Figure 2. Deletion of internal intron 1 sequences affects splicing of tva receptor gene.

(A) Schematic diagram of intron 1 in tva pre-mRNA showing the 5′ splice site, 3′ splice site and the branch point sequence (corresponding bases are indicated by dots). The adenosine residue which is required for the first cleavage-ligation step of the splicing reaction¹⁸ is marked by an arrow. (B) Schematic diagram of RT-PCR strategy. The use of PCR primers TVA3 and TVA4 generated easily discernible whole cDNA fragments that were amplified from the longer and shorter tva forms, respectively, as well as the longer and shorter tva forms with intron 1 retention. Sizes of diagnostic PCR products are indicated. Exons are drawn as boxes, retained introns are shown as black lines and spliced introns as diagonal lines. The vertical white bar indicates the position of the intronic deletion. (C) RT-PCR of RNA isolated from DF-I cells and samples from lives of defined origin. Lane 1–16 indicated the RT-PCR products from DF-I cells and the tva^s/s, tva^s/r3, tva^s/r4, tva^s/r5, tva^s/r6, tva^r3/r3, tva^r4/r4, tva^r5/r5, tva^r6/r6, tva^r3/r4, tva^r3/r5, tva^r3/r6, tva^r4/r5, tva^r4/r6 and tva^r5/r6 samples from lives, respectively. Positions of full-length and intron 1 retention PCR products are indicated on the right, and sizes of diagnostic PCR products are indicated on the left. Spliced products from the full-length longer and shorter tva forms (Full-length-L and Full-length-S) migrated slightly faster than the corresponding RNAs of the mutants (Full-length-L-Int1 and Full-length-S-Int1). The gels have been run under the same experimental conditions, and the cropped gels are used. The full-length gel images are presented in the supplementary information. (D) Separate sequence analysis of PCR products revealed normal splicing of longer and shorter tva forms of tva^s/s homozygotes, while splicing of longer and shorter tva forms of tva^r5/r5 and tva^r6/r6 homozygotes containing intron 1 with the corresponding deletion mutation, however, the tva^s/r3, tva^s/r4, tva^s/r5 and tva^s/r6 heterozygotes generate both the normal splicing of shorter tva forms and abnormal splicing of longer and shorter tva forms. Stars represent premature TGA stop codons identified in the alternative transcript.