JCS027862 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Sequence comparison of EST1 proteins. (A) Multiple ClustalW alignment of EST1-TPR domains. The following protein sequences were used for the alignment: Caenorhabditis elegans CeEST1 (AAK27880), Drosophila melanogaster DmEST1 (CAD89221), human hSMG6/EST1A (AAN46114), hSMG5/EST1B (NP_056142), hSMG7/EST1C (NP_775179), Xenopus tropicalis XtEST1C (AAH63908), Arabidopsis thaliana AtSMG7L (AAF98429), AtSMG7 (NP_197441), Oryza sativa OsCIG3 (BAD30942), Schizosaccharomyces pombe SpEst1-like (CAB10152), SpEst1 (CAA21171) and Saccharomyces cerevisiae Est1p (NP_013334) and Ebs1p (NP_010492). Putative tertratricopeptide repeats (TPR) are indicated. The alignment of the EST1 domains was performed with BioEdit Sequence Alignment Editor (Hall, 1999). The following amino acid color code is used: green Y, F, A, I, L, M, V, pale green W, violet G, yellow P, red S, D, E, T, blue R, N, K, pale blue H, gray Q and brown C. (B) Conserved regions between A. thaliana SMG7 and SMG7L proteins and human SMG6 and SMG7. Identities and similarities are indicated for each region. Nuclease PIN domain is indicated in red, EST1-TPR domain with putative TPR motives are depicted in blue and green, respectively, and EST1-CD is indicated in orange. (C) Phylogenetic relationship of plant SMG7 proteins. Selected homologues were aligned using Probcons (Do et al., 2005) and the phylogenetic tree was prepared with PHYLO_WIN (Galtier et al., 1996) using the neighbor-joining method with observed divergence distances. The scale bar corresponds to 0.051 estimated amino acid substitutions per site. The following sequences were used for the alignment: hSMG5, hSMG6, hSMG7, AtSMG7, AtSMG7L, OsCIG3, Nicotina tabacum NtCIG3 (BAB82502), Medicago truncatula MtCIG3 (ABD32367), Hordeum vulgare HvSMG7 (AK248878), Vitis vinifera VvCIG3 (CAN78121). V. vinifera VvSMG7 and M. truncatula MtSMG7 were derived from unannotated genome sequences (AM465968 and CT033769, respectively). Populus trichocarpa PpSMG7 and PpSMG7L, and Physcomitrella patens PpSMG7 sequences were obtained from the land plant genome sequence database “Phytozome” (http://dev.phytozome.net/index.php).

• Supplemental Figure S2 -

  Fig. S2. Molecular characterization of smg7-1, smg7-3 and smg7-5 alleles. (A) Schematic representation of the SMG7 gene with smg7-1, smg7-3 and smg7-5 insertions. Position of primers used for characterization of the alleles is indicated by arrowheads. (B) The smg7-1 locus consists of at least two T-DNAs that are inserted in a head-to-head orientation. Sequence analysis of the insertion junctions revealed filler sequences at both the 5′ and 3′ ends of the insertion (shown in red). LB repeats derived from T-DNA borders are indicated. (C) The smg7-3 locus represents an insertion composed of one T-DNA, with the LB oriented towards the 5′ end of SMG7, and the RB oriented towards the 3′ end of SMG7. Approximately 33 nt of LB sequence was located immediately between the RB sequence and the 5′ end of SMG7, with 5 nt of microhomology at the RB-LB junction. Sequence analysis of the insertion revealed microhomology (shown in green) at the 5′ end of the insertion, and filler sequences (red) at the 3′ end of the T-DNA insertion.

• Supplemental Figure S3 -

  Fig. S3. Structure of the meiosis 1 spindle in wild-type and smg7-1 mutant plants. Microtubules detected by indirect immunolabeling with an antibody against α-tubulin are stained green. DNA is counterstained with DAPI (red). Bars: 5 µm.

• Supplemental Figure S4 -

  Fig. S4. Histone H3 phosphorylation on the Ser10 residue (green signals) in the course of meiosis in wild-type and smg7-1 mutants. DNA is counterstained with DAPI (red). The figures represent meiotic stages: M1 (A,G), T1 (B,H), M2 (C,I), A2 (D,E), T2 (F), irregular A2 (J), scattered chromatids (K) and polyad (L).

• Supplemental Figure S5 -

  Fig. S5. Anaphase 2 in megaspore mother cells. Regular A2 in a wild-type MMC contains four compact clusters of chromatin each representing 5 chromatids (indicated by arrowheads in left panel). The smg7-1 mutation causes random distribution of separated chromatids in MMCs (indicated by arrowheads in the middle and right panels). Chromosomes were stained with DAPI and analyzed by deconvolution microscopy. Bars: 5 µm.