A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations.
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 of SuRA, 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.
Beetham
P R
PR
Boyce Thompson Institute for Plant Research, Cornell University, Tower Road, Ithaca, NY 14853-1801, USA.
Kipp
P B
PB
Sawycky
X L
XL
Arntzen
C J
CJ
May
G D
GD
D000095:Acetolactate Synthase / Q000235:genetics; D002478:Cells, Cultured; D018744:DNA, Plant / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D016368:Frameshift Mutation; D005786:Gene Expression Regulation; D018390:Gene Targeting / Q000379:methods; D017343:Genes, Plant / Q000235:genetics; D049452:Green Fluorescent Proteins; D006540:Herbicides; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics*; D010641:Phenotype; D010947:Plants, Toxic; D018749:RNA, Plant / Q000235:genetics*; D013449:Sulfonamides; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic; D019076:Transgenes; D014227:Triazines
D018744:DNA, Plant; D006540:Herbicides; D008164:Luminescent Proteins; D009841:Oligonucleotides; D018749:RNA, Plant; D013449:Sulfonamides; D014227:Triazines; D049452:Green Fluorescent Proteins; D000095:Acetolactate Synthase; C037137:chlorsulfuron