Cloning and characterization of 13 novel transcripts and the human RGS8 gene from the 1q25 region encompassing the hereditary prostate cancer (HPC1) locus.

The aim of this study was to develop a saturated transcript map of the region encompassing the HPC1 locus to identify the susceptibility genes involved in hereditary prostate cancer (OMIM 176807) and hyperparathyroidism-jaw tumor syndrome (OMIM 145001). We previously reported the generation of a 6-Mb BAC/PAC contig of the candidate region and employed various strategies, such as database searching, exon-trapping, direct cDNA hybridization, and sample sequencing of BACs, to identify all potential transcripts. These efforts led to the identification and precise localization on the BAC contig of 59 transcripts representing 22 known genes and 37 potential transcripts represented by ESTs and exon traps. Here we report the detailed characterization of these ESTs into full-length transcript sequences, their expression pattern in various tissues, their genomic organization, and their homology to known genes. We have also identified an Alu insertion polymorphism in the intron of one of the transcripts. Overall, data on 13 novel transcripts and the human RGS8 gene (homologue of the rat RGS8 gene) are presented in this paper. Ten of the 13 novel transcripts are expressed in prostate tissue and represent positional candidates for HPC1.

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.

A 6-Mb high-resolution physical and transcription map encompassing the hereditary prostate cancer 1 (HPC1) region.

Several hereditary disease loci have been genetically mapped to the chromosome 1q24-q31 interval, including the hereditary prostate cancer 1 (HPC1) locus. Here, we report the construction of a 20-Mb yeast artificial chromosome contig and a high-resolution 6-Mb sequence-ready bacterial artificial chromosome (BAC)/P1-derived artificial chromosome (PAC) contig of 1q25 by sequence and computational analysis, STS content mapping, and chromosome walking. One hundred thirty-six new STSs, including 10 novel simple sequence repeat polymorphisms that are being used for genetic refinement of multiple disease loci, have been generated from this contig and are shown to map to the 1q25 interval. The integrity of the 6-Mb BAC/PAC contig has been confirmed by restriction fingerprinting, and this contig is being used as a template for human chromosome 1 genome sequencing. A transcription mapping effort has resulted in the precise localization of 18 known genes and 31 ESTs by database searching, exon trapping, direct cDNA hybridization, and sample sequencing of BACs from the 1q25 contig. An additional 11 known genes and ESTs have been placed within the larger 1q24-q31 interval. These transcription units represent candidate genes for multiple hereditary diseases, including HPC1.

A modified two-step phage display selection for isolation of polycystin-1 ligands.

The identification of proteins that interact with polycystin-1, the product of the autosomal dominant polycystic kidney disease gene, is an important step towards understanding the molecular pathogenesis of the disease. We have developed a two-step approach for the efficient identification of potential polycystin-1 ligands using the T7 phage display system. The first enrichment step of 4-5 rounds of biopanning is followed by a second step of reverse protein overlay assay. Thus, the sequencing efforts are minimized to the analysis of only positive rather than randomly chosen clones from the enriched population as in the standard phage display approach. Most importantly, the modified approach immediately provides the confirmation of the specificity of interaction and discriminates between strong and weak interactions. Here we present several potential interactors with distinct regions of polycystin-1, representing high-affinity binding partners.

Combining serial analysis of gene expression and array technologies to identify genes differentially expressed in breast cancer.

