Introns in the 3'-untranslated region can inhibit chimeric CAT and beta-galactosidase gene expression.
The expression of a cyc::cat [cytochrome c/chloramphenicol acetyltransferase (CAT)] chimeric gene was stimulated 100-fold by the inclusion of a cyc intron in the 5'-untranslated region. In contrast, a single intron in the 3'-untranslated region was at best only slightly stimulatory, and surprisingly, inhibited expression of cat when an intron was also included in the 5'-untranslated region. This inhibition was independent of the identity of the downstream intron, occurring when either the simian virus 40 (SV40) small t intron or a cyc intron was located downstream from the cat coding region. Analysis of CAT mRNA levels, using a riboprobe spanning the 5' end of the CAT message, revealed that the stimulatory effect of a 5'-noncoding region intron were manifest at both the protein and RNA levels, whereas the inhibitory effects of 3'-noncoding region introns were detectable only at the protein level. The effects of intron position on chimeric gene expression were observed in both primate and rodent cell lines and also when the beta-galactosidase coding region was substituted for that of cat. Therefore, the common placement of an intron in the 3'-noncoding region is not the most beneficial to the expression of cyc chimeric genes. The position of introns within a transcriptional unit is a major factor to be considered when optimizing the efficiency of animal cell expression vectors.
Evans
M J
MJ
Department of Molecular Biology, Northwestern University Medical School, Chicago, IL 60611.
Scarpulla
R C
RC
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D004274:DNA, Recombinant / Q000378:metabolism; D005696:Galactosidases / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007438:Introns; D051379:Mice; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics*
D004274:DNA, Recombinant; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase; D005696:Galactosidases; D001616:beta-Galactosidase