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Cloning of the Arabidopsis RSF1 gene by using a mapping strategy based on high-density DNA arrays and denaturing high-performance liquid chromatography.
Mapping genes by chromosome walking is a widely used technique applicable to cloning virtually any gene that is identifiable by mutagenesis. We isolated the gene responsible for the recessive mutation rsf1 (for reduced sensitivity to far-red light) in the Arabidopsis Columbia accession by using classical genetic analysis and two recently developed technologies: genotyping high-density oligonucleotide DNA array and denaturing high-performance liquid chromatography (DHPLC). The Arabidopsis AT412 genotyping array and 32 F(2) plants were used to map the rsf1 mutation close to the top of chromosome 1 to an interval of approximately 500 kb. Using DHPLC, we found and genotyped additional markers for fine mapping, shortening the interval to approximately 50 kb. The mutant gene was directly identified by DHPLC by comparing amplicons generated separately from the rsf1 mutant and the parent strain Columbia. DHPLC analysis yielded polymorphic profiles in two overlapping polymorphic amplicons attributable to a 13-bp deletion in the third of five exons of a gene encoding a 292-amino acid protein with a basic helix-loop-helix (bHLH) domain. The mutation in rsf1 results in a truncated protein consisting of the first 129 amino acids but lacking the bHLH domain. Cloning the RSF1 gene strongly suggests that numerous phytochrome A-mediated responses require a bHLH class transcription factor.
Alleles, Amino Acid Sequence, Arabidopsis, Arabidopsis Proteins, Base Sequence, Basic Helix-Loop-Helix Transcription Factors, Chromatography, High Pressure Liquid, Chromosome Walking, Cloning, Molecular, DNA Primers, DNA, Complementary, Genome, Plant, Molecular Sequence Data, Nucleic Acid Denaturation, Oligonucleotide Array Sequence Analysis, Sequence Homology, Amino Acid, Transcription Factors
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