Selfing mutants link Ku proteins to mating type determination in Tetrahymena
Fig 3
Unusual mat gene structures are formed during conjugation.
(A) Detection of the unusual II+IV and IV+V mixed-type genes in the macronucleus during conjugation. Left panels illustrate the presumed unusual mat gene structures and the PCR primer locations. Cellular DNA of ΔTKU80 selfers, normal cells, conjugating ΔTKU80 normal cells at different time points (0, 8, 10, 12, 14, 16, 18, 20, 24, and 36 hours post-mixing) and after conjugation and feeding (3 and 6 hours) was examined by PCR. These unusual products were detected at late conjugation in both normal and mutant cells, suggesting that they could be unfinished mat rearrangement intermediates [10]. (B) Unusual mat gene products persist long after conjugation in mutants but not normal cells. Left panels illustrate the unusual mat gene structures and the locations of the PCR primers. DNA of ΔTKU80, normal cells, conjugating ΔTKU80 and normal cells at different hours during conjugation (16 and 24 hours post-mixing) and at different numbers of fission after conjugation (1F, 4F, 10F, 22F, 34F, 40F, 52F, 64F, 82F, and 100F) was examined by PCR. α-tub gene served as the template control. These likely unfinished intermediates were detected at late conjugation and were lost later during vegetative growth in normal cells. In ΔTKU80 cells, these unusual structures were maintained in vegetative growth. (C) Examination of unusual mat genes during and after conjugation by Southern hybridization. Left panels illustrate the mat locus in the micronucleus and the normal and unusual structures in the macronucleus. IESs in the micronucleus are indicated by gray rectangles and the probe by the black bar. Bs and Ps indicate the Bgl II and Pst I sites, respectively. Cellular DNA from normal cells, TKU80 germline KO strains, ΔTKU80 selfer E4, and normal and TKU80 mutant cells during and after conjugation was analyzed. In normal cells, these unusual structures were observed in late conjugation and disappeared at approximately 10 fissions after conjugation and feeding. However, they were abundantly present and did not disappear in progeny of ΔTKU80 cells. Raw images associated with this figure can be found in S1 Raw Images. Conj, conjugation; F, fissions; IES, internal eliminated sequence; Mac, macronucleus; Mic, micronucleus; MT, mating type.