PARENT SESSION
Posters P7A Mechanisms of water oxidation. Abstracts (347-381)

Targeted screening for PsbO mutations in Chlamydomonas reinhardtii. Haijun Liu^*,1, Jyoti Iyer¹, Ajay Yekkirala¹, Ron Hutchison¹, ¹ Dept of Biological Science North Dakota State University, Fargo, ND, USA

ABSTRACT- Although chemical mutagenesis is efficient, reliable and well understood, it has not been widely used in reverse genetic methodology to study protein structure and function. To develop a method for the targeted recovery of mutation, we combine EMS chemical mutagenesis regimen with mutation detection by induced targeted gene rescuing. Ethyl methanesulfonate (EMS) generates primarily GC-to-AT transitions, most likely a result of unrepaired O6-ethyl guanine adducts that mispair with thymine during replication. In this study, we constructed rescue plasmid pND107 in which Nit promoter is ligated to the upstream of psbO gene. Chlamydomonas strain CC-2454 (nit1-305 cw15 mt-) was cotransformed by pND107 and p387 by glass beads method. Transformants were screened by SGII/NO3 and PCR. The introduced copy psbO is under the control of Nit promoter, i.e. in the presence of ammonia, Nit promoter will switch off the transcription of downstream gene and psbO can not be expressed. In the absence of ammonia and in the presence of nitrate, psbO can be induciblely expressed. We treated the transformants Chlamydomonas cell with EMS (0.1M) for 90 min and enriched the photosynthesis defective cells selectively by metronidazol. Those cells that survive in N (NO3) medium but die in M (ammonia) medium are potential mutants. We have isolated many potential mutants and characterization of these mutants is in progress. These strains with defective OEE1 protein will be useful in elucidating the biochemistry of the manganese stabilizing protein. We have also begun characterizing the extrinsic proteins using site directed spin labeling. Results from these experiments will be presented.

KEY WORDS: psbO, Chlamydomonas, Manganese Stabilizing Protein (MSP)