Fig. 3. The s30 and sk8 mutations disrupt the foggy/spt5 locus. (A) The s30 mutation deletes spt5. The first and second lanes demonstrate that a 145 bp fragment of the 5' end of spt5 genomic DNA can not be amplified by PCR from s30 genomic DNA. The third and fourth lanes demonstrate a similar result for a 172 bp fragment of the 3' end of spt5 genomic DNA. Similar results were obtained for all regions of spt5 genomic DNA (data not shown). A control fragment (225 bp) can be amplified from all samples. (B) spt5 cDNA from sk8 mutants is missing 31 bp. PCR primers that flank a 310 bp fragment of wild-type spt5 cDNA amplify a 279 bp fragment from sk8 mutant cDNA. This smaller fragment represents the major splice isoform of spt5 in sk8 mutants at 24 hpf, although we have infrequently detected trace amounts of normally spliced cDNA that could represent a low level of maintained maternal mRNA or a low level of normally spliced mutant mRNA. (C) Comparison of wild-type and sk8 mutant spt5 cDNA. Labeled regions are proposed to encode discrete protein features (see E). The location of the missing nucleotides is indicated following nucleotide 520 in sk8 mutant cDNA. (D) Structure of spt5 genomic DNA indicates that the missing bases correspond to a 31 bp exon, flanked by a 88 bp intron and a 103 bp intron. In wild-type embryos, this region is spliced normally and the 31 bp exon is located between nucleotides 520 and 552 in spt5 cDNA. In sk8 mutants, incorrect splicing results in the omission of the exon and both introns from spt5 cDNA. sk8 genomic DNA contains a point mutation (boxed) in the first nucleotide of the intron following the 31 bp exon. (E) Predicted protein structure for wild-type Spt5 protein (1084 amino acids) and truncated sk8 mutant Spt5 protein (184 amino acids). The acidic region, KOW motifs, and hexapeptide repeats are indicated (Guo et al., 2000). The omission of an exon in sk8 mutant cDNA creates a frameshift that is predicted to result in the addition of 10 mis-sense amino acids after residue 174 before reaching a premature in-frame stop codon. Therefore, the sk8 mutant Spt5 protein would lack all KOW motifs and hexapeptide repeats.