Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining
Three IESs are presented: 1 = 51A1835 (28bp), 3 = 51A4404 (77 bp) from surface antigen A gene and 6 = 51G4404 (222 bp) from surface antigen G gene. Gene names are indicated on the left of panels A and B. IESs are drawn as grey boxes and MAC flanking sequences as black lines. Vertical arrows indicate the position of DNA cleavage in each experiment. All details about the linkers and primers used in this experiment are provided in Table S1. In both time-courses, cells were transferred to RNAi medium at day 0 (D0). The T0-time point is the time when 50% of cells have a fragmented old MAC. RNAi against LIG4 was obtained using mixed bacterial cultures producing dsRNA from LIG4a (pLIG4a-R) and LIG4b (pLIG4b-R). Histograms show the progression of autogamy. V: vegetative cells. F: fragmented parental MAC. NM: cells with two visible new developing MACs. PA: post-autogamous cells with one MAC and surrounding fragments. A. LMPCR detection of free broken MAC ends at IES excision sites during autogamy of strain 51 submitted to control or LIG4 RNAi. B. LMPCR detection of free broken IES ends at the left boundary of IES #6 during the same time-course.