An oligo selection procedure for identification of sequence‐specific DNA‐binding activities associated with the plant defence response

• Published in: The Plant journal : for cell and molecular biology Vol. 16; no. 4; pp. 515 - 522
• Main Authors: Wang, Zeping, Yang, Peizhen, Fan, Baofang, Chen, Zhixiang
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.11.1998; Blackwell Science
• Subjects: Amino Acid Sequence; Arabidopsis - genetics; Base Sequence; Biological and medical sciences; Cell Nucleus - metabolism; Cloning, Molecular - methods; Conserved Sequence; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gene Library; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Nicotiana - genetics; Nicotiana - physiology; Nicotiana tabacum; Oligodeoxyribonucleotides - chemistry; Oligonucleotide Probes - chemistry; Pathology. Damages, economic importance; Phytopathology. Animal pests. Plant and forest protection; Plant viruses and viroids; Plants, Toxic; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Tobacco mosaic virus; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing; Sequence specificity; Plant pathogen; Nucleotide sequence; Hypersensitivity; DNA binding protein; Tobamovirus; Plant leaf; Gene expression; Nicotiana tabacum; Structure function relationship; Virus; Regulation(control); Microorganism resistance; Tobacco mosaic virus; Dicotyledones; Angiospermae; Isolation; Defense mechanism; Spermatophyta; Solanaceae; Transcription factor
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1046/j.1365-313x.1998.00311.x

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.