Strong homophilic interactions of the Ig-like domains of polycystin-1, the protein product of an autosomal dominant polycystic kidney disease gene, PKD1.

The 14 kb mRNA of the polycystic kidney disease gene PKD1 encodes a novel large (approximately 460 kDa) protein, polycystin-1, of unknown function that is responsible for autosomal dominant polycystic kidney disease (ADPKD). The unique organization of multiple adhesive domains of polycystin-1, including 16 Ig-like domains (or PKD domains) suggests that it may play an important role in cell-cell/cell-matrix interactions. Here we demonstrate the localization of polycystin-1 to epithelial cell-cell contacts in culture. These results along with structural predictions prompted us to propose that polycystin-1 is involved in cell-cell adhesion through its cluster of Ig-like repeats. We show that Ig-like domains II-XVI are involved in strong calcium-independent homophilic interactions in vitro. Domains XI-XVI form interactions with high affinity (K(d) = 60 nM) and domains II-V exhibit the lowest binding affinity (K(d) = 730 nM) in these studies. Most importantly, we show that antibodies raised against Ig-like domains of polycystin-1 disrupt cell-cell interactions in MDCK cell monolayers, thus indicating that polycystin-1 is directly involved in the cell-cell adhesion process. Collectively, these data suggest that interactions of the Ig-like repeats of polycystin-1 play an important role in mediating intercellular adhesion. We suggest that the loss of these interactions due to mutations in polycystin-1 may be an important step in cystogenesis.

Polycystin: in vitro synthesis, in vivo tissue expression, and subcellular localization identifies a large membrane-associated protein.

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.

The region surrounding the PKD1 gene: a 700-kb P1 contig from a YAC-deficient interval.

As part of an effort to identify the gene responsible for the predominant form of polycystic kidney disease (PKD1), we used a gridded human P1 library for contig assembly. The interval of interest, a 700-kb segment on chromosome 16p13.3, can be physically delineated by the genetic markers D16S125 and D16S84 and chromosomally characterized as a GC-rich isochore enriched for CpG islands, genes, and Alu-like repeats. Our attempts to recover CEPH YACs that encode this region of chromosome 16 were unsuccessful. However, we screened an arrayed P1 library using 15 distinct probes from the D16S125-D16S84 interval and identified 56 independent P1 clones. Only one probe from the interval was unsuccessful in identifying a P1 clone. Forty-four P1 clones were determined to be unique based on restriction enzyme analysis, and 42 of these were found to originate from chromosome 16p13.3, based on FISH to metaphase chromosomes. The 700-kb interval could be defined by a single sequence-ready contig comprised of 12 P1 clones and 1 cosmid clone. Our studies support the use of multiple libraries to generate the requisite physical reagents for positional cloning and encourage the use of Escherichia coli-based large-insert cloning systems to recover clones from YAC-deficient chromosomal intervals.

Generation of a transcriptional map for a 700-kb region surrounding the polycystic kidney disease type 1 (PKD1) and tuberous sclerosis type 2 (TSC2) disease genes on human chromosome 16p3.3.

A 700-kb region of DNA in human chromosome 16p13.3 has been shown to contain the polycystic kidney disease 1 (PKD1) and the tuberous sclerosis type 2 (TSC2) disease genes. An estimated 20 genes are present in this region of chromosome 16. We have initiated studies to identify transcribed sequences in this region using a bacteriophage P1 contig containing 700 kb of DNA surrounding the PKD1 and TSC2 genes. We have isolated 96 unique exon traps from this interval, with 23 of the trapped exons containing sequences from five genes known to be in the region. Thirty exon traps have been mapped to additional transcription units based on data base homologies, Northern analysis, or their presence in cDNA or reverse transcriptase (RT)-PCR products. We have mapped the human RNPS gene to the cloned interval. We have obtained cDNAs or RT-PCR products from eight novel genes, with sequences from seven of these genes having homology to sequences in the data bases. Two of the newly identified genes represent human homologs for rat and murine genes identified previously. We have isolated three exon traps with homology to sequences in the data bases but have been unable to confirm the presence of these exon traps in expressed sequences. In addition, we have isolated 43 exon traps that do not map to our existing cDNAs or PCR products and have no homology to sequences in the data bases. In this report we present a transcriptional map for the 700 kb of DNA surrounding the PKD1 and TSC2 genes.

