frizzled 9 is expressed in neural precursor cells in the developing neural tube.

The wnt signaling pathway has important functions in nervous system development. To better understand this process we have cloned and analyzed the expression of the wnt receptor, frizzled 9, in the developing nervous system in mouse, chick and zebrafish. The earliest expression of mouse frizzled 9 mRNA expression begins at E8.5 with expression throughout the entire rostral-caudal neuraxis. This early expression pattern within the neural tube appears to be conserved between chick and zebrafish. Expression becomes restricted to a ventral domain in the mouse ventricular zone at E11.5, a region specified to give rise to neurons and glia. Using a polyclonal antibody to MFZ9 further shows expression limited to neural restricted precursors cells.

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 NTN2L gene encoding a novel human netrin maps to the autosomal dominant polycystic kidney disease region on chromosome 16p13.3.

The netrins define a family of chemotropic factors that have been shown to play a central role in axon guidance. We identified two exon traps encoding netrin-like sequences during the assembly of a transcriptional map for the genomic interval surrounding the polycystic kidney disease type 1 and tuberous sclerosis type 2 genes. We describe the characterization of a novel human netrin-2-like gene, designated NTN2L, and its transcript. The genomic interval containing the NTN2L gene was sequenced, and the coding region was predicted based on computer analysis. The structure of the NTN2L gene has been confirmed utilizing nested RT-PCR. The NTN2L gene is predicted to encode a 580-amino-acid protein having homology to the chicken and Drosophila netrins and to Caenorhabditis elegans UNC-6. The NTN2L gene has a restricted pattern of expression; its transcript is undetectable by Northern analysis in all tissues examined, but can be recovered from spinal cord RNA by RT-PCR. This report represents the first description and characterization of a human netrin.

The cloning of a human ABC gene (ABC3) mapping to chromosome 16p13.3.

The ATP binding cassette (ABC) transporters, or traffic ATPases, constitute a large family of proteins responsible for the transport of a wide variety of substrates across cell membranes in both prokaryotic and eukaryotic cells. We describe a human ABC protein with regions of strong homology to the recently described murine ABC1 and ABC2 transporters. The gene for this novel protein, human ABC3, maps near the polycystic kidney disease type 1 (PKD1) gene on chromosome 16p13.3. The ABC3 gene is expressed at highest levels in lung compared to other tissues.

A novel ribosomal protein L3-like gene (RPL3L) maps to the autosomal dominant polycystic kidney disease gene region.

A full-length cDNA encoding a novel ribosomal protein L3 gene was isolated and sequenced. The deduced protein sequence is 407 amino acids long and shows 77% identity to other known mammalian ribosomal protein L3 genes, which are themselves highly conserved. Southern blot analysis of human genomic DNA suggests that this novel gene is single copy. While the previously identified human ribosomal protein L3 gene has ubiquitous expression in all tissues surveyed, the novel gene described herein is strongly expressed in skeletal muscle and heart tissue, with low levels of expression in the pancreas. This novel gene, RPL3L, is located in a gene-rich region near the PKD1 and TSC2 genes on chromosome 16p13.3.

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.

A 2.5 kb polypyrimidine tract in the PKD1 gene contains at least 23 H-DNA-forming sequences.

A pyrimidine-rich element (PyRE), present in the 21st intron of the PKD1 gene, posed a significant obstacle in determining the primary structure of the gene. Only cycle sequencing of nested, single-stranded phage templates of the CT-rich strand enabled complete and accurate sequence data. Similar attempts on the GA-rich strand were unsuccessful. The resulting primary structure showed the 3 kb 21st intron to contain a 2.5 kb PyRE, whose sense-strand is 97% C + T. The PKD1 PyRE does not appear to be polymorphic based on RFLP analysis of DNA from 6 unrelated individuals digested with 9 different restriction enzymes. This is the largest pyrimidine tract sequenced to date, being over twice as large as those previously identified and shows little homology to other polypyrimidine tracts. Additional analysis of this PyRE revealed the presence of 23 mirror repeats with stem lengths of at least 10 nucleotides. The 23 H-DNA-forming sequences in the PKD1 PyRE exceed the cumulative total of 22 found in 157 human genes that have been completely sequenced. The mirror repeats confer this region of the PKD1 gene with a strong probability of forming H-DNA or triplex structures under appropriate conditions. Based on studies with PyRE found in other eukaryotic genes, the PKD1 PyRE may play a role in regulating PKD1 expression, and its potential for forming an extended triplex structure may explain some of the observed instability in the PKD1 locus.

Mitochondrial DNA diversity in an apomictic Daphnia complex from the Canadian high arctic.

Cyclic parthenogenesis is the ancestral mode of reproduction in the cladoceran crustacean, Daphnia pulex, but some populations have made the transition to obligate parthenogenesis and this is the only mode of reproduction known to occur in arctic populations. Melanism and polyploidy are also common in arctic populations of this species. Prior allozyme studies of arctic D. pulex revealed substantial levels of clonal diversity on a regional scale. Clonal groupings based on cluster analysis of allozyme genotypes do not conform to groupings based on the presence/absence of melanin or on ploidy level. In order to further elucidate genetic relationships among arctic D. pulex clones, mitochondrial DNA (mtDNA) variation was examined in 31 populations from two Canadian high-arctic sites. The data were also compared to a previous study of mtDNA variation in populations from a Canadian low-arctic site. Cladistic analysis of restriction site variation of the entire mitochondrial genome and nucleotide sequence variation of the mitochondrial control region was used to construct genetic relationships among mitochondrial genotypes. Three distinct mitochondrial lineages were detected. One lineage was associated with diploid, nonmelanic clones and is the same as the lineage that is found in temperate populations of D. pulex. The other two lineages (A & B) were associated with polyploid, melanic clones. Sequence divergence between the A and B lineages was 2.4%. Sequence divergence between D. pulex and either of these two lineages exceeded 3%.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Partial mitochondrial DNA sequence of the crustacean Daphnia pulex.

A 3667-base pair (bp) fragment of the mitochondrial genome of the crustacean Daphnia pulex has been sequenced and found to contain the complete genes for the small subunit ribosomal RNA, ND2, seven tRNAs and the control region. This organization is identical to that found in Drosophila yakuba mtDNA yet D. pulex mtDNA exhibits several unique features when compared to other mitochondrial sequences. The sequenced fragment is only 62.6% A + T which is much lower than that of any other arthropod mtDNA sequenced to date. D. pulex mtDNA also exhibits length conservation having shorter coding and non-coding regions. The putative control region is 689 bp in length and includes a sequence that has the potential to fold into a hairpin structure with a perfect 20-bp pair stem and a 22-base loop.