Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal.
We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.
Lassner
M W
MW
Calgene Inc., Davis, CA 95616.
Jones
A
A
Daubert
S
S
Comai
L
L
D015153:Blotting, Western; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000201:enzymology*; D002478:Cells, Cultured; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D066298:In Vitro Techniques; D010947:Plants, Toxic; D011993:Recombinant Fusion Proteins; D013539:Simian virus 40 / Q000235:genetics; D013604:T-Phages / Q000201:enzymology / Q000235:genetics; D014026:Nicotiana / Q000235:genetics; D014764:Viral Proteins
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases