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Isolation of a Calmodulin-binding Transcription Factor from Rice (Oryza sativa L.)

Choi, Man-Soo (Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center) , Cho, Moo-Je| (Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center) , Yoo, Jae-Hyuk (Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center) , Moon, Byeong-Cheol (Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center) , Koo, Sung-Cheo (Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center) , Park, Byung-Ouk (Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center) , Lee, Ju-Huck (Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center) , Koo, Yoon-Duck (Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center) , Han, Hay-Ju (Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center) , Lee, Sang-Yeol (Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Environmental Biotechnology National Core Research Center) , , ,

Plant molecular biology and biotechnology research center Annual report, 2005, 2005, 243-254

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Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expressi...
Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horseradish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca^(2+)-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5'-TWCG(C/T)GTKKKKTKCG-3' (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate β-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.