Figure 4.

Immunoprecipitation of HHO1p–2HA-associated chromatin. (A) Ethidium stained agarose gel of restriction digested whole cell DNA. M, λ Eco130I + MluI digested DNA marker (sizes in bp are to left of figure). Lane 1, EcoRI; lane 2, HindIII; lane 3, BglII; lane 4, XhoI. Repeated DNA sequences can be seen as bands on the gel. (B) Southern blot of left panel using immunoprecipitated DNA as probe. All the bands that appear were identified in Foley et al. (41) as encoding rRNA. No other repeated sequences were detected. (C) (Left) rDNA and CUP1 sequences (primers IF9–IF14) were multiplex PCR amplified from total genomic and immunoprecipitated DNA as described in Materials and Methods. The sizes of the amplicons are listed in parentheses. Lane 1, total genomic DNA; lane 2, immunoprecipated DNA. Duplicate lanes are duplicate PCR reactions. Band intensities are quantified in Table 2. (Right) LYS9, CTR1, SAG1, FET3 and FRE1 (primers IF27–IF34) were PCR amplified as in the left panel. All these genes are single copy in the genome. Neither rDNA nor CUP1 were included in the right panel as they are in multiple copies in the genome and they overwhelm the PCR reaction when amplified in the same tube. While none of the DNA fragments in the right panel is specifically immunoprecipitated one relative to the other, it is evident in the left panel that only the rDNA sequences are preferentially immunoprecipitated. About 10-fold more DNA template was used in the multiplex PCR reaction for the single copy genes (right) as for the multicopy genes (left).