Identification of Single Nucleotide Mutations That Prevent Developmentally Programmed DNA Elimination in Paramecium tetraurelia

Abstract

The excision of internal eliminated sequences (IESs) occurs during the differentiation of a new somatic macronuclear genome in ciliated protozoa. In Paramecium tetraurelia, IESs show few conserved features with the exception of an invariant 5'-TA-3' dinucleotide that is part of an 8-bp inverted terminal repeat consensus sequence with similarity to the ends of mariner/Tc1 transposons. We have isolated and analyzed two mutant cell lines that are defective in excision of individual IESs in the A-51 surface antigen gene. Each cell line contains a mutation in the flanking 5'-TA-3' dinucleotide of IES6435 and IES1835 creating a 5'-CA-3' flanking sequence that prevents excision. The results demonstrate that the first position of the 5'-TA-3' is required IES excision just as previous mutants have shown that the second position (the A residue) is required. Combining these results with other Paramecium IES mutants suggests that there are few positions essential for IES excision in Paramecium. Analysis of many IESs reveals that there is a strong bias against particular nucleotides at some positions near the IES termini. Some of these strongly biased positions correspond to known IES mutations, others correlate with unusual features of excision.