Analysis of the genomic sequence for the autosomal dominant polycystic kidney disease (PKD1) gene predicts the presence of a leucine-rich repeat. The American PKD1 Consortium (APKD1 Consortium).

The complete genomic sequence of the gene responsible for the predominant form of polycystic kidney disease, PKD1, was determined to provide a framework for understanding the biology and evolution of the gene, and to aid in the development of molecular diagnostics. The DNA sequence of a 54 kb interval immediately upstream of the poly(A) addition signal sequence of the PKD1 transcript was determined, and then analyzed using computer methods. A leucine-rich repeat (LRR) motif was identified within the resulting predicted protein sequence of the PKD1 gene. By analogy with other LRR-containing proteins, this may explain some of the disease-related renal alterations such as mislocalization of membrane protein constituents and changes in the extracellular matrix organization. Finally, comparison of the genomic sequence and the published partial cDNA sequence showed several differences between the two sequences. The most significant difference detected predicts a novel carboxy-terminus for the PKD1 gene product.

High resolution mapping of overlapping cosmids by fluorescence in situ hybridization.

The constituents of two cosmid contigs were analyzed by high resolution mapping using two-color fluorescence in situ hybridization (FISH) to extended DNA molecules. Samples were prepared by lysing the nuclei in situ followed by histone depletion. This treatment results in elongate DNA filaments appropriate for high resolution mapping. The hybridization signals appeared as a strong of fluorescent spots separated by non-fluorescing gaps. Probe-specific features of the hybridization patterns were detected and some of the non-fluorescing gaps were found to represent regions of repetitive DNA suppressed during hybridization.

Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: clinical experience with 4,500 specimens.

Detection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH). We herein describe the results of the first clinical program which utilized FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes. FISH was performed on physician request, as an adjunct to cytogenetics in 4,500 patients. Region-specific DNA probes to chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. A sample was considered to be euploid when all autosomal probes generated two hybridization signals and when a normal sex chromosome pattern was observed in greater than or equal to 80% of hybridized nuclei. A sample was considered to be aneuploid when greater than or equal to 70% of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. Of the attempted analyses, 90.2% met these criteria and were reported as informative to referring physicians within 2 d of receipt. Based on these reporting parameters, the overall detection rate for aneuploidies was 73.3% (107/146), with an accuracy of informative results for aneuploidies of 93.9% (107/114). Compared to cytogenetics, the accuracy of all informative FISH results, euploid and aneuploid, was 99.8%, and the specificity was 99.9%. In those pregnancies where fetal abnormalities had been observed by ultrasound, referring physicians requested FISH plus cytogenetics at a significantly higher rate than they requested cytogenetics alone. The current prenatal FISH protocol is not designed to detect all chromosome abnormalities and should only be utilized as an adjunctive test to cytogenetics. This experience demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies.

Chromosomal localization of glutamate receptor genes: relationship to familial amyotrophic lateral sclerosis and other neurological disorders of mice and humans.

Receptors for the major excitatory neurotransmitter glutamate may play key roles in neurodegeneration. The mouse Glur-5 gene maps to chromosome 16 between App and Sod-1. The homologous human GLUR5 gene maps to the corresponding region of human chromosome 21, which contains the locus for familial amyotrophic lateral sclerosis. This location, and other features, render GLUR5 a possible candidate gene for familial amyotrophic lateral sclerosis. In addition, dosage imbalance of GLUR5 may have a role in the trisomy 21 (Down syndrome). Further characterization of the murine glutamate receptor family includes mapping of Glur-1 to the same region as neurological mutants spasmodic, shaker-2, tipsy, and vibrator on chromosome 11; Glur-2 near spastic on chromosome 3; Glur-6 near waltzer and Jackson circler on chromosome 10; and Glur-7 near clasper on chromosome 4.

Multicolor fluorescence in situ hybridization for the simultaneous detection of probe sets for chromosomes 13, 18, 21, X and Y in uncultured amniotic fluid cells.

