Full-length cDNA cloning and genomic organization of the mouse liver-specific organic anion transporter-1 (lst-1).

We have cloned a cDNA that codes for mouse liver-specific transporter-1, mouse lst-1. The cDNA is comprised of 3296 base pairs and it contains a coding sequence for a protein of 689 amino acids with 12 putative transmembrane domains. The deduced amino acid sequence of the mouse lst-1 shares 64 and 77% identities with the reported human and rat lsts, respectively. Northern blot analysis demonstrates that mouse lst-1 mRNA is expressed exclusively in liver. We also report here the structural organization of the mouse lst-1 gene as the first evidence for the structure of a gene encoding an lst. The mouse lst-1 gene spans approximately 60 kbp in length and consists of 16 exons, including two noncoding exons. All the introns are flanked by GT-AG consensus splice sequences. 5'-Rapid Amplification of cDNA Ends (RACE) analyses demonstrate three splice variant mRNAs involving the noncoding exon 2 and exon 3. The 5'-flanking region of the gene contains consensus CAAT and TATA boxes and several potential binding sites for transcription factors for CAAT enhancer binding protein (C/EBP) and hepatocyte nuclear factors (HNF-3beta, HFH-1, and HFH-2), transcription factors important for liver-specific gene expression.

Ogura K ，Choudhuri S ，Klaassen CD 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Genomic organization and tissue-specific expression of splice variants of mouse organic anion transporting polypeptide 2.

cDNAs that code for mouse organic anion transporting polypeptide 2 (oatp2) have been cloned. At least three forms of mouse oatp2 cDNAs containing the same coding sequence were isolated. The common coding sequence is for a protein of 670 amino acids with 12 putative transmembrane domains. The deduced amino acid sequence of the mouse oatp2 shares 89% identity with the reported rat oatp2. Cloning and analysis of mouse oatp2 gene indicates that these isoforms are alternatively spliced products from the same gene. Heterogeneity was observed in the 5'-untranslated region of the cDNAs. Two of the three isoforms lacked the noncoding exon 3 sequence. Northern-blot hybridization analysis using the exon 3-specific probes demonstrated that mouse oatp2 mRNA containing exon 3 sequence is expressed in heart and lung, whereas exon 1-, 2-, and 17-specific probes detected mRNA only in brain and liver. The mouse oatp2 gene consists of 17 exons, including three noncoding exons, and 16 introns. All of the introns are flanked by GT-AG splice sequences except for intron 10 that is flanked by GC-AG splice sequence.

Ogura K ，Choudhuri S ，Klaassen CD 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Cloning of the full-length coding sequence of rat liver-specific organic anion transporter-1 (rlst-1) and a splice variant and partial characterization of the rat lst-1 gene.

The full-length coding sequence of rat liver-specific organic anion transporter-1 (lst-1) and its splice variant have been cloned. The full-length rat lst-1 (designated rlst-1a) encodes a protein containing 687 amino acids and has 12-putative transmembrane domains, multiple potential N-glycosylation and phosphorylation sites. Therefore, rat lst-1a has 35 additional amino acid residues compared to the previously reported rat lst-1. A splice variant (designated rlst-1c) reported in this communication encodes a protein containing 654 amino acids and has 10-putative transmembrane domains. PCR analysis suggests that rlst-1a is the most abundant form in liver. Phylogenetic analysis reveals that rat lst-1a is an ortholog of human LST-1 (hLST-1) and mouse lst-1 (mlst-1). The rlst-1 gene is composed of 15 exons and 14 introns. Analysis of exon-intron boundary reveals that the splice variant rlst-1c lacks the entire exon 7, while the previously reported rat lst-1 (designated herein as rlst-1b) lacks approximately half of exon 10, and the splicing has occurred through alternative usage of a splice donor site within exon 10.

Choudhuri S ，Ogura K ，Klaassen CD 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

The human Nramp2 gene: characterization of the gene structure, alternative splicing, promoter region and polymorphisms.

