The human renal sodium sulfate cotransporter (SLC13A1; hNaSi-1) cDNA and gene: organization, chromosomal localization, and functional characterization

Sulfate plays an essential role during growth, development, bone/cartilage formation, and cellular metabolism. In this study, we have determined the structure of the human Na+-sulfate cotransporter (hNaSi-1) cDNA (Human Genome Nomenclature Committee-approved symbol SLC13A1) and gene (NAS1). hNaSi-1 encodes a protein of 595 amino acids with 13 putative transmembrane domains. hNaSi-1 mRNA expression was exclusive to the human kidney. Expression of hNaSi-1 protein in Xenopus oocytes demonstrated a high-affinity Na+-sulfate cotransporter that was inhibited by selenate, thiosulfate, molybdate, tungstate, citrate, and succinate. Antisense inhibition experiments suggest hNaSi-1 to represent the major Na+-sulfate cotransporter in the human kidney. NAS1 was localized on human chromosome 7, mapped to 7q31-q32, near the sulfate transporter genes, DRA and SUT-1. The NAS1 gene contains 15 exons, spanning over 83 kb in length. Knowledge of the structure, function, and chromosomal localization of hNaSi-1 will permit the screening of NAS1 mutations in humans with disorders in renal sulfate reabsorption and homeostasis.     

Genomic organization and chromosomal localization of the human CD27 gene.

CD27 is a lymphocyte-specific member of a recently identified receptor family with at least 10 members that includes the receptors for nerve growth factor and TNF, CD40, and Fas. Several members of this family play a role in cell differentiation, proliferation, and survival. Within the amino terminal ligand binding domain of these receptors, repeat motifs have been identified. These repeats contain many cysteine residues in a conserved pattern, characteristic of this family. We have isolated and characterized the human CD27 gene to gain insight into the evolution of this type of receptor domain. The gene was localized on chromosome 12, band 12p13. Sequence analysis showed no correlation between the intron/exon organization and the subdivision of the protein into distinct domains. Structural information for the cysteine-rich domain is contained within three exons. In addition, the splice sites in the CD27 gene are located in a different position from those in the related nerve growth factor receptor gene. However, a comparison of the splice sites within the regions encoding the respective ligand-binding domains of the CD27 and nerve growth factor receptor genes identifies the archetypal cysteine-rich building blocks, from which the members of this family may have arisen during the course of evolution. From this observation, we propose a new organization of the repeat motifs.     

Exon-intron organization, expression, and chromosomal localization of the human motilin gene

The human motilin gene has been isolated and characterized. The gene spans about 9 kilobase pairs (kb) and the 0.7 kb motilin mRNA is encoded by five exons. The 22-amino-acid motilin sequence is encoded by exons 2 and 3. The human motilin gene was mapped to the p21.2----p21.3 region of chromosome 6 by hybridization of the cloned cDNA to DNAs from a panel of reduced human-mouse somatic cell hybrids and by in situ hybridization to human prometaphase chromosomes. RNA blotting using RNA prepared from various regions of the human gastrointestinal tract revealed high levels of motilin mRNA in duodenum and lower levels in the antrum of the stomach; motilin mRNA could not be detected by this procedure in the esophagus, cardia of the stomach, descending colon or gallbladder.     

Human biotin-containing subunit of 3-methylcrotonyl-CoA carboxylase gene (MCCA): cDNA sequence, genomic organization, localization to chromosomal band 3q27, and expression

