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We have identified a novel cochlear gene, designated OTOR, from a comparative sequence analysis of over 4000 clones from a human fetal cochlear cDNA library. Northern blot analysis of human and chicken organs shows strong OTOR expression only in the cochlea; very low levels are detected in the chicken eye and spinal cord. Otor and Col2A1 are coexpressed in the cartilaginous plates of the neural and abneural limbs of the chicken cochlea, structures analogous to the mammalian spiral limbus, osseous spiral lamina, and spiral ligament, and not in any other tissues in head and body sections. The human OTOR gene localizes to chromosome 20 in bands p11.23-p12.1 and more precisely to STS marker WI-16380. We have isolated cDNAs orthologous to human OTOR in the mouse, chicken, and bullfrog. The encoded protein, designated otoraplin, has a predicted secretion signal peptide sequence and shows a high degree of cross-species conservation. Otoraplin is homologous to the protein encoded by CDRAP/MIA (cartilage-derived retinoic acid sensitive protein/melanoma inhibitory activity), which is expressed predominantly by chondrocytes, functions in cartilage development and maintenance, and has growth-inhibitory activity in melanoma cell lines.

We constructed and screened a human fetal cochlear cDNA library to identify genes involved in hearing and deafness. From this library we isolated a cDNA corresponding to the highly conserved ancient gene antiquitin (ATQ1). The plant homolog ofATQ1is thought to be involved in regulating turgor pressure, a function that also would be essential for cells of the mammalian cochlea. Northern blots of 13 human fetal tissues show antiquitin to be highly expressed in cochlea, ovary, eye, heart, and kidney. Using RT-PCR of rat cochlear hair cell-specific cDNA libraries, we detect antiquitin expression in outer hair cells, but not in inner or vestibular type 1 hair cells, suggesting that antiquitin is not expressed ubiquitously in the cochlea. HumanATQ1was mapped to human chromosome region 5q31 using fluorescencein situhybridization, and mouseATQ1was mapped to mouse chromosome 18 by single-strand conformation polymorphism mapping of interspecific backcross progeny DNAs. Four human antiquitin-like sequences, possibly pseudogenes, were also identified and mapped.

Eotaxin is a CC chemokine that is a specific chemoattractant for eosinophils and is implicated in the pathogenesis of eosinophilic inflammatory diseases, such as asthma. We describe the genomic organization, complete sequence, including 1354 bp 5' of the RNA initiation site, and chromosomal localization of the human eotaxin gene. Fluorescence in situ hybridization analysis localized eotaxin to human chromosome 17, in the region q21.1-q21.2, and the human gene name SCYA11 was assigned. We also present the 5' flanking sequence of the mouse eotaxin gene and have identified several regulatory elements that are conserved between the murine and the human promoters. In particular, the presence of elements such as NF-kappa B, interferon-gamma response element, and glucocorticoid response element may explain the observed regulation of the eotaxin gene by cytokines and glucocorticoids.

Cathepsin K is a recently identified lysosomal cysteine proteinase that is the major protease responsible for bone resorption and remodeling. Mutations in this gene cause the sclerosing osteochondrodysplasia pycnodysostosis. To assess its evolutionary relatedness to other cysteine proteases and to facilitate mutation identification in patients with pycnodysostosis, a genomic clone, 74e16, containing the cathepsin K gene was isolated from a human PAC library, and the cathepsin K genomic structure was determined. The cathepsin K gene contained eight exons and spanned approximately 9 kb. The transcription initiation site, determined by primer extension analysis, was 169 nucleotides upstream from the translation initiation site. The 5'-flanking region lacked a TATA box but contained two AP1 sites. Comparison of genomic and cDNA sequences suggested that this flanking sequence may be the major promoter in osteoclasts and macrophages. Cathepsin K was mapped to chromosome 1q21 by fluorescence in situ hybridization and found to reside within 150 kb of an evolutionarily related cysteine protease, cathepsin S. These findings expand our understanding of the papain family lysosomal cysteine proteases and should facilitate mutation analysis in pycnodysostosis.

