| Summary: | 碩士 === 國立陽明大學 === 遺傳學研究所 === 92 === Human phenylalanine hydroxylase (PAH) is a liver-specific enzyme, which involved in the rate-limiting step of phenylalanine catabolism. Deficiency of PAH activity results in phenylketonuria, an autosomal recessive disorder.
Study has shown that PAH exon 11 skipping occurs in a fraction of PAH mRNA when illegitimate PAH RNA from lymphoblasts was analyzed by RT-PCR. And, exon 11 is totally skipped when the exon 11 mutation, c.1197A>T, occurs. By S1 nuclease mapping, we confirmed that exon 11 skipping indeed occurs in RNA isolated from liver cells. To study the mechanism of exon 11 skipping, we compared sequence of splice site to that of consensus, and found that the splicing acceptor site of intron 10 has poor pyrimidine content. When we modified such site in the minigene to become optimal by site-directed mutagenesis, we found that exon 11 recognition improved greatly. Thus, the poor exon 11 recognition in the human PAH gene may be mainly due to the sub-optimal splice acceptor site in intron 10.
Because of weak splice site, one would predict the existence of splicing enhancer on exon 11 and/or intron 10 to facilitate exon 11 recognition. Two strategies were used to search for such sequences: (1) to correlate between SR protein score matrices and PAH exon 11 splicing; (2) to evaluate naturally occurring missense, nonsense, frameshift and silent mutations on PAH exon 11 recognition. The nucleotide substitutions of predicted SR protein binding site or sequences correspond to the nature mutations were introduced into minigene, RNA splicing pattern was then analyzed by RT-PCR. Our results show the lack of the predictive capacity of SR protein score matrices on the PAH exon 11 recognition. On the other hand, several nature mutations induce changes in the splicing pattern with either positive or negative effect on exon 11 recognition. The results suggest that human PAH exon 11 recognition may act through multiple sequence motifs.
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