Timing of developmentally programmed excision and circularization of Paramecium internal eliminated sequences

Paramecium internal eliminated sequences (IESs) are short AT-rich DNA eleme nts that are precisely eliminated from the germ line genome during developm ent of the somatic macronucleus, They are flanked by one 5'-TA-3' dinucleot ide on each side, a single copy of which remains at the donor site after ex cision. The timing of their excision was examined in synchronized conjugati ng cells by quantitative PCR. Significant amplification of the germ line ge nome was observed prior to IES excision, which starts 12 to 14 h after init iation of conjugation and extends over a 2- to 4-h period. Following excisi on, two IESs were shown to form extrachromosomal circles that can be readil y detected on Southern blots of genomic DNA from cells undergoing macronucl ear development. On these circular molecules, covalently joined IES ends ar e separated by one copy of the flanking 5'-TA-3' repeat. The similar struct ures of the junctions formed on the excised and donor molecules point to a central role for this dinucleotide in IES excision.