spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/jcs.00160

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Santos, A. P.
Right arrow Articles by Shaw, P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Santos, A. P.
Right arrow Articles by Shaw, P. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

The architecture of interphase chromosomes and gene positioning are altered by changes in DNA methylation and histone acetylation

Ana Paula Santos1,2, Rita Abranches1, Eva Stoger3, Alison Beven1, Wanda Viegas2 and Peter J. Shaw1,*

1 John Innes Centre, Colney, Norwich NR4 7UH, UK
2 Instituto Superior de Agronomia, Tapada da Ajuda, Lisbon, Portugal
3 Institute for Molecular Biotechnology RWTH, Warringer Weg 1, D-52074 Aachen, Germany



View larger version (70K):

[in a new window]
  		Fig. 1. Genomic in situ hybridisation using total genomic rye DNA as a probe in wheat root sections followed by confocal microscopy imaged entire chromosomes during interphase. Projections of consecutive confocal sections of entire rye chromosome pairs are shown. (A) Two consecutive confocal sections of control seedlings germinated on water. The interphase chromosomes appear as elongated domains with the two arms very close together and only rarely distinguishable (arrows). Bar, 10 µm. (B,C) Stereo pairs of nuclei after germination in the presence of 5-AC. The two chromosome arms can be seen separated and the chromosome structure appears very irregular with a more complex and broken configuration. (D,E) Stereo pairs of nuclei after germination in the presence of TSA. Similar changes to those produced by 5-AC are seen. However, the overall Rabl configuration is maintained, with the centromeres and subtelomeric heterochromatic blocks (arrows in B,D) remaining at the nuclear periphery. Bar, 10 µm (B-E).

 


View larger version (142K):

[in a new window]
  		Fig. 2. Genomic in situ hybridisation in interphase nuclei from wheat root sections labelled an individual chromosome arm in the 1R wheat/rye translocation line. Projections of consecutive confocal sections are shown. The rye telomeric knobs (arrows in 2A) are clearly seen as intensely labelled regions at one end of each arm, located at the nuclear periphery (line in 2A). (A) A group of interphase nuclei from control seedlings germinated on water. A gap is usually seen at the position of the decondensed nucleolar organisers (NOR), where the rDNA genes are decondensed into the nucleolus (n). Bar, 10 µm. (B,C) Two nuclei from control seedlings. (D,E) Two nuclei after treatment with 5-AC. The chromosome arms are much more irregular, and regions of strong labelling are interrupted by several gaps of decondensed chromatin. (F,G) Two nuclei after treatment with TSA. Again the chromosome arms are interrupted by four to five gaps, but the overall configuration appears more regular than after 5-AC. Bar, 10 µm, in B-G.

 


View larger version (74K):

[in a new window]
  		Fig. 3. Effect of TSA on histone acetylation. Total extracted proteins from roots germinated in water (lane 1), in 15 µM TSA changed daily (lane 2) or in 15 µM TSA without changes (lane 3) were electrophoresed on a 15% SDS gel. (A) Total proteins stained with Coomassie, showing equivalent loading in the three lanes. (B) The equivalent gel to A was western blotted with antibody AHP 418, specific to acetylated histone H4. (C) Equivalent gel to A, western blotted with antibody AHP 416, specific to histone H4 acetylated at lysine 12. There is an increase in the labelling of the histone H4 band at ~10 kDa in lanes 2 and 3 with both antibodies (arrows in B,C), showing increases in the amount of acetylated histone H4, but no difference in intensity between daily changes (lane 2) and a single application of TSA (lane 3).

 


View larger version (111K):

[in a new window]
  		Fig. 4. Transgene sites visualised in root interphase nuclei of transgenic wheat lines after germination in water (control) or in the presence of 5-AC or TSA. Two lines are illustrated: line 6 (A-F), which carries five transgene copies at two sites on metaphase chromosomes; and line 2 (G-L), which carries more than 10 transgene copies at four sites on metaphase chromosomes (Abranches et al., 2000Go). The number of sites was determined by computer modelling of the entire 3D stacks as described by Abranches et al. (Abranches et al., 2000Go). Note that in the single confocal sections, only some of the sites present in the full 3D data from which the models were made are visible. The sites clearly visible on the single optical sections shown are indicated by arrows. (A) Single confocal section from line 6: control seedling germinated on water. (B) Single confocal section from line 6 after 5-AC. (C) Single confocal section after TSA. (D) Model from complete 3D data stack shown in A. The four nuclei each show two sites — one per homologue. (E) Model from the 3D stack shown in B. Three G1 nuclei (a,b,c) each show four sites, the G2 nucleus (d) shows eight sites. (F) Model from 3D stack shown in C. Each of the four nuclei show four sites. (G) Single confocal section from line 2 control seedlings germinated on water. (H) Single confocal section from line 2 after 5-AC. (I) Single confocal section from line 2 after TSA. (J) Model from the 3D stack shown in G. Three nuclei (a,c,d) show one site each and nucleus b shows two sites. (K) Model from the 3D stack shown in H. Four sites are shown in nucleus a and two or three sites are shown in nuclei b and c, respectively. (L) Model from the 3D stack shown in I. Two nuclei (a,d) show four sites each and the other two (b,c) show two or three sites each, respectively. Bar.10 µm (A-C,G-I).

 


View larger version (83K):

[in a new window]
  		Fig. 5. Visualisation of transcription sites in interphase nuclei by BrUTP incorporation into unfixed wheat root seedling sections. Stereo pairs of 3D confocal stacks are shown. (A) Control seedling germinated in water. (B) Seedling germinated in 5-AC. (C) Seedlings germinated in TSA. The BrUTP labelling comprises many small punctate foci dispersed throughout the nucleus (Abranches et al., 1998Go), and there is no obvious difference in the overall organisation of the nuclear transcription sites after either treatment. The labelling of transcription is particularly strong in the nucleolus and comprises intranucleolar foci that are closely associated, much like closely packed beads on a string, and organised in a network of strands throughout the nucleolus. Bar, 10 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2002