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In preliminary experiments, the optimal drug concentrations to employ in the deletion mutant screening were determined using the wild-type 972 h- and mutant rad3 strain because rad3 is hypersensitive to cisplatin (IC50 = 0.01 ± 0.009 μM) and 972 h- is the strain from which rad3 mutant was generated.
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EMS treatment and mutant screening was performed as described previously (Abe et al. 2012; Rao 1977).
A mutant screening was carried out previously to look for new genes related to the Cucumber mosaic virus infection response in Arabidopsis.
It is reassuring that most genes identified in the mutant screens were also found to be differentially expressed by microarray analysis.
As indicated above, only 4 out of the 16 genetic determinants identified in a transposon mutant screen were differentially expressed in our biofilm conditions.
The identification of genes through mutant screens is beginning to reveal the structure of a number of signaling pathways in plants.
However, gene identification via forward mutant screen is complex, as FN-induced mutagenesis hits multiple targets.
These 4 mutants, which represent key pathways identified in the mutant screens, are involved in colanic acid synthesis (rcsB), iron homeostasis (fur), DNA repair/SOS response (ruvA), and a small lipoprotein component of the outer membrane assembly complex (smpA).
A similar mutant screen was conducted as part of the work reported here.
The mutant screen was repeated three times with 10 plants per repeat.
The duration of antibiotic exposure in the mutant screen is critical.
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