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Volumn 285, Issue 5427, 1999, Pages 582-584

Transcriptional activation of APETALA1 by LEAFY

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; GENE EXPRESSION REGULATION; GROWTH REGULATION; NONHUMAN; PLANT GROWTH; PRIORITY JOURNAL; PROTEIN SYNTHESIS REGULATION; TRANSCRIPTION REGULATION;

EID: 0033597851     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5427.582     Document Type: Article
Times cited : (380)

References (31)
  • 11
    • 0345227070 scopus 로고    scopus 로고
    • note
    • This system allows us to posttranslationally activate the fusion protein without nonspecific effects on the plant because the fusion protein is held in the cytoplasm until hormone treatment and because the treatment itself has no apparent effects on Arabidopsis [our data and (20, 24, 27)].
  • 12
    • 0345227069 scopus 로고    scopus 로고
    • note
    • 2-terminal extension consisting of a FLAG epitope and a HMK site. Both constructs were introduced into the ter ecotype of Arabidopsis. 355:LFY exhibited the same gain-of-function phenotypes as previously described (3). AP3::GUS was created by transformation of Ler with the plasmid described in (28) (J. L. Riechmann and E. M. Meyerowitz, unpublished data).
  • 13
    • 0344796167 scopus 로고    scopus 로고
    • note
    • Polyclonal antibodies against LFY were produced in rabbits with recombinant hexa-histidine-tagged LFY protein after Nickel-NTA column (Qiagen, Santa Clarita, CA) purification and size separation on denaturing gels. Crude serum was affinity purified against recombinant glutathione S-transferase-tagged LFY protein. Later bleeds of the crude serum were indistinguishable from the affinity-purified antibody and were used at 1:2000 dilution on immunoblots and at 1:6000 dilution in immunolocalization experiments. Immunotocalization was as in (15).
  • 14
    • 0345658927 scopus 로고    scopus 로고
    • note
    • Plants were grown at 18°C in continuous light For immunolocalization and GUS analyses, plants were treated as described for the in situ analyses. In situ protocols were previously described (29). The probes used were as in (19) (AP1) and (3) (LFY). To monitor AP1 induction at the transition to flowering, we removed the central shoot and treated young axillary inflorescences before visible bolting. In addition, 15-to 17-day-old seedlings were treated, and after sectioning, primary shoots that had just started to initiate flowers were mounted for in situ hybridization. For LFY-GR activation before the floral transition, seedlings were 8 or 11 days old. Inflorescences and seedlings were treated twice, at time 0 and again after 4 hours. They were harvested 6 or 8 hours after the first treatment and processed immediately. The hormone or protein synthesis inhibitor, or both, was applied to inflorescences in ethanol and 0.015% Silwet L-77 (OSI Specialties, Danbury, CT) for increased penetration, and seedlings were submerged in hormone or protein synthesis inhibitor, or both, in 0.1% ethanol. The primers used for RT-PCR were as follows: AP1 5′, GCAATGAGCCCTAAAGAGCT-TCAG; AP1 3′, CATGTAAGGATGCTGGATTTGGTGC; UB 5′, GGTGCTAAGAAGAGGAAGAAT; and UB 3′, CTC-CTTCTTTCTG-GTAAACGT. Products were in the linear range of the response as determined by phosphorimager quantitation (with 22 cycles for AP1 and 20 cycles for ubiquitin amplification).
  • 22
    • 0345227066 scopus 로고    scopus 로고
    • note
    • These findings suggest that after the initial induction of AP1 by LFY, full levels of AP1 expression require either the presence of a preexisting, unstable protein or de new protein synthesis. Alternatively, prolonged inhibition of protein synthesis (14) may cause depletion of general transcription machinery proteins and explain the failure to see high levels of AP1 expression in the presence of cycloheximide.
  • 31
    • 0344364342 scopus 로고    scopus 로고
    • note
    • We thank J. Fletcher, J. D. Wagner, and D. Weigel for stimulating discussion of the manuscript and C. C. Baker, X. Chen, T. Ito, C. Ohno, and E. Ziegelhoffer for critical review of the manuscript. Supported by the Helen Hay Whitney Foundation (D.W.) and U.S. Department of Energy grant FG03-88ER13873 (E.M.M.).


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.