s441 is a mutation in pard6γb. (A) Positional cloning of s441. s441 was linked to a region on Chromosome 19 between the SSLP markers z9059 and z6079. Using an additional SSLP marker (labeled CA-1) the position of the mutation was narrowed to a region flanked by two recombinants, each unique to one side. The red asterisk represents the location of pard6γb. pard6γb contains three exons (rectangles) and two introns (lines). The black asterisk represents the lesion. The red bar represents the region targeted by the splice-blocking morpholino. (B–E) Expression of pard6γb. (B) pard6γb mRNA is maternally deposited. (C) At 10 hpf, pard6γb appears to be expressed ubiquitously. (D, E) At 18 and 24 hpf, pard6γb appears to be expressed in all tissues and shows heightened expression in the neural tube (arrows). (F, G) Sequence analysis of wildtype versus s441 pard6γb cDNA revealed a T to A (gray highlight in panels H and I) transition 192 base pairs following the start of translation. (H) Schematic of the PB1, CRIB (abbreviated C), and PDZ domains and the predicted protein-protein interactions with the wildtype protein. The premature stop codon caused by the s441 mutation leads to a protein truncated at amino acid 63. Numbers shown represent amino acid position. (I) Injecting 2 ng of a splice morpholino designed against the first exon-intron boundary phenocopied the s441 mutation. The morphants showed defects in heart development (red arrow), failure of the brain ventricles to inflate (black arrow), and dorsal curvature.
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