Formation of high molecular weight <i>mip1</i> promoter- protein complexes is dependent on pitx3 presence.

Abstract

<p><b>A.</b> 48-hpf wild-type and <i>pitx3</i> morphant embryos showing normal body length and morphology that were selected for EMSA experiments. <b>B.</b> Results of RT-PCR performed with RNA extracted from the pooled tail tissues from wild-type and <i>pitx3</i> morphant embryos shown in C. A sharp reduction in normal <i>pitx3</i> transcript (black arrowhead) and the presence of abnormally spliced product (red arrowhead) are evident in <i>pitx3</i> morphant embryos. <b>C.</b> Coomassie Blue R-250 stained polyacrilamide gel demonstrating equal protein concentration in nuclear extracts obtained from wild-type (lane 1) and <i>pitx3</i> morphant (lane 2) embryos that were used in EMSA experiments shown in A. <b>D.</b> Electrophoretic mobility shift assays (EMSA) show formation of a DNA-protein complex when an oligonucleotide corresponding to the −44/−76 region of zebrafish <i>mip1</i> promoter and nuclear extracts from 48-hpf wild-type zebrafish embryos are used. Please note a presence of a specific slow migrating complex, which is formed by wild-type <i>mip1</i> probe and proteins extracted from nuclei of 48-hpf wild-type zebrafish embryos (lane 2), absence of this complex in lane 3 when the same nuclear extracts were combined with a mutant <i>mip1</i> probe where the pitx3-binding <i>bicoid</i> site GGATTA was replaced by AAATTA, and sharp reduction of this complex in lane 4 containing a combination of a wild-type <i>mip1</i> probe and nuclear extracts obtained from <i>pitx3</i> morphants (red arrow).</p