SPA1, a WD-repeat protein specific to phytochrome A signal transduction

Science

Volumn 284, Issue 5413, 1999, Pages 496-499

  Hoecker, Ute ^a   Tepperman, James M ^a   Quail, Peter H ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

AMINO ACID SEQUENCE; ARTICLE; GENE EXPRESSION REGULATION; GENETIC ANALYSIS; MOLECULAR CLONING; MORPHOGENESIS; NONHUMAN; NUCLEOTIDE SEQUENCE; PHOTOCHEMISTRY; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN LOCALIZATION; SENSORY SYSTEM; SEQUENCE ANALYSIS; SIGNAL TRANSDUCTION;

AMINO ACID SEQUENCE; ARABIDOPSIS; ARABIDOPSIS PROTEINS; CELL CYCLE PROTEINS; CELL NUCLEUS; CLONING, MOLECULAR; DARKNESS; GENE EXPRESSION REGULATION, PLANT; LIGHT; MOLECULAR SEQUENCE DATA; MORPHOGENESIS; MUTATION; NUCLEAR LOCALIZATION SIGNALS; PHYTOCHROME; PHYTOCHROME A; PLANT PROTEINS; PROTEIN KINASES; REPETITIVE SEQUENCES, AMINO ACID; REPRESSOR PROTEINS; RNA, MESSENGER; SEQUENCE ALIGNMENT; SIGNAL TRANSDUCTION;

ARABIDOPSIS THALIANA;

EID: 0033574739     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5413.496     Document Type: Article

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40. The Nla coding region in the plasmid pRTL2-GUS/ NlaΔBam (23) was replaced by an expanded multiple cloning site: Bgl II-Cla I-Not I-Sal I-BamH I-Xho I. The SPA1 cDNA clone was amplified with primers to create a Cla I site at the ATG and a Sal I site at the TAG, digested with these enzymes, and inserted into the modified pRTL2-GUS/NlaΔBam plasmid to create GUS:SPA1. This plasmid was then digested with Bgl II to remove a fragment containing the first 1773 bp of the SPA1 coding region and religated to create GUS: SPA1ΔN591.
41. Total RNA was extracted from 3-day-old seedlings that had been transferred to light for the indicated time using the Qiagen RNeasy Plant Miniprep kit. We separated 5 μg of total RNA on a Mops-RNA gel containing 6.7% formaldehyde and subsequently transferred the RNA to MSI Nylon membrane. The membranes were hybridized with a random prime-labeled PCR product containing the full-length SPA1 cDNA and washed with a final wash with 0.2X saline sodium citrate and 0.1% SDS at 65°C. Membranes were subsequently hybridized with a pea 185 rRNA probe.
42. We thank Y. Kang and S. Moran for excellent technical assistance, M. Hudson for critical reading of the manuscript, D. Aubert for providing aliquots of a binary cosmid library, and the Arabidopsis Biological Resource Center in Ohio for providing seeds, a cDNA library, and clones. Supported by NIH grant GM-47475 and the U.S. Department of Agriculture, Current Research Information Service number 5335-21000-006-00D.