Evaluation of a cDNA scanning method concerning the fidelity and efficiency of cDNA selection using the YAC CIC3B1-S region of Arabidopsis thaliana chromosome 5.
We previously reported a cDNA selection method using DNA latex particles to identify expressed genes in specific regions of genomes and named this cDNA scanning method (Hayashida et al., 1995 Gene 155 161). We applied the cDNA scanning method to the YAC CIC3B1-S DNA on Arabidopsis thaliana chromosome 5, and constructed a region-specific sublibrary in which cDNAs for genes on the YAC CIC3B1-S DNA were concentrated. We isolated 545 cDNA clones from the sublibrary, and determined partial sequence of them to produce expressed sequence tags (ESTs) derived from the YAC region. In total, 74 nonredundant groups of cDNAs were obtained from 545 cDNA clones. Forty-seven percent of these EST clones had significant homology to functional proteins such as protein kinases, LON protease, nucleic acid binding protein and chloride channel protein. We compared the cDNA sequences isolated by the cDNA scanning method to the Arabidopsis genomic sequence corresponding to the YAC CIC3B1-S region, and found that 69% of the selected cDNAs are located in the region. We discuss the fidelity and efficiency of the cDNA scanning method for cloning region-specific cDNAs and its useful application in positional cloning.
Motohashi
R
R
Laboratory of Plant Molecular Biology, Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), Ibaraki, Japan.
Ito
T
T
Seki
M
M
Ichimura
K
K
Yamaguchi-Shinozaki
K
K
Shinozaki
K
K
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D020224:Expressed Sequence Tags; D008969:Molecular Sequence Data; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary