Eukaryotic Cell doi:10.1128/EC.00447-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Creation of a chloroplast microsatellite reporter for detection of replication slippage in Chlamydomonas reinhardtii
Monica GuhaMajumdar,
Ethan Dawson-Baglien,
and
Barbara B. Sears*
Department of Plant Biology, Michigan State University, East Lansing, MI 48824-1312
* To whom correspondence should be addressed. Email:
sears{at}msu.edu.
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Abstract |
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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 by 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 can be deduced to be 4.5 x 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 E.coli during maintenance of the plasmid, where duplications were the rule.
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