The primary structure of polycystin predicts a large integral membrane protein with multiple cell recognition motifs, but its function remains unknown. Insight into polycystin's normal function and its role in the development of autosomal dominant polycystic kidney disease (PKD1) requires the assembly of an extensive collection of molecular reagents to examine its expression and create model systems for functional studies. Development of these crucial reagents has been complicated due to the presence of transcriptionally active homologous loci. We have assembled the authentic full-length PKD1 cDNA and demonstrated expression of polycystin in vitro. Polyclonal antibodies directed against distinct extra-and intracellular domains specifically immunoprecipitated in vitro translated polycystin. The panel of antibodies was used to determine localization of polycystin in renal epithelial and endothelial cell lines and tissues of fetal, adult, and cystic origins. In normal adult kidney and maturing fetal nephrons, polycystin expression was confined to epithelial cells of the distal nephron and vascular endothelial cells. Expression in the proximal nephron was only observed after injury-induced cell proliferation. Polycystin expression was confined to ductal epithelium in liver, pancreas, and breast, and restricted to astrocytes in normal brain. We report clear evidence for the membrane localization of polycystin by both tissue sections and by confocal microscopy in cultured renal and endothelial cells. Interestingly, when cultured cells made cell-cell contact, polycystin was localized to the lateral membranes of cells in contact. These data suggest that polycystin is likely to have a widespread role in epithelial cell differentiation and maturation and in cell-cell interactions.

SummaryDetection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH

Protein S is a cofactor of activated protein C; together they function as a regulator of blood coagulation. A human liver cDNA library constructed in bacteriophage Xgtll was screened with DNA fragments from a full-length bovine cDNA clone encoding protein S. Several cDNA clones were isolated and sequenced. The combined cDNA sequences encoded the mature protein and 15 residues ofthe leader sequence when compared to bovine protein S. Human protein S is a single-chain protein consisting of 635 amino acids with 82% homology to bovine protein S. After an NH2-terminal y carboxyglutamic acid-containing region, there is a short region with thrombin-sensitive bond(s), followed by a region with four repeat sequences that are homologous to the precursor of mouse epidermal growth factor. In contrast to the other vitamin K-dependent plasma proteins, the COOH-terminal portion of human protein S does not show any resemblance to serine proteases.Protein S is a single-chain plasma glycoprotein that undergoes vitamin K-dependent y-carboxylation during its biosynthesis (1-4). The concentration of protein S in human blood plasma is -25 mg/liter (4). Both human and bovine protein S have been purified from plasma, and recently the amino acid sequence of the bovine protein was established (5). Unlike the vitamin K-dependent clotting factors, protein S is not a proenzyme to serine protease but functions as a cofactor to activated protein C (6, 7). Patients with hereditary protein S deficiency, as well as those with protein C deficiency, suffer from a predisposition to venous thrombosis (8, 9). In addition to its established role as a cofactor to activated protein C, a regulatory role for protein S in the complement system has been suggested based on the observation that half of protein S in plasma is in a 1:1 complex with complement component C4b-binding protein (10,11).Bovine protein S has 11 y-carboxyglutamic acid (Gla in sequences) residues (12). In addition, acid hydrolysates ofthe bovine protein have been found to contain 03-hydroxyaspartic acid (Hya in sequences) (13-15), which is located in regions homologous to the epidermal growth factor (EGF) precursor (16) in all vitamin K-dependent plasma proteins except prothrombin (13-15, 17, 18); its function is unknown. We now report the isolation and sequence of human cDNA clones that code for mature human protein S. MATERIALS AND METHODSA human fetal liver cDNA library in phage Xgtll was prepared by a modification of the procedure of Gubler and Hoffman (19) similar to that described by Lapeyre and Amalric (20). The library contained >6 x 107 recombinants with inserts averaging 1800 nucleotides in length. Approximately 2 x 107 plaques from the amplified library were screened by standard techniques (21,22). RNA blot-hybridization analysis was conducted by standard methods (23). Nick-translated DNA fragments from a bovine protein S cDNA clone, pBLS-2400 (5), and a human protein C cDNA clone (N. Capalucci and R.W., unpublished data) were used as probes.For sequence analysis, 200...