Developmentally regulated excision of a 28-base-pair sequence from the Paramecium genome requires flanking DNA

The micronuclear DNA of Paramecium tetraurelia is estimated to contain over 50,000 short DNA elements that are precisely removed during the formation of the transcriptionally active macronucleus. Each internal eliminated sequence (IES) is bounded by 5'-TA-3' dinucleotide repeats, a feature common to some classes of DNA transposons. We have developed an in vivo assay to analyze these highly efficient and precise DNA excision events. The microinjection of a cloned IES into mating cells results in accurately spliced products, and the transformed cells maintain the injected DNA as extrachromosomal molecules. A series of deletions flanking one side of a 28-bp IES were constructed and analyzed with the in vivo assay. Whereas 72 bp of DNA flanking the eliminated region is sufficient for excision, lengths of 31 and 18 bp result in reduced excision and removal of all wild-type sequences adjacent to the TA results in complete failure of excision. In contrast, nucleotide mutations within the middle of the 28-bp IES do not prevent excision. The results are consistent with a functional role for perfect inverted repeats flanking the IES.