Characterization of three novel human cadherin genes (CDH7, CDH19, and CDH20) clustered on chromosome 18q22-q23 and with high homology to chicken cadherin-7

Full-length coding sequences of two novel human cadherin cDNAs were obtained by sequence analysis of several EST clones and 5' and 3' rapid amplification of cDNA ends (RACE) products. Exons for a third cDNA sequence were identified in a public-domain human genomic sequence, and the coding sequence was completed by 3' RACE. One of the sequences (CDH7L1, HGMW-approved gene symbol CDH7) is so similar to chicken cadherin-7 gene that we consider it to be the human orthologue. In contrast, the published partial sequence of human cadherin-7 is identical to our second cadherin sequence (CDH7L2), for which we propose CDH19 as the new name. The third sequence (CDH7L3, HGMW-approved gene symbol CDH20) is almost identical to the mouse "cadherin-7" cDNA. According to phylogenetic analysis, this mouse cadherin-7 and its here presented human homologue are most likely the orthologues of Xenopus F-cadherin. These novel human genes, CDH7, CDH19, and CDH20, are localized on chromosome 18q22-q23, distal of both the gene CDH2 (18q11) encoding N-cadherin and the locus of the six desmosomal cadherin genes (18q12). Based on genetic linkage maps, this genomic region is close to the region to which Paget's disease was linked. Interestingly, the expression patterns of these three closely related cadherins are strikingly different.     

The prepro vasoactive intestinal contractor (VIC)/endothelin-2 gene (EDN2): structure, evolution, production, and embryonic expression

Murine vasoactive intestinal contractor (VIC) and its human analog endothelin-2 (ET2) are potent vasoactive hormones composed of 21 amino acids. To study the structural characteristics of the VIC/ET2 gene (HGMW-approved symbol EDN2), we isolated the full length of the mouse VIC gene. Sequence analysis indicates that a biologically active mature VIC peptide is produced from a 175-residue precursor protein; preproVIC (PPVIC). Several remarkable similarities of the PPVIC gene to the human preproendothelin-1 gene strongly suggest that the two genes have arisen from a common progenitor by gene duplication. Transfection of ACHN adenocarcinoma cells with the cDNA resulted in the production of VIC peptide. VIC production was increased by the deletion of the 3'-untranslated region, which contains an AU-rich mRNA destabilizing sequence. Increased PPVIC gene expression during the late embryonic stage suggests an important function in development. This study provides the basis for disruption and regulation analysis of the gene, which may lead to a better understanding of VIC/ET2's physiological significance.     

Characterization of TNFRSF19, a novel member of the tumor necrosis factor receptor superfamily

By searching the expressed sequence tag database, a novel murine tumor necrosis factor receptor designated TNFRSF19 was identified. TNFRSF19 cDNA encodes a putative membrane protein of 348 amino acids with one incomplete and two complete cysteine-rich motifs within its extracellular region and a large cytoplasmic domain. TNFRSF19 mRNA can be detected in most murine tissues examined, particularly in brain, reproductive organs, and late developmental stages of murine embryo, but not in tissues of the immune system. The cell surface expression of the ligand of TNFRSF19 is highly restricted. Of 22 human and murine cell lines examined by FACS analysis, only Raji (B cell lymphoma cell line), GM847 (fibroblast cell line), 293 (embryonic kidney cell line), and K562 (chronic myeloid leukemia) were positive. TNFRSF19 did not bind newly cloned TNF ligands, including TWEAK (HGMW-approved symbol TNFSF12), VEGI/TL1 (HGMW-approved symbol TNFSF15), TL6/endokine (HGMW-approved symbol TNFSF18), APRIL (HGMW-approved symbol TNFSF13), OPGL (HGMW-approved symbol TNFSF11), LIGHT (HGMW-approved symbol TNFSF14), or BAFF/THANK (HGMW-approved symbol TNFSF13B) by enzyme-linked immunosorbent assay and FACS analyses. Overexpression of TNFRSF19 transduced neither apoptotic signaling nor signals leading to NF-kappaB induction. Taken together with the data that the TNFRSF19 extracellular domain-immunoglobulin fusion protein did not affect the allogeneic mixed lymphocyte reaction, our data indicate that TNFRSF19 is not involved in the modulation of immune responses.     

