Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors.
The transcriptional and translational signals required for efficient expression of the chloramphenicol acetyltransferase, beta-galactosidase, and tissue plasminogen activator genes, under the control of the polyhedrin promoter in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus, were investigated by SDS-PAGE and RNA dot blot analysis. The recombinant baculoviruses all contained alterations in the leader sequence or 5' proximal coding region of the polyhedrin gene. Highest levels of foreign proteins and polyhedrin-linked mRNAs were observed when portions of the coding sequence of the polyhedrin gene were fused in phase with the foreign gene. Recombinant viruses in which the foreign gene was inserted upstream from the polyhedrin ATG start codon expressed nonfused products but at lower levels than contructs which produced fusion proteins. A corresponding decrease in the levels of mRNAs produced by such constructs was also observed. Some constructs in which the foreign gene was inserted out of phase downstream from the polyhedrin start codon expressed nonfused protein products at low levels but produced polyhedrin-linked mRNA at levels comparable to vectors which produced protein fusions. These data suggest that reinitiation of translation can take place at AUG start codons a short distance downstream from the primary polyhedrin start codon. These results indicate that sequences immediately upstream from the polyhedrin start codon are important for regulation of transcription and that additional sequences near the AUG start codon can have a dramatic influence on the levels of translation observed.
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003062:Codon / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D010957:Plasmids; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D003062:Codon; D004279:DNA, Viral; D012333:RNA, Messenger; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; D010959:Tissue Plasminogen Activator