Mouse vesicular GABA transporter gene: genomic organization, transcriptional regulation and chromosomal localization.
The vesicular GABA transporter (VGAT) loads GABA from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons that contain GABA and/or glycine. To elucidate the molecular mechanisms of mouse VGAT (mVGAT) gene expression, we have isolated and characterized the mVGAT gene. The mVGAT gene was found to be 4.7 kilobases in size and to contain three exons and two introns by comparison of the cloned genomic DNA with the cDNA (termed mVGATa) sequence reported by Sagne et al. [FEBS Lett. 417 (1997) 177]. Analysis of transcripts and genomic DNA revealed an alternatively spliced mVGAT isoform (termed mVGATb) that retains intron 2 of mVGATa as an exon. This alternative transcript specifies 514 amino acid residues identical to VGATa followed by a unique C-terminal sequence of 11 amino acids encoded by intron 2. Fluorescent in situ hybridization studies showed that the mVGAT gene is localized on chromosome 2. One major transcription start site of the mVGAT gene is an A residue 209 bp upstream from the translational initiation site, as shown using the 5'-RACE method. RT-PCR analysis revealed that the mVGAT gene was expressed at a high level in retinoic acid-treated P19 embryonal carcinoma cells, at a very low level in non-treated P19 cells, and not detectably expressed in Neuro-2a neuroblastoma cells. Sequence analysis of the 5'-flanking region revealed a number of putative regulatory elements including Sp1, Egr-1 and Pitx binding sites. In transient transfection assays, 2 kilobases of the mVGAT 5'-flanking region generated similar levels of luciferase reporter activity in three kinds of cultured cells. Deletion analysis and gel mobility shift assays demonstrated that the region -161 to +155 contained the basal promoter activity of the mVGAT gene and that an activating region from -49 to -27 bound an Sp1-like protein. These results suggest a possible mechanism for regulation of the expression of the mVGAT gene.
Ebihara
Satoe
S
Laboratory of Neurochemistry, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan.
Obata
Kunihiko
K
Yanagawa
Yuchio
Y
D024506:5' Flanking Region / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002874:Chromosome Mapping; D054278:Embryonal Carcinoma Stem Cells; D050485:GABA Plasma Membrane Transport Proteins; D017353:Gene Deletion; D015870:Gene Expression; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D008969:Molecular Sequence Data; D014411:Neoplastic Stem Cells; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology / Q000502:physiology; D009687:Nuclear Proteins / Q000378:metabolism; D027361:Organic Anion Transporters; D011401:Promoter Regions, Genetic / Q000235:genetics; D013572:Synaptic Vesicles / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology*; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009687:Nuclear Proteins; D027361:Organic Anion Transporters; D008156:Luciferases