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Study of SCI1 and its protein-protein interactions that result in cell cycle inhibition


In our laboratory it was identified the SCH gene (Stigma/style Cell cyde Inhibitor 1), capable of controlling cell proliferation on the upper part of the pistil (DePaoli et ai., 2011), probably through the inhibrtron of the cyclin-CDK (Cyclin Dependent protein Kinase) complex. To exert its function, the protein SCH has to interact with other proteins. In a previous project (process no. 10/50213-2), some candidates for protein interaction were identified, like the CDKG;2 and two proteins of the 14-3-3 family. Therefore, the objectives of this project are: 1) To identify/confirm the cyclin(s) that interacts with SC/1; 2) To identify partners/substrates of the cyclin dependent kinase (CDKG;2), by the screening of a stigma/style library in the two hybrid vector; 3) To study the hypothesis of SCH regulating cell division through the inhibition of CDKG.2. a. To test the interaction of the proteins cyclin L and other cyclins (cyclin B1 and/or cyclin-related) with CDKG;2 by two hybrid and/or BiFC (Bimolecular Fluorescence Complementation). b. In the case a cyclin capable of interacting with CDKG;2 is identified in the directed experiments (item 3.a) or in the screening, to test CDKG;2 activity on histones and/or another possible substrate identified on the screening (item 2); c. If a and b are positive, to test the inhibition of CDKG;2 activity by SCI1; 4) To test the hypothesis of tissue-specificity on the expression of SCH interaction partners; 5) To analyze the subcellular localization of the CDKG;2,14-3-3A and 14-3-3D proteins; 6) To perform two hybrid and/or BiFC experiments to test the formation of homo and heterodimers between the 2 proteins of the 14-3-3 family and their interactions with SCH; 7) To characterize the phosphorylation of SCH protein, by the comparison of phosphorylated residues in SCH extracted from leaves and pistils of 35Sprom::SCI1-GFP transgenic plants; 8) To confirm SCI1 interaction with the new candidates identified by pull-down or two hybrid screening performed. (AU)

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