Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe

Abstract

Insertion of a 36-base-pair (bp) synthetic oligonucleotide comprising the sequence 5'-GTAGGT(19N)CTAAT (4N)AG-3' into several different positions within the coding region of the naturally intronless ura4 gene of Schizosaccharomyces pombe leads to an efficiently spliced gene producing a functional product. This suggests that the proper signals within an intron are sufficient to initiate and complete a splicing event independent of the location of the intron in the gene. Point mutations in the 5' junction (5'-GTAGGT-3') and in the putative branch sequence (5'-CTAAT-3') affect splicing efficiency significantly. A G-to-A transition at the first nucleotide at the 5' splice junction (5'-ATAGGT-3') abolishes the use of the authentic splice junction and leads to the increased use of an alternative splice site. No functional product is produced from this transcript. An A-to-G transition of the second A in the putative branch sequence (5'-CTAGT-3') lowers the splicing efficiency drastically, but still results in a functional gene product. Furthermore, extension of the 36-bp intron to introns more than 180 bp in size abolishes splicing, suggesting that the splicing apparatus might be restricted to very short introns. We discuss the possibility that S. pombe introns represent a simple type of eucaryotic intron.