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First published online 21 June 2005
doi: 10.1242/jcs.02443

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Characterization of Arabidopsis thaliana SMC1 and SMC3: evidence that AtSMC3 may function beyond chromosome cohesion

Wing See Lam^*, Xiaohui Yang^* and Christopher A. Makaroff

The Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA

View larger version (18K): [in a new window]   Fig. 1. Schematic representation of the AtSMC1 and AtSMC3 loci. Genomic maps showing the relative positions of AtSMC1, PTPG and AtSMC3, as well as cDNA fragments that were analyzed, are shown. The black boxes represent exons. Arrows (not drawn to scale) mark the position and orientation of primers used in this study. Broken arrows represent the main AtSMC1 and AtSMC3 transcriptional units. Bars separated by broken lines represent the cDNA fragment obtained from AtSMC1, PTPG, and an EST from GenBank. Bars, 0.4 kb.

View larger version (49K): [in a new window]   Fig. 2. AtSMC1 and AtSMC3 transcript and protein patterns differ in plant tissues. (A) AtSMC1, AtSMC1-3'UTR, AtSMC3 and PTPG expression patterns were examined by RT-PCR analysis of oligo(dT)-generated total cDNA synthesized from RNA isolated from different plant organs: roots, stems, leaves, flower buds. Transcripts for ACT8 were used as a control. RT-PCR products were analyzed by Southern blotting. The number of cycles used for PCR is shown to the right. (B) Relative levels of AtSMC1, AtSMC3 and PTPG transcripts as determined by real-time PCR. Transcript levels were standardized relative to the level of ACT8 cDNA in the sample. (C) AtSMC3 total protein (10 µg) from Arabidopsis root, stem, leaf, flower bud tissues and suspension culture cells was isolated and analyzed by western blotting using anti-AtSMC3 antibody.

View larger version (60K): [in a new window]   Fig. 3. AtSMC3 is present in the cytoplasm, nucleus and nuclear matrix of Arabidopsis suspension cells. (A) Images of anti-AtSMC3 labeling of interphase cells after various extractions and fixing with 4% paraformaldehyde. Cells were sequentially extracted with salt (CSK) followed by detergent extraction buffer to remove soluble cytoplasmic proteins, and subsequently treated with DNase. Untreated cells and buffer without DNAse (mock) were used as controls. DNA was stained with DAPI. AtSMC3 was detected with anti-AtSMC3 antibody followed by treatment with Alexa Fluor 488-labeled secondary antibody. Scale bar: 10 µm. (B) Western blot analysis of AtSMC3 eluted by the series of extractions as described for A. Proteins from these extractions were analyzed by western blotting with anti-AtSMC3 antibody. Cells before treatment were used as the control.

View larger version (67K): [in a new window]   Fig. 4. AtSMC3 localizes to the chromosomes and spindles in Arabidopsis somatic cells. Fluorescence immunolocalization using anti-AtSMC3 antibody (red), anti-ß-tubulin antibody (green), DAPI stained DNA (blue), and the merged images (right column). (A) interphase; (B) metaphase; (C) anaphase; (D) telophase; (E) tapetal cell after nuclear division. Bar, 10 µm.

View larger version (53K): [in a new window]   Fig. 5. AtSMC3 localizes to chromosomes and spindles in Arabidopsis meiocytes. Fluorescence immunolocalization using anti-AtSMC3 antibody (red), anti-ß-tubulin antibody (green), DNA stained with DAPI (blue), and the merged images (right column). (A) Interphase; (B) pachytene; (C) diplotene; (D) diakinesis; (E) metaphase I; (F) anaphase I; (G) telophase I; (H) telophase II. Bar, 10 µm.

View larger version (69K): [in a new window]   Fig. 6. AtSMC3 and SYN1 localize to axial elements of pachytene chromosomes. Immunolocalization of AtSMC3 (A, green) and SYN1 (B, green) on meiotic spreads of pachytene chromosomes. DNA was counterstained with DAPI (red). The merged images are shown on the right. Bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 7. SYN1 is required for proper localization of AtSMC3. Immunolocalization of AtSMC3 (green) in meiocytes of wild-type (A-D) and syn1 (E-L) plants. DNA was counter-stained with DAPI (red). (A,E) interphase; (B,F) zygotene; (C,G) pachytene; (D,H) diakinesis; (I) metaphase I; (J) telophase I; (K) anaphase II; (L) telophase II. Arrows indicate chromosome regions not labeled with anti-AtSMC3 antibody. Bar, 10 µm.