We report a partial cDNA sequence that encodes a protein, dubbed “polycystwin,” with 21% identity and 46% similarity to amino acids 3688–4109 of the carboxyl terminus of polycystin, the gene product of the autosomal dominant polycystic kidney disease locus located on chromosome 16 at band p13 (PKD1). Northern analysis demonstrates that the R48321 gene is expressed in all tissues examined, including both adult and fetal kidneys. Finally, in situ hybridization studies localize this novel gene to 4q22, where PKD2, the second most common locus for ADPKD, is known to map. Therefore, R48321 is an excellent candidate gene for PKD2.

Uterine leiomyomata are the most common tumors in women and can cause abnormal uterine bleeding, pelvic pain, and infertility. Approximately 200,000 hysterectomies are performed annually in the U.S. to relieve patients of the medical sequelae of these benign neoplasms. Our efforts have focused on cloning the t(12;14)(q14-q15;q23-q24) breakpoint in uterine leiomyoma to further our understanding of the biology of these tumors. Thirty-nine YACs and six cosmids mapping to 12q14-q15 have been mapped by fluorescence in situ hybridization to tumor metaphase chromosomes containing a t(12;14). One YAC spanned the translocation breakpoint and was mapped to tumor metaphases from a pulmonary chondroid hamartoma containing a t(12;14)(q14-q15;q23-q24) and a lipoma containing a t(12;15)(q15;q24); this YAC also spanned the breakpoint in these two tumors, suggesting that the same gene on chromosome 12 may be involved in the pathobiology of these distinct benign neoplasms.

Cartilage oligomeric matrix protein (COMP) is a 524,000-Da protein that is expressed at high levels in the territorial matrix of chondrocytes. The sequences of rat and bovine COMP indicate that it is a member of the thrombospondin gene family. In this study, we have cloned and sequenced human COMP. Phylogenetic analysis using progressive sequence alignment and two parsimony-based algorithms indicates that the COMP gene and a precursor of the thrombospondin-3 and -4 genes were produced by a gene duplication that occurred 750 million years ago. An interspecific backcross mapping panel has been used to map the murine COMP gene to the central region of mouse chromosome 8. Southern blot analysis of a somatic cell hybrid DNA panel and in situ hybridization to human metaphase chromosomes indicate that the human COMP gene is located on chromosome 19 in band p13.1. These data confirm and extend the known regions of homology between human and mouse chromosomes and establish that COMP, like thrombospondin-1, -2, -3, and -4, is present in the human and mouse genomes.

Nitric oxide (NO) is an important intercellular signaling molecule synthesized in diverse human tissues by proteins encoded by a family of NO synthase (NOS) genes. The similarity of sequence and cofactor binding sites has suggested that the NOS genes may also be related to cytochrome P450 reductase, as well as to plant and bacterial oxidoreductases. Endothelial NOS activity is a major determinant of vascular tone and blood pressure, and in several important (and sometimes hereditary) disease states, such as hypertension, diabetes, and atherosclerosis, the endothelial NO signaling system appears to be abnormal. To explore the relationship of the endothelial NOS gene to other similar genes, and to delineate the genetic factors involved in regulating endothelial NOS activity, we isolated the human gene encoding the endothelial NOS. Genomic clones containing the 5′ end of this genie were identified in a human genomic library by applying a polymerase chain reaction (PCR)-based approach. Identification of the human gene for endothelial NOS (NOS3) was confirmed by nucleotide sequence analysis of the first coding exon, which was found to be identical to its cognate cDNA. The NOS3 gene spans at least 20 kb and appears to contain multiple introns. The transcription start site and promoter region of the NOS3 gene were identified by primer extension and ribonuclease protection assays. Sequencing of the putative promoter revealed consensus sequences for the shear stress-response element, as well as cytokine-responsive cis regulatory sequences, both possibly important to the roles played by NOS3 in the normal and the diseased cardiovascular system. We also mapped the chromosomal location of the NOS3 gene. First, a chromosomal panel of human-rodent somatic cell hybrids was screened using PCR with oligonucleotide primers derived from the NOS3 genomic clone. The specificity of the amplified PCR product was confirmed by human and hamster genomic DNA controls, as well as by Southern blot analysis, using the NOS3 cDNA as probe. Definitive chromosomal assignment of the NOS3 gene to human chromosome 7 was based upon 0% discordancy; fluorescence in situ hybridization sublocalized the NOS3 gene to 7q36. The identification and characterization of the NOS3 gene may lead to further insights into heritable disease states associated with this gene product.

