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An oligo selection procedure for identification of sequence‐specific DNA‐binding activities associated with the plant defence response
Author(s) -
Wang Zeping,
Yang Peizhen,
Fan Baofang,
Chen Zhixiang
Publication year - 1998
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1998.00311.x
Subject(s) - dna , biology , tobacco mosaic virus , genetics , dna binding site , computational biology , dna sequencing , dna replication , cloning (programming) , gene , population , transcription factor , microbiology and biotechnology , virus , gene expression , promoter , demography , sociology , computer science , programming language
Summary Sequence‐specific DNA‐binding (SSDB) factors play central roles in transcription, DNA replication, recombination and repair. This report describes a simple procedure for high‐throughput identification of SSDB activities without prior knowledge of their target genes or binding sequences. The procedure starts with a population of completely random oligo(nucleotide) sequences and selects for those oligo molecules that specifically bind to cellular SSDB factors by use of common gel‐retardation assays and PCR. Amplification and subsequent cloning of these selected oligo molecules result in the establishment of oligo DNA libraries enriched in DNA molecules containing specific sequences recognized by SSDB factors. These oligo libraries can be rapidly screened to identify a large number of SSDB activities, including those that are differentially regulated by developmental and environmental signals. With identified oligo DNA as probes, the corresponding SSDB factors can be isolated and analysed with respect to their structures, regulation and functions. Using this procedure, we have identified approximately 100 SSDB activities from tobacco leaves, including seven that are differentially regulated during the tobacco mosaic virus‐induced hypersensitive response in resistant tobacco plants.