Transcriptional regulation of cell line-dependent, baculovirus-mediated expression of foreign genes.
Cell line-dependent expression of foreign genes in the baculovirus system was investigated using a recombinant vAc beta hCG-luc virus carrying two reporter genes--beta subunit of human chorionic gonadotropin (beta hCG) and luciferase (luc)--placed under the transcriptional control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoters. Five different lepidopteran cell lines derived from Spodoptera frugiperda (Sf21 and Sf9), Bombyx mori (BmN and Bm5), and Trichoplusia ni (TN368) were used as host cells. TN368 expressed both beta hCG and LUC to maximum levels, followed by BmN, Sf21, and Sf9 in descending order. Bm5 did not show any evidence of synthesis of the two proteins. Dot blot analysis of DNA from the vAc beta hCG-luc-infected cells revealed that the level of entry of viral DNA was the same for all the five cell lines. After the completion of viral DNA replication (18 hr post infection), the level of viral DNA was the same for all the cell lines except for Bm5 where viral DNA replication did not take place and the residual virus was cleared from the cells. Analysis of RNA from the four expressing cell lines revealed a direct correlation between protein levels and levels of mRNA, suggesting transcriptional control. Differences in mRNA stability between cell lines was also evident. Gel retardation analysis of a host factor binding to transcriptionally important sequence motifs within the AcNPV polyhedrin gene promoter revealed an inverse correlation between the levels of this polyhedrin promoter-binding protein (PPBP) and reporter gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Mukherjee
B
B
Eukaryotic Gene Expression Laboratory, National Institute of Immunology, New Delhi, India.
Burma
S
S
Talwar
G P
GP
Hasnain
S E
SE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000096:biosynthesis / Q000235:genetics*; D018997:Chorionic Gonadotropin, beta Subunit, Human; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D007181:Inclusion Bodies, Viral; D019476:Insect Proteins; D007915:Lepidoptera; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000254:growth & development; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013045:Species Specificity; D014158:Transcription, Genetic
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D004268:DNA-Binding Proteins; D019476:Insect Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins; C085605:polyhedrin promoter-binding protein, insect; D004247:DNA; D008156:Luciferases