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Fig. 3. The Bst mutation. (A) Map of the Rpl24 gene showing the
informative PCR, the 4 bp deletion within the intron 1 branchpoint, the first
codons of the open reading frame, and the premature stop codon in Bst
(red). (B) Sequence chromatograms comparing +/+ and Bst/+ PCR
products. The exon 2 splice acceptor (arrowheads) and 4 bp Bst
deletion (boxed) are indicated. (C) Allele-specific PCR assay used to
distinguish Bst and wild-type Rpl24 alleles. The deletion is
unique to the C57BLKS Bst/+ strain. (D) Bst causes abnormal
Rpl24 splicing with retention of intron 1. RT-PCR was performed using
the indicated primers. A common downstream primer (*) was
end-labeled with 32P using T4 DNA kinase. Product A originates from
correctly spliced Rpl24 transcripts, whereas A' and B indicate
inclusion of intron 1. The relatively low abundance of product A' is due
to PCR competition and nonsense-mediated decay. The low level of product B in
the wild-type lanes most probably reflects amplification from hnRNA. (E) Some
Bst transcripts are correctly spliced. RT-PCR was performed using
(Bst/+ x SPRET) F1 RNA and the indicated primers. The upstream
primer spans the exon 1-2 junction. Product C (388 bp) was end-labeled and
digested with AluI to distinguish between musculus (M, 365
bp) and spretus (S, 135 bp) alleles. The molar ratio of M and S
products is 1.27 for +/+ mice and 0.26 for Bst/+ littermates, giving
a normalized expression level for Bst of 0.21 (±0.02). (F)
Similar fluorometric assay performed using a HEX-labeled reverse primer and
ABI sequencer following AluI digestion. The Bst expression
level is 0.25 (±0.03) relative to wild-type musculus Rpl24.
(G) Sequence of correctly spliced Rpl24 cDNAs amplified from F1 mice,
showing the informative AluI site in exon 4 (overlined, sense
strand). The normalized ratio of Bst to wild-type peak areas is 0.22
(±0.01).
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