Functional Roles for Tetrahymena RAD51 During Conjugation and the Cell Cycle

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The ciliated protozoa possess an unusual genome organization that effectively divides the labor of somatic and germline genetic functions between two distinct nuclei. The micronucleus is the germline nucleus, divides mitotically during vegetative growth, is diploid (2N = 10 for Tetrahymena), and is transcriptionally silent. In contrast, the macronucleus is the somatic nucleus, divides amitotically, contains multiple copies of all genes, and is transcriptionally active. Sexual reproduction, or conjugation, is a tightly controlled process in Tetrahymena whose progression follows a predictable pattern over approximately twelve hours. Soon after two Tetrahymena cells of opposite mating type form a conjugating pair, the micronuclei undergo a series of meiotic and mitotic divisions. Following these nuclear divisions, conjugants exchange haploid nuclei, which then fuse to produce a zygotic, diploid nucleus. Late in conjugation the parental macronuclei are destroyed, and a new macronucleus develops from a copy of the zygotic micronucleus. Macronuclear development proceeds in a pre-programmed and controlled manner that involves a series of site-specific chromosome breakage and DNA deletion events.A functional study of the highly conserved DNA strand transfer protein Rad51 (the eukaryotic homolog to the bacterial recA recombinase) from Tetrahymena thermophila has been initiated. Tetrahymena RAD51 expression varies under a number of different environmental conditions and developmental stages. RAD51 expression increases following exposure to DNA damaging agents. In addition, RAD51 levels vary during both the vegetative cell cycle and conjugation. Disruption of the somatic RAD51 locus results in failure of conjugating cells to complete meiosis, and in vegetative cell cycle defects that lead to an accumulation of hypodiploid cells. Conjugation of germline, homozygous rad51 null strains lead to a block of the first vegetative cell division following conjugation, resulting in an arrest at the exconjugant developmental stage. In order to more precisely decipher the role of Rad51p during conjugation, a conditional, temperature sensitive (t.s.) allele of Tetrahymena RAD51 will be identified. Plasmid-encoded Tetrahymena Rad51p will be tested for complementation of a yeast rad51 null strain. Successful complementation will make it possible to directly screen for Tetrahymena RAD51 t.s. alleles in yeast. If the Tetrahymena RAD51 homolog fails to complement a yeast rad51 null strain, plasmid-encoded yeast RAD51 will be mutagenized, introduced into a rad51 null strain, and screened for a t.s. allele. Missense mutations of conserved amino acid(s) that confer a t.s. phenotype to the yeast Rad51p will be introduced to the Tetrahymena homolog, which will subsequently be expressed in Tetrahymena rad51 null exconjugants. A RAD51 conditional mutant that can be inactivated during conjugation at the restrictive temperature will provide insight into how developmentally controlled genome rearrangements in Tetrahymena, including those leading to rDNA palindrome formation, are mediated.A more complete understanding of these developmentally controlled recombination will provide insight into how similar processes are achieved in other organisms, such as the rearrangement of vertebrate immunoglobulin genes. Mechanisms by which DNA sequences are rearranged can be more easily explored and understood using a single-celled model organism such as Tetrahymena.