DOI: 10.1073/pnas.96.15.8774. A tool for functional plant genomics: Chimeric RNA/DNA oligonucleotides cause <i>in vivo</i> gene-specific mutations

A tool for functional plant genomics: Chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations

10.1073/pnas.96.15.8774

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

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.

Bibliography

Beetham, P. R., Kipp, P. B., Sawycky, X. L., Arntzen, C. J., & May, G. D. (1999). A tool for functional plant genomics: Chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations. Proceedings of the National Academy of Sciences, 96(15), 8774â8778.

Authors 5

Peter R. Beetham (first)
Peter B. Kipp (additional)
Xenia L. Sawycky (additional)
Charles J. Arntzen (additional)
Gregory D. May (additional)

References 14 Referenced 186

10.1093/genetics/149.2.473
10.1073/pnas.93.5.2071
10.1126/science.273.5280.1386
10.1038/nm0398-285
10.1038/4322
10.1093/nar/27.5.1323
10.1126/science.223.4641.1148
10.1126/science.224.4656.1443
10.1002/j.1460-2075.1988.tb02937.x
J R Bedbrook, R S Chalef, S C Falco, B J Mazur, C R Somerville, N S Yadav U.S. Patent 5, 013,659, 1991). / U.S. Patent 5, 013,659 by Bedbrook J R (1991)
10.1126/science.227.4691.1229
10.1016/S0960-9822(02)00483-9
10.1093/carcin/17.4.849
10.1016/0014-5793(93)80979-5

Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:41 a.m.)
Deposited	3 years, 4 months ago (April 13, 2022, 4:39 p.m.)
Indexed	4 weeks ago (Aug. 6, 2025, 9:22 a.m.)
Issued	26 years, 1 month ago (July 20, 1999)
Published	26 years, 1 month ago (July 20, 1999)
Published Online	26 years, 1 month ago (July 20, 1999)
Published Print	26 years, 1 month ago (July 20, 1999)

Funders 0

None

BibTeX

@article{Beetham_1999, title={A tool for functional plant genomics: Chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations}, volume={96}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.96.15.8774}, DOI={10.1073/pnas.96.15.8774}, number={15}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Beetham, Peter R. and Kipp, Peter B. and Sawycky, Xenia L. and Arntzen, Charles J. and May, Gregory D.}, year={1999}, month=jul, pages={8774–8778} }

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