Preparation of transcriptionally active nuclei from etiolated Arabidopsis thaliana

被引:23
作者
Folta, KM [1 ]
Kaufman, LS [1 ]
机构
[1] Univ Illinois, Dept Biol Sci, Mol Biol Lab, Chicago, IL 60607 USA
关键词
Arabidopsis; nuclei; run-on; transcription; light regulation;
D O I
10.1007/s002990050764
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
Despite its emergence as the plant model system, there are few reports that describe protocols for the isolation of functional nuclei from Arabidopsis thaliana and their use in nuclear run-on assays or in preparation of nuclear extracts. This is especially true for etiolated seedlings. Here we report conditions, optimized for use in Arabidopsis, which allow for the isolation of enriched fractions of functional nuclei from less than 2 g of etiolated or light-grown tissue. The nuclei are capable of incorporating H-3-UTP into TCA-insoluble RNA, and also incorporate P-32-CTP into transcripts that can subsequently be hybridized to specific filter-bound DNA target sequences. The functional nuclei are sensitive to the transcriptional inhibitors actinomycin D and alpha-amanitin, confirming that the transcription observed is both template dependent and relies on nuclear polymerase II.
引用
收藏
页码:504 / 510
页数:7
相关论文
共 14 条
[1]   Blue light-directed destabilization of the pea Lhcb1*4 transcript depends on sequences within the 5′ untranslated region [J].
Anderson, MB ;
Folta, K ;
Warpeha, KM ;
Gibbons, J ;
Gao, J ;
Kaufman, LS .
PLANT CELL, 1999, 11 (08) :1579-1589
[2]   INCREASED RIBOSOMAL-RNA GENE ACTIVITY DURING A SPECIFIC WINDOW OF EARLY PEA LEAF DEVELOPMENT [J].
BAERSON, SR ;
KAUFMAN, LS .
MOLECULAR AND CELLULAR BIOLOGY, 1990, 10 (02) :842-845
[3]  
BRUSSLAN JA, 1993, PLANT CELL, V5, P667, DOI 10.1105/tpc.5.6.667
[4]   Regions of the pea Lhcb1*4 promoter necessary for blue-light regulation in transgenic Arabidopsis [J].
Folta, KM ;
Kaufman, LS .
PLANT PHYSIOLOGY, 1999, 120 (03) :747-755
[5]   LIGHT-STIMULATED TRANSCRIPTION OF GENES FOR 2 CHLOROPLAST POLYPEPTIDES IN ISOLATED PEA LEAF NUCLEI [J].
GALLAGHER, TF ;
ELLIS, RJ .
EMBO JOURNAL, 1982, 1 (12) :1493-1498
[6]  
GUILANO G, 1988, EMBO J, V7, P3635
[7]   BLUE-LIGHT REGULATION OF TRANSCRIPTION FOR NUCLEAR GENES IN PEA [J].
MARRS, KA ;
KAUFMAN, LS .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1989, 86 (12) :4492-4495
[8]  
MARRS KA, 1991, PLANTA, V183, P327, DOI 10.1007/BF00197729
[9]  
MILLAR AJ, 1991, PLANT CELL, V3, P541, DOI 10.1105/tpc.3.5.541
[10]   DEMONSTRATION OF TRANSCRIPTIONAL REGULATION OF SPECIFIC GENES BY PHYTOCHROME ACTION [J].
SILVERTHORNE, J ;
TOBIN, EM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1984, 81 (04) :1112-1116