3-Methylcrotonyl-CoA carboxylase (MCCase; EC 6.4.1.4) is a mitochondrial biotin enzyme and plays an essential role in the catabolism of leucine and isovalerate in animals, bacterial species, and plants. MCCase consists of two subunits, those that are biotin-containing and non-biotin-containing. The genes responsible for these subunits have been isolated in soybean, Arabidopsis thaliana, and tomatoes, but not in mammals. In humans, MCCase deficiency has been thought to be a rare metabolic disease, but the number of patients with MCCase deficiency appears to be increasing with a wide range of clinical presentations, some that result in a lethal condition and others that are asymptomatic. In this report, we have isolated and carried out chromosomal mapping of the gene for the biotin-containing subunit (A subunit) of the human MCCase gene, MCCA. The cDNA predicts an open reading frame coding for a 725-amino-acid protein with mitochondrial signal peptide, biotin carboxylase, and biotin-carrier domains. The gene is composed of at least 19 exons and covers more than 70 kb of sequence on band q27 of chromosome 3. MCCA was abundantly expressed in mitochondria-rich organs, such as the heart, skeletal muscles, kidney, and liver. In exon 13, we observed a His/Pro polymorphism at codon 464 (an A to C transition at nucleotide position 1391 in the cDNA sequence). Then, we determined the DNA sequences of the 5' untranslated region and entire coding regions in two patients with MCCase deficiency, but no sequence substitution was detected, suggesting that the gene mutations might be in the non-biotin-containing subunit (B subunit) gene, MCCB, in these patients.     

The human T-type amino acid transporter-1: characterization, gene organization, and chromosomal location

System T is a Na+-independent transport system that selectively transports aromatic amino acids. Here, we determined the structure of the human T-type amino-acid transporter-1 (TAT1) cDNA and gene (SLC16A10). The human TAT1 cDNA encoded a 515-amino-acid protein with 12 putative membrane-spanning domains. Human SLC16A10 was localized on human chromosome 6, mapped to 6q21-q22. SLC16A10 contains six exons spanning 136 kb. In contrast to rat TAT1, which is mainly present in the intestine, human TAT1 was strongly expressed in human kidney as well as in human intestine. Expression of human TAT1 in Xenopus laevis oocytes demonstrated the Na+-independent transport of tryptophan, tyrosine, phenylalanine, and L-dopa, indicating that human TAT1 is a transporter subserving system T. Because human TAT1 is proposed to be crucial to the efficient absorption of aromatic amino acids from intestine and kidney, its defect could be involved in the disruption of aromatic amino-acid transport, such as in blue diaper syndrome.     

The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization

Characterization of the human glucagon-receptor-encoding gene (GGR) should provide a greater understanding of blood glucose regulation and may reveal a genetic basis for the pathogenesis of diabetes. A cDNA encoding a complete functional human glucagon receptor (GGR) was isolated from a liver cDNA library by a combination of polymerase chain reaction and colony hybridization. The cDNA encodes a receptor protein with 80% identity to rat GGR that binds [¹²⁵I] glucagon and transduces a signal leading to increases in the concentration of intracellular cyclic adenosine 3′,5′-monophosphate. Southern blot analysis of human DNA reveals a hybridization pattern consistent with a single GGR locus. In situ hybridization to metaphase chromosome preparations maps the GGR locus to chromosome 17q25. Analysis of the genomic sequence shows that the coding region spans over 5.5 kb and is interrupted by 12 introns.     

The human vigilin gene: identification,chromosomal localization and expression pattern

Chick vigilin cRNA clones were used to isolate the cognate human gene, by screening a pWE15 genomic library. Three independent cosmid clones were isolated and characterized by restriction mapping. The gene was identified by sequencing an internal EcoRI fragment containing two exons homologous to exon 24 and 25 of the chicken vigilin gene and corresponding to nucleotides 1973–2104 of the human HBP-cDNA. The homology between the chicken and human sequences was 77% and 82% at the cDNA level, and 91% and 100% at the amino acid level. In addition, the analyzed intron/exon boundaries were invariantly conserved. The 5 and 3 regions of the human gene were mapped by Southern analysis of the respective clones with synthetic oligonucleotides. The entire vigilin gene spans a region of about 50 kb and has been assigned to chromosome 2q36–q37.2 (FL-pter value of 0.96 ± 0.03) by fluorescence in situ hybridization to metaphase spreads from normal peripheral blood lymphocytes. The vigilin gene is localized in a chromosomal region comprising a cluster of collagen genes (COLIVA3, COLVIA3) and the locus of the Waardenburg syndrome I. Only one mRNA species of 4.4 kb is transcribed from the human vigilin gene. In accordance with previous observations on chicken mRNA, the expression of the human vigilin mRNA depends on the stage of cytodifferentiation both in vitro and in situ.     

cDNA cloning, expression and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene(1).

