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.
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/CIC2B9 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.
Seki
M
M
Laboratory of Plant Molecular Biology, Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), Japan.
Ito
T
T
Shibata
D
D
Shinozaki
K
K
D017360:Arabidopsis / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods*; D018244:Chromosomes, Artificial, Yeast; D003433:Crosses, Genetic; D004251:DNA Transposable Elements; D018076:DNA, Complementary; D005819:Genetic Markers; D018745:Genome, Plant; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods
D004251:DNA Transposable Elements; D018076:DNA, Complementary; D005819:Genetic Markers