Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining
A. Maps of LIG4 and XRCC4 genes. XRCC4 accession number in ParameciumDB is GSPATG00029540001. The fragments used in the construction of RNAi plasmids are shown in hatched boxes and designated in italics (L or R). Probes used for Northern blot hybridization are displayed as black boxes: we used the first 510 bp of LIG4a open reading frame and a 302-bp fragment encompassing bp 508–809 for XRCC4. Restriction sites: P = PstI, S = SpeI, K = KpnI, H = HindIII. B. Survival and genetic analysis of F1 progeny from a cross between a3093 (mt7 pwB) and d4-502 (mt8 pwA). The histogram shows the number of cells surviving conjugation and the genotype of their MAC, out of 18 cells for the control experiment and 17 for the LIG4 knock-down. NC: cells resulting from a conjugation failure within a mating pair (mutant phenotype in F1 and F2). Zyg. MAC: cells containing a functional zygotic nucleus. Regen: MAC regenerants in F1 (mutant phenotype in F1 and recovery of wild-type clones in post-autogamous F2). Control RNAi was carried out with an empty vector (no insert). The segregation of pwA (A-) and pwB (B-) markers in F1 MICs is shown below the histogram. The cell phenotype determined by the MAC is indicated between square brackets. C. MAC regeneration in post-autogamous progeny of 51 cells injected with a GFP transgene. Cells were transferred at day 0 in control (no RNAi or RNAi against ND7) or LIG4-silencing medium and, following starvation, 100% autogamy was observed at day 2. Survival tests were performed on 12-cell samples at day 2 (I) or day 4 (II). Regen: MAC regenerants, as judged by GFP fluorescence. D. Survival of post-autogamous progeny following LIG4 or XRCC4 silencing (experiments are independent of the one presented in B). Regen: MAC regenerants. The inserts cloned in plasmids used for feeding experiments are displayed in A. The histogram represents a compilation of several independent experiments, in each of which the progeny of 30 individual autogamous cells was assayed for survival.