Fig. 2.
Nonspecific binding of the fusion proteins to DNA. Bacteriophage Φ×174 DNA was cleaved by HaeIII and end-labelled using T4 polymerase48. The labelled DNA (about 30 ng) was incubated for 30 min at 0 °C in 25 μ1 of binding buffer (BB) (50 mM NaCl, 20 mM Tris pH 7.6, 0.25 mM EDTA, 1 mM DTT, 10% glycerol) in the presence of a bacterial extract containing the fusion protein (about 10 μg of total protein extract, see Fig. 1 legend). The complexes formed between DNA and the fusion proteins were then immunoprecipitated by 30 min further incubation at 0 °C with an anti-β-galactosidase monoclonal antibody (provided by Tom Mason and Judy Partaledis, courtesy of Mike Hall) preadsorbed on cross-linked Staphylococcus (Pansorbin, Calbiochem). The pellet was washed twice in BB, phenol extracted and the DNA ethanol precipitated before polyacrylamide gel electrophoresis and autoradiography. The autoradiogram shows the results of precipitations performed in the presence of extracts containing the full-length fusion (FL), the homoeo domain fusion (HD), the non-homoeo domain fusion (NHD) or β-galactosidase (lacZ). Lane T shows the labelled digest before immunoprecipitation, representing 25% of the counts added to the incubation mixture in the immunoprecipitation experiments.