FIG. 7.

CgCbf1p binds in vivo at the centromere. (A) An HA-tagged version of CgCBF1 was created by employing a PCR-based strategy (see Materials and Methods for details). (B) Coimmunoprecipitation (ChIP) of CEN DNA and CgCbf1-HA. Formaldehyde-cross-linked chromatin (15 min of fixation) prepared from CgCbf1p-HA epitope-tagged strains CgTS5 (wild type) and CgTS7 (ΔI) was immunoprecipitated with anti-HA antibody or mock treated (no antibody [AB]). Experimental reaction mixtures were prepared in duplicate. Total input material (1 μl of chromatin solution) and coimmunoprecipitated DNA (2 μl of chromatin solution) were analyzed by PCR using primers specific to CgCEN (180 bp) and two noncentromeric loci, CgACT1 (308 bp) and CgHIS3 (224 bp) (see Table 2 for primers). Aliquots of the PCRs were loaded on 2% agarose gels and analyzed. α, anti.