Several methods have been used recently to determine gene expression profiles of cell populations. Here we demonstrate the strength of combining two approaches, serial analysis of gene expression (SAGE) and DNA arrays, to help elucidate pathways in breast cancer progression by finding genes consistently expressed at different levels in primary breast cancers, metastatic breast cancers, and normal mammary epithelial cells. SAGE profiles of 21PT and 21MT, two well-characterized breast tumor cell lines, were compared with SAGE profiles of normal breast epithelial cells to identify differentially expressed genes. A subset of these candidates was then placed on an array and screened with clinical breast tumor samples to find genes and expressed sequence tags that are consistently expressed at different levels in diseased and normal tissues. In addition to finding the predicted overexpression of known breast cancer markers HER-2/neu and MUC-1, the powerful coupling of SAGE and DNA arrays resulted in the identification of genes and potential pathways not implicated previously in breast cancer. Moreover, these techniques also generated information about the differences and similarities of expression profiles in primary and metastatic breast tumors. Thus, combining SAGE and custom array technology allowed for the rapid identification and validation of the clinical relevance of many genes potentially involved in breast cancer progression. These differentially expressed genes may be useful as tumor markers and prognostic indicators and may be suitable targets for various forms of therapeutic intervention.

Two novel genes in the center of the 11p15 imprinted domain escape genomic imprinting.

We previously reported the isolation of a 2.5 Mb tumor-suppressing subchromosomal transferable fragment (STF) from human chromosome 11p15 and the identification of nine known genes and four novel genes within this STF. We now report the isolation of two novel cDNAs, designated here as TSSC4 and TSSC6 (tumor-suppressing STF cDNA 4 and 6), located within the STF. TSSC4 and TSSC6 encode predicted proteins of 329 and 290 amino acids, respectively, with no close similarity to previously reported proteins. TSSC4 and TSSC6 are both located in the center of a 1 Mb imprinted domain, which contains the imprinted genes TSSC3, TSSC5, p57(KIP2), KVLQT1, ASCL2, IGF2 and H19. However, we found that neither TSSC4 nor TSSC6 was significantly imprinted in any of the fetal or extra-embryonic tissues examined. Based on this result, the imprinted gene domain of 11p15 appears to contain at least two imprinted subdomains, between which TSSC4 and TSSC6 substantially escape imprinting, due either to lack of initial silencing or to an early developmental relaxation of imprinting.

Comparative genomic hybridization and its application to Wilms' tumorigenesis.

Eighty sporadic Wilms' tumor samples were analyzed by comparative genomic hybridization (CGH) to identify chromosomal regions involved in the etiology of the disease. Twenty percent of the samples showed chromosomal gains or losses. The majority of chromosomal gains and losses were similar to those identified through molecular and cytogenetic studies. Gains were observed on chromosomes 1q, 7q, 8, and 12, whereas losses were found on chromosomes 1p, 4p, 4q, 7p, 16q, 18q, 21q, and 22q. Other genetic aberrations identified in this study included deletions of chromosomes 5p and 15q, as well as gains of discrete loci on chromosomes 3p and 3q. These latter regions have not been previously implicated in Wilms' tumorigenesis and may contain novel genes relevant to the development and/or progression of this disease.

A 2.5-Mb transcript map of a tumor-suppressing subchromosomal transferable fragment from 11p15.5, and isolation and sequence analysis of three novel genes.

11p15.5 is an important tumor-suppressor gene region, showing loss of heterozygosity in Wilms tumor, rhabdomyosarcoma, adrenocortical carcinoma, and lung, ovarian, and breast cancer. We previously mapped directly by genetic complementation a subtransferable fragment (STF) harboring an embryonal tumor-suppressor gene and spanning about 2.5 Mb. We have now mapped the centromeric end of this STF between D11S988 and D11S12 and its telomeric end between D11S1318 and TH. We have isolated a complete contig of PAC, P1, BAC, and cosmid genomic clones spanning the entire 2.5-Mb region defined by this STF, as well as more than 200 exons from these genomic clones using exon trapping. We have isolated genes in this region by directly screening DNA libraries as well as by database searching for ESTs. Nine of these genes have been reported previously by us and by others. However, the initial mapping of most of those genes was based on FISH or somatic cell hybrid analysis, and here we precisely define their physical location. These genes include RRM1, GOK (D11S4896E), Nup98, CARS, hNAP2 (NAP1L4), p57KIP2 (CDKN1C), KVLQT1 (KCNA9), TAPA-1, and ASCL2. In addition, we have identified several novel genes in this region, three of which, termed TSSC1, TSSC2, and TSSC3, are reported here. TSSC1 shows homology to Rb-associated protein p48 and chromatin assembly factor CAF1, and it is located between GOK and Nup98. TSSC2 is homologous to Caenorhabditis elegans beta-mannosyl transferase, and it lies between Nup98 and CARS. TSSC3 shows homology to mouse TDAG51, which is implicated in FasL-mediated apoptosis, and it is located between hNAP2 and p57KIP2. Thus, these genes may play a role in malignancies that involve this region.

