Expression of intron-containing GUS constructs is reduced due to activation of a cryptic 5 ' splice site

An intron-containing beta -glucuronidase (GUS) gene has been used widely in promoter analyses and as a plant transformation marker. Maximal plant gene expression requires accurate and efficient removal of the intron from the expressed pre-mRNA transcripts by splicing. Detailed analysis of splicing o f potato ST-LS1 and pea legumin introns from GUS constructs revealed the ac tivation of a cryptic 5 ' splice site in the GUS coding sequence 4 nt upstr eam from the authentic intron 5 ' splice site. About 40% of transcripts uti lised the cryptic 5 ' splice site in tobacco protoplasts, reducing the tran slational potential of expressed pre-mRNA. The same cryptic splicing event was evident in transgenic tobacco leaves but at reduced levels. Mutations t hat removed the cryptic 5 ' splice site are associated with a two-fold enha ncement in GUS activity in tobacco protoplasts, highlighting the need for c areful examination of introns and their sites of insertion into gene constr ucts to minimise variability in gene activity and maximise gene expression.