Control of gene expression in plant cells using a 434:VP16 chimeric protein.
The herpes simplex virus type 1 VP16 polypeptide is a potent trans-activator of viral gene expression. We have tested the ability of the VP16 activation domain to activate gene expression in plant cells. A plasmid encoding a translational fusion between the full-length 434 repressor and the C-terminal 80 amino acids of VP16, was constructed. When expressed in Escherichia coli, the chimeric protein binds efficiently to 434-binding motifs (operators). For expression in plant cells, the chimeric activator gene was placed between the cauliflower mosaic virus (CaMV) 35S promoter and nos terminator sequences in a pUC-based plasmid. The 434 operators were placed upstream of a minimal CaMV 35S promoter linked to the E. coli gus reporter gene. This reporter-expression cassette was then incorporated into the same plasmid as the 434 cI/VP16 activator-expression cassette. Two control plasmids were also constructed, one encoding the 434 protein with no activator domain and the second a chimeric activator with no DNA-binding domain. The chimeric activator was tested for its ability to activate gene expression in a tobacco protoplast transient assay system. Results are presented to show that we can obtain in plant cells significant activation of gene expression that is dependent on both DNA-binding and the presence of the activator domain.
Wilde
R J
RJ
Zeneca Seeds, Plant Biotechnology Section, Bracknell, UK.
Cooke
S E
SE
Brammar
W J
WJ
Schuch
W
W
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D059828:Plant Cells; D010944:Plants / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins
C063367:434-repressor protein, Bacteriophage 434; D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D004247:DNA