title: Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.).
abstract: 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:horse-radish 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 beta-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.
authors.affiliation: Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Korea.
authors.forename: Man Soo
authors.initials: MS
authors.lastname: Choi
authors.forename: Min Chul
authors.initials: MC
authors.lastname: Kim
authors.forename: Jae Hyuk
authors.initials: JH
authors.lastname: Yoo
authors.forename: Byeong Cheol
authors.initials: BC
authors.lastname: Moon
authors.forename: Sung Cheol
authors.initials: SC
authors.lastname: Koo
authors.forename: Byung Ouk
authors.initials: BO
authors.lastname: Park
authors.forename: Ju Huck
authors.initials: JH
authors.lastname: Lee
authors.forename: Yoon Duck
authors.initials: YD
authors.lastname: Koo
authors.forename: Hay Ju
authors.initials: HJ
authors.lastname: Han
authors.forename: Sang Yeol
authors.initials: SY
authors.lastname: Lee
authors.forename: Woo Sik
authors.initials: WS
authors.lastname: Chung
authors.forename: Chae Oh
authors.initials: CO
authors.lastname: Lim
authors.forename: Moo Je
authors.initials: MJ
authors.lastname: Cho
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000235:genetics; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D015723:Gene Library; D005966:Glucuronidase / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000737:chemistry* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016415:Sequence Alignment; D020080:Tandem Repeat Sequences; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D014162:Transfection
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D018076:DNA, Complementary; D018744:DNA, Plant; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D005966:Glucuronidase