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 (Sh1) 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 engineering experiments and because of the paucity of information concerning the mechanism(s) governing increased expression, we examined how placement and orientation of this cassette, intron sequence and length, and interactions of intron and exon sequences affect expression levels. The data reveal two significant features: (1) substantial alterations within the intron portion of the cassette do not affect its stimulatory effect 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 complete cassette to a construct already containing the Sh1 first intron cassette nor is stimulation affected by an 87% increase or a 63% decrease in the size of the intron. Placement of an additional cassette within a Sh1 first intron cassette does not significantly alter the stimulatory effect; however, the orientation of sequences is important, since placement of an inverted cassette within another cassette abolishes the stimulatory effect. A significant finding is that the exon portion of the Sh1 intron 1 cassette boosts gene expression approximately 7-fold in the absence of the intron, while exon sequences from the alcohol dehydrogenase-1 (Adh1) gene inhibit gene expression. Placement of Adh1 exon sequences adjacent to the Sh1 first intron, however, elevates gene expression above the level observed with the Sh1 first intron cassette. These mixing experiments strongly suggest that the exon and intron components of the Sh1 first intron cassette act at different steps in gene expression.