Verification that T27B1.2 is pat-9. A) The sequence of T27B1.2 amplified from pat-9 mutant animals shows a G→A transversion mutation in the splicing donor site of the third exon/intron junction. DNA sequence alignment of the amplified pat-9 cDNA from N2 and pat-9 animals compared with the predicted T27b1.2 gene model from WormBase shows six additional nucleotides in exon 3 and predicts a truncated protein in pat-9 animals. The single mutated base change in pat-9 is highlighted in red and the in-frame stop codon is boxed in red. The remaining sequence alignments are identical (not shown). B) The point mutation at the splicing donor site predicts the subsequent incorporation of intron 3, disrupting the third Zn finger and generating a premature in-frame stop codon at amino acid 164. C) RT-PCR from RNA extracted from N2 and pat-9 (P) worms was performed using oligonucleotide primers specific for exons 1, 2, 3, or 4 and shows that intron 3 is incorporated into the mRNA of pat-9 animals (red arrow) but not N2 animals. Black arrows indicate properly spliced products. D) Analysis of the predicted PAT-9 amino acid sequence indicates that pat-9 animals produce a truncated protein of 163 amino acids.