Developmentally controlled DNA rearrangements commonly occur in eukaryotic cells and include examples such as the somatic recombination of immunoglobulin genes in mammals and mating type switching in yeast. Ciliated protozoa provide a unique and rich source of developmentally controlled DNA rearrangements. They occur on a massive scale during each round of sexual reproduction in which the macronuclear genome is formed from the germline micronuclear DNA. The entire genome is remodeled by events that include site specific chromosome breakage, de novo telomere addition and the precise removal of DNAs called internal eliminated sequences (IESs). More than 10,000 IESs are excised from the Paramecium genome during the few hours required to form a new macronucleus. These relatively small DNA sequences (27-882 bp) are located in open reading frames as well as in non-coding regions. Recent studies of Paramecium IESs have identified a consensus inverted terminal repeat that has similarity to the ends of transposons in the Tc1/mariner family. This is the most widespread of all class II (DNA) transposon families. Members have been found in nematodes, insects, fungi, and mammals. It is possible that the precise and highly efficient excision of IESs in Paramecium evolved from transposons. The primary objective of this proposal is to identify the cis acting DNA elements that regulate IES excision. This will be accomplished with two methods. 1) The isolation and analysis of mutant cell lines that are defective for IES excision, and 2) the use of an in vivo assay to analyze mutations inside and flanking a 28 bp IES. Mutants will be isolated by treating mutagenized cells with anti-serum against the A surface protein. The A gene contains seven IESs within the coding region and failure to eliminate any IES prevents A protein expression. A-mutants surviving the treatment with anti-serum will be isolated and the subclass with defects in IES excision will be identified. This procedure has proven successful for the isolation of mutants containing defects in IES excision. The in vivo assay will allow any nucleotide to be analyzed for its function in DNA excision. Site-directed mutations will test the hypothesis that the consensus inverted terminal repeat is required for IES excision. Deletions of the flanking DNA sequence will determine whether sequences outside the 28 bp IES are required for excision.
Studies of the extensive DNA elimination events during Paramecium macronuclear development may uncover novel molecular mechanisms or provide insight into similar but less common DNA rearrangements in other organisms. The relationship between Paramecium IESs and the Tc1/mariner transposons could reveal the evolutionary transfer of an activity originally encoded by a transposon into a function used by the host cell.
