Chloroplast lysates support directed mutagenesis via modified DNA and chimeric RNA/DNA oligonucleotides.
Plant J
; 27(3): 267-74, 2001 Aug.
Article
em En
| MEDLINE
| ID: mdl-11532172
ABSTRACT
Chimeric RNA/DNA and modified DNA oligonucleotides have been shown to direct gene-conversion events in vitro through a process involving proteins from several DNA-repair pathways. Recent experiments have extended the utility of these molecules to plants, and we previously demonstrated that plant cell-free extracts are competent to support oligonucleotide-directed genetic repair. Using this system, we are studying Arabidopsis DNA-repair mutants and the role of plant proteins in the DNA-repair process. Here we describe a method for investigating mechanisms of plastid DNA-repair pathways. Using a genetic readout system in bacteria and chimeric or modified DNA oligonucleotides designed to direct the conversion of mutations in antibiotic resistance genes, we have developed an assay for genetic repair of mutations in a spinach chloroplast lysate system. We report genetic repair of point and frameshift mutations directed by both types of modified oligonucleotides. This system enables the mechanistic study of plastid gene repair and facilitates the direct comparison between plant nuclear and organelle DNA-repair pathways.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cloroplastos
/
Mutagênese
/
RNA de Plantas
/
DNA de Plantas
/
Hibridização de Ácido Nucleico
Idioma:
En
Revista:
Plant J
Ano de publicação:
2001
Tipo de documento:
Article