By Detlef Weigel; Jane Glazebrook
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Use disposable containers and pipettes and rinse them with 1 N NaOH before disposal. Use I N NaOH to wipe down all surfaces that may be contaminated with EMS, and then rinse them with water. Procedure 1. Place 5 , 0 0 0 - 5 0 , 0 0 0 seeds in a disposable container that has a volume at least 50 times that of the dry seed. A 50-ml Falcon tube w o r k s well. Add at least 25 volumes of 0 . 2 % EMS (v/v) in water. Use a fresh bottle of EMS. Seal the tube verv well with Parafilm. Rotate the tube end-over-end with a tube rotator, or lay it flat and rock it on a rocking platform for 15 hours.
Mapping FRI, a locus controlling flowrering time and vernalization response in Arabidopsis thaliana. Mol. Gen. Genet. 242:81-89. L. 1999. Distinct mechanisms promote polarity establishment in carpels oí Arabidopsis. Cell 99: 199-209. , and Moore I. 2000. The DNAbinding activity of GAL4 is inhibited by methylation of the GAE4 binding site in plant chromatin. Plant J. 23: 143-157. , and Waiden R. 1992. Activation of a plant gene by T-DNA tagging: Auxin-independent growth in vitro. Science 2 5 8 : 1 3 5 0 - 1 3 5 3 .
If the transformants display a phenotype similar to that of the original imitant, this confirms that the overexpressed gene is the cause of the dominant mutation. The activation-tagging vectors arc constructed in such a way that it is often possible to recover such a piece of genomic DNA by plasmid rescLie. Alternatively, a piece of wild-type genomic DNA that spans the gene of interest can be inserted into vectors that contain multimeric 35S enhancers, such as the pMN19 and pMN20 vectors (Weigel et al.
Arabidopsis : a laboratory manual by Detlef Weigel; Jane Glazebrook