High-frequency T-DNA-mediated gene tagging in plants.

Abstract

An insertion element [transferred DNA (T-DNA)], transferred by soil agrobacteria into the nuclear genome of plants, was used for induction of gene fusions in Arabidopsis thaliana, Nicotiana tabacum, and Nicotiana plumbaginifolia. A promoterless aph(3')H (aminoglycoside phosphotransferase II) reporter gene was linked to the right end of the T-DNA and transformed into plants along with a plasmid replicon and a selectable hygromycin-resistance gene. Transcriptional and translational reporter gene fusions were identified by screening for APH(3')ll enzyme activity in diverse tissues of transgenic plants. The frequency of gene fusions, estimated by determination of the copy number of T-DNA insertions, showed that on average 30% of T-DNA inserts induced gene fusions in Arabidopsis and Nicotiana. Gene fusions were rescued from plants by transformation of the T-DNA-linked plasmid and flanking plant DNA into Escherichia coli. By dissection of gene fusions and construction of chimeric genes, callus-and root-specific promoters were identified that showed an altered tissue specificity in the presence of a 3'-downstream-located 35S promoter. Transcript mapping of a gene fusion and expression of a non-frame transcriptional fusion of bacterial luciferase luxA and luxB genes demonstrated that dicistronic transcripts are translated in tobacco.

Promoter tagging with a promoterless ipt gene leads to cytokinin‐induced phenotypic variability in transgenic tobacco plants: implications of gene dosage effects

“…The high percentage of clones that show morphological symptoms of cytokinin overproduction (21%) indicates that most of the promoter-tagging events led to phenotypic alterations. Koncz et al (1989) who used a similar vector reported that 24% of transformed tobacco clones contained detectable transcriptional reporter gene fusions. However, we cannot exclude that a positive selection for cytokinin-synthesizing clones occurred, nor that some tagging events were not detected because the transcription was too low or expression occurred in cytokinin-insensitive tissues.…”

Identification of molecular markers of embryogenesis in Arabidopsis thaliana by promoter trapping

“…Chemical mutagenesis studies have greatly improved our understanding of the genetic control of embryo morphogenesis in Arabidopsis (Mayer et al, 1991(Mayer et al, , 1993Meinke, 1986), but T-DNA-mediated gene tagging has the advantage of facilitating gene cloning. Promoter trapping has the further advantage over other mutagenic treatments in that it does not rely necessarily on the identification of mutant phenotypes (which may be low-frequency events) fop successful gene tagging, since the generation of functional gene fusions in situ allows the detection of tagged regulatory elements at high frequency (Koncz et al, 1989;. To date, however, no embryonic genes have been successfully isolated by the use of T-DNA tags (Errampalli et al, 1991;Meinke, 1992;Patton et al, 1991).…”

An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration

“…The incoming T-DNA may have to interact with this nucleosome structure during the integration process. However, T-DNA may preferentially integrate into transcribed regions of the genome (32,33). These regions are believed to be temporarily free of histones.…”