Fig. S4

Fig. S4 The slo phenotype is caused by mutations in hsp90a. (A) Schematic of LG20 around the slo^u45 mutation. slo^u45 was mapped to a region between SSLP markers z7568 and z9959 (SSLP primers sequences from MGH: http://zebrafish.mgh.harvard.edu/zebrafish/index.htm). Genes/ESTs located between these markers were identified and enzyme-scorable SNPs within these genes were found by sequencing genomic DNA from mutants and siblings using primers designed from public genome sequence (www.ensembl.org). These SNPs were tested for linkage: hsp90a mapped very close to the slo locus. (B-D) Sequence traces from embryos of genotypes as shown above each trace illustrating mutations (asterisks). Both hsp90a and hsp90a2 were sequenced from genomic DNA for all genotypes; primers were designed using the LIMSTILL program (http://limstill.niob.knaw.nl/); all primer sequences available on request. (E) Diagram of the genomic region around the slo locus showing BACs that all rescued the phenotype of slo^u45 mutants. The shaded area indicates the genomic region responsible for the phenotype found through use of overlapping BACs. The three genes within are: hsp90a2, orange; hsp90a, purple; si:dkey-241I7.6, blue. Black boxes are neighbouring genes. (F-H) Polarised light micrographs of trunk muscle from BAC4 rescue experiments. Panels show normally moving (F), paralysed (H) and partially moving (G) embryos. Striated birefringence, where present, is shown in insets. Arrowheads point to birefringent pigment cells. Scale bars: ruler divisions in E, 20 kb; 100 μm in F-H, 5 μm in insets.