|
|
|
|
|
|
|
||
Article |
Address correspondence to Andrew S. Belmont, Dept. of Cell and Structural Biology, University of Illinois, Urbana-Champaign, B107 CLSL, 601 S. Goodwin Ave., Urbana, IL 61801. Tel.: (217) 244-2311. Fax: (217) 244-1648. E-mail: asbel{at}uiuc.edu
Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an
250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.
Key Words: mitotic chromosome; large-scale structure of chromatin; scaffold-associated regions; transgenes; chromosome scaffold
* Abbreviations used in this paper: DHFR, dihydrofolate reductase; dSAR, double SAR construct; MAR, matrix-associated region; MTX, methotrexate; SAR, scaffold-associated region.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
Related Article
This article has been cited by other articles:
|
|
