Fig. 1

Mutant sqt RNAs are expressed and localized in MZsqt mutant zebrafish embryos. (A,B) Whole-mount in situ hybridization to detect localization of maternal sqt RNA (arrowheads) in 8-cell stage wild-type (WT) (A) and MZsqt^cz35 mutant (B) embryos. (C) The genotype of wild-type (A) and MZsqt^cz35 (B) embryos was confirmed by PCR to detect either wild-type (primer pair 1) or mutant sqt (primer pair 2) alleles. (D) Quantitative PCR to detect sqt RNA shows that maternal sqt transcript levels in MZsqt mutants are similar to those of wild-type embryos at the 1-cell and 4-cell stages, and that reduced sqt transcript levels are observed at gastrula stages in the mutant. Error bars indicate s.d. between three independent experiments. (E) Schematic of constructs to express lacZ, wild-type sqt (WT Sqt), sqt^cz35 (Sqt^cz35), sqt with a terminator codon in exon 1 (Sqt^STOP), a 52 truncation of sqt (T-Sqt), and FLAG epitope-tagged wild-type sqt (FLAG WT Sqt) and sqt^STOP (FLAG Sqt^STOP) in rabbit reticulocyte lysates (as shown in F). sqt coding sequences are in cyan (exons are indicated as E1-3), with the sqt^cz35 insertion in yellow. Black line indicates globin 32UTR sequences and the blue line indicates the sqt UTRs. Red octagons indicate the position of the terminator codon in sqt^STOP and FLAG sqt^STOP and black flags mark the position of the FLAG epitope tags. SS, signal sequence. (F) In vitro translation to express Sqt proteins from the constructs described in E showing the expected 44 kDa wild-type Sqt protein, a C-terminus truncated 17 kDa Sqt^cz35 peptide, and that both sqt^STOP and T-sqt produce the predicted 39 kDa protein from Met35. Scale bar: 100 μm.