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. 2001 Feb 15;29(4):886–894. doi: 10.1093/nar/29.4.886

Figure 4.

Figure 4

Role of the splice sites of the constitutive introns in the splicing of the 116 nt alternative intron. (A) Diagram of the TPO cDNA+IVS2 Δss and TPO cDNA+IVS3 Δss constructs in which the 5′ and the 3′ splice sites of introns 2 and 3 were deleted, respectively. (B) Analysis of pre-mRNA splicing of TPO cDNA+IVS2 Δss and TPO cDNA+IVS3 Δss constructs in Hep3B cells. TPO cDNA+IVS2 Δss transfections showed that the intron 2 was retained (upper band). The faint band observed below, after cloning and sequencing, was shown to present simultaneous retention of intron 2 and splicing of the 116 nt sequence (arrow). The proportion of the 116 nt sequence splicing was 2% of the total amount of PCR products (upper band + lower band). In TPO cDNA+IVS3, the splicing pattern was more complex with a main splicing product corresponding to the TPO cDNA retaining IVS3 (upper band). The additional bands present arise from the activation of cryptic splice sites. In the first lane 1 kb DNA molecular weight marker was loaded. RT +/–, presence/absence of MMLV enzyme in the RT mixture.