The plant exon finder: a tool for precise detection of exons using a T-DNA-based tagging approach.
We have developed a new tool, named the plant exon finder (PEF), for identifying exons in plant genome sequences as an applied technique of T-DNA insertional mutagenesis. The T-DNA constructs contain a heat-shock gene promoter or the cauliflower mosaic virus (CaMV) 35S promoter, followed by the first exon of an Arabidopsis gene with its start codon and the intron donor sequence facing the T-DNA left border (LB) in order to trap exons in the genome. The constructs were used to make transgenic Arabidopsis plants. We generated 280 transgenic lines and identified 156 T-DNA-tagged readthrough transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) using an oligo(dT)-linker primer and a T-DNA-specific primer. Sequence analysis of the RT-PCR products revealed that 18 of them carried cDNAs processed by the use of an intron acceptor sequence adjacent to T-DNA insertion sites and 11 of them were in-frame fusions. In one case, the readthrough transcript trapped an exon located 1.6 kb downstream of the site of the insertion.
Jingu
Fumito
F
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.
Shirase
Toshiyuki
T
Ohtomo
Ichiro
I
Imai
Akihiro
A
Komeda
Yoshibumi
Y
Takahashi
Taku
T
D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D005091:Exons / Q000235:genetics*; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics; D018745:Genome, Plant; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010944:Plants / Q000235:genetics*; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; C446287:HSP18.2 protein, Arabidopsis; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; C036483:T-DNA