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note
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The ETR1(1-128)GST fusion protein consists of the first 128 amino acids of ETR1, fused to the coding region of GST obtained from the bacterial expression vector pGEX4T1 (Pharmacia). The fusion protein was expressed in yeast with the use of the expression vector pYcDE-2 (6).
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12
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0345037631
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note
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Cultures of Saccharomyces cerevisiae strain LRB 520 (6) were grown to mid-log phase at 30°C and harvested by centrifugation at 1500g for 5 min. Cell pellets were washed and resuspended in 50 mM tris-HCl (pH 7.4), 10% glycerol, and 1.0 mM phenylmethylsulfonyl fluoride (PMSF) at a concentration of 0.5 g of cells per milliliter. Cell suspension: were mixed with an equal volume of glass beads and disrupted in a bead beater with a cooling bath (BioSpec, Bartlesville, OK). Cell debris was separated from the homogenate by centrifugation at 10,000g and total cell membranes were harvested by centrifugation at 100,000g for 30 min and then resuspended in assay buffer [10 mM MES (pH = 5.5), 20% sucrose, 1% dimethyl sulfoxide, and 1 mM PMSF]. Membrane preparations obtained from 2 g of cells were diluted with assay buffer to 1 ml and tested for ethylene binding (11).
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13
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0344606659
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note
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4 trapped in the mercuric perchlorate was then determined as described (7).
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note
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4 incubation.
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0030712081
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GAF is an acronym for a domain present in guanosine 3′,5′-cydic monophosphate phosphodiesterases, Anabaena adenylate cyclases, and Escherchia coli FhlA [as described by L. Aravin and C. P. Ponting, Trends Biochem. Sci. 22, 458 (1997)].
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0021115563
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note
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4 in the extraction buffer. Ethylene-binding activity was solubilized as described by R. Serrano [FEBS Lett. 156, 11 (1983)], using a solubilization buffer consisting of 50 mM tris (pH 8.0) and 0.5% (w/v) α-lysophosphatidylcholine (LPC). Solubilized samples were purified by affinity chromatography as described (18) using buffers including 0.5% LPC (w/v) previously purified in Chelex-100 columns. The purified extracts were desalted through Sephadex G-25 columns.
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0345468719
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Samples were treated with 100 mM dithiothreitol and separated by SDS-polyacrylamide gel electrophoresis, and immunoblots were performed as described (6) using antibodies to GST (Sigma).
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27
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0030884102
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PAS is an acronym for a domain present in Drosophila Per, mammalian Arnt, and Drosophila Sim [as described by I. B. Zhulin, B. L. Taylor, R. Dixon, Trends Biochem. Sci. 22, 331 (1997)].
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Zhulin, I.B.1
Taylor, B.L.2
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0345037629
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Amino acid sequences were aligned by means of the clustal method with the PAM 250 residue weight table (Megalign-DNASTAR, DNASTAR, Madison, WI, 1993).
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0344174879
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We thank J. Spanbauer and A. Hahr for their contributions during the initial stages of this work; J. Burstyn and members of her laboratory for their helpful advice and assistance; and C. Chang and S. E. Patterson for their comments and help on the manuscript. Supported by NSF (grant 9513463), the U.S. Department of Energy (DOE) (grant DEFG02-91ER20029), and the DOE-NSF-USDA Collaborative Research in Plant Biology Program (grant DBI960-2222).
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