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.

ABSTRACT: A rapid and quantitative DNA-binding assay was developed based on the translational fusion of a DNA-binding protein (DBP) with a Neocallimastix patriciarum beta-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 identif

SUBMITTER: Xue GP 

PROVIDER: S-EPMC137095 | biostudies-literature | 2002 Aug

REPOSITORIES: biostudies-literature