FIGURE 3.
Zebrafish synucleins: transcripts, promoters, and genomic organization. A, Northern blots containing total RNA from adult zebrafish brain were hybridized with cRNA probes complementary to sncb (left) or sncg1 (right). The positions of molecular size markers (kb) are shown to the left of the blots, and the positions of the 28 and 18 S ribosomal RNA bands are shown to the right. B, total RNA derived from adult brain or embryos at 48 hpf was subjected to RT-PCR using primers complementary to exons 3 and 5 of sncg2. The picture shows an ethidium-stained agarose gel under UV illumination following electrophoretic separation of the products. Bands corresponding to sncg2 tv1 (lacking exon 4) and sncg2 tv2 (containing exon 4) are labeled to the right of the picture. C, 5′-RACE was used to map the transcriptional start sites of sncb (lanes 1, 3, 5, and 7) and sncg1 (lanes 2, 4, 6, and 8). The picture shows an ethidium-stained agarose gel under UV illumination following electrophoretic separation of PCR products. 5′-RACE (lanes 1–4) and RT-PCR (lanes 5–8) were carried out using cDNA template derived from samples in which RNA was either treated with tobacco acid pyrophosphatase (TAP+; lanes 1, 2, 5, and 6) or received no treatment (TAP−; lanes 3, 4, 7, and 8) prior to adapter ligation. D and E, the promoter regions of sncb (D) and sncg1 (E) are shown. The arrows indicate transcriptional start sites identified by 5′-RACE; initiator sequences are highlighted in gray, and the major start sites of each transcript are highlighted in black. The splice donor consensus of sncb exon 1 is underlined. A pyrimidine-rich sequence in the sncg1 5′-flanking region is underlined. F–H, the genomic organization of sncb (F), sncg1 (G), and sncg2 (H) are shown schematically. The lower part of each diagram depicts the genomic sequence showing the position and number of each exon. The upper part shows the major transcripts arising from each gene and the locations of the open reading frames (shaded in black and labeled ATG→TAG). *, splice donor sequences in sncb and sncg1 that were targeted by antisense morpholino oligonucleotides.