Functional Roles for Tetrahymena RAD51 During Conjugation and the Cell Cycle

University Of Minnesota-Twin Cities, Minneapolis MN

Investigators

Abstract

The regulation of genome dynamics has been evolutionarily conserved. A key factor involved in maintaining the genome's integrity is the ubiquitous DNA strand transfer protein Rad51p. Despite a full decade of research on RAD51 from model systems ranging from yeast to mammals, it is still unclear how Rad51p mediates its effects. This project focuses on RAD51 from the ciliated protozoan Tetrahymena thermophila. Due to Tetrahymena's unique cell biology and nuclear organization, genetic manipulations can showcase the separable functions of Rad51p, including roles unwitnessed in other model organisms. Ciliates possess an unusual genome organization that effectively divides the somatic and germ-line genetic functions between two distinct nuclei. The micronucleus (MIC) is the germline nucleus, dividing mitotically during vegetative growth, and transcriptionally silent. The macronucleus (MAC) is the somatic nucleus, dividing amitotically, and transcriptionally active. The MICs of a conjugating pair undergo a series of meiotic and mitotic divisions, followed by an exchange of haploid nuclei, which fuse to produce a zygotic nucleus. Late in conjugation the parental MACs are destroyed, and new MACs develop in a programmed manner from copies of the zygotic MIC. Tetrahymena RAD51 expression varies under a number of different environmental conditions and developmental stages. Constitutively high Rad51p expression during vegetative growth decouples MAC division from the cell cycle, leading to a high percentage of amacronucleate progeny. This project has two goals. First, a cDNA expression library will be screened for Tetrahymena gene products that suppress the severe amacronucleate phenotype exhibited by strains that constitutively express Rad51p. This experimental approach will help identify factors that directly and/or indirectly interact with Rad51p during macronuclear division. Second, randomly mutagenized Rad51p will be constitutively expressed in an effort to identify missense mutations that alleviate the amacronucleate phenotype in transformants. This project, taking advantage of genetic manipulations possible only within the Tetrahymena experimental system, should extend our understanding of Rad51p structure and function and thus will contribute to the analysis of this highly important protein in other eukaryotic model systems. Both undergraduate and graduate students will help carry out this research.