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》

Genomic organization, chromosomal mapping and promoter analysis of the mouse selenocysteine tRNA gene transcription-activating factor (mStaf) gene.

Adachi K ，Katsuyama M ，Song S ，Oka T ... -  《-》

Structural and functional characterizations of the 5'-flanking region of the mouse glucagon receptor gene: comparison with the rat gene.

A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5'-rapid amplification of cDNA ends from mouse liver tissue. The 5'-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5' untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5' region of the rat GR gene. The mouse distal promoter region, extending up to -1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5'-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.

Geiger A ，Decaux JF ，Burcelin R ，Le Cam A ，Salazar G ，Charron MJ ，Girard J ，Kervran A ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Genomic organization of the JEM-1 (BLZF1) gene on human chromosome 1q24: molecular cloning and analysis of its promoter region.

The Jem-1 (JEM-1, HGMW-approved symbol BLZF1) gene mapping to human chromosome 1q24 codes for a ubiquitously expressed 3-kb mRNA, translated in a 45-kDa nuclear protein. Recent studies have shown a deficient expression of this gene in acute promyelocytic leukemia (APL). However, treatment with retinoids was able to upregulate JEM-1 mRNA in maturing NB4 leukemia cells. Here, we report the characterization of the structural organization of JEM-1. By hybridization screening of a human genomic library derived from blood mononuclear cells, five overlapping genomic DNA clones were isolated. These clones extend over 34 kb of the human genome and comprise the complete JEM-1 gene and a 4-kb 5'flanking region. Determination of the exon-intron structure of Jem-1 revealed seven exons whose junctions with introns exhibited typical splice sequences. A shorter transcript (Jem-1s, 1.3 kb) generated by exon 3 extension and polyadenylation was identified. Its translation generated a 23-kDa protein that exhibited a cytoplasmic localization. 5'RACE-PCR identified a major transcription start site (TSS) located at 403 nt upstream of the ATG. Computer analysis of the 1. 8-kb 5'flanking region showed that it lacks a TATA box, Inr motifs or DPE motifs, but it contains a typical CCAAT box located 95 bp upstream of the TSS. Sequencing also revealed potential cis-acting elements for multiple transcription regulators including Sp1, GATA, C/EBP, AP-1, and Pu1. No retinoic acid receptor elements or retinoic X receptor elements were detected. This 1.8-kb DNA sequence showed a strong constitutive promoter activity determined by a luciferase-reporter gene assay in transiently transfected HeLa cells. Retinoids further increased luciferase expression 2.7-fold. We demonstrated that the 1-kb distal sequence contains yet unidentified elements reducing constitutive transcription. Thus, the maximal constitutive promoter activity was assigned to a -432 + 101 region overlapping the TSS. These data support the idea of a constitutive expression of JEM-1, but a negative regulation in APL released by retinoids.

Tong JH ，Fant X ，Benoit G ，Chen SJ ，Chen Z ，Lanotte M ... -  《GENOMICS》

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》