Fig. 5

Molecular analysis of the paics^hi2688 locus. (A,B) The approximate position of the proviral insertion and RT-PCR primers mapped onto a schematic of the mutated gene (www.ensembl.org/Danio_rerio/). (A) paics^LONG is encoded by at least 15 exons, and all of the conserved enzymatic domains of the Paics protein are found in the sequence encoded by exons 7-15. The proviral insert in paics mutants is located in exon 7 of paics^LONG. (B) paics^SHORT is composed of the latter nine exons of paics^LONG, in addition to a tenth noncoding first exon. The proviral insert in paics mutants is located in exon 2 of paics^SHORT. (C) RT-PCR using primer sets that amplify the region between exons 4 and 9 of paics^LONG (3F/4R), and exons 1 and 4 of paics^SHORT (UTR/4R) indicates that both transcripts are expressed in wild-type embryos. Amplification of the region surrounding the site of proviral insertion (3F/4R) in paics^LONG indicates that the proviral insert leads to an alteration in mRNA splicing in the paics^LONG transcript. (D) RT-PCR using primer sets that assay for the presence of the proviral insert in paics^SHORT (UTR/V) indicates that the viral insert is present in paics^SHORT transcripts. RT-PCR amplification using paics^SHORT-specific exon 2 primers located downstream of the site of proviral insertion (7F/4R and 7-8F/4R) indicates that the virus sequence in paics^SHORT does not lead to detectable changes in transcript levels. WT, wild-type AB; SIB, wild-type sibling mixture (+/+ and +/-); M, homozygous mutant.