The most frequent aneuploidies in newborns involve the autosomes 13, 18 and 21 as well as both sex chromosomes. Fluorescence in situ hybridization readily allows the detection of numerical chromosomal aberrations throughout all stages of the cell cycle. Using a multicolor fluorescence in situ hybridization approach based on combinatorial probe labeling and digital imaging microscopy we demonstrate the simultaneous visualization of probe sets specific for chromosomes 13, 18, 21, X and Y. This approach enables one to evaluate aberrations of multiple chromosomes in a single hybridization experiment using metaphase chromosomes and interphase nuclei from a variety of cell types, including lymphocytes and amniocytes.

Molecular analysis of human chromosome 16 cosmid clones containing NotI sites.

To test the feasibility of using cloned NotI sites as markers for physical mapping, we have screened for cosmid clones spanning the NotI sites on human Chromosome (Chr) 16. Fluorescence in situ hybridization analysis of these clones confirms the previously reported cluster of NotI sites on 16p13.3. Methylation status of the cloned NotI sites on genomic DNA was established by hybridization of the cosmids to Southern blots containing EcoRI and EcoRI/NotI digest of genomic DNA. These results indicated that four of six clones included in our study can be used as linking clones for physical mapping. Two clones have NotI sites which are not cleavable in the cell lines tested. In one clone, the NotI site exists as an isolated rare-cutting restriction enzyme site, whereas in the other clone the NotI site appears to be island-related.

Expression of completely gamma-carboxylated and beta-hydroxylated recombinant human vitamin-K-dependent protein S with full biological activity.

Human anticoagulant vitamin-K-dependent protein S was expressed in mouse C127 cells using a bovine papilloma virus vector system. A full-length cDNA construct was introduced into the vector in the 5' untranslated region of the mouse metallothionein-I gene. Transfected cells expressed approximately 10 micrograms/ml of the recombinant protein which was purified by ion-exchange chromatography followed by affinity chromatography using Ca2(+)-dependent monoclonal antibodies against the region of protein S containing 4-carboxyglutamic acid. Recombinant protein S was structurally and functionally similar to protein S purified from plasma. On SDS/polyacrylamide-gel electrophoresis recombinant protein S had a slightly higher molecular mass than plasma protein S. After treatment with endoglycosidase F, the proteins comigrated suggesting the observed molecular mass difference to be due to alterations in the N-linked carbohydrate side chains. Recombinant and plasma protein S demonstrated identical amino-terminal sequences, similar amino acid composition and number of 4-carboxyglutamyl and 3-hydroxyaspartyl/asparaginyl residues. Recombinant protein S had the same affinity for Ca2+ as protein S from plasma and the two proteins had the same activated protein C cofactor activity in a functional assay. In addition, both forms of protein S formed complexes with C4b-binding protein with the same apparent Kd. Protein S is the most extensively post-translationally modified vitamin-K-dependent protein, and all the modifications were carried out in the recombinant DNA system yielding a recombinant protein S with full biological activity.

Identification of a putative isoform of the Na,K-ATPase beta subunit. Primary structure and tissue-specific expression.

We have isolated cDNA clones from rat brain and human liver encoding a putative isoform of the Na,K-ATPase beta subunit. The rat brain cDNA contains an open reading frame of 870 nucleotides coding for a protein of 290 amino acids with a calculated molecular weight of 33,412. The corresponding amino acid sequence shows 98% identity with its human liver counterpart. The proteins encoded by the rat and human cDNAs exhibit a high degree of primary sequence and secondary structure similarity with the rat Na,K-ATPase beta subunit. We have therefore termed the polypeptides these cDNAs encode a beta 2 subunit with the previously characterized rat cDNA encoding a beta 1 subunit. Analysis of rat tissue RNA reveals that the beta 2 subunit gene encodes a 3.4-kilobase mRNA which is expressed in a tissue specific fashion distinct from that of rat beta 1 subunit mRNA. Cell lines derived from the rat central nervous system shown to lack beta 1 subunit mRNA sequences were found to express beta 2 subunit mRNA. These results suggest that different members of the Na,K-ATPase beta subunit family may have specialized functions.

