Alison Meyer
Marquette University
Milwaukee, WI
Faculty Mentor: Dr. Kathleen Karrer

The single celled protozoan Tetrahymena thermophila has two nuclei: the transcriptionally active macronucleus and the germ-line micronucleus. During the mating of two cells, the micronucleus undergoes meiosis and the two cells reciprocally exchange these haploid meiotic products. A new micronucleus and macronucleus develop in each cell from the mitotic products of this zygotic nucleus. A family of approximately 30 DNA sequences (referred to as Tetrahymena long repeats, or TLR elements) is present within the micronuclear DNA, but is completely eliminated from the developing macronucleus. TLR elements are composed of an internal, highly conserved region and are thought to terminate with long inverted repeat sequences. Previously, transformations were done using a micronuclear genomic library, and several TLR clones were isolated and sequenced. Together, the sequences span the entire internal conserved region of the TLR elements. While most of these clones were 90-97% similar to each other in sequence, two clones contained inserts that have open reading frames encoding a deduced protein with similarity to homing endonucleases-enzymes that can copy their coding sequence into an allele lacking the sequence. Sequences encoding homing endonucleases can, therefore, replicate and increase in number in the genome. Since the elements containing homing endonuclease genes are introns, they do not disrupt the coding sequences of the host. Southern blot analyses have already shown that TLR elements are present in several copies throughout the micronuclear genome, but are completely eliminated from the macronucleus. While the endonuclease sequence is present within mic-limited DNA, it is uncertain whether the sequence exists only in TLR elements, or whether it is also present within other mic-limited elements. To answer this question, two probes were utilized. One probe (available in the lab) contained the homing intron region from clone 8E1, while a second probe was created through PCR amplification of a portion of TLR clone 4C1, which lacks the homing endonuclease sequence. The portion amplified was the region homologous to the homing intron's insertion site. Electrocompetent bacteria cells were transformed with a micronuclear DNA library and duplicate colony lifts were done. One filter was hybridized with the 4C1 insertion site probe while the duplicate filter was hybridized with the 8E1 homing intron probe. Positive colonies were subjected to a second screening to confirm the presence of the probe sequence. Two classes of colonies were found following these two screens: those that had an endonuclease sequence present within a TLR element, and those that appeared to have empty insertion sites. From these results, all sequences with homology to the 8E1 homing intron seem to be present within TLR elements. Mini plasmid preps were done on four clones that appeared to contain the homing intron. Restriction enzyme digests show different band patterns from 8E1 and from each other, providing evidence that all four are different, previously uncharacterized clones. Southern blots of plasmid DNA were done to verify the presence of the endonuclease sequence in each clone.