Figure 1

brd2b transcript variants are differentially regulated during development. (A) Schematic of predicted proteins from the brd2b locus, full-length Brd2b-L and truncated Brd2b-S. Bromodomains BD1 and BD2 (ovals), nuclear localization signal (NLS, triangle), and a C-terminal extraterminal (ET) domain (rectangle). (B) Schematic of the long (brd2b-L) and short (brd2b-S) transcript variants encoded by the brd2b locus, with numbered exons. UTRs of 5? and 3? are shown in white; protein-coding regions are shown in orange. The short transcript retains part of intron 5 (shown in yellow), which introduces a premature stop codon (darker yellow shows part of intron that is translated). Primer pairs flanking exon?exon junctions and used in RT-PCR are shown as dark blue arrows. (C) RT-PCR products produced from mRNA of staged oocytes (top gels) and 4, 24, and 48 hpf embryos (bottom gels), using various primer pairs, as shown in B. Lanes labeled with the same number display the PCR product obtained using the same primer pairs, as shown in the key: (1) 1 kb ladder (NEB); (2) ex5/in5 primers (brd2b-S 250 bp predicted product); (3) ex5/6 primers (brd2b-L 400 bp predicted product); (4) ex9/10 primers (brd2b-L 300 bp predicted product); (5) ex11/12 (brd2b-L 404 bp predicted product); (6) ex1/in5 primers (full-length brd2b-S 1447 bp predicted product); (7) ex1/ex12 primers (full-length brd2b-L 4037 bp predicted product); (8) primers for ?-actin (+) control (322 bp predicted product); (9) primers internal to intron 1 (not shown in B), as (?) control for genomic DNA (784 bp predicted product). Maternal brd2b-L predicted PCR products are detected in all stages of ooctye tested (blue arrowheads, lanes labeled 3,4,5, top gels), but brd2b-S predicted products are not present (lanes labeled 2, top gels). The bands observed in stage 1, 2 and 3 oocytes in lanes labeled 2 are either smaller than the 250 bp predicted product for brd2b-S (s1, s2 gels), or are very faint products among several faint products (s3 gel), so are likely a spurious. Zygotic brd2b-S predicted product is detected in 24 hpf and 48 hpf embryos (red asterisk, lanes labeled 2, bottom gels), but not in 4 hpf embryos, while zygotic brd2b-L is detected in all stages of embryos tested (blue arrowheads, lanes labeled 3,4,5, bottom gels).