Developmentally regulated DNA rearrangements in Paramecium tetraurelia

The germline chromosomes of Paramecium and other ciliates contain DNA sequences that are eliminated during the development of a new macronuclear genome. In Paramecium, internal eliminated sequences (IESs) are invariably flanked by a 5′-TA-3′ dinucleotide that is part of a larger 8 bp terminal inverted-repeat consensus sequence. These are the only known conserved sequence features between IESs. It is unknown how Paramecium recognizes an IES for excision. I took a classic genetics approach to identify mutations in cis-acting elements which disrupt IES excision. The A51 surface protein gene, which contains 7 IESs within its coding region, is an excellent target for mutagenesis. Failure to remove an IES will create either a frameshift or a stop codon and prevent A51 surface protein expression. I selected mutants that were unable to express the A51 surface antigen, then analyzed the resulting collection of A51 - cell lines for mutants defective in the removal of IESs from the A51 gene coding region. The four mutants I isolated and characterized each contained a single nucleotide change within the consensus sequence. This means that nucleotides within the consensus are necessary for IES excision. Furthermore, two of the four mutants revealed the presence of internal IESs, reminiscent of the reiterative integration of transposable elements. No alternate excision was observed between internal and external IES ends, and the internal IESs are removed independently during macronuclear development 6–24 hours after mixing cells of complementary mating types. Taken together, these data suggest that Paramecium IESs may have evolved from transposons