Flanking regulatory sequences of the Tetrahymena R deletion element determine the boundaries of DNA rearrangement

In the ciliate Tetrahymena thermophila, thousands of DNA segments of variab le size are eliminated from the developing somatic macronucleus by specific DNA rearrangements. It is unclear whether rearrangement of the many differ ent DNA elements occurs via a single mechanism or via multiple rearrangemen t systems. In this study, we characterized in vivo cis-acting sequences req uired for the rearrangement of the 1.1-kbp R deletion element. We found tha t rearrangement requires specific sequences flanking each side of the delet ion element. The required sequences on the left side appear to span roughly a 70-bp region that is located at least 30 bp from the rearrangement bound ary. When we moved the location of the left cis-acting sequences closer to the eliminated region, we observed a rightward shift of the rearrangement b oundary such that the newly formed deletion junction retained its original distance from this flanking region. Likewise,when we moved the flanking reg ion as much as 500 bp away from the deletion element, the rearrangement bou ndary shifted to remain in relative juxtaposition. Clusters of base substit utions made throughout this critical flanking region did not affect rearran gement efficiency or accuracy, which suggests a complex nature for this reg ulatory sequence. We also found that the fight flanking region effectively replaced the essential sequences identified on the left side, and thus, the two flanking regions contain sequences of analogous function despite the l ack of obvious sequence identity. These data taken together indicate that t he R-element flanking regions contain sequences that position the rearrange ment boundaries from a short distance away. Previously, a 10-bp polypurine tract flanking the M-deletion element was demonstrated to act from a distan ce to determine its rearrangement boundaries. No apparent sequence similari ty exists between the M and R elements. The functional similarity between t hese different cia-acting sequences of the two elements is firm support for a common mechanism controlling Tehahymena rearrangement.