Creation of a Chloroplast Microsatellite Reporter for Detection of Replication Slippage in Chlamydomonas reinhardtii

Abstract

Microsatellites are composed of short tandem direct repeats; deletions or duplications of those repeats through the process of replication slippage result in microsatellite instability relative to other genomic loci. Variation in repeat number occurs so frequently that microsatellites can be used for genotyping and forensic analysis. However, an accurate assessment of the rates of change can be difficult because the presence of many repeats makes it difficult to determine whether changes have occurred through single or multiple events. The current study was undertaken to experimentally assess the rates of replication slippage that occur in vivo in the chloroplast DNA of Chlamydomonas reinhardtii. A reporter construct was created in which a stretch of AAAG repeats was inserted into a functional gene to allow changes to be observed when they occurred at the synthetic microsatellite. Restoration of the reading frame occurred through replication slippage in 15 of every million viable cells. Since only one-third of the potential insertion/deletion events would restore the reading frame, the frequency of change could be deduced to be 4.5 ؋ 10 ؊5 . Analysis of the slippage events showed that template slippage was the primary event, resulting in deletions rather than duplications. These findings contrasted with events observed in Escherichia coli during maintenance of the plasmid, where duplications were the rule.Microsatellite sequences, also called simple sequence repeats (SSRs), are short tandem DNA repeats, 1 to 6 bases long, commonly found in the genomes of eukaryotes and some prokaryotes (4,6,12,40). These repeats, which are present in both the noncoding and coding regions of genomes, are unstable, undergoing additions or deletions of one or more repeat units, leading to variations in the length of the microsatellite (17,35,38). In most cases, the insertions or deletions (indels) occur as a consequence of slippage of the template or daughter strand at the replication fork (16,18). Although a similar indel could theoretically occur through intra-or intermolecular recombination, homologous recombination does not appear to be involved in microsatellite variability, since the variation is independent of homologous recombination factors, such as the RecA protein (18).Because of their abundance and variability, microsatellite loci have been used extensively as genetic markers in evolutionary and ecological studies of natural populations in eukaryotes (14, 37) and also as highly polymorphic markers for forensics and genotyping of animals (39). Well-saturated microsatellite maps have been developed for nuclear genomes from a number of plants, including rice, maize, barley, Arabidopsis, and soybean (14,23). In some cases, nuclear microsatellite instability is remarkably high: in chickpea, indels were found at an average rate of 2.9 ϫ 10 Ϫ3 to 10 ϫ 10 Ϫ3 per (TAA) n locus per generation (36). An even higher rate of somatic instability was found for mononucleotide microsatellites in reporter genes in Arabidopsis thaliana, wi...

A Dynamic Tandem Repeat in Monocotyledons Inferred from a Comparative Analysis of Chloroplast Genomes in Melanthiaceae

“…Albeit the sequence data supported our hypothetical scenarios, there was not essential evidence of in vivo experiment in this study. However, GuhaMajumdar et al ( 2008 ) previously attempted to trace replication slippage in vivo and successfully confirmed this event from results of deletion and duplication in C. reinhardtii and Escherichia coli . Although, this study employed only one type of short tandem repeat, it fundamentally supported the reliability of three hypothetical scenarios in our study.…”

Two Distinct Plastid Genome Configurations and Unprecedented Intraspecies Length Variation in the accD Coding Region in Medicago truncatula

“…Unlike in M. truncatula , the repeats in the Oenothera ptDNA are found in intergenic regions. 38 , 39 …”