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Molecular and Cellular Biology, March 2000, p. 1553-1561, Vol. 20, No. 5
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Mireille Bétermier,* Sandra Duharcourt,dagger Hervé Seitz, and Eric Meyer

UMR 8541 Centre National de la Recherche Scientifique, Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 75005 Paris, France

Received 27 July 1999/Returned for modification 3 September 1999/Accepted 29 November 1999

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.

* Corresponding author. Mailing address: UMR 8541 Centre National de la Recherche Scientifique, Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France. Phone: (0) 1 44 32 39 47. Fax: (0) 1 44 32 39 41. E-mail: betermie{at}wotan.ens.fr.

dagger Present address: Fred Hutchinson Cancer Research Center, Seattle, WA 98109-4417.

Molecular and Cellular Biology, March 2000, p. 1553-1561, Vol. 20, No. 5
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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