Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (396K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Xue, G.-P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xue, G.-P.
Related Collections
Right arrow Protein-nucleic acid interaction
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2002, Vol. 30, No. 15 e77
© 2002 Oxford University Press

Characterisation of the DNA-binding profile of barley HvCBF1 using an enzymatic method for rapid, quantitative and high-throughput analysis of the DNA-binding activity

Gang-Ping Xue*

CSIRO Plant Industry, 120 Meiers Road, Indooroopilly, Brisbane, Qld 4068, Australia

*Tel: +61 7 3214 2763; Fax: +61 7 3214 2881; Email: gang.ping.xue{at}pi.csiro.au

A rapid and quantitative DNA-binding assay was developed based on the translational fusion of a DNA-binding protein (DBP) with a Neocallimastix patriciarum ß-1,4-D-glucanase, CelD. CelD releases a fluorescent 4-methylumbelliferyl product from 4-methylumbelliferyl cellobioside. This hydrolysis activity was used to quantify the amount of DBP–CelD bound to an immobilised biotin-labelled target sequence. The DNA-binding assay can be performed in a 96-well plate format for high- throughput analysis of putative DBPs. This method was applied to analysis of the binding properties and sequence selectivity of a cold-inducible transcription factor HvCBF1 from barley containing an AP2 DNA-binding domain. A base-scanning approach using degenerate oligonucleotide probes was employed for rapid identification of the conserved core motif of the HvCBF1 binding site. Quantitative analysis of the binding site of HvCBF1 using systematic base substitution revealed that a (G/a) (C/t)CGAC sequence was sufficient to constitute a functional motif, where the lower-case letters represent less efficient bases. The method enables us to provide accurate and quantitative data on a comprehensive DNA-binding profile for a cold-inducible AP2 transcription factor as well as information on environmental parameters potentially influencing the DNA-binding activity. The accurate binding sequence data facilitate identification of candidate genes regulated by HvCBF1 from genome sequence databases.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Plant Physiol.Home page
J. T. Svensson, C. Crosatti, C. Campoli, R. Bassi, A. M. Stanca, T. J. Close, and L. Cattivelli
Transcriptome Analysis of Cold Acclimation in Barley Albina and Xantha Mutants
Plant Physiology, May 1, 2006; 141(1): 257 - 270.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
Y. Jiang, F. Qin, Y. Li, X. Fang, and C. Bai
Measuring specific interaction of transcription factor ZmDREB1A with its DNA responsive element at the molecular level
Nucleic Acids Res., July 12, 2004; 32(12): e101 - e101.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.