Intron-mediated enhancement of heterologous gene expression in maize

Abstract

Chimeric genes containing the coding sequence for bacterial chloramphenicol acetyl transferase (CAT) have been introduced by electroporation into maize protoplasts (Black Mexican Sweet) and transient expression monitored by enzyme assays. Levels of CAT expression were enhanced 12-fold and 20-fold respectively by the inclusion of maize alcohol dehydrogenase-1 introns 2 and 6 in the chimeric construct. This enhancement was seen when the intron was placed within the 5' translated region but not when it was located upstream of the promoter or within the 3' untranslated region. Deletion of exon sequences adjacent to intron 2 abolished its ability to mediate enhancement of CAT gene expression. Northern analysis of protoplasts electroporated with intron constructs revealed elevated levels of CAT mRNA. However, this elevation was insufficient to account for the increased enzyme activity. One explanation of these results is that splicing affects both the quantity and quality of mRNA.

Splicing of the Maize Sh1 First Intron Is Essential for Enhancement of Gene Expression, and a T-Rich Motif Increases Expression without Affecting Splicing

“…Not all plant introns enhance gene expression. Three dicot introns do not enhance gene expression in transgenic plants (Chee et al, 1986;Kuhlemeier et al, 1988;Vancanneyt et al, 1990) and two maize introns are nonenhancing in transient assays: Adh1 intron 9 (Mascarenhas et al, 1990) and Hsp81 intron 1 (Sinibaldi et al, 1992).…”

“…The observation that some introns stimulate gene expression was first made in animal systems and extended to plants when Callis et al (1987) demonstrated that the maize (Zea mays) Adh1 first intron increased expression of several genes. Other maize introns that have been shown to increase expression include the Bz1 intron (Callis et al, 1987), Hsp82 first intron (Silva et al, 1988), Sh1 first intron (Vasil et al, 1989), Adh1 introns 2 and 6 (Mascarenhas et al, 1990), actin third intron (Luehrsen and Walbot, 1991), GapA1 first intron (Donath et al, 1995), Ubi1 first intron (Vain et al, 1996), and the RpoT fourth intron (Bourdon et al, 2001).Plant introns that stimulate expression have been documented in petunia (Petunia hybrida; Dean et al, 1989;Vain et al, 1996) Leon et al, 1991;Fu et al, 1995aFu et al, , 1995b, Arabidopsis (Rose and Last, 1997; ChaubetGigot et al, 2001), soybean (Glycine max; Kato et al, 1998), and tobacco (Nicotiana tabacum; Plesse et al, 2001). The approximately 2-to 10-fold range of intron-mediated enhancement usually seen in dicots is much less than increases observed in monocots, which can be more than 100-fold (for review, see Simpson and Filipowicz, 1996).…”

“…Not all plant introns enhance gene expression. Three dicot introns do not enhance gene expression in transgenic plants (Chee et al, 1986;Kuhlemeier et al, 1988;Vancanneyt et al, 1990) and two maize introns are nonenhancing in transient assays: Adh1 intron 9 (Mascarenhas et al, 1990) and Hsp81 intron 1 (Sinibaldi et al, 1992).Intron-mediated enhancement of gene expression has been studied in plants for about 15 years, and although the underlying mechanisms have not been identified, several features are known. Positioning of the intron in the natural orientation within the 5Ј portion of the transcription unit is typically required for enhancement (Callis et al, 1987;Mascarenhas et al, 1990;Maas et al, 1991;Clancy et al, 1994;Snowden et al, 1996;Bourdon et al, 2001;Chaubet-Gigot et al, 2001), establishing that introns do not function as traditional transcriptional enhancers.…”

“…The observation that some introns stimulate gene expression was first made in animal systems and extended to plants when Callis et al (1987) demonstrated that the maize (Zea mays) Adh1 first intron increased expression of several genes. Other maize introns that have been shown to increase expression include the Bz1 intron (Callis et al, 1987), Hsp82 first intron (Silva et al, 1988), Sh1 first intron (Vasil et al, 1989), Adh1 introns 2 and 6 (Mascarenhas et al, 1990), actin third intron (Luehrsen and Walbot, 1991), GapA1 first intron (Donath et al, 1995), Ubi1 first intron (Vain et al, 1996), and the RpoT fourth intron (Bourdon et al, 2001).…”

“…Positioning of the intron in the natural orientation within the 5Ј portion of the transcription unit is typically required for enhancement (Callis et al, 1987;Mascarenhas et al, 1990;Maas et al, 1991;Clancy et al, 1994;Snowden et al, 1996;Bourdon et al, 2001;Chaubet-Gigot et al, 2001), establishing that introns do not function as traditional transcriptional enhancers. Length (Sinibaldi and Mettler, 1992) and composition of flanking sequences influence the degree of stimulation (Luehrsen and Walbot, 1991;Maas et al, 1991;Clancy et al, 1994).…”