Role of the 3' untranslated region of baculovirus p10 mRNA in high-level expression of foreign genes.
The p10 gene of Autographa californica nucleopolyhedrovirus has two putative AATAAA polyadenylation signals. The downstream signal is used predominantly, as was determined by analysing 3' cDNA ends. This downstream motif is followed by a GT-rich sequence, known to be important for efficient polyadenylation in mammalian systems. To analyse the importance of polyadenylation for p10 gene expression, recombinant viruses with altered 3' untranslated regions (UTRs) were tested using chloramphenicol acetyltransferase (CAT) as a reporter. Surprisingly, after inactivation of the downstream AATAAA motif, CAT expression remained at the same high level as observed with a wild-type 3' UTR. Polyadenylation occurred 24-28 nucleotides further downstream, probably due to an ATTAAA sequence motif. Replacing the p 10 3' UTR with the SV40 early terminator sequence as part of an hsp70-lacZ-SV40 gene cassette, which is commonly used in baculovirus expression vectors, resulted in a reduction in reporter gene expression. Polyadenylation occurred far more efficiently in the original p10 3' UTR than in the SV40 terminator sequence, as was shown by testing the SV40 terminator separately. These results indicate that in order to obtain high levels of foreign gene expression, vectors that provide a wild-type p10 3' UTR are to be preferred over those containing the hsp70-lacZ-SV40 gene cassette. Comparison of the p10 genes of various baculoviruses showed the presence of at least one AATAAA or ATTAAA motif in combination with a GT-rich sequence in the 3' UTR, suggesting an evolutionary conservation of these two elements, thereby maintaining the high level of p10 gene expression.
van Oers
Monique M
MM
Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands1.
Vlak
Just M
JM
Department of Virology, Wageningen Agricultural University, The Netherlands2.
Voorma
Harry O
HO
Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands1.
Thomas
Adri A M
AAM
Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands1.
D020413:3' Untranslated Regions / Q000502:physiology*; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005822:Genetic Vectors; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011061:Poly A; D012333:RNA, Messenger / Q000502:physiology; D012367:RNA, Viral / Q000502:physiology*; D014764:Viral Proteins / Q000235:genetics*
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D011061:Poly A; C078072:p10 protein, Baculovirus; D015500:Chloramphenicol O-Acetyltransferase