Measurement of transcription rates in Arabidopsis chloroplasts.
Methods Mol Biol
; 774: 171-82, 2011.
Article
em En
| MEDLINE
| ID: mdl-21822839
ABSTRACT
The regulation of gene expression is still one of the major issues in modern plant molecular biology. The amount of RNA in a cell is regulated by both transcriptional and posttranscriptional events. Methods to determine these steady-state levels of RNAs, such as Northern analysis, ribonuclease protection assay (RPA), and quantitative real-time PCR, do not discriminate between regulation by de novo RNA synthesis and the influence by degradation or stabilization. To assess the rate of transcription of individual genes, run-on transcription is utilized. To this end, isolated chloroplasts are used in brief in vitro transcription reactions in the presence of radiolabeled nucleotides, with a subsequent hybridization of the isolated RNA with DNA fragments spotted on membranes. Here, we describe a protocol for run-on transcription in chloroplasts isolated from Arabidopsis leaves and present data on the transcriptional activity of several plastid genes in detached leaves of different Arabidopsis ecotypes.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Transcrição Gênica
/
Cloroplastos
/
Técnicas Genéticas
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Arabidopsis
/
RNA de Cloroplastos
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Alemanha