Journal Article

Regional Insertional Mutagenesis of Specific Genes on the CIC5F11/CIC2B9 Locus of <i>Arabidopsis thaliana Chromosome</i> 5 Using the <i>Ac</i>/<i>Ds</i> Transposon in Combination with the cDNA Scanning Method

Motoaki Seki, Takuya Ito, Daisuke Shibata and Kazuo Shinozaki

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 40, issue 6, pages 624-639
Published in print January 1999 | ISSN: 0032-0781
Published online January 1999 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/oxfordjournals.pcp.a029586
Regional Insertional Mutagenesis of Specific Genes on the CIC5F11/CIC2B9 Locus of Arabidopsis thaliana Chromosome 5 Using the Ac/Ds Transposon in Combination with the cDNA Scanning Method

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We have recently developed a novel cDNA selection method (the cDNA scanning method) to select cDNAs for expressed genes in specific regions of the genome [Hayashida et al. (1995) Gene 165: 155, Seki et al. (1997) Plant J. 12: 481]. The gene Ds is known to transpose mainly in its neighborhood. By combining the cDNA scanning method with this trait of Ds, we started functional analysis of region-specific expressed genes on the Arabidopsis thaliana genome. DNA fragments of yeast artificial chromosome (YAC) clones CIC5F11 and CIC2B9 on A. thaliana chromosome 5 were used for the selection of region-specific cDNAs. In total, 50 and 68 cDNA clones were selected from CIC5F11 and CIC2B9, respectively. In parallel, we transposed Ds from a donor T-DNA line, which was mapped on the CIC5F11/C1C2B9 locus of chromosome 5, and obtained Ds-transposed lines. To isolate Ds insertion mutants in the 10 specific genes identified by the cDNA scanning method, we carried out PCR-based screening of 100 Ds-transposed lines and found that 2 lines contain Ds mutations in the genes isolated. We also isolated Ds-flanking genomic DNAs by thermal asymmetric interlaced PCR (TAIL-PCR) in 153 Ds transposon-tagged lines. Southern blot analysis showed that 14% of the lines contained the transposed Ds in the CIC5F11/2B9 region. This suggests that this Ac/Ds transposon system is effective for region-specific insertional mutagenesis.

Keywords: Ac/Ds; cDNA selection; Functional analysis; Insertional mutagenesis; Reverse genetics; Transcriptional map

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Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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