Nramp2 is a gene encoding a transmembrane protein that is important in metal transport, in particular iron. Mutations in nramp2 have been shown to be associated with microcytic anemia in mk/mk mice and defective iron transport in Belgrade rats. Nramp2 contains a classical iron responsive element in the 3' untranslated region that confers iron dependent mRNA stabilization. In this report, we describe a splice variant form of human nramp2 that has the carboxyl terminal 18 amino acids substituted with 25 novel amino acids and has a new 3' untranslated region lacking a classical iron-responsive element. This splice form of nramp2, nramp2 non-IRE, was found to be derived from splicing of an additional exon into the terminal coding exon. The nramp2 gene is comprised of 17 exons and spans more than 36 kb. It contains an additional 5' exon and intron (exon and intron 1) and an additional 3' exon (exon 17) and intron (intron 16) as compared to nramp1, a homologous gene. The additional exons and introns account for much of the difference in length between nramp2 (> 36 kb) and nramp1 (12 kb). The exon-intron borders of nramp2 exons 3-15 are homologous to nramp1 exons 2-14. The nramp2 5' regulatory region contains two CCAAT boxes but lacks a TATA box. The 5' regulatory region of nramp2 also contains five potential metal response elements (MRE's) that are similar to the MRE's found in the metallothionein-IIA gene, three potential SP1 binding sites and a single gamma-interferon regulatory element. Five single nucleotide mutations or polymorphisms were identified within the nramp2 gene. One of these, 1303C-->A, occurs in the coding region of nramp2 and results in an amino acid change from leucine to isolecine. A polymorphism, 1254T/C, also occurs in the coding region of nramp2 but does not cause an amino acid change. The other 3 polymorphisms are within introns (IVS2 + 11A/G, IVS4 + 44C/A, and IVS6 + 538G/Gdel). In addition, a polymorphic microsatellite TATATCTATATATC (TA)6-7 (CA)10-11 CCCCCTATA (TATC)3 (TCTG)5 TCCG (TCTA)6 was identified in intron 3. Analysis of cDNA derived by direct amplification of reversed transcribed RNA or cDNA clones isolated from a library provide evidence of skipping of exons 10 and 12 of nramp2. Deletion of either of these exons would result in a sequence that remains in frame yet would generate a protein that would lack transmembrane spanning region 7 or 8 respectively. The deletion of a single transmembrane domain would have severe topological consequences. The coding region of the nramp2 gene of hemochromatosis patients with or without mutations in the hemochromatosis gene, HFE, were examined and found to be normal. One hemochromatosis patient, with a normal HFE genotype, was heterozygous for the 1303C-->A mutation. Furthermore, in an examination of hemochromatosis patients with mutant HFE and normal HFE genes, we did not observe a linkage disequilibrium of either group with a particular nramp2 haplotype. These data suggest that mutations in nramp2 are not commonly associated with hemochromatosis.

Lee PL ，Gelbart T ，West C ，Halloran C ，Beutler E ... -  《BLOOD CELLS MOLECULES AND DISEASES》

Genomic organization and expression of the mouse equilibrative, nitrobenzylthioinosine-sensitive nucleoside transporter 1 (ENT1) gene.

We have cloned and characterized the genomic structure of the mouse gene for the NBMPR-sensitive equilibrative nucleoside transporter (mENT1), which is located on chromosome 17C. About 12-kb of genomic DNA was sequenced including the promoter region, 12 exons, 11 introns, and the 3'-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. Primer extension analysis demonstrated a transcription initiation site located 252 bp upstream of the translation start site. Analysis of the 2.5-kb 5'-flanking sequence shows putative binding sites for several transcription factors, including GATA-1, IRF-2, Pit-1, myogenin, CREB, Sp-1, Ap-2, MAZ, and GR. We demonstrated that mouse ENT1 mRNA was highly expressed in heart, spleen, lung, liver, and testis. Lower levels of expression were detected in brain and kidney. Functional analysis of the 5'-flanking region showed that the nucleotide sequence from -652 to -111 contains cis-regulatory elements that promote gene expression. We found two Sp-1 binding sites (-296/-303, -307/-313) and two MAZ binding sites (-353/-359, -522/-528) in this region. Luciferase assay results suggest that MAZ and Sp-1 transcription factors are important positive regulators of transcription for the ENT1 gene in NG108-15 cells.

Choi DS ，Handa M ，Young H ，Gordon AS ，Diamond I ，Messing RO ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》