heavy chain (236 amino acids), and these two chains are held together by a disulfide bond(s). The active site residues including histidine, aspartate, and serine are located in the heavy chain at positions 221, 270, and 366, respectively. These amino acids are homologous with His57, AspIO, and Ser'95 in the active site of chymotrypsin. Two potential carbohydrate binding sites (Asn-X-Thr) were identified in the activation peptide, and these were located at Asn157 and Asn167. The homology in the amino acid sequence between human and bovine factor IX was found to be 83%.Factor IX (Christmas factor)* is a vitamin K-dependent plasma protein that plays an important role in the middle phase of blood coagulation (1). Individuals lacking this protein may bleed spontaneously into their skin, soft tissues, and joints, and this bleeding is often serious in patients after minor injury. A deficiency of factor IX (Christmas disease or hemophilia B) affects primarily males because it is transmitted as a sex-linked recessive trait. Factor IX has been extensively purified from bovine and human plasma (2, 3). At the present time, approximately 20% of the amino acid sequence for the human molecule has been determined (4), and the entire sequence for the bovine molecule has been established (5). Both proteins are single-chain glycoproteins (Mr 55,000-57,000) with an amino-terminal sequence of Tyr-Asn-Ser-Gly-Lys. The human and bovine proteins also contain 12 y-carboxyglutamic acid residues in their amin...

A B S T R A C T Human Factor IX (Christmas factor) is a single-chain plasma glycoprotein (mol wt 57,000) that participates in the middle phase of the intrinsic pathway of blood coagulation. It is present in plasma as a zymogen and is converted to a serine protease, Factor IXao, by Factor XIa (activated plasma thromboplastin antecedent) in the presence of calcium ions. In the activation reaction, two internal peptide bonds are hydrolyzed in Factor IX. These cleavages occur at a specific arginyl-alanine peptide bond and a specific arginyl-valine peptide bond. This results in the release of an activation peptide (mol wt-11,000) from the internal region of the precursor molecule and the generation of Factor IXaO (mol wt -46,000). Factor IXa, is composed ofa light chain (mol wt -18,000) and a heavy chain (mol wt -28,000), and these chains are held together by a disulfide bond(s). The light chain originates from the amino terminal portion of the precursor molecule and has an amino terminal sequence of Tyr-Asn-Ser-Gly-Lys. The heavy chain originates from the carboxyl terminal region of the precursor molecule and contains an amino terminal sequence of Val-Val-Gly-Gly-Glu. The heavy chain of Factor IXa, also contains the active site sequence of Phe-Cys-Ala-Gly-Phe-His-Glu-Gly -Gly -Arg -Asp -SerCys-Gln-Gly-Asp-SER-Gly-Gly-Pro. The active site serine residue is shown in capital letters. Factor IX is also converted to Factor IXa,,: by a protease from Russell's viper venom. This activation reaction, however, occurs in a single step and involves only the cleavage of the internal arginyl-valine peptide bond. Human Factor IX,,3 was inhibited by human antithrombin III by the formation of a one-to-one complex of enzyme and inhibitor. In this reaction, the inhibitor was tightly bound to the heavy chain of the enzyme. These data indicate that the mechanism of activation of human Factor IX and its inhibition by antithrombin III is essentially identical to that previously shown for bovine Factor IX.