Comparative Chloroplast Genomics
Sears, Barbara , GuhaMajumdar, Monica .
Mutation events in chloroplast DNA: replication slippage and base substitution.
The frequency and types of mutation events that affect chloroplast DNA have been deduced primarily through evolutionary comparisons. To experimentally assess whether these evolutionary observations reflect on-going mutation processes, we used the unicellular green alga Chlamydomonas reinhardtii to develop reporter systems that allow the observation of base substitution or replication slippage in chloroplast DNA. For characterizing base substitutions, forward mutations to spectinomycin resistance were isolated, which also caused the loss of an Aat II restriction endonuclease cut site within the 16S rRNA gene. Mutations were recovered in three bases within the cut site, with a rate of about one in a billion viable cells, and transversions were recovered more often than transitions. The prominence of A/T to C/G transversions suggests that oxidative damage may be the major cause of base substitution mutations within the chloroplast. As a reporter system for replication slippage, a series of microsatellite repeats were inserted into the sixth codon position of the rbcL gene, disrupting the reading frame. Deletion or duplication of repeats can restore the reading frame leading to photosynthetic function, allowing easy detection of the event. Depending on the size of the repeat, the rate of replication slippage ranged from one in a thousand cells to one in a million. Deletions were found to be the most common event, indicating that template slippage occurs more readily than slippage of the daughter strand. In contrast, when the same substrates were maintained in E. coli, the frequency of slippage was much higher, and duplications occurred. These tangible differences suggest that endosymbiosis may have provided chloroplasts with improved replication proof-reading or DNA repair to minimize replication slippage.
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1 - Michigan State University, Department of Plant Biology, 166 Plant Biology, East Lansing, Michigan, 48824, USA
Presentation Type: Symposium or Colloquium Presentation
Location: Salon K - Austin Grand Ballroom/Hilton
Date: Monday, August 15th, 2005
Time: 10:45 AM