Chloroplast Microsatellite Mutators

Michigan State University, East Lansing MI

Investigators

Abstract

Microsatellites are composed of simple sequence repeats that have been found in both nuclear and chloroplast DNA. In both genetic compartments, they are sites of genomic instability due to replication slippage, which causes duplications or deletions of individual repeats. Although the numbers of repeats in microsatellites are intrinsically unstable, mechanisms exist for the recognition and repair of slipped and misaligned DNA strands. Studies in bacteria and yeast have shown that mutations in genes that encode components of mismatch repair or replication can have a mutator effect on microsatellites by causing their further destabilization. The goal of this research is to isolate mutator alleles that increase the instability of chloroplast microsatellites, using Chlamydomonas reinhardtii as a model plant system. To enable rapid recognition and quantification of microsatellite variation, specific microsatellite repeats will be inserted into a reporter gene, which will produce a selectable phenotype when one or more repeats are deleted or duplicated. The microsatellite "targets" will be placed in the alga chloroplast using standard procedures for transformation, relying on a linked selectable marker for the detection of transformants. After basal levels of chloroplast replication slippage are quantified, another plasmid will be used for nuclear transformation to accomplish gene disruptions. The nuclear transformants will be tested for elevated levels of chloroplast microsatellite instability. The second plasmid will serve as a tag to enable recovery of adjacent sequences for identification of the disrupted nuclear loci that contribute to microsatellite stability. In parallel, chemical mutagenesis will be used to induce chloroplast microsatellite mutators, which will be mapped genetically and physically. These mutators will be tested to determine whether they target a variety of microsatellites and whether they increase the frequency of base substitutions. This research will yield the first true library of nuclear mutators of the chloroplast genome in any plant system. This should complement and extend what has been learned from the very few chloroplast mutators that have been isolated by chance in assorted plants. This approach should also identify a different range of loci from those that have been recovered through complementation of specific microbial recombination/repair defects by plant cDNA clones.