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Fig. 1. Cloning and characterization of a zebrafish tfap2b. (A)
Phylogenetic tree of AP2 genes shows zebrafish tfap2b more closely
related to other vertebrate AP2b genes than to other zebrafish AP2 genes.
Putative protein sequences of AP2 genes were aligned using ClustalX, edited by
eye and used to generate a maximum likelihood tree by quartet puzzling using
the PUZZLE program. Values at the nodes are likelihood values, showing support
for that node out of 1000; branch lengths represent sequence divergence. (B)
tfap2b contains the highly conserved transactivation (red),
DNA-binding (blue) and heterodimerization domains (yellow) found in all AP2
proteins. Exon-intron boundaries are identical in tfap2a and
tfap2b, and both have three alternative first exons (1a-c). A
morpholino oligonucleotide directed against the splice acceptor site before
exon 5 (ap2b-x5.1-'ap2bMO') should create a larger, unspliced product. (C)
Splicing defects in tfap2b transcripts following ap2bMO injection.
tfap2b was amplified from pools of ap2bMO-injected and control
uninjected animals at 24 hpf using primers tfap2bf and tfap2br directed to
exons 4 and 6, respectively. Uninjected animals showed PCR products of
210 bp in size in comparison with ap2bMO-injected animals in which one
larger product (424 bp) was observed because of aberrant splicing. (D)
PCR amplification of tfap2b from RNA isolated from different stages
of zebrafish gastrulation fails to detect expression before 10 hpf.
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