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Fig. 5. ryr mutant embryos have a mutation in ryr1b due to a
DNA insertion that leads to aberrant splicing of ryr1b mRNA.
(A) Meiotic mapping placed the ryr locus between z737 and
z8343 in chromosome 18. No recombination was found with a polymorphic marker
in the ryr1b gene (0/1110). (B) ryr mutants contained
a 32-bp insertion (red) in ryr1b mRNA that added a nonsense codon
5' to the transmembrane domains. (C) Reverse transcriptase
(RT)-PCR using primers #1 and #2 (see B) amplified both a longer mutant band
(169 bp) and a shorter wild-type band (137 bp) from a group of wild-type
siblings (lane 1), whereas the mutant band was predominant in ryr
mutants (lane 2). The wild-type band can be seen faintly in the mutant lane.
Only the shorter band was amplified from wild-type AB embryos (lane 3).
(D) ryr mutants carry a 4046-bp insertion (red) in the intron
between exons 48 and 49 of ryr1b that includes the additional 32-bp
sequence found in the mutant cDNA. (E) Injection of an antisense
morpholino (MO1, see D) into recently fertilized wild-type embryos effectively
decreases normal ryr1b mRNA at 24, 36 and 48 hpf, whereas control MO
does not. A fragment of normal ryr1b mRNA was examined by RT-PCR with
primers #1 and #2 (see D). (F) Genomic PCR and RT-PCR with primers #1
and #2 using individual embryos as a template. Genotypes, as shown, were
identified by genomic PCR. Notice that, in mutants, the intensity of the
wild-type (short) band amplified by RT-PCR was comparable to that of the
mutant (long) band at 24 hpf (lane 3) but was diminished at 48 hpf (lane
6).
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