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Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes.

Strukov YG, Wang Y, Belmont AS - J. Cell Biol. (2003)

Bottom Line: 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.Specifically, an approximately 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.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell and Structural Biology, University of Illinois Urbana-Champaign, B107 CLSL, 601 S. Goodwin Ave., Urbana, IL 61801, USA.

ABSTRACT
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 approximately 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.

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Mitotic chromosomes have normal large-scale structure at the resolution of EM. Mitotic chromosomes of all four clones showed no change in the chromosome diameter at the sites of vector insert. Arrows indicate immunogold-stained insert sites. (A) Con-610 clone. (B) Con-1 clone. (C and D) dSAR-d11 clone. C shows smaller insert, D shows larger insert. (E and F) dSAR-g12 clone. E shows the smaller insert and F shows the larger insert. Bars: 1 μm for images, 0.5 μm for inserts.
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fig6: Mitotic chromosomes have normal large-scale structure at the resolution of EM. Mitotic chromosomes of all four clones showed no change in the chromosome diameter at the sites of vector insert. Arrows indicate immunogold-stained insert sites. (A) Con-610 clone. (B) Con-1 clone. (C and D) dSAR-d11 clone. C shows smaller insert, D shows larger insert. (E and F) dSAR-g12 clone. E shows the smaller insert and F shows the larger insert. Bars: 1 μm for images, 0.5 μm for inserts.

Mentions: The above observations were based on light microscopy. To examine the labeled chromosome regions at higher resolution, cell lines not expressing lac repressor were studied with EM. Mitotic cells were collected by shakeoff after a 3-h nocodazole block and were deposited on coverslips before fixation with formaldehyde. Precise localization of the inserts was provided by lac repressor staining followed by pre-embedding immunogold staining and silver amplification of the gold-labeled secondary antibody. For all insert regions in all cell clones, chromosome diameters were similar to surrounding chromosome regions (Fig. 6)Figure 6.


Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes.

Strukov YG, Wang Y, Belmont AS - J. Cell Biol. (2003)

Mitotic chromosomes have normal large-scale structure at the resolution of EM. Mitotic chromosomes of all four clones showed no change in the chromosome diameter at the sites of vector insert. Arrows indicate immunogold-stained insert sites. (A) Con-610 clone. (B) Con-1 clone. (C and D