Cloning and characterization of promoter and 5'-UTR of the NMDA receptor subunit epsilon 2: evidence for alternative splicing of 5'-non-coding exon.
Using rapid amplification of cDNA ends (RACE), we have cloned the 5'-untranslated region (5'-UTR) of the N-methyl-D-aspartate receptor subunit epsilon 2 from murine forebrain-derived mRNA. We identified two distinct types of cDNA species differing in the presence or absence of one exon sequence. Sequencing of the 5'-non-coding region of the epsilon 2 gene revealed that the epsilon 2 5'-UTR consists of three untranslated exons located at least 20 kb upstream of exon 4 that contains the ATG codon for initiation of translation. This genomic organization shows a close similarity to the epsilon 3 gene. The transcriptional start site was determined by primer extension assays. Expression of the alternative exon sequence was shown by in situ hybridization in the murine brain. Basal transcriptional activity of the epsilon 2 promoter was detected in different neuronal and non-neuronal cell lines with transient reporter gene expression assays. Potential SP1 and CREB binding sites were found in the promoter region. Specific binding of these transcription factors was demonstrated in electrophoretic mobility shift assays.
Klein
M
M
Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
Pieri
I
I
Uhlmann
F
F
Pfizenmaier
K
K
Eisel
U
U
D016475:3T3 Cells; D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D005091:Exons; D014644:Genetic Variation; D017403:In Situ Hybridization; D007739:L Cells; D046911:Macromolecular Substances; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D016548:Prosencephalon / Q000378:metabolism; D014176:Protein Biosynthesis; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D017931:DNA Primers; D046911:Macromolecular Substances; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins