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doi: 10.1242/10.1242/jcs.00048

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Asy1, a protein required for meiotic chromosome synapsis, localizes to axis-associated chromatin in Arabidopsis and Brassica

Susan J. Armstrong^*, Anthony P. Caryl^*, Gareth H. Jones and F. Christopher H. Franklin

School of Biosciences, The University of Birmingham, Birmingham B15 2TT, UK

View larger version (67K): [in a new window]   Fig. 1. (A) Nucleotide sequence of the Brassica oleracea BoASY1 cDNA including 5' and 3' untranslated regions. The deduced amino-acid sequence of the BoAsy1 protein is presented below the cDNA. These sequence data are available from GenBank/EMBL/DDBJ under accession no. AF410429. (B) Full-length alignment of the BoAsy1 and Asy1 proteins. Strictly conserved residues are highlighted in grey blocks, and gaps are indicated by dashes. Sequences were aligned with BLAST (Tatusova and Madden, 1999) using the BLOSUM62 comparison tables (Henikoff and Henikoff, 1992).

View larger version (77K): [in a new window]   Fig. 2. Expression of Asy1 and BoAsy1 proteins in Arabidopsis and B. oleracea, respectively. (A) Western analysis using the anti-Asy1 antibody of an Arabidopsis bud series from a single inflorescence. Lane 1 represents pooled small (pre-meiotic) buds. Lanes 2 through 12 represent buds of increasing size. Expression is first detected in lane 4 corresponding to early male meiosis. The signal then declines, rising again in lane 10, which corresponds to early female meiosis (late male meiosis). (B) Western analysis using an anti-Asy1 antibody of a meiotically staged anther series and vegetative tissues from B. oleracea. Lanes 1-6 represent interphase, leptotene, zygotene, pachytene, diplotene and tetrad stage, respectively. Lane 7, stem; lane 8, leaf.

View larger version (74K): [in a new window]   Fig. 3. Fluorescence immunolocalization of Asy1 antibody (green) to Arabidopsis PMCs, counterstained with DAPI. (A,B) Semi-thin wax-embedded anther sections showing immunolocalization to PMCs at pachytene (A) and absence of signal at the tetrad stage (B). Bar, 10 µm.

View larger version (29K): [in a new window]   Fig. 4. Immunolocalization of Asy1 antibody (green) to spread Arabidopsis PMCs, counterstained with DAPI. (A) Interphase/early leptotene showing numerous punctate foci. (B,C) Zygotene and pachytene showing continuous signals along chromosomal axes. Bar, 10 µm.

View larger version (75K): [in a new window]   Fig. 5. (A-H) Flourescence immunolocalization of Asy1 antibody (green) to spread B. oleracea PMCs, counterstained with DAPI. The antibody signal is initially punctate to the axial elements at early leptotene (A), becoming progressively continuous through leptotene (B), zygotene (C) and pachytene (D). By early diplotene (E), the signal becomes fragmented, coinciding with desynapsis of the chromosomal axes (arrow); the inset highlights the association of the Asy1 signal with the desynapsing chromosome axes, and by late diplotene (F) the bivalents are unlabelled, although the antibody forms aggregates in the nucleoplasm. Antibody labelling is not detected in PMCs at the diakinesis (G) to tetrad (H) stages. Bar, 10 µm.

View larger version (62K): [in a new window]   Fig. 6. (A) Simultaneous immunolocalization of Asy1 antibody (green) to chromosomal axes and FISH to telomeres (red) of a B. oleracea pachytene PMC. Bar, 10 µm. (B-E) Enlarged termini of individual pachytene bivalents showing that the Asy1 labelling stops short of the telomere repeats. (B) DAPI channel; (C) DAPI + telomere probe; (D) DAPI + Asy1 antibody (E) merged image; note that Asy1 localizes to the chromosome axes but is absent from the surrounding chromatin. Bar, 5 µm.

View larger version (94K): [in a new window]   Fig. 7. Immunogold localization of Asy1 antibody to spread PMCs of B. oleracea. (A) Meiotic interphase showing localization of 5 nm gold particles to chromatin in the absence of axial elements. Linear arrays of gold particles are occasionally visible. Preparation stained with uranyl acetate. (B) Leptotene showing localization of 5 nm gold particles to chromatin associated with unsynapsed axial elements. The preparation was stained with osmium tetroxide. (C) Low power survey of pachytene chromosomes for orientation purposes; the hatched box indicates the region shown in (D). (D) Pachytene showing localization of 5 nm gold particles to chromatin associated with synaptonemal complex. Chromosome axes are indicated with arrows; the boxed area (line) shows an example of gold particles on chromatin (grey area) associated with the axes; the hatched box shows absence of gold particles on regions free of chromatin (white) Preparation stained with PTA. Bar, 200 nm.