Conservation of post-translational modification (PTM) targets in Stylonychia H3 variants and the accumulation of H3 variant mRNAs during macronuclear differentiation. (A) A similarity matrix of sequence motifs adjacent to well-characterized PTM target sites exhibited similarities and differences between several H3 variants. A match score was calculated between two aligned amino acids using an amino acid class hierarchy diagram . (B) The relative abundance of several H3 variant mRNAs changed over time. Accumulation of Stylonychia H3 variants mRNA during macronuclear development was assessed by quantitative PCR (qPCR). Prior to cDNA synthesis, RNA was isolated from synchronized cells at several developmental stages, which corresponded to the time line (x-axis) as follows: 1) during vegetative growth phase; 2) from cells after conjugation, when an early anlagen nucleus was visible; 3) from cells with polytene chromosome anlagen nuclei prior to bulk DNA elimination; 4) from cells containing polytene anlagen nuclei at the onset of bulk DNA elimination; and 5) from cells within the DNA-poor anlagen stage. Values represent mean and standard deviation (SD), and only the upper error bar is shown. All values were normalized to H3.3 mean mRNA levels during vegetative growth. Extensive enrichment of H3.7 and H3.4 mRNAs was observed during the first round of DNA amplification leading to polytene chromosomes. Intermediate levels of H3.5 mRNA were measured during chromosome polytenization, whereas H3.1 mRNAs accumulated during the second round of DNA amplification, leading to the final copy numbers of mature nanochromosomes.