Timing of Developmentally Programmed Excision and Circularization of Paramecium Internal Eliminated Sequences

Abstract

Paramecium internal eliminated sequences (IESs) are short AT-rich DNA elements that are precisely eliminated from the germ line genome during development of the somatic macronucleus. They are flanked by one 5′-TA-3′ dinucleotide on each side, a single copy of which remains at the donor site after excision. The timing of their excision was examined in synchronized conjugating cells by quantitative PCR. Significant amplification of the germ line genome was observed prior to IES excision, which starts 12 to 14 h after initiation of conjugation and extends over a 2- to 4-h period. Following excision, two IESs were shown to form extrachromosomal circles that can be readily detected on Southern blots of genomic DNA from cells undergoing macronuclear development. On these circular molecules, covalently joined IES ends are separated by one copy of the flanking 5′-TA-3′ repeat. The similar structures of the junctions formed on the excised and donor molecules point to a central role for this dinucleotide in IES excision.

ACKNOWLEDGMENTS

We thank Kevin Williams, Tom Doak, and Glenn Herrick for communicating their results prior to publication; Janine Beisson, Anne-Marie Keller, and Françoise Ruiz for their advice in Paramecium culture handling; Rémi Dillys and Anne Turbé for their participation in the sequencing of IES circular junctions; and Robert Alazard for the gift of plasmid pRA593. Many thanks go to all members of the group for stimulating discussions and critical reading of the manuscript.

This work was supported by the Association pour la Recherche sur le Cancer (grant 1374), the Centre National de la Recherche Scientifique (Programme Génome), and the Ministère de l'Education Nationale de la Recherche et de la Technologie (Programme de Recherche fondamentale en Microbiologie et Maladies infectieuses et parasitaires). S.D. was a recipient of doctoral fellowships from the Association pour la Recherche sur le Cancer and from the Fondation pour la Recherche Médicale.