A novel genetic system to isolate a dominant negative effector on DNA-binding activity of Oct-2.
Recent studies have revealed that interactions between transcription factors play an important role in regulation of gene expression in eukaryotic cells. To isolate cDNA clones that dominantly inhibit the DNA-binding activity of Oct-2, chosen as a representative factor, we have developed a novel screening system. This employs an Escherichia coli tester strain carrying a modified lac operon as a reporter gene, with the lac operator sequence replaced by an octamer sequence. Oct-2 expressed in this tester strain represses the expression of the reporter gene and changes the phenotype of the cell from Lac+to Lac-. Introduction of a cDNA expression library prepared from a human T-cell line into the Oct-2-harboring tester strain allowed selection of three Lac+clones out of 1 x 10(5) transformants. One of them, hT86, encoding a putative zinc finger protein was found to derepress beta-galactosidase activity in the Oct-2-harboring tester strain at the transcriptional level. In gel mobility shift assays, hT86 attenuated the intensity of the retarded band composed of the octamer probe and Oct-2, suggesting a dominant negative effect on the DNA-binding activity of Oct-2. The strategy described here provides a new approach for studying protein-protein interactions that govern the complex regulation of gene expression.
Terunuma
A
A
Department of Cell Biology, The Cancer Institute, Japanese Foundation for Cancer Research, Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan.
Shiba
K
K
Noda
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D007763:Lac Operon; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D050813:Octamer Transcription Factor-2; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D013601:T-Lymphocytes; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D016335:Zinc Fingers; D001616:beta-Galactosidase / Q000096:biosynthesis
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050813:Octamer Transcription Factor-2; D009838:Oligodeoxyribonucleotides; C058435:POU2F2 protein, human; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C106242:ZNF593 protein, human; D001616:beta-Galactosidase