Sequence comparison and branchpoint analysis of introns with tissue-specific adjacent alternative 3′ splice sites. (A) WebLogo sequence comparison of regions upstream of a randomly selected set of 3′ splice sites. Upstream frequencies were measured from the distal splice site to 40 nt upstream. A logo displays the frequencies of bases at each position as the relative heights of letters, along with the degree of sequence conservation as the total height of a stack of letters, measured in bits of information. (B–D) WebLogo sequence comparisons of regions upstream of tissue-specific alternative 3′ splice sites that are 6 nt, 9 nt, and 12 nt apart. (E) WebLogo sequence comparison of tissue-specific alternative 3′ splice sites 6 nt apart in which the proximal AG dinucleotide is used <10% in the germline. Note the similarity of this sequence logo to that in which the dominant 3′ splice site in the germline is the proximal AG dinucleotide (Fig. 7B). (F) WebLogo sequence comparison of the 25 most used AG dinucleotide proximal 3′ splice sites in glp-4(bn2ts) when found 6 nt upstream of an intron-terminating AG dinucleotide. Note the signature of TTTCAG at the proximal 3′ splice site that is lacking in the tissue-specific proximal 3′ splice site (Fig. 7B). (G) Tissue-specific alternative 3′ splice site usage in the cdk-12 gene. (H) Orientation of divergent primers used to map branchpoints within an intron. (I) Lariat structure with alternative 3′ splice sites, the convergent orientation of the primers within the structure, and the branchpoint through which reverse transcriptase passes. Reverse transcription was first performed using the A1 primer, amplified with A1/B1 primers, purified using a Qiagen PCR clean-up kit, amplified a second time using the A2/B2 primers, and gel-purified. (J) Terminal 27 nt of the cdk-12 intron containing the tissue-specific alternative 3′ splice site with the proximal and distal 3′ splice sites and the location of the only branchpoint identified in our analysis. Also shown are the distances from this branchpoint to the proximal and distal 3′ splice sites.