A sequence-ready physical map of the region containing the human natural killer gene complex on chromosome 12p12.3-p13.2

We developed a sequence-ready physical map of a part of human chromosome 12p12.3-p13.2 where the natural killer gene complex (NKC) is located. The NKC includes a cluster of genes with structure similar to that of the Ca(2+)-dependent lectin superfamily of glycoproteins that are expressed on the surface of most natural killer (NK) cells and a subset of T cells. These killer cell lectin-like receptors (KLR) are involved in NK target cell recognition, leading to activation or inhibition of NK cell function. We used a number of sequence-tagged site (STS) markers from this region to screen two large insert bacterial artificial chromosome (BAC) libraries and a bacteriophage P1-derived (PAC) chromosome library. The clones were assembled into contiguous sets by STS content analysis. The 72-BAC and 11-PAC contig covers nearly 2 Mb of DNA and provides an average marker resolution of 26 kb. We have precisely localized 17 genes, 5 expressed sequence tags, and 49 STSs within this contig. Of this total number of STS, 30 are newly developed by clone-end sequencing. We established the order of the genes as tel-M6PR-MAFAL (HGMW-approved symbol KLRG1)-A2M-PZP-A2MP-NKRP1A (HGMW-approved symbol KLRB1)-CD69-AICL (HGMW-approved symbol CLECSF2)-KLRF1-OLR1-CD94 (HGMW-approved symbol KLRD1)-NKG2D (HGMW-approved symbol D12S2489E)-PGFL-NKG2F (HGMW-approved symbol KLRC4)-NKG2E (HGMW-approved symbol KLRC3)-NKG2A (HGMW-approved symbol KLRC1)-LY49L (HGMW-approved symbol KLRA1)-cen. This map would facilitate the cloning of new KLR genes and the complete sequencing of this region.     

Chemokine PARC gene (SCYA18) generated by fusion of two MIP-1alpha/LD78alpha-like genes

Two loci in the human genome, chromosomes 4q12-q21 and 17q11.2, contain clusters of CXC and CC chemokine subfamily genes, respectively. Since mice appear to contain fewer chemokine genes than humans, numerous gene duplications might have occurred in each locus of the human genome. Here we describe the genomic organization of the human pulmonary and activation-regulated CC chemokine (PARC), also known as DC-CK1 and AMAC-1. Despite high sequence similarity to a CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha)/LD78alpha, PARC is chemotactic for lymphocytes and not for monocytes and does not share its receptor with MIP-1alpha. Analyses of the BAC clones containing the human PARC gene indicated that the gene is located most closely to MIP-1alpha (HGMW-approved symbol SCYA3) and MIP-1beta (HGMW-approved symbol SCYA4) on chromosome 17q11.2. Dot-plot comparison suggested that the PARC gene had been generated by fusion of two MIP-1alpha-like genes with deletion and selective usage of exons. Base changes accumulated before and after the fusion might have adapted the gene to a new function. Since there are variably duplicated copies of the MIP-1alpha gene called LD78beta (HGMW-approved symbol SCYA3L) in the vicinity of the MIP-1alpha gene, the locus surrounding the MIP-1alpha gene seems to be a "hot spring" that continuously produces new family genes. This evidence provides a new model, duplication and fusion, of the molecular basis for diversity within a gene family.     

The expanded human kallikrein gene family: locus characterization and molecular cloning of a new member, KLK-L3 (KLK9)

In rodents, kallikreins are encoded by a large multigene family but in humans, only three kallikrein genes were thought to exist. Based on the homology between the human and the rodent kallikrein loci, we defined a 300-kb human kallikrein gene region on chromosome 19q13. 3-q13.4. By using linear sequence information, restriction analysis, PCR, and blotting techniques, we were able to construct the first detailed map of the human kallikrein gene locus. Comparative analysis of genes located in this area enabled us to expand the human kallikrein multigene family with some recently identified serine proteases and establish common structural features. We further identified a new kallikrein-like gene, named kallikrein-like gene 3 (KLK-L3; HGMW-approved symbol KLK9). We describe the structural characterization of the KLK-L3 gene, together with its precise chromosomal localization in relation to other kallikreins and its tissue expression pattern and hormonal regulation.     

