Afm. Ibrahim et al., Expression of intron-containing GUS constructs is reduced due to activation of a cryptic 5 ' splice site, MOL GENET G, 265(3), 2001, pp. 455-460
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.