Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining
A. Northern blot hybridization of total RNA extracted during conjugation of d4-110 mt7 and mt8. LIG4 and XRCC4 probes (Figure 4A) were used successively. Mac. Dev.: MAC development. B. Localization of a Lig4a-GFP fusion during conjugation. Reactive mt7 cells transformed with plasmid pLIG401GC, and exhibiting a wild-type swimming behavior (see Materials and Methods), were mated with non injected d4-502 (mt8 pwA) at t = 0 hrs. At indicated time-points, cells were fixed and stained with DAPI before observation with a standard fluorescence microscope (GFP filter: a–f; DAPI filter: a’–f’). Developing new MACs are indicated with arrows. C. Quantification of intracellular GFP fluorescence. White circles represent the signal from strongly fluorescent cells originating from the transformed mt7 parent, black triangles represent exconjugants produced by the non-transformed mt8 partner, into which fluorescence has diffused during mating. The calculated total amount of Lig4a-GFP produced by a single transformant is represented as the sum of the two signals (dotted line). Bar = standard deviation.