Repositorium
A tool for functional plant genomics: Chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations
167
Journal Article / 1999
PETER R. BEETHAM,PETER B. KIPP,XENIA L. SAWYCKY,CHARLES J. ARNTZEN, GREGORY D. MAY
Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site-specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 ofSuRA, a tobacco acetolactate synthase (ALS) gene. An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) flanked by regions each composed of 10 ribonucleotides. After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196. To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green fluorescent protein transgene that had been designed to contain a single base deletion. Recovery of fluorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
359 |
May, Gregory D. USA |
Nicotiana tabacum |
Oligonucleotide-directed mutagenesis ALS |
herbicide tolerance PM |
Basic research Basic research |
360 |
May, Gregory D. USA |
Nicotiana tabacum |
Oligonucleotide-directed mutagenesis GFP |
green fluorescence protein expression PM |
Basic research Basic research |