Characterization of glyoxylate cycle mutants of Chlamydomonas reinhardtii
Kumar Shah, Lee McGowan, Alyssa Lombardi, You Ding Chang, Renée Roberts, and Michael Kuchka
Dept. of Biol. Sciences, Lehigh University, Bethlehem, PA 18018
We have identified a collection of spontaneous mutants that fail to grow on acetate medium in the dark, i.e., they are obligate photoautotrophs. These strains have normal rates of respiration and photosynthesis. They have greatly diminished ability to consume and/or metabolize acetate, as measured by cellular assimilation of [14C] labeled acetate. Two mutant strains, "did1" (derived from the 137c; mt+ wild type strain CC125) and CC67 (er-u-37; mt-), are deficient in isocitrate lyase activity, one of the key enzymes of the glyoxylate cycle. One isocitrate lyase encoding gene (ICL1) is situated on linkage group (LG) VI. The Chlamydomonas genome sequence predicts two additional isocitrate lyase genes on LG III. We have determined that the mutation in the did1 strain is linked to molecular markers on LG III, close to one of these isocitrate lyase genes. Using gene specific primers and total cellular RNA, we have isolated a cDNA clone of this gene which we denote ICL2. Quantitative real-time PCR experiments are underway to measure relative expression levels of ICL1 and ICL2 in wild type and mutant cells, under various growth conditions. Although they are defective in acetate assimilation, both did1 and CC67 are sensitive to the toxic analog of acetate, fluoroacetate, at concentrations as low as 0.5 mM. We have isolated a new collection of fluoroacetate resistant mutants of Chlamydomonas, only some of which are obligate photoautotrophs. We expect that the characterization of these strains will help to elucidate the details of acetate uptake and metabolism, as well as the regulation of the glyoxylate cycle in Chlamydomonas.
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