Characterization of Three Novel Human Cadherin Genes (CDH7, CDH19, and CDH20) Clustered on Chromosome 18q22–q23 and with High Homology to Chicken Cadherin-7

Full-length coding sequences of two novel human cadherin cDNAs were obtained by sequence analysis of several EST clones and 5′ and 3′ rapid amplification of cDNA ends (RACE) products. Exons for a third cDNA sequence were identified in a public-domain human genomic sequence, and the coding sequence was completed by 3′ RACE. One of the sequences (CDH7L1, HGMW-approved gene symbol CDH7) is so similar to chicken cadherin-7 gene that we consider it to be the human orthologue. In contrast, the published partial sequence of human cadherin-7 is identical to our second cadherin sequence (CDH7L2), for which we propose CDH19 as the new name. The third sequence (CDH7L3, HGMW-approved gene symbol CDH20) is almost identical to the mouse “cadherin-7” cDNA. According to phylogenetic analysis, this mouse cadherin-7 and its here presented human homologue are most likely the orthologues of Xenopus F-cadherin. These novel human genes, CDH7, CDH19, and CDH20, are localized on chromosome 18q22–q23, distal of both the gene CDH2 (18q11) encoding N-cadherin and the locus of the six desmosomal cadherin genes (18q12). Based on genetic linkage maps, this genomic region is close to the region to which Paget's disease was linked. Interestingly, the expression patterns of these three closely related cadherins are strikingly different.     

The human cystatin C gene (CST3) is a member of the cystatin gene family which is localized on chromosome 20

The fourth gene from the human cystatin gene family of salivary-type cysteine-proteinase inhibitors has been isolated and partially characterized by DNA analysis. The gene, which we name CST3, codes for human cystatin C, and has the same organization as the CST1 gene for cystatin SN and the CST2 gene for cystatin SA. Southern analysis of EcoR I digested DNAs from 32 independent somatic cell hybrid clones hybridized to a probe from CST1 demonstrated that all members of the cystatin gene family segregate with human chromosome 20. These results indicate that the genes for salivary-type cystatins and cystatin C are members of a multigene family--the cystatin gene family.     

Localization of the gene for autosomal recessive congenital hereditary endothelial dystrophy (CHED2) to chromosome 20 by homozygosity mapping.

Congenital hereditary endothelial dystrophy (CHED) is a corneal disorder that presents with diffuse bilateral corneal clouding. Vision may be severely impaired, and many patients require corneal transplantation. Both autosomal dominant (AD) and autosomal recessive (AR) forms of the disorder have been described. The gene responsible for AD CHED (HGMW-approved symbol CHED1) has been mapped to the pericentromeric region of chromosome 20. Investigating a large, consanguineous Irish pedigree with autosomal recessive CHED, we have previously excluded linkage to this AD CHED locus. We now describe a genome-wide search using homozygosity mapping and DNA pooling. Evidence of linkage to chromosome 20p was demonstrated with a maximum lod score of 9.30 at a recombination fraction of 0.0 using microsatellite marker D20S482. A region of homozygosity in all affected individuals was identified, narrowing the disease gene locus to an 8-cM region flanked by markers D20S113 and D20S882. This AR CHED (HGMW-approved symbol CHED2) disease gene locus is physically and genetically distinct from the AD CHED locus.     

The human serum deprivation response gene (SDPR) maps to 2q32-q33 and codes for a phosphatidylserine-binding protein

The serum deprivation response gene (SDPR, alias sdr) has been previously isolated for its high mRNA expression in serum-starved cells compared to contact-inhibited NIH3T3 cells; such regulation is not observed in single-oncogene transformed NIH3T3 cells after serum starvation. More recently Sdpr has been identified as a substrate of protein kinase C (PKC): this interaction determines the compartimentalization of PKC to caveolae, a plasma membrane invagination of which Sdpr is a major component. Lack of Sdpr-PKC interaction in transformed cells has been proposed to be involved in the alteration of PKC subcellular localization and substrate specificity. Here we report the cloning of the human SDPR homologue (HGMW-approved symbol SDPR) and its mapping to 2q32-q33 in the human genome. In analogy with the murine system, SDPR mRNA expression is increased when human fibroblasts are serum starved, it becomes down-regulated during synchronous cell-cycle reentry, but it is not induced in cells arrested by contact inhibition. Analysis of SDPR expression in human tissues reveals a near ubiquitous expression, with highest levels found in heart and lung. We show that human SDPR encodes PS-p68, a previously characterized phosphatidylserine-binding protein purified from human platelets. Accordingly, recombinant Sdpr is able to specifically bind phosphatidylserine in the absence of Ca2+. SDPR is homologous to two genes in the databank, one of which, srbc, is similarly regulated during growth arrest and encodes a phosphatidylserine-binding protein that is a substrate of PKC.     

