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Arabidopsis thaliana Landsberg erecta suspension culture was maintained by weekly 20-fold dilution (12, 13). Early stationary phases (day 7 after previous subculture) were filtered and washed five times with 200 ml of Murashige and Skoog (MS) medium (containing 3% sucrose, but lacking auxin and cytokinin) and maintained in this medium for 24 hours before addition of inducing agents at time T = O and sampling at times indicated. Identical results were obtained with exponential (day 3) cells similarly treated. Chx (100 μM) was added 1 hour before other inducers and effectively prevents protein synthesis in Arabidopsis [S. Abel, M. D. Nguyen, A. Theologis. J. Mol. Biol. 251, 533 (1995); (22)].
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0345011361
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note
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Transgenic Arabidopsis seedlings carrying the CycD3 promoter linked to a GUS reporter gene treated with zeatin also showed twofold to sevenfold induction of GUS but no change in its spatial distribution.
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20
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35S-uridine triphosphate-labeled riboprobes (Promega), were viewed in dark field (autoradiographic signal) or fluorescence (calcofluor staining). For cytokinin induction, 50 μM BAP (Sigma) was applied daily to the apical bud for five successive days.
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note
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Calli were induced by Agrobacterium-mediated transformation of Arabidopsis roots as described (20) with a construct expressing CycD3 under the CaMV 35S promoter. Calli expressed high levels of CycD3 by Northern (RNA) blot (22) but could not be regenerated.
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Kilby, N.J.1
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Murray, J.A.H.4
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24
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0345011359
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in preparation
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The CycD3 cDNA without 5′ untranslated region (UTR) was cloned in conditional vector pFLP-SWITCH (G. D. Davies, N. J. (Kilby, C. Riou-Khamlichi, J. A. H. Murray, in preparation) and crossed to an hsp-FLP line (20). F1 embryos were heat shocked 3 days after pollination, and F2 seeds from individual F1 plants were analyzed for reduced GUS transmission. F2 plants from such parents were tested for transmission of activated CycD3 expression and absence of hsp-FLP (see supplementary material available at www.sciencemag.org/feature/data/985481.shl).
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Davies, G.D.1
Kilby, N.J.2
Riou-Khamlichi, C.3
Murray, J.A.H.4
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26
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0344148854
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note
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Explants cut from rosette leaves of 3-week-old F3 CycD3-expressing and wild-type plants were placed on MS medium with 0.45 μM 2,4-dichlorophenoxy-acetic acid (2,4-D) or 2,4-D and 0.44 μM BAP (control). Calli were propagated and trimmed every 10 days; trimming was unnecessary for wild-type calli on medium lacking cytokinin.
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note
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RNA blots on GeneScreen Plus (NEN) were stained with methylene blue to confirm equal loading, hybridized at 42°C, and washed (CycD3 blots: 0.1% standard saline citrate and 0.1% SDS at 42°C for 20 min and at 65°C for 10 min; Arabidopsis histone H4 blots: 42°C for 10 min and 55°C for 10 min).
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34
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0000139540
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Isoprenoid cytokinin determinations by enzyme-linked immunosorbent assay (ELISA) were carried out as described [H. Kraigher, A. Grayling, T. L. Wang, D. E. Hanke, Phytochemistry 30, 2249 (1991)] with SepPak purified extracts of 10-day-old seedlings grown in liquid MS medium. Five ELISA determinations were carried out for at least two separate extractions.
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0345011350
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note
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3H]thymidine into acid-insoluble material was measured in triplicate relative to background as described (10).
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0345442435
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note
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We thank colleagues for helpful suggestions; N. Kilby, G. Davies, and A. Sessions for advice on the FLP activation system; L. Dehon for in situ hybridizations; D. Hanke for advice and assistance with cytokinin assays; I. Furner for Arabidopsis mutants; and A. Inskip for technical assistance. Support of Biotechnology and Biological Sciences Research Council grant P05114 and Pôles d'attraction interuniversitaires belges (Service du Premier Ministre, Services fédéraux des Affaires scientifiques, techniques et culturelles) P4/15 is acknowledged.
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