We have mapped the mouse protein tyrosine phosphatase epsilon (PTP epsilon, gene symbol Ptpre) gene to the distal region of chromosome 7 by linkage analysis using two sets of multilocus genetic crosses. The human PTP epsilon gene (gene symbol PTPRE) was mapped to chromosome 10q26 by fluorescence in situ hybridization. We have previously documented the existence of two isoforms of PTP epsilon--a transmembranal, receptor-type isoform and a shorter, cytoplasmic one. Both isoforms have been suggested to arise from a single gene through the use of alternative promoters and 5' exons. The identification of a single PTP epsilon locus in both organisms is consistent with this suggestion.

We report localization of the human cone transducin (GNAT2) gene using fluorescence in situ hybridization on chromosome 1 in band p13. The recent assignment of a gene for Stargardt disease to the same chromosomal region by linkage analysis prompted us to investigate the possible role of GNAT2 in the pathogenesis of this disease. We investigated 66 unrelated patients for mutations in the coding region of the GNAT2 gene using polymerase chain reaction-single strand conformation polymorphism analysis (SSCP) and direct sequencing. No disease-specific mutations were found, indicating that GNAT2 is probably not involved in the pathogenesis of most cases of Stargardt disease. 19 refs., 1 fig., 1 tab.

Polycystin-1 and polycystin-2 are the products of PKD1 and PKD2, genes that are mutated in most cases of autosomal dominant polycystic kidney disease. Since the first two polycystins were cloned, three new members, polycystin-L, -2L2, and -REJ, have been identified. In this study, we describe a sixth member of the family, polycystin-1L1, encoded by PKD1L1 in human. The full-length cDNA sequence of PKD1L1, determined from human testis cDNA, encodes a 2849-amino-acid protein and 58 exons in a 187-kb genomic region. The deduced amino acid sequence of polycystin-1L1 has significant homology with all known polycystins, but the longest stretches of homology were found with polycystin-1 and -REJ over the 1453- and 932-amino-acid residues, respectively. Polycystin-1L1 is predicted to have two Ig-like PKD, a REJ, a GPS, a LH2/PLAT, a coiled-coil, and 11 putative transmembrane domains. Several rhodopsin-like G-protein-coupled receptor (GPCR) signatures are also found in polycystin-1L1. Dot-blot analysis and RT-PCR revealed that human PKD1L1 is expressed in testis and in fetal and adult heart. In situ hybridization analysis showed that the most abundant and specific expression of Pkd1l1 was found in Leydig cells, a known source of testosterone production, in mouse testis. We have assigned PKD1L1 to the short arm of human chromosome 7 in bands p12--p13 and Pkd1l1 to mouse chromosome 11 in band A2 by fluorescence in situ hybridization. We hypothesize a role for polycystin-1L1 in the heart and in the male reproductive system.

We determined the chromosomal location of the mouse gene Stk25, encoding a member of the Ste20/PAK family of serine/threonine kinases, by interspecific backcross analysis. We mapped Stk25 to the central region of mouse chromosome 1 linked to Chrng (formerly Acrg) and En1. This central region of mouse chromosome 1 shares a region of homology with the long arm of human chromosome 2, suggesting that the human homologue of Stk25 would also map to 2q. We proved this prediction of syntenic homology correct by mapping human STK25 to 2q37. Deletion of the 2q37 region has been implicated in the expression of pseudopseudohypoparathyroidism (PPHP), a disease which shares features of the Albright hereditary osteodystrophy (AHO) phenotype. To investigate a pathogenetic relationship between STK25 and PPHP, we carried out fluorescence in situ hybridization (FISH) using an STK25 gene probe and chromosomes from PPHP patients characterized as having small deletions near the distal end of 2q. PPHP patient DNA showed no hybridization to STK25 genomic DNA, indicating that STK25 is contained within the deleted chromosomal region. This finding, in conjunction with previous studies demonstrating the role of Ste20/PAK kinases in heterotrimeric G protein signaling, suggests that STK25 is a positional candidate gene for PPHP.

