LARGE EXON SIZE DOES NOT LIMIT SPLICING IN-VIVO

Exon sizes in vertebrate genes are, with a few exceptions, limited to less than 300 bases. It has been proposed that this limitation may der ive from the exon definition model of splice site recognition. In this model, a downstream donor site enhances splicing at the upstream acce ptor site of the same exon. This enhancement may require contact betwe en factors bound to each end of the exon; an exon size limitation woul d promote such contact. To test the idea that proximity was required f ar exon definition, we inserted random DNA. fragments from Escherichia coli into a central exon in a three-exon dihydrofolate reductase mini gene and tested whether the expanded exons were efficiently spliced. D NA from a plasmid library of expanded minigenes was used to transfect a CHO cell deletion mutant lacking the dhfr locus. PCR analysis of DNA isolated from the pooled stable cotransfectant populations displayed a range of DNA insert sizes from 50 to 1,500 nucleotides. A parallel a nalysis of the RNA from this population by reverse transcription follo wed by PCR showed a similar size distribution. Central exons as large as 1,400 bases could be spliced into mRNA. We also tested individual p lasmid clones containing exon inserts of defined sizes. The largest ex on included in mRNA was 1,200 bases in length, well above the 300-base limit implied by the sun-ey of naturally occurring exons. We conclude that a limitation in exon size is not part of the exon definition mec hanism.