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.

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.

Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias.

Genetic factors contribute to the risk of sudden death from cardiac arrhythmias. Here, positional cloning methods establish KVLQT1 as the chromosome 11-linked LQT1 gene responsible for the most common inherited cardiac arrhythmia. KVLQT1 is strongly expressed in the heart and encodes a protein with structural features of a voltage-gated potassium channel. KVLQT1 mutations are present in affected members of 16 arrhythmia families, including one intragenic deletion and ten different missense mutations. These data define KVLQT1 as a novel cardiac potassium channel gene and show that mutations in this gene cause susceptibility to ventricular tachyarrhythmias and sudden death.

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.

Increased exon-trapping efficiency through modifications to the pSPL3 splicing vector.

Exon trapping allows for the rapid identification and cloning of coding regions from cloned eukaryotic DNA. In preliminary experiments, we observed two phenomena which limited the exon-trapping efficiency of pSPL3-based systems. The first factor that affected performance was revealed when we found that up to 50% of the putative trapped exons contained sequences derived from the intron of the pSPL3 trapping vector. Removal of the DNA sequences responsible for the cryptic splice event from the original splicing vector resulted in a new vector, pSPL3B. We demonstrate that pSPL3B virtually eliminates pSPL3-only spliced products while maximizing the proportion of exon traps containing genomic DNA (> 98%). The other step which impacted performance was our observation that a majority of the ampicillin-resistant (APR) clones produced after shotgun subcloning from ApR cosmids into pSPL3 were untrappable, pSPL3-deficient, recircularized cosmid vector fragments. Replacement of the pSPL3 ApR gene with the CmR cassette encoding chloramphenicol (Cm) acetyltransferase enabled selection for only pSPL3-containing CmR clones. We show a 30-40-fold increase in the initial subcloning efficiency of cosmid-derived fragments with pSPL3-CAM, when compared to pSPL3. The collective vector alterations described improve the overall exon-trapping efficiency of the pSPL3-based trapping system.

Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family.

The casein kinase I (CKI) gene family is a rapidly enlarging group whose members have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair. We report here the cloning and characterization of a novel isoform of CKI from a human placental cDNA library. The cDNA for this isoform, hCKI epsilon, predicts a basic polypeptide of 416 amino acids and a molecular mass of 47.3 kDa. It encodes a core kinase domain of 285 amino acids and a carboxyl-terminal tail of 123 amino acids. The kinase domain is 53-98% identical to the kinase domains of other CKI family members and is most closely related to the delta isoform. Localization of the hCKI epsilon gene to chromosome 22q12-13 and the hCKI delta gene to chromosome 17q25 confirms that these are distinct genes in the CKI family. Northern blot analysis shows that hCKI epsilon is expressed in multiple human cell lines. Recombinant hCKI epsilon is an active enzyme that phosphorylates known CKI substrates including a CKI-specific peptide substrate and is inhibited by CKI-7, a CKI-specific inhibitor. A budding yeast isoform of CKI, HRR25, has been implicated in DNA repair responses. Expression of hCKI epsilon but not hCKI alpha rescued the slow-growth phenotype of a Saccharomyces cerevisiae strain with a deletion of HRR25. Human CKI epsilon is a novel CKI isoform with properties that overlap those of previously described CKI isoforms.

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.