?(Fig.8), 8), competes very efficiently with the authentic site when the G-rich sequence is placed downstream. these sites, respectively. Also, sequences that enhance cryptic splice site use must be absent from this large exon. Intro How vertebrate exons are properly recognized from amongst the sequence complexity of a pre-mRNA has been the subject of much attention over the past 20 years. It is obvious that multiple guidelines can contribute to exon acknowledgement, including the sequence of the splice junctions bordering the exon, internal exon sequences, sequences in the surrounding introns and exon size. Most vertebrate exons are between 50 and 400 nt long, the average becoming 137 nt; 1% of internal exons are 400 nt (1,2). This observation, along with practical studies of small and large exons, has suggested that a practical exon size limit is present when an exon is definitely surrounded by large (greater than 500 nt) introns Iloperidone (observe for example 3C6). Relationships among factors bound in the splice sites bordering an exon is an important first step in exon acknowledgement and these relationships are Iloperidone ideal when the exon is at least 50 nt and less than 500 nt (2,6,7). Consequently, exons that fall outside these size constraints are more likely to be option exons or to have additional features that enhance their acknowledgement from the splice machinery. Indeed, naturally happening small exons are often on the other hand processed such that the exon is included or excluded in different cell types and sequences within the exon and surrounding introns contribute to this rules (8C11). In several instances, when the splice sites were improved to better match the consensus sequences the small exons became constitutively spliced (4,11,12). Only a few abnormally large internal exons have been examined to day, all of which are on the other hand processed; these include the 3500, 1090 and 800 nt exons of the breast malignancy 1 (13), caldesmon (14) and neural cell adhesion molecule (NCAM) genes (15), respectively. Improving the 5 splice site of the NCAM option exon resulted in constitutive splicing of this exon (16) and sequences in the upstream exon were shown to contribute to its controlled splicing (17). However, additional sequences in and surrounding these or additional large exons have not been examined for splice enhancing activity. One might presume that exons 400C500 nt, whether they are on the other hand or constitutively spliced, would also require specific polymerase for 25 cycles of 94C for 1 min, 58C for 1 min and 72C for 2 min. Southern blot analysis was performed by standard methods after separating 1C10 l of RTCPCR products on a 1.2% agarose gel. Filters were probed having a labeled em Pst /em IC em Ppu /em MI DdCpIgR cDNA fragment and were visualized having a phosphorimager. S1 nuclease analysis The S1 probe used to differentiate the DdCpIgR transcripts was derived from a DdCpIgR cDNA clone; the sequence between the Dd Ex lover3 PCR primer and the em Ppu /em MI site in pIgR exon 4 was subcloned into pGEM4. The probe was 3-end-labeled at an em Msp /em I site using Klenow and [-32P]dCTP and extends to the em Eco /em RI site in the vector (Fig. ?(Fig.3).3). A total of 100 g RNA, a combination of 50C100 g specific RNA and carrier RNA, was hyridized over night at 50C with the labeled probe. Digestion was at 37C for 30 min with 60 U S1 nuclease (Pharmacia) per reaction. The safeguarded fragments were separated on a 6% acrylamide, 7 M urea gel and quantitated by phosphorimager analysis. Multiple safeguarded fragments are observed with the cryptically spliced RNAs due Iloperidone to fortuitous partial homology between the probe and the Dd exon 4 sequences to which the cryptic site is definitely spliced; by changing the S1 digestion temperature all bands could be combined into one. Open in a separate window Number 3 S1 nuclease PDGFC quantitation of DdCpIgR spliced products. (A).