Direct targeting of light signals to a promoter element-bound transcription factor

Science

Volumn 288, Issue 5467, 2000, Pages 859-863

  Martínez García, Jaime F ^a,b   Huq, Enamul ^a,b   Quail, Peter H ^a,b  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b WESTERN REGIONAL RESEARCH CENTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

HELIX LOOP HELIX PROTEIN; PHYTOCHROME; TRANSCRIPTION FACTOR;

ARABIDOPSIS; ARTICLE; DNA SEQUENCE; GENE EXPRESSION; GENE TARGETING; NONHUMAN; PHOTORECEPTOR; PHOTOSTIMULATION; PRIORITY JOURNAL; TRANSCRIPTION REGULATION;

ARABIDOPSIS; ARABIDOPSIS PROTEINS; BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTORS; CELL CYCLE PROTEINS; DNA, PLANT; DNA-BINDING PROTEINS; GENE EXPRESSION PROFILING; GENE EXPRESSION REGULATION, PLANT; GENES, PLANT; HELIX-LOOP-HELIX MOTIFS; LIGHT; MODELS, GENETIC; OLIGODEOXYRIBONUCLEOTIDES; PHOTORECEPTORS; PHOTOSYNTHETIC REACTION CENTER COMPLEX PROTEINS; PHYTOCHROME; PHYTOCHROME B; PLANT PROTEINS; PROMOTER REGIONS (GENETICS); RECOMBINANT FUSION PROTEINS; SIGNAL TRANSDUCTION; TRANSCRIPTION FACTORS;

ARABIDOPSIS;

EID: 0041029474     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.288.5467.859     Document Type: Article

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42. 6-tag at the NH2-terminal end and was used throughout the work (referred to as PIF3 in the figures). G:bhPIF3 corresponds to GST (glutathione S-transferase) (Fig. 1D, gray box) fused to the PIF3 bHLH domain (from residue 340 to 397; cross-hatched box) The PIF3 bHLH domain was amplified by PCR and cloned in pGEX-4T-1. The resulting coding region was amplified with oligonucleotides that added the T7 promoter sequence upstream of the first ATG, and the PCR product was directly used as a template in the TnT reaction. The full-length Arabidopsis PHYB apoprotein was produced by TnT reaction, and the chromophore was autocatalytically attached (28).
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46. -1).
47. The selected LREs used [GTI, 4X (5′-TGTGTGGTTA-ATATG-3′); Z, 2X (5′-ATCATTCGTATACGTGTCAC-3′); G, 4X (5′-TGACACGTGGCA-3′); and GATA, 4X (5′-AAGATAAGATT-3′)] have been described elsewhere (33).
48. -1). Total RNA was isolated with the RNeasy Plant Miniprep kit (Qiagen). For RNA analyses, 5 μg of total RNA were loaded per lane, and then transferred to MSI Nylon membranes. The membranes were hybridized in Church buffer at 65°C overnight with random primer-labeled fragments (CCA1, LHY, CHS, SPA1). CCA1 and LHY probes were amplified by PCR from Arabidopsis DNA with the use of specific primers, cloned, and confirmed by sequencing. The CCA1 probe (amplified with the primers 5′-GCAGCTGCTAGTGCTTGGTGGGCT-3′ and 5′-TCA-TGTGGAAGCTTGAGTTTCCAA-3′) corresponded to positions 2082 to 3010 in the 3′ region of the main open reading frame (ORF) (38, 39); the LHY probe (amplified with the primers 5′-CATGCTGCAGCTACATTCGCT-GCT-3′ and 5′-TCATGTAGAAGCTTCTCCTTCCAATCG-3′) corresponded to positions 1271 to 2275 in the 3′ region of the main ORF (40). Southern blot analysis showed no detectable cross-hybridization between CCA1 and LHY probes under the washing conditions used (34). The SPA1 (14) and CHS [R. L. Feinbaum and F. M. Ausubel, Mol. Cell. Biol. 8, 1985 (1988)] probes have been described elsewhere.
49. Relative levels of transcripts were normalized to 18S ribosomal RNA levels (44) after Phosphorlmager Storm 860 (Molecular Dynamics) quantification.
50. We thank Y. Kang for technical assistance; M. Ni for A22 seeds and original PIF3 clones; C. Fairchild for the phycocyanobilin; U. Hoecker for SPA1 cDNA; N. Wei for the CHS probe; E. Monte and M. Rodriguez-Concepción for support and discussion; all the lab members for discussion and support; and the Arabidopsis Biological Resource Center (Columbus, Ohio) for providing hyS (hy5-1 allele) seeds. Supported by grants from the U.S. Department of Energy Basic Energy Sciences (DE-FG03-87ER13742) and U.S. Department of Agriculture Current Research Information Service (5335-21000-010-00D).