Cytosolic thioredoxin (Trx) and thioredoxin reductase (TrxR) comprise a ubiquitous system that uses the reducing power of NADPH to act as a general disulfide reductase system as well as a potent antioxidant system. Human and rat mitochondria contain a complete thioredoxin system different from the one present in the cytosol. The mitochondrial system is involved in the oxidative stress protection through a mitochondrial thioredoxin-dependent peroxidase. We report here the cDNA cloning and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene (TrxR2). The mouse TrxR2 cDNA encodes for a putative protein of 527 amino acid residues with a calculated molecular mass of 57 kDa, that displays high homology with the human and rat counterparts. The N-terminus of the protein displays typical features of a mitochondrial targeting sequence with absence of acidic residues and abundance of basic residues. Mouse TrxR2 also contains a stop codon in frame at the C-terminus of the protein, necessary for the incorporation of selenocysteine that is required for enzymatic activity. The typical stem-loop structure (SECIS element) that drives the incorporation of selenocysteine is identified in the 3'-UTR. Northern analysis of the mouse TrxR2 mRNA shows a similar pattern of expression with the human homologue, with higher expression in liver, heart and kidney. Finally, we have assigned the mouse TrxR2 gene to chromosome 16 mapping at 11.2 cM from the centromer and linked to the catechol-o-methyltransferase (comt) gene.     

Cloning,characterization and chromosomal localization of the Sus scrofa SLC31A1 gene

Copper is an essential element necessary for normal function of numerous enzymes in all living organisms. Uptake of copper into the cell is thought to occur through the membrane protein, SLC31A1 (CTR1), which has been described in a variety of species including yeast, human and mouse. In this study, we present cloning, gene structure, chromosomal localization and expression pattern of the Sus scrofa SLC31A1 gene, which encodes a 189 amino acid protein. The (SSC) SLC31A1 gene is organized in four exons and spans an approximately 2.3 kb genomic region. We have localized the gene to chromosome 1q28-q2.13 using a somatic cell hybrid panel. This region shows conservation of synteny with human chromosome 9, where the human SLC31A1 (CTR1) gene has been localized. Expression studies suggest that SLC31A1 mRNA is transcribed in all tissues examined.     

Human indolethylamine N-methyltransferase: cDNA cloning and expression, gene cloning, and chromosomal localization.

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K(m) value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exon-intron splice junctions conformed to the "GT-AG" rule, and no canonical TATA or CAAT sequences were present within the 5'-flanking region of the gene. Human INMT mapped to chromosome 7p15.2-p15.3 on the basis of both PCR analysis and fluorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identified within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps toward future studies of the function and regulation of this methyltransferase enzyme in humans.     

Human substance P receptor (NK-1): organization of the gene, chromosome localization, and functional expression of cDNA clones.

The gene for the human substance P receptor (NK-1) was cloned using cDNA probes made by the polymerase chain reaction from primers based on the rat sequence. The gene spans 45-60 kb and is contained in five exons, with introns interrupting at sites homologous to those in the NK-2 receptor gene. Analysis of restriction digests of genomic DNA from mouse/human cell hybrids indicates the NK-1 receptor is a single-copy gene located on human chromosome 2. Polymerase chain reaction using primers based on the 5' and 3' ends of the coding sequence was used to generate full-length cDNAs from human lung and from IM9 lymphoblast cells. When transfected into COS-7 cells, the NK-1 receptor binds 125I-BHSP with a Kd of 0.35 +/- 0.07 nM and mediates substance P induced phosphatidylinositol metabolism. The receptor is selective for substance P; the relative affinity for neurokinin A and neurokinin B is 100- and 500-fold lower, respectively. Human IM9 lymphoblast cells express relatively high levels of the NK-1 receptor, and Northern blot analysis indicates modulation of mRNA levels by glucocorticoids and growth factors, suggesting that this cell line may be useful as a model for studying the control of NK-1 receptor gene expression.