Members of the DEAD/H box-containing helicase superfamily include proteins essential to genome replication, repair, and expression. We report here the cloning and initial characterization of a novel human member of this protein family, designated hHel1 (human helicase 1), now designated SMARCAD1 by HUGO. This DEAD/H box-containing molecule has seven highly conserved sequence regions that allow us to place it in the SNF2 family of the helicase superfamily. Uniquely, though, hHel1 contains two DEAD/H box motifs, a property not reported to be shared by any other SNF2 family members. This defines a new subfamily consisting of hHel1 and its homologues. In addition to these DEAD/H box/ATP-binding motifs, hHel1 has a putative nuclear localization signal and several regions that may mediate protein–protein interactions. Expression analysis indicates that hHel1 transcripts are ubiquitous, with particularly high levels in endocrine tissue. We have mapped the gene for hHel1 to human chromosome 4q22–q23; this region is rich in breakpoints and deletion mutants of genes involved in several human diseases, notably soft tissue leiomyosarcoma, hepatocellular carcinoma, and hematologic malignancies. Our observation that human Hel1 gene overexpression is present in an E1A-expressing cell line with increased capacity for gene reactivation events by genomic rearrangement suggests that human Hel1 may play a role in genetic instability development.

Polycystins-1, -2, -L, and -REJ are the four known members of the polycystin family of proteins. In this study, we describe a fifth member of the family, polycystin-L2, encoded by PKD2L2 in human and Pkd2l2 in mouse. Full-length cDNA sequences for both mouse and human polycystin-L2 were obtained from testis cDNA. Sequence analysis predicts that the mouse and human polycystin-L2 proteins consist of 621 and 624 amino acid residues, respectively. Polycystin-L2 has significant homology with polycystins-L and -2, with similarities of 58 and 59%, respectively. Both human and murine polycystin-L2 proteins are predicted to have seven putative transmembrane (TM) domains, and, by comparison with transient receptor potential channels, the six carboxyl-terminal TM domains are likely to constitute an ion channel subunit. Northern blot analysis indicated that mouse Pkd2l2 has an abundant approximately 2.5-kb transcript in testis and an approximately 2.2-kb transcript in heart. RT-PCR analysis showed that the full-length transcript is expressed in human brain, kidney, testis, and HepG2 cells, and there are three alternatively spliced variants that were differentially expressed. PKD2L2 consists of 17 exons spanning approximately 50 kb of genomic DNA. PKD2L2 was mapped to human chromosome 5q31 and Pkd2l2 to mouse chromosome 18 in band C.

GDP-dissociation inhibitors (GDIs) play a primary role in modulating the activity of GTPases. We recently reported the identification of a new GDI for the Rho-related GTPases named RhoGDIgamma. This gene is now designated ARHGDIG by HUGO. Here, in a detailed analysis of tissue expression of ARHGDIG, we observe high levels in the entire brain, with regional variations. The mRNA is also present at high levels in kidney and pancreas and at moderate levels in spinal cord, stomach, and pituitary gland. In other tissues examined, the mRNA levels are very low (lung, trachea, small intestine, colon, placenta) or undetectable. RT-PCR analysis of total RNA isolated from exocrine pancreas and islets shows that the gene is expressed in both tissues. We also report the genomic structure of ARHGDIG. The gene spans over 4 kb and is organized into six exons and five introns. The upstream region lacks a canonical TATA box and contains several putative binding sites for ubiquitous and tissue-specific factors active in central nervous system development. Using FISH, we have mapped the gene to chromosome band 16p13.3. This band is rich in deletion mutants of genes involved in several human diseases, notably polycystic kidney disease, alpha-thalassemia, tuberous sclerosis, mental retardation, and cancer. The promoter structure and the chromosomal location of RhoGDIgamma suggest its importance and underscore the need for further investigation into its biology.

Previously we identified a partial human cDNA for a novel cochlear transcript, hCoch-5B2 (HGMW-approved symbol D14S564E), using subtractive hybridization techniques. Herein we report isolation and characterization of both human and mouse (D12H14S564E) cDNAs for Coch-5B2. Full-length Coch-5B2 deduced amino acid sequences reveal a very high degree of conservation in the coding region (89% nucleotide and 94% amino acid identity) and a potential signal peptide and two regions of extensive homology to the collagen-binding type A domains of von Willebrand factor, also present in other secreted proteins, including extracellular matrix components. High levels of hCoch-5B2 expression are seen only in human fetal inner ear structures, cochlea, and vestibule, among a large panel of human fetal and adult tissues. Coch-5B2 expression in the mouse is more widespread than in the human, with message detected in mouse adult spleen, cerebrum, cerebellum/medulla, and thymus. In both species very low level expression is detected in total eye. More specifically, mouse retina shows a higher level of mCoch-5B2 message than sclera and choroid. We have mapped hCoch-5B2 to human 14q11.2–q13 by somatic cell hybrid analysis and FISH and, more precisely, using radiation hybrids to a region of markers linked to DFNA9, a nonsyndromic autosomal dominant sensorineural hearing loss with vestibular defects. Furthermore, we detect hCoch-5B2 on three overlapping YACs, two of which also contain one of the markers linked to DFNA9. mCoch-5B2 was genetically mapped in the mouse to chromosome 12, in a region of homologous synteny with human 14q11.2–q13, which contains theasp1(audiogenic seizure prone) locus in the mouse.

Two novel cDNAs, DNAS1L2 and DNAS1L3, are predicted to encode proteins of 299 and 305 amino acids with 56 and 46% residue identity (71 and 63% similarity), respectively, to deoxyribonuclease I (DNase I). DNAS1L2 is located on a 16p13.3 cosmid, while DNAS1L3 maps to 3p14.3-p21.1 by fluorescence in situ hybridization and by PCR analysis of a radiation hybrid panel. Northern analysis revealed DNAS1L3 expression nearly exclusively in liver, while DNAS1L2 expression was detected in brain by RT-PCR. The previously defined DNL1L or DNAS1L1 is expressed highest in heart and skeletal muscle, while DNase I is expressed in the pancreas, parotid gland, and kidney. Thus, to date, four DNase I-like genes that show different tissue expression patterns are known. A comparison of DNAS1L1, DNAS1L2, and DNAS1L3 with the well-characterized DNase I suggests that the DNAS1L proteins are unlikely to be glycosylated or bind actin; however, catalytic and calcium- and DNA-binding residues are conserved, and potentially cleavable signal peptides are present among all these proteins. This analysis also identifies regions of high conservation among these proteins with no currently assigned function.

Transcriptional Enhancer Factor-1 (TEF-1) is a transcription factor required for cardiac muscle gene activation. Since ablation of TEF-1 does not abolish cardiac gene expression, we sought to identify a human gene related to TEF-1 (RTEF-1) that might also participate in cardiac gene regulation. A human heart cDNA library was screened to obtain a full-length RTEF-1 cDNA. Fluorescence in situ hybridization assigned the RTEF-1 gene to chromosome 12p13.2-p13.3. In contrast, PCR screening of human/rodent cell hybrid panels identified TEF-1 on chromosome 11p15.2, between D11S1315 and D11S1334, extending a region of known synteny between human chromosomes 11 and 12 and arguing for an ancient divergence between these two closely related genes. Northern blot analysis revealed a striking similarity in the tissue distribution of RTEF-1 and TEF-1 mRNAs; skeletal muscle showed the highest abundance of both mRNAs, with lower levels detected in pancreas, placenta, and heart. Phylogenetic analysis of all known TEF-1-related proteins identified human RTEF-1 as one of four vertebrate members of this multigene family and further suggests that these genes diverged in the earliest metazoan ancestors.

