RT-PCR analysis of splice morpholino function at 26-28 hpf. (A) Changes in size and intensity of the amplification product between wild-type (wt) and MO-injected (MO) embryos indicates effectiveness of splice morpholinos. The dock1 wild-type band decreased in intensity when the embryos were injected with dock1 splice MO, although a 40 bp deletion was detectable by sequencing. dock5 splice 1 morpholino caused inclusion of 273 bp of intron. dock5 splice 2 morpholino caused a 75 bp deletion. crk morpholino caused excision of 578 bp. crkl morpholino mainly caused inclusion of 57 bp of intron, although a deletion of 472 bp was also detected by sequencing. All changes created premature stop codons. (B) Diagrammatic representation of the Dock and Crk protein structures and the truncations produced by the splice morpholinos. Sites recognised by morpholinos are marked with arrows. Affected exon boundaries are shown with red vertical lines. Dock proteins of the DOCK180 subfamily possess two CZH domains (green) and one or two C-terminal proline-rich PXLPXK motifs (*), which bind Crk. The adaptor proteins Crk and Crkl contain an SH2 domain (dark blue) and two SH3 domains (pale blue). The N-terminal SH3 domain binds to DOCK180 proteins.
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