| Abstract: |
Using a modified 5'RACE approach, we isolated a novel exon (exon 11), which led to identify three additional exons (exons 12, 13 and 14) and eight novel splice variants (MOR-1G, -1H, -1I, -1J, -1K, -1L, -1M and MOR-1N) of the mouse MOR-1 gene. Exon 11 was mapped to upstream of exon 1 and the others between exons 11 and 2. Exon 11 is the first 5'-end exon for all eight new variants. They all underwent different splicing patterns yielding transcripts for a number of predicted proteins, including the original MOR-1. A single putative transcription start point was determined. Functional characterization of the 5'-flanking region of exon 11 by a SEAP reporter system in several cell lines revealed neuronal-specific promoter activities in the 5' flanking region, which contains a TATA box, two CAAT boxes and several binding sites including C/EBPbeta and AP-1 and cMyc/max. Northern blot analysis with an exon 11 probe shows a diffuse band from 4 to 7 kb. The differential regional distributions of the variant mRNAs, determined by RT-PCR, implied region-specific and/or cell-specific RNA processing. Antisense mapping using two probes targeting exon 11 had different actions spinally and supraspinally, blocking either morphine spinally or M6G supraspinally. Since exon 11 is present in all eight variants, it is not possible to assign a specific variant to these actions, but these findings do suggest that these new variants are pharmacologically relevant. |
