Escherichia coli RecA protein modified with a nuclear location signal binds to chromosomes in living mammalian cells.
We tried to make a well-characterized bacterial protein function in mammalian cell nuclei. For this purpose we chose Escherichia coli RecA protein and fused its carboxy terminus to the nuclear location signal of SV40 large T-antigen by oligonucleotide-dependent modification of the gene. When injected into the cytoplasm, the modified RecA protein (T-RecA for the T-antigen signal) accumulated efficiently in the nuclei, whereas the wild-type RecA protein remained in the cytoplasm. The T-RecA protein retained its original in vivo activity, judging from the finding that uv-sensitive bacteria (recA- E. coli) became uv-resistant on transformation with the T-recA plasmid as well as the recA plasmid. For expression of the T-recA gene in mammalian cells, the 5' region was replaced by the chicken beta-actin promoter and Kozak's initiation signal. A high level of expression was observed when Chinese hamster ovary (CHO-K1) cells were transfected with this plasmid. Indirect immunofluorescence examination revealed that the T-RecA protein in nuclei of mammalian cells bound to chromatin.
Kido
M
M
Institute for Molecular and Cellular Biology, Osaka, Japan.
Yoneda
Y
Y
Nakanishi
M
M
Uchida
T
T
Okada
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002875:Chromosomes / Q000378:metabolism*; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D008845:Microinjections; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011938:Rec A Recombinases