|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
| ||||||||||||||||||||
Files in this Data Supplement:
Fig. S1. Schematic representation of putative mutant proteins produced by new alleles of mea, fie, dme and msi1. (A) MEDEA is a SET domain protein composed of an acidic region (A), five conserved cysteines (C5) a putative nuclear localisation sequence (N), a cysteine-rich region (CXC) and a SET domain (SET) (Grossniklaus et al., 1998). In mea-5 mutant, a point mutation (T to G at position 5229 relative to the sequence in GenBank, AF096094) leads to cysteine substitution by a glycine inside the CXC domain. In mea-6, sequencing of MEA showed a 10 bp deletion in exon 3, at positions 3108-3117, and two point mutations, at positions 3096 (T to A) and 3105 (A to G). The deletion leads to a new Stop codon after 52 amino acids (aa). In mea-7, one point mutation has been sequenced in MEA gene at position 5760 (exon 15: T to A). This substitution creates a Stop codon at the very beginning of MEA SET domain. When aberrant aa are produced, the corresponding region is boxed. (B) FIE protein contains seven WD40 repeat domains (Ohad et al., 1999). fie-10 allele presents a single base deletion in exon 3 at position 11344 (A) on BAC MOE17 (AB025629) leading to a frameshift and production of a truncated protein of 130 aa. We detected in fie-11 a 5 bp deletion in the intron 3 at positions 11244-11248, associated with two point mutations, at positions 11241 (A to T) and 11242 (T to C). Netplantgene software (Hebsgaard et al., 1996), (www.cbs.dtu.dk/services/NetPGene/) predicts that this mutation creates an acceptor site for splicing 7 bp before the WT acceptor site, leading to a frameshift that creates a non-sense codon 27 aa after splicing. Both the FIE-10 and FIE-11 truncated proteins contain two intact WD40 repeat domains. (C) In DME, the DNA glycosylase domain (DNA glyc.) is followed by 4 conserved cysteine residues (C), and the protein contains a N-terminal basic region including the Nuclear Localisation Sequence (NLS) (Choi et al., 2002). After detection of a bandshift in the homozygous mutant PCR products for amplification of DME, sequencing the gene in dme-4 line revealed a point mutation (A to G) at position 72428 (on BAC T32M21 AL162875) and a 29 bp deletion, at positions 72429-72457. These mutations are located at the junction between intron 9 and exon 10. Netplantgene (Hebsgaard et al., 1996) predicts in this case a splicing 33 bp before deletion, allowing 7 aa translation before a stop codon. The putative truncated protein contains the full DNA glycosylase domain, but not the following cysteines. (D) MSI1 is, like FIE, a 7 WD40 repeats protein (Ach et al., 1997). We identified in msi1-2 1 base deletion in exon 1 of MSI1 (C at position 304 in AF016846), causing a frameshift and stop codon after 79 aa. This truncated protein does not contain any WD40 motif.
Movie 1. NCD migration at the posterior pole of wild-type endosperm. The endosperm endosplasmic reticulum is labelled with mGFP5 expressed under the control of the specific enhancer KS22 (Boisnard-Lorig et al., 2001). Hence each NCD is labelled individually and the cyst appears as a large fluorescent mass at the posterior pole (top of the picture). Confocal sections were acquired every 10 minutes. Time is indicated in hours and minutes. The seventh mitotic division takes place at time 1:20. After mitosis, NCDs migrate toward the cyst, which is followed by fusion of NCDs, indicated by red dots. Multiple fusion of NCDs produces multinucleate nodules that eventually fuse with the cyst.
Movie 2. Absence of NCD migration at the posterior pole of fis2-3 endosperm. The seed was selected as fis2-3 at the mid-globular stage on the basis of its strong fluorescence persistent in all domains of the endosperm provided by the activity of the enhancer KS117 that drives expression of mGFP5 (Sørensen et al., 2001). Confocal sections were acquired every 10 minutes. Time is indicated in hours and minutes. A mitotic division takes place at time 2:50. No posterior migration of NCDs is observed in comparison to wild type. A gap indicated by arrows appears between the cyst and peripheral NCDs. Unusually large nodules assemble as a result of ‘passive’ engulfing of NCDs by growing cytoplasm.
| ||||||||||||||||||||
