Figure 6

Confirmation of predicted methylation by bisulfite sequencing. (a) Use of C-to-T converted primers specifically amplifies the non-methylated chromosomes from bisulfite-treated 40 h (hour) or vegetative DNA (labeled 'Non-methylated'), while standard, cytosine-retaining primers amplify methylated DNA (labeled 'Methylated'). PCR of TEBP β was performed on native DNA to demonstrate functionality of the oligos. (b) A repeat of the experiment in (a), but with a 46 h, not 40 h, sample, and with additional methylation cohort chromosomes, as well as TEBP α and TEBP β. Strains JRB310 (310) and JRB510 (510) are two mating types of Oxytricha trifallax whose mixing induces conjugation; the 40 h and 46 h samples are an equal combination of both mating types. (c) Bisulfite sequencing of eleven Contig4414.0 clones. Cytosines in bold are methylated. Note that methylation occurs in all sequence contexts and can have runs of consecutive skipped residues. Two CC motifs occur in this region of the chromosome, as marked. (d) Three aberrantly spliced, and methylated/hydroxymethylated, versions of TEBP α identified by bisulfite-PCR of 46 h DNA. MDS 12 would normally never be fused directly to MDS 2, as observed in these products; 3 to 4 bp cryptic pointers (marked 'cp' in red arrowheads) are present at recombination junctions. Normal unscrambling entails fusion of MDS 1 to MDS 2 and MDS 12 to MDS 13; wild-type pointers for these events are marked in turquoise arrowheads. Products 2 and 3 (recovered 1 and 2 times, respectively) appear heavily methylated, while product 1 is more lightly methylated (G to A substitutions indicate C to T conversions on the opposite strand, highlighted in pink). Colored nucleotides differ from the WT sequence (top). PCR primers are marked by purple arrowheads. MDS, Macronuclear Destined Sequence; TEBP α, Telomere End-Binding Protein α; Veg, vegetative.