Fig. 2.

Application of RITE to endogenous histone H3. (A) One of the two genes encoding histone H3 in yeast (HHT2) was tagged with a RITE cassette (H3-RITE) containing short epitope tags: HA (old) and T7 (new). The other gene encoding histone H3 (HHT1) was deleted. A Hygromycin resistance gene (Hygro) was used to select against illegitimate recombinants. The tag switch was under control of a constitutively expressed hormone-dependent Cre recombinase (Cre-EBD78). (B) Growth of wild-type and H3 RITE-tagged [before (HA) and after (T7) the switch] yeast cells spotted in a 10-fold dilution series. (C) The efficiency of recombination in the cell population was determined by Southern blot analysis of genomic DNA digested with HindIII (H) before (Pre) and after (Post) addition of the hormone β-estradiol. An invariant fragment was used as a control (Ctrl). (D) Detection of old (HA) and new (T7) histone H3 by quantitative immunoblot analysis of whole cell lysates of equal numbers of starved switched cells released into fresh media. The number of population doublings was calculated by staining the cells with N-hydroxysuccinimide-tetra-ethylrhodamine (NHS-TER) (SI Materials and Methods). (E) The percentage of old H3-HA plotted against the number of population doublings. The measured HA/T7 ratios of the blot in D were converted into H3-HA percentages by using standard curves of samples with known percentages of H3-HA and H3-T7 (SI Materials and Methods).