| Summary: | To facilitate the characterisation of SCL regulatory elements, 33 kb of sequence encompassing the SCL genomic locus of the pufferfish, <I>Fugu rubripes</I> and 84 kb from the locus of the zebrafish, <I>Danio rerio</I> were sequenced and analysed. Sequence comparisons with the SCL genes of human, mouse and chicken identified four conserved non-coding regions. The most striking of these was a 150 bp stretch corresponding to the mouse SCL promoter 1a. Five conserved blocks of sequence were identified including two new putative transcription factor binding motifs, CS1 and CS2. <I>In vitro</I> analysis of mouse promoter constructs showed that mutation of CS1 and CS2 reduced promoter activity to 67% and 63% respectively within the erythroid cell line, J2E. Two protein complexes were shown to bind CS1 but the nature of the proteins within these complexes remains unclear. Expression studies in transgenic mice demonstrated that two conserved GATA motifs are necessary for full midbrain expression of SCL, the first demonstration that GATA acts upstream of SCL in neural development. Although the pattern of SCL expression is highly conserved from mammals to teleost fish, the genes flanking the pufferfish SCL gene were unrelated to those known to flank both avian and mammalian SCL genes. These data suggest that SCL regulatory elements are confined to the region between the upstream and downstream flanking genes, a region of 65 kb in moue and 8.5 kb in pufferfish. Consistent with this hypothesis, the entire 33 kb pufferfish SCL locus directed appropriate haemopoietic and neural expression in transgenic zebrafish embryos as did a 10.4 kb fragment containing the SCL gene and extending to the 5' and 3' flanking genes.
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