DEV01400 Supplementary Material

DataThe molecular basis of the emf2-10 mutation is described in Fig. S1. To map the regions of CLF and SWN that interacted with FIE, we expressed the N-terminal 110 amino acids of MEA, SWN and CLF as ‘prey’ fusions to the Gal4 transcriptional activation (TA) domain. These were tested for interaction in yeast two-hybrid assays with a FIE bait previously described (Spillane et al., 2000). All three prey constructs were able to interact with MEA in yeast (Fig. 2S), although there is little sequence conservation between these regions of the MEA, CLF and SWN proteins.

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Molecular basis of emf2-10 mutation. (A) Structure of EMF2 locus showing location of primers (red arrows) used for RT-PCR analysis. Exons are shown as filled boxes, introns as lines. The location of the emf2-10 deletion (red triangle) relative to the first in-frame ATG codon is shown. (B) RT-PCR analysis of EMF2 transcripts in seedlings of wild-type progenitor and emf2-10. Three independent wild-type samples and two emf2-10 samples were analysed. A single transcript is detected in wild type, the same size (530 bp) as predicted from the EMF2 cDNA. At least five novel transcripts were detected in emf2-10, which lacked the wild-type product. (C) Sequencing of emf2-10 genomic DNA revealed a 17 bp lesion at the exon 2-intron 2 splice junction (highlighted with a yellow box) followed by a nucleotide substitution from cytosine to guanine. Sequence analysis of the emf2-10 RT-PCR products identified five novel splicing products. In the most common transcript, a cryptic 5¢-splice site splice junction in the untranslated region of exon 2 is used (green triangle) causing the ATG start site to be removed. It is likely that a downstream, in-frame ATG start codon in exon 3 is used instead (highlighted in black), resulting in a 20 amino acid truncation at the N terminus of EMF2. In three other classes of transcripts, alternative cryptic 5¢-splice sites are used (orange triangles), all of which are predicted to introduce frame-shift mutations near the 5¢ end of the coding sequence. In the final class of EMF2 transcript identified, intron 2 is not spliced, which is also predicted to cause premature termination of translation.

• Supplemental Figure 2 -

  Fig. S2. Interaction of FIE and Arabidopsis E(Z) homologues in yeast. Two hybrid assays were performed in yeast strain HF7c. For nutritional assays, four independent transformant were stamped onto –LW and –LWH media. Growth on –LW selects for markers carried on the bait and prey plasmids, whereas growth on –LWH also indicates activity of the HIS3 reporter gene. b-Galactosidase activity was quantified using the assay and units of Miller (Miller, 1992). Each value is an average from assays of three independent transformants, the s.e.m. is also indicated.