PgmNr C34: Repair of a fragile site in the mating type genes using an episomal template in Tetrahymena.

Authors:
Marcella D. Cervantes 1 ; Michael J. Lawson 2 ; Linying Zhang 1 ; Eileen Hamilton 3 ; Eduardo Orias 3 ; Geoffrey Kapler 1


Institutes
1) Texas A&M Health Science Center, College Station, TX; 2) Uppsala University, Uppsala, Sweden; 3) University of California Santa Barbara, Santa Barbara, California.


Abstract:

The Tetrahymena thermophila somatic mating type genes offer an opportunity to understand an unusual mechanism for repairing fragile sites using episomal DNA as a template. Tetrahymena has seven possible mating types, and each is determined by the MTA and MTB gene pair. In the somatic nucleus, the gene pair is oriented head to head, sharing a presumed promoter region. Both genes are transcribed at a very low level during growth, but are induced to a high level of transcription by starvation. In the germline nucleus, the mating type locus contains an array of truncated gene pairs. While the terminal exons of each gene are conserved between mating types, each copy of the conserved terminal exons contains polymorphic nucleotides unique to that particular copy. The germline array of genes is rearranged during somatic nuclear development by homologous recombination. PCR experiments suggests that recombination produces circular episomal byproducts that are retained in progeny for more than 120 fissions. Homologous recombination reduces the mating type locus to one complete gene pair in the new somatic nucleus. The pattern of polymorphic nucleotides shows up to 89% of the newly assembled MTB genes are produced by a single recombination event. Unexpectedly, older vegetative cells, do not exhibit a single recombination junction because the terminal exons have a mosaic of polymorphic nucleotides - presumably resulting from multiple gene conversion events. I propose that the template for these gene conversion events are episomal circular byproducts generated during mating type determination. I am addressing two questions regarding the episomes. The first question is whether the promoter region of the mating type gene pair can serve as an autonomously replicating sequence (ARS). To test the promoter region as an ARS, a plasmid with an E.coli backbone, a Tetrahymena selection cassette and regions of interest was transformed into vegetative Tetrahymena. We have obtained stable transformants that harbor a plasmid with the mating type VII promoter region. The second question is whether gene conversion within the conserved terminal exons occurs at a particular site due to replication fork pausing. Although the MTA and MTB genes acquire a mosaic of polymorphic nucleotides, they do not acquire mutations. This suggests homologous recombination. A pause site of DNA replication could explain the propensity for gene conversion in the terminal exons of MTA and MTB. The presence of pause sites in the terminal exons is being determined by 2D gel electrophoresis.