A simple and rapid method for nuclear run-on transcription assays in plants

Plant Molecular Biology Reporter - Tập 21 - Trang 65-71 - 2012
Ling Meng1, Peggy G. Lemaux1
1Department of Plant and Microbial Biology, University of California, Berkeley, USA

Tóm tắt

Nuclear run-on transcription assays allow researchers to determine if a gene is regulated at the transcriptional level. Existing methods to perform this procedure involve the application of a large variety of biochemical reagents, numerous steps, and time-consuming procedures. Here we report a method to perform this assay on plant tissue that involves a minimal number of reagents and steps, is time-efficient, and produces clean definitive results within 24 h.

Tài liệu tham khảo

Breukelen FV, Maier R, and Hand S (2000) Depression of nuclear transcription and extension of mRNA half-life under anoxia inArtemia franciscana embryos. J Exp Biol 203: 1123–1130. Chen ZJ and Pikaard CS (1997) Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar dominance. Genes and Devel 11(16): 2124–2136. Christensen AH and Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5: 1–6. Clark RA, Li SL, Pearson DW, Leidal KG, Clark JR, Denning GM, Reddick R, Krause KH, and Valente AJ (2002) Regulation of calreticulin expression during induction of differentiation in human myeloid cells. Evidence for remodeling of the endoplasmic reticulum. J Biol Chem 277(35): 32369–32378. Cox KH and Goldberg RB (1988) Analysis of plant gene expression. In: Shaw CH (ed), Plant Molecular Biology: A Practical Approach, pp 1–35, IRL press, Oxford. Gaal T, Bartlett MS, Ross W, Turnbough CL, and Gourse RL (1997) Transcription regulation by initiating NTP concentration: rRNA synthesis in bacteria. Science 278: 2092–2097. Grummt I and Grummt F (1976) Control of nucleolar RNA synthesis by the intracellular pools of ATP and GTP. Cell 7: 447–453. Gubler F, Chandler PM, White RG, Llewellyn DJ, and Jacobsen JV (2002) Gibberellin signaling in barley aleurone cells. Control of SLN1 and GAMYB expression. Plant Physiol 129: 191–200. Hamilton RH, Kunsch U, and Temperli A (1972) Simple rapid procedures for the isolation of tobacco leaf nuclei. Anal Biochem 49: 48–57. Korn T, Kühlkamp T, Track C, Schatz I, Baumgarten K, Gorboulev V, and Koepsell H (2000) The plasma membrane-associated protein RS1 decreases transcription of the transporter SGLT1 in confluent LLC-PK1 cells. J Biol Chem 276: 45330–45340. Lohr D and Ide GI (1983)In vitro initiation and termination of ribosomal RNA transcription in isolated yeast nuclei. J Bio Chem 258: 4668–4671. Rees BB, Ropson I, and Hand SC (1989) Kinetic properties of hexokinase under near-physiological conditions. Relation to metabolic arrest inArtemia embryos during anoxia. J Biol Chem 264: 15410–15417. Strand R, Boe R, and Flatmark T (1994) The choice of resuspension medium for isolated rat liver nuclei: effects on nuclear morphology andin vitro transcription. Mol Cell Biochem 139(2): 149–157. Thompson WF, Beven AF, and Shaw PJ (1997) Sites of rDNA transcription are widely dispersed through the nucleus inPisum sativun and can comprise single genes. Plant J 12: 571–581. Wan Y and Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104: 37–48. Wang H, Jiang X, Yang F, Chapman GB, Durante W, Sibinga NE, Schafer AI, and Cyclin A (2002) Transcriptional suppression is the major mechanism mediating homocysteine-induced endothelial cell growth inhibition. Blood 99(3): 939–45. Weber J, Jelinek W, and Darnell JE (1977) The definition of a large viral transcription unit late in Ad2 infection of HeLa cells: Mapping of nascent RNA molecules labeled in isolated nuclei. Cell 10: 611–616. Zhang MY, Wang X, Wang JT, Compagnone NA, Mellon SH, Olson JL, Tenenhouse HS, Miller WL, and Portale AA (2002) Dietary phosphorus transcriptionally regulates 25-hydroxyvitamin D-1 alpha-hydroxylase gene expression in the proximal renal tubule. Endocrinol 143(2): 587–595.