Figure 8

Mutations that insert AG dinucleotides in a large AGEZ impair gene expression. (a) Rat α-tropomyosin (TM) minigene constructs and sequence between exon 3 branch point (in bold) and 3' splice site CAG. The underlined Ts are positions where mutagenesis to A created a new AG dinucleotide in mutants 3a and b. (b) In vitro spliced [³²P]labelled RNA was analyzed by phosphorimaging after denaturing PAGE. The fully spliced 134 product is indicated by the black diamonds, and the intron lariat resulting from excision of the intron between exons 1 and 3 by the open circles. The sizes of these two bands varied, consistent with use of the first AG downstream of the dBP for splicing of exon 3. (c) Reverse transcriptase polymerase chain reaction analysis of transiently expressed constructs in HeLa cells. No bands corresponding to skipping or inclusion of exon 3 using either AG dinucleotide were observed in mutants 3a and 3b. The wild type construct shows a band corresponding to spliced exons 1-3-4. WT, wild type;