Novel mutations to spectinomycin resistance in Chlamydomonas reinhardtii
Barbara B. Sears, Allison Blaine, Monica Guha-Majumdar, Matthew R. Enell, Ashita Nagoria, Miriam Kleinman, Ryan Mayle, and Ngoc Nguyen
Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA
Resistance to spectinomycin in Chlamydomonas reinhardtii is known to occur by base substitution at three contiguous bases in the chloroplast 16S rRNA gene, which lies in the large inverted repeat of the chloroplast DNA. However, we have found that half of the spontaneous spectinomycin resistance mutations are not located at that site. We have identified Mendelian mutations in the rps5 gene, which is the main site responsible for spectinomycin resistance in bacteria. We also have identified another region of 16S rRNA in which a 12-base deletion confers resistance to spectinomycin. In recombination-deficient cells expressing a dominant negative recA gene, the 16S rRNA mutations are recovered at a greatly reduced frequency. This observation is consistent with: 1) the recessive nature of spectinomycin resistance, such that both copies of the 16S rRNA gene must contain the mutation before the cells become resistant to the antibiotic; and 2) the dependence of copy correction on homologous recombination between the two copies of the large inverted repeat of the chloroplast DNA.
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