The gene for protein S maps near the centromere of human chromosome 3.

Two different mapping approaches were used to determine the human chromosomal location of the gene for protein S. A human protein S cDNA was used as a hybridization probe to analyze a panel of somatic cell hybrids containing different human chromosomes. Cosegregation of protein S-specific DNA restriction fragments with human chromosome 3 was observed. Three cell hybrids containing only a portion of chromosome 3 were analyzed in order to further localize protein S. Based on the somatic cell hybrid analysis, protein S is assigned to a region of chromosome 3 that contains a small part of the long arm and short arm of the chromosome including the centromere (3p21----3q21). In situ hybridization of the protein S cDNA probe to human metaphase chromosomes permitted a precise localization of protein S to the region of chromosome 3 immediately surrounding the centromere (3p11.1----3q11.2). Protein S is the first protein involved in blood coagulation that has been mapped to human chromosome 3.

Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation.

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 lambda gt11 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 of the 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 gamma-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.

Expression of active human uterine tissue plasminogen activator in yeast.

Tissue-type plasminogen activator (tPA) cDNA derived from human uterine mRNA was inserted into different yeast expression vectors. All such expression plasmids carried a yeast acid phosphatase (PHO5) promoter, a 2-micron plasmid replication origin, transcription termination signals, and a selectable TRP1 gene. Plasmid pYBDT-10 contained the entire tPA coding region ("pre-pro-tPA"), pYBDT-10-PRO contained a sequence encoding the putative pro-tPA precusor, and pYBDT-6 contained only a mature tPA cDNA fused precisely in frame to the sequence encoding the entire signal peptide of acid phosphatase. All constructions directed the synthesis of single-chain tPA proteins that were readily precipitated with a specific antibody directed against human uterine tPA. Electrophoretic mobilities were approximately the same as those of the Bowes melanoma single-chain tPA and a 68-kD protein marker. Treatment of immunoprecipitates with endoglycosidase H resulted in increased electrophoretic mobilities, suggesting that these yeast products are glycosylated. Despite the use of either human or yeast signal sequences, however, tPA produced in yeast was not secreted into the culture medium, but rather was found only in cells following disruption with glass beads. Although this cellular tPA exhibited fibrinolytic activity, most of the activity was associated with large cellular debris.

Somatically generated mouse myeloma variants synthesizing IgA half-molecules.

Whereas mouse myelomas that secrete IgA half-molecules have been shown to arise in vivo, their origin has not been definitely established. We show that somatic variants secreting phenotypically similar molecules can arise directly from the normal IgA-secreting myelomas S107 and W3082. In addition to being improperly assembled, the variant proteins have distinct carboxy-terminal deletions and an aberrant heavy-light chain disulfide bond. For at least one of the variants, variable region serology and affinity for hapten are both unaffected by these changes. Southern and Northern blot analyses indicate normal size DNA restriction fragments and mRNA, suggesting premature termination as the mechanism of deletion. These results are discussed in relation to possible mutational hot spots and long-range interdomain interactions.

Two alpha heavy chain disease proteins with different genomic deletions demonstrate that nonexpressed alpha heavy chain genes contain methylated bases.

Two independently arising alpha heavy chain mutants have been found to synthesize heavy chains with CH1 deletions of approximately equal extent. Both were isolated from heavy chain-producing variants of the mouse myeloma W3129 and demonstrate that it is possible to arrive at the heavy chain disease phenotype by the pathway H + L leads to H leads to delta H. Analysis of genomic DNA by digestion with restriction endonucleases followed by molecular hybridization showed that one mutant (delta 37) had a deletion of approximately 0.2 kilobase and the second mutant (delta 15) had a deletion of approximately 0.5 kilobase. Mouse myeloma cells contain several alpha chain alleles but only one is expressed; the presence of the deletion in delta 37 and delta 15 made it possible to identify the restriction fragments from the expressed allele. Analysis of the fragments produced after cleavage with an isoschizomeric pair of restriction enzymes, Msp I and Hpa II, indicated that, in the W3129 cell line and its variants, the unexpressed alpha alleles contain methylated bases. The influence of methylation on gene expression remains to be elucidated.