Abstract
We present a mini-scale method for nuclear run-on transcription assay. In our method, all the centrifuge steps can be carried out by using micro-tubes for short time (5 min each) throughout the process, including isolation of transcriptionally active nuclei and purification of labeled RNA after synthesis of RNA in isolated nuclei. The assay can be performed using a small amount of plant tissue, which enables analysis of developmental changes in transcriptional status of given genes in a single individual plant. Successful results were obtained using the tissues of flower and leaf of petunia and embryo of pea, suggesting that the method is potentially applicable to a variety of plant tissues.
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Abbreviations
- CaMV:
-
cauliflower mosaic virus
- CHS-A:
-
chalcone synthase A
References
Celano P, Berchtold C and Casero Jr A (1989) A simplification of the nuclear run-off transcription assay. Biotech 7: 942–943.
Cox KH and Goldberg RB (1988) In: Shaw CH (ed), Plant Molecular Biology: A Practical Approach. pp 1–35, IRL press, Oxford.
Greenberg ME (1988) Identification of newly transcribed RNA. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Smith JA Seidman JG and Strahl K (eds), Current Protocols in Molecular Biology. John Wiley and Sons, New York.
Hamilton RH, Kunsch U and Temperli A (1972) Simple rapid procedures for isolation of tobacco leaf nuclei. Anal Biochem 49: 48–57.
Higgins TJV, Chandler PM, Randall RJ, Spencer D, Beach LR, Blagrove RJ, Kortt AA and Inglis AS (1986) Gene structure, protein structure and regulation of the synthesis of sulphur-rich protein in pea seeds. J Biol Chem 261: 11124–11130.
Higgins TJV, Beach LR, Spencer D, Chandler PM, Randall PJ, Blagrove RJ, Kortt AA and Guthrie RE (1987) cDNA and protein sequence of a major pea seed albumin (PA2: Mr∼26000). Plant Mol Biol 8: 37–45.
Luthe DS and Quatrano RS (1980) Transcription in isolated wheat nuclei. II. Characterization of RNA synthesizedin vitro. Plant Physiol 65: 309–313.
Marzluff WF (1978) Transcription of RNA in isolated nuclei. Methods Cell Biol 19: 317–331.
Marzluff WF and Huang RCC (1985) In: Hames BD and Higgins SJ (eds), Transcription and Translation: A Practical Approach. pp 89–129, IRL Press, Oxford.
Metzlaff M, O’Dell M, Cluster PD and Flavell RB (1997) RNA-mediated RNA degradation and chalcone synthase A silencing in petunia. Cell 88: 845–854.
O’Dell M, Metzlaff M and Flavell RB (1999) Post-transcriptional gene silencing of chalcone synthase in transgenic petunias, cytosine methylation and epigenetic variation. Plant J 18: 33–42.
Thompson WF, Beven AF and Shaw PJ (1997) Sites of rDNA transcription are widely dispersed through the nucleolus inPisum sativum and can comprise single genes. Plant J 12: 571–581.
Van Blokland R, Van der Geest N, Mol JNM and Kooter JM (1994) Transgene-mediated suppression of chalcone synthase expression inPetunia hybrida results from an increase in RNA turnover. Plant J 6: 861–877.
Van der Krol A, Mur LA, Beld M, Mol JNM and Stuitje AR (1990) Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2: 291–299.
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Kanazawa, A., O’Dell, M., Hellens, R.P. et al. Mini-scale method for nuclear run-on transcription assay in plants. Plant Mol Biol Rep 18, 377–383 (2000). https://doi.org/10.1007/BF02825066
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DOI: https://doi.org/10.1007/BF02825066