Employing libraries of zinc finger artificial transcription factors to screen for homologous recombination mutants in Arabidopsis.
A library of genes for zinc finger artificial transcription factors (ZF-ATF) was generated by fusion of DNA sequences encoding three-finger Cys(2)His(2) ZF domains to the VP16 activation domain under the control of the promoter of the ribosomal protein gene RPS5A from Arabidopsis thaliana. After introduction of this library into an Arabidopsis homologous recombination (HR) indicator line, we selected primary transformants exhibiting multiple somatic recombination events. After PCR-mediated rescue of ZF sequences, reconstituted ZF-ATFs were re-introduced in the target line. In this manner, a ZF-ATF was identified that led to a 200-1000-fold increase in somatic HR (replicated in an independent second target line). A mutant plant line expressing the HR-inducing ZF-ATF exhibited increased resistance to the DNA-damaging agent bleomycin and was more sensitive to methyl methanesulfonate (MMS), a combination of traits not described previously. Our results demonstrate that the use of ZF-ATF pools is highly rewarding when screening for novel dominant phenotypes in Arabidopsis.
Lindhout
Beatrice I
BI
Clusius Laboratory, Department of Molecular and Developmental Genetics, Institute of Biology Leiden, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.
Pinas
Johan E
JE
Hooykaas
Paul J J
PJ
van der Zaal
Bert J
BJ
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D001761:Bleomycin / Q000494:pharmacology; D017931:DNA Primers; D009154:Mutation; D011995:Recombination, Genetic; D014157:Transcription Factors / Q000235:genetics*; D016335:Zinc Fingers
D017931:DNA Primers; D014157:Transcription Factors; D001761:Bleomycin