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.