Sequence and analysis of the 5′ flanking and 5′ untranslated regions of the rat N-methyl- d-aspartate receptor 2A gene

The 5′ flanking region and the 5′untranslated region (5′ UTR) of the rat N-methyl- d -aspartate receptor subunit 2A were cloned and sequenced using polymerase chain reaction-mediated chromosome walking. The complementary DNA (cDNA) was obtained by rapid amplification of 5′cDNA ends (5′RACE). The comparison of the cDNA and the genomic sequences showed that the 5′ UTR contained two introns and three exons, the third exon overlapping the beginning of the coding region. Transcriptional initiation sites were identified by 5′RACE and RNA-protection assays, using total rat brain RNA. The main start sites were found at −591, −577, −560 and −541 nucleotides 5′ of the AUG. The promoter region lacked TATA and CAAT positioning elements. A CpG island of about 700 bp overlapped the 5′ flanking sequences and the 5′ UTR. The CpG island was inside a wider GC-rich region (66% GC) spanning the entire 5′ UTR. Comparison of the rat sequences with the human sequences from the Human Genome Data Bank revealed that the 5′ UTR exon 2 was extremely conserved with 95.8% sequence identity, as were the initial 640 bp of 5′ flanking sequences, with 78% sequence identity. Beyond this point, sequence identity dropped abruptly to 44%. Putative recognition sequences for the transcription factors S8, Sp1, GATA, AML1 and NF-κB were identified in both the rat and human promoter sequences.