The mitogen-induced gene, DUSP2, encodes a nuclear protein, PAC1, that acts as a dual-specific protein phosphatase with stringent substrate specificity for MAP kinase. MAP kinase phosphorylation and consequent enzymatic activation is a central and often obligatory component in signal transduction initiated by growth factor stimulation or resulting from various types of oncogenic transformation. DUSP2 downregulates intracellular signal transduction through the dephosphorylation/inactivation of MAP kinases. To facilitate assessment of the possible role of DUSP2 in growth processes, the genomic structure and chromosomal location of the gene have been determined. DUSP2 has been localized to the pericentromeric region of human chromosome 2 (2p11.2-q11) by analysis of somatic cell hybrids, in situ chromosome hybridization, and genetic linkage analysis using a single-strand conformational polymorphism (SSCP) that has been identified in the 3' UTR of the gene. No consistent translocations or deletions at this chromosomal site have been reported in hematopoietic neoplasias or other tumors.

Biotinidase (EC 3.5.1.12) recycles the vitamin biotin by catalyzing the hydrolysis of biocytin, the product of biotin-dependent carboxylase degradation, to biotin and lysine. Biotinidase deficiency is a metabolic disorder that is inherited as an autosomal recessive trait and can be successfully treated with biotin supplementation. Children with biotinidase deficiency who are not treated usually exhibit neurological and cutaneous abnormalities. We have cloned and sequenced the cDNA for human serum biotinidase and now report the chromosomal localization of the gene encoding the enzyme. Fluorescence in situ hybridization (FISH) techniques using a genomic fragment mapped the locus of the biotinidase gene to chromosome 3 in band p25. 4 refs., 1 fig.

Functional defects of high-affinity glutamate transporters have been implicated in the pathophysiology of neurodegenerative diseases such as amyotrophic lateral sclerosis. In small intestine and kidney, in which the high-affinity glutamate transporter mediates net absorption of glutamate and aspartate across epithelial cells, an inborn error of glutamate transport is thought to cause dicarboxylic aminoaciduria. This disorder is characterized by increased urinary excretion of glutamate and aspartate and is, in general, associated with neurologic and developmental abnormalities. Recently, the authors isolated a cDNA encoding a high-affinity glutamate transporter (EAAC1) that also transports aspartate but not other amino acids. EAAC1 is ubiquitously expressed throughout the body, particularly in brain (neurons), intestine, and kidney. Here, the authors present mapping of the chromosome location of EAAC1 using Southern analysis of a panel of human/rodent somatic cell hybrids and fluorescence in situ hybridization (FISH). Southern analysis of EcoRI-digested DNA gave bands at 6.5, 5.6, 5.1, and 1.2 kb for human genomic DNA; 7.5 kb for mouse genomic DNA; and 7.3, 3.2, and 1 kb for hamster genomic DNA. All four human EAAC1-specific bands were observed in the lane corresponding to the human/Chinese hamster hybrid containing chromosome 9 but not in lanes corresponding to any other hybrid.more » Because the human/Chinese hamster hybrid is the only one retaining chromosome 9, this result unambiguously assigns human EAAC1 to chromosome 9. For precise chromosome assignment of the human EAAC1 gene, they employed FISH. Map position of the EAAC1 probe was assigned by visual inspection of the fluorescent signal on the DAPI-stained metaphase chromosomes. The human EAAC1 gene was assigned to 9p24.« less

The ribonuclease inhibitor from human placenta is a tight-binding inhibitor of alkaline and neutral ribonucleases, including the blood vessel-inducing protein, angiogenin. The location of the inhibitor gene within the human genome has now been determined. Utilizing human-rodent hybrid cell lines, it was found on chromosome 11. The localization was refined to chromosome band 11p15 by in situ hybridization of the ribonuclease inhibitor cDNA to normal metaphase chromosomes. A further refinement was obtained by in situ hybridization of the probe to metaphase chromosomes from RPMI 8402 cells, a line containing a well-characterized translocation t(11;14)(p15;q11) with a chromosome 11 breakpoint between the insulin-like growth factor 2 (IGF2) and Harvey rat sarcoma viral oncogene homolog genes. This analysis has localized the ribonuclease inhibitor gene to chromosome subband 11p15.5, distal to the IGF2 gene.