Molecular cloning and characterization of two novel retinoic acid-inducible orphan G-protein-coupled receptors (GPRC5B and GPRC5C)

Using homology searching of public databases with a metabotropic glutamate receptor sequence from Caenorhabditis elegans, two novel protein sequences (named RAIG-2 (HGMW-approved symbol GPRC5B) and RAIG-3 (HGMW-approved symbol GPRC5C) were identified containing seven putative transmembrane domains characteristic of G-protein-coupled receptors (GPCRs). RAIG-2 and RAIG-3 encode open reading frames of 403 and 442 amino acid polypeptides, respectively, and show 58% similarity to the recently identified retinoic acid-inducible gene-1 (RAIG-1, HGMW-approved symbol RAI3). Analysis of the three protein sequences places them within the type 3 GPCR family, which includes metabotropic glutamate receptors, GABA(B) receptors, calcium-sensing receptors, and pheromone receptors. However, in contrast to other type 3 GPCRs, RAIG-1, RAIG-2, and RAIG-3 have only short N-terminal domains. RAIG-2 and RAIG-3 cDNA sequences were cloned into the mammalian expression vector pcDNA3 with c-myc or HA epitope tags inserted at their N-termini, respectively. Transient transfection experiments in HEK239T cells using these constructs demonstrated RAIG-2 and RAIG-3 expression at the cell surface. Distribution profiles of mRNA expression obtained by semiquantitative Taq-Man PCR analysis showed RAIG-2 to be predominantly expressed in human brain areas and RAIG-3 to be predominantly expressed in peripheral tissues. In addition, expression of RAIG-2 and RAIG-3 mRNA was increased following treatment with all-trans-retinoic acid in a manner similar to that previously described for RAIG-1. Finally, RAIG-2 was mapped to chromosome 16p12 (D16S405-D16S3045) and RAIG-3 to chromosome 17q25 (D17S1352-D17S785). These results suggest that RAIG-1, RAIG-2, and RAIG-3 represent a novel family of retinoic acid-inducible receptors, most closely related to the type 3 GPCR subfamily, and provide further evidence for a linkage between retinoic acid and G-protein-coupled receptor signal transduction pathways.     

Identification of a new EGF-repeat-containing gene from human Xp22: a candidate for developmental disorders

Epidermal growth factor (EGF) repeat-containing proteins constitute an expanding family of proteins involved in several cellular activities such as blood coagulation, fibrinolysis, cell adhesion, and neural and vertebrate development. By using a bioinformatic approach, we have identified a new member of this family named MAEG (MAM- and EGF-containing gene; HGMW-approved gene symbol and gene name). Sequence analysis indicates that MAEG encodes a secreted protein characterized by the presence of five EGF repeats, three of which display a Ca(2+)-binding consensus sequence. In addition, a MAM domain is also present at the C-terminus of the predicted protein product. The human and murine full-length cDNAs were identified and mapped to human Xp22 and to the mouse syntenic region. Northern analysis indicates that MAEG is expressed early during development. Taken together, these data render MAEG a candidate for human and murine developmental disorders.     

The human fatty acid transport protein-1 (SLC27A1; FATP-1) cDNA and gene: organization, chromosomal localization, and expression

Uptake of fatty acids into cells is a controlled process in part regulated by fatty acid transport proteins (FATPs), which facilitate the transport of fatty acids across the cell membrane. In this study the structure of the human FATP-1 (HGMW-approved symbol SLC27A1) cDNA and gene was determined, and the expression of its mRNA in human was characterized. Muscle and adipose tissue have the highest levels of FATP-1 mRNA, small intestine has intermediate levels, and FATP-1 mRNA is barely detectable in liver. The human FATP-1 gene has 12 exons and extends over more than 13 kb of genomic DNA. The FATP gene maps to chromosome 19p13.1 by fluorescence in situ hybridization, a region previously suggested to be implicated in the determination of small dense low-density lipoprotein (LDL). Knowledge of the gene structure and chromosomal localization will allow screening for FATP mutations in humans with metabolic disorders, whereas knowledge of its expression pattern and factors regulating its expression could be of importance in understanding its biology.