Cristina Montero Díez

AN EXAMINATION OF THE SEQUENCE REQUIREMENTS FOR THE ELIMINATION OF THE R-ELEMENT, AN INTERNALLY ELIMINATED SEQUENCE IN TETRAHYMENA THERMOPHILA. Cristina Montero Díez ¹, Joshua C. L. Trein¹, Douglas L. Chalker¹. Biology Department, Washington University, St. Louis, MO¹.

Tetrahymena thermophila is a bi-nucleate ciliated protozoan that contains a transcriptionally-active somatic macronucleus (MAC) and a transcriptionally-silent germ-line micronucleus (MIC). During conjugation, new germline and somatic nuclei are derived from the parental MIC, and the old MAC is destroyed. Formation of the new MAC involves extensive DNA rearrangements that occur via a homology-directed mechanism guided by small RNAs (sRNAs) that removes 10-15% of the germline genome by excising ~6000 internal elimination sequences (IESs). Two such IESs are the R- and M-IESs located on micronuclear chromosome four, which have no marked sequence similarity but seem to share a rearrangement mechanism dependent on different, yet functionally equivalent, cis-acting flanking regulatory sequences. The R-IES is eliminated by a ~1.1 kilobase pair (kbp) deletion event; the M-IES undergoes two alternative deletion events, eliminating 0.6 or 0.9 kbp.

Previous studies on the M-IES established that its flanking regulatory sequences determine the boundaries for deletion whilst internal MIC-limited sequences promote deletion. A linear relationship exists between rearrangement efficiency and the length of MIC-limited sequence present, with a threshold value of 300 bp. Size, rather than specific sequences within the IES, appears to be the key determinant of rearrangement efficiency for the M-IES, with non-overlapping regions of the IES contributing fairly equally.

The flanking sequences for the R-IES have also been identified and, while markedly different from the M-IES’s, likewise delineate deletion boundaries. It is thus of interest to investigate the MIC-limited sequences to ascertain whether the R-IES exhibits similar size-dependent and sequence-independent deletion, which would support a global homology-dependent mechanism controlling Tetrahymena DNA rearrangement. To do this, we have introduced cloned variants of the R-IES (full-length and left, middle, and right halves and thirds) into a Tetrahymena rDNA vector that was electroporated into mating cells just prior to DNA rearrangement. Successful transformants were selected for by the drug resistance conferred by the vector and allowed to undergo normal nuclear development, then genomic DNA was extracted and Southern hybridization with an R-IES-specific probe performed to ascertain the extent of rearrangement of each region of the IES relative to the full-length.

Initial results show high efficiency for the full-length control, indicating that the experimental vector system does not significantly interfere with rearrangement. The ~600bp halves appear to show, on average, higher rearrangement than the ~370bp thirds, and each half shows greater rearrangement than its corresponding third (e.g. 96% for left half vs. 79% for left third), suggesting some size dependence. The middle of the IES appears to be the exception, with the third displaying significantly higher rearrangement than the half (86% vs. 17%). Experimental duplication using the same subclones will serve to corroborate or refute the unexpected behavior of the middle of the R-IES, and the inclusion of other known IESs and use of sRNA blots to determine the distribution and accumulation of sRNAs homologous to different regions of the IESs will help further elucidate the details of homology-dependent DNA deletion and, more generally, coordinated DNA recognition and processing in Tetrahymena.