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).