MAIZE SHRUNKEN-1 INTRON AND EXON REGIONS INCREASE GENE-EXPRESSION IN MAIZE PROTOPLASTS

A DNA cassette containing the first intron of the maize Shrunken-1 (Sh 1) and adjacent exon sequences stimulates reporter gene expression in maize protoplasts up to 100-fold. Because this highly active cassette is being used to elevate gene expression in numerous genetic engineeri ng experiments and because of the paucity of information concerning th e mechanism(s) governing increased expression, we examined how placeme nt and orientation of this cassette, intron sequence and length, and i nteractions of intron and exon sequences affect expression levels. The data reveal two significant features: (1) substantial alterations wit hin the intron portion of the cassette do not affect its stimulatory e ffect and (2) the stimulation appears to occur at more than one level of gene expression. Specifically, we show that the magnitude of intron -mediated stimulation is not increased by the tandem addition of a com plete cassette to a construct already containing the Sh1 first intron cassette nor is stimulation affected by an 87% increase or a 63% decre ase in the size of the intron. Placement of an additional cassette wit hin a Sh1 first intron cassette does not significantly alter the stimu latory effect; however, the orientation of sequences is important, sin ce placement of an inverted cassette within another cassette abolishes the stimulatory effect. A significant finding is that the exon portio n of the Sh1 intron 1 cassette boosts gene expression approximately 7- fold in the absence of the intron, while exon sequences from the alcoh ol dehydrogenase-1 (Adh1) gene inhibit gene expression. Placement of A dh1 exon sequences adjacent to the Sh1 first intron, however, elevates gene expression above the level observed with the Sh1 first intron ca ssette. These mixing experiments strongly suggest that the exon and in tron components of the Sh1 first intron cassette act at different step s in gene expression.