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Fig. 2. Identification of an internal deletion in the ntn mutant
cct3 gene and expression of the cct3 mRNA in wild-type
embryos during development. (A) A 143 bp internal deletion in the
cct3 transcript. The zebrafish CCT 
subunit is schematically
shown above; the equatorial, apical and intermediate regions are indicated in
boxes. The nucleotide sequences of a putative ATPase motif in the wild-type
(wt) and mutant (ntn) cct3 transcripts are shown below. The
143 bp deletion corresponding to the nucleotide residues 1205-1347 of the
zebrafish cct3 transcript is in the coding sequence for a putative
ATPase motif of CCT. The deletion causes frame-shift and aberrant
termination of translation. (B) RT-PCR analysis of the cct3 gene
transcripts from pools of 
1190 wild-type embryos of the AB strain (AB),
650 wild-type siblings (wt) and 200 mutant (ntn) embryos
with (+) or without (–) a reverse transcriptase using primers flanking
the 143 bp deletion. Lane M shows 100 bp DNA ladder as size markers. Black and
white arrowheads on the right indicate the 221 bp and 78 bp PCR products
representing intact and deleted transcripts, respectively. (C) PCR analysis of
genomic DNA from pools of 100 wild-type embryos of AB strain (AB),
300 wild-type siblings (wt), 100 mutant (ntn) and three
individual mutant (N151, N150 and S20) embryos using primers flanking the 143
bp deletion. Lane M shows 100 bp DNA ladder as size markers. Black and white
arrowheads on the right indicate the 221 bp and 78 bp PCR products
representing the intact and deleted cct3 genes, respectively. (D)
Whole-mount in situ hybridization analysis of cct3 mRNA at 0.2 hpf
(one-cell stage), 6 hpf (shield stage), 9 hpf (90%-epiboly stage), 12 hpf, 24
hpf and 36 hpf. A section through the retina was shown for embryos at 36 hpf.
mhb, midbrain-hindbrain boundary; ov, optic vesicle. Scale bars: 100
µm.
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