Detection of Z DNA binding proteins in tissue culture cells. |
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Authors: | I R Leith R T Hay and W C Russell |
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Institution: | Department of Biochemistry and Microbiology, University of St Andrews, Fife, UK. |
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Abstract: | A gel electrophoresis DNA binding assay to detect Z DNA binding proteins has been developed utilising 32P] labelled poly d(G-C)] which was converted to the Z form by incubation in 100 microM Co(NH3)6Cl3. The parameters of the assay were established using a Z DNA antibody as a model system and then applied to extracts of Hela and BHK21 cells. Using an anti-Z DNA antibody conditions were established which allowed resolution of antibody-DNA complexes and free DNA in the presence of 100 microM Co(NH3)6Cl3. The inclusion of unlabelled complementary homopolymers eliminated non-specific binding to the labelled Z-DNA probe. Competition experiments demonstrated that the assay was highly specific for double stranded non-B DNA. Application of the technique to extracts of mammalian cells demonstrated that human and hamster cells contain Z-DNA binding proteins; further characterisation by a blotting technique indicated that a 56,000 molecular weight cell protein preferentially binds Z-DNA. |
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