Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining
A. DAPI staining of single cells fixed during autogamy in the macronuclear variant 51ΔA. Cells were not treated with RNase prior to staining. For ND7 & LIG4 silencing, cells were fixed 3 days following transfer to RNAi medium. For XRCC4, samples were treated at day 4. White arrows: new MAC. Other stained nuclei are old MAC fragments. B. Quantification of DNA content in propidium iodide-stained exconjugants following RNAi against LIG4 (pLIG4b-L). Fixed cells were treated with RNase A prior to staining. For each time-point, the average DNA amount (in arbitrary units) contained in the new MACs is displayed in the curve. C. DNA under-amplification in the new developing MACs of XRCC4-silenced cells depends on the presence of wild-type levels of PiggyMac transposase. P. tetraurelia strain 51 was silenced for the expression of ND7 (Control), XRCC4 or PiggyMac (PGM) individual genes, or cosilenced for XRCC4 and PGM. During autogamy, cells were stained with DAPI and observed using a Zeiss fluorescence microscope (magnification: 630X). Except for the ND7 control, no viable sexual progeny was recovered from RNAi-treated cells.