High-affinity potassium uptake in plants.

نویسندگان

  • N A Walker
  • D Sanders
  • F J Maathuis
چکیده

oocyte]. Oocytes were incubated for 24 to 48 hours, treated with coelenterazine [10 ,uM coelenterazine (Molecular Probes, Eugene, OR) and 30 p.M reduced glutathione in OR-2 media (no calcium)] for 2 to 3 hours with gentle orbital shaking in the dark at 18°C, and retumed to ND-86 medium with calcium (maintained in the dark with shaking) until measurements were initiated. For luminometer measurements, oocytes (singly or in pairs) were transferred to plastic tubes (75 mm by 12 mm, Sarstedt) containing 2.9 ml of Ca2+-free OR-2 medium. Each cRNA pool was tested in triplicate. Measurements (duration 2 min) were triggered by the injection of 0.1 ml of 30 p.M MK-0677. 19. Additional GHS-R clones from the swine cDNA library were identified by hybridization of the clone 7-3 32P-labeled insert to slot-blot pools of plasmid DNA (500 cDNAs per pool). Filters were prehybridized [at 42°C for 4 hours in 5x standard saline citrate (SSC) with 5x Denhardt's solution, 250 p.g/ml of tRNA, 1 % glycine, 0.075% SDS, 50 mM NaPO4 (pH 6), and 50% formamide], and hybridizations were done at 42°C for 20 hours in 5x SSC, with 1 x Denhardt's solution, 0.1 % SDS, 50 mM NaPO4, and 50% formamide. Clonal isolates were identified by colony hybridization. Human pituitary homologs of the swine GHS-R were obtained by screening a cDNA library [lambda ZAP II (Stratagene); -2 x 1 06 phages gave 21 GHS-R clones]. DNA sequencing was done on both strands [automated Applied Biosystems instrument (ABI model 373); manually by dideoxy chain termination (Sequenase version 2.0; U.S. Biochemical, Cleveland, OH)]. Database searches [GenBank 92, EMBL 43, Swiss-Prot 31, PIR 45, dEST (Gbest 92), and Prosite 12], sequence alignments, and analysis of the GHS-R nucleotide and protein sequences

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عنوان ژورنال:
  • Science

دوره 273 5277  شماره 

صفحات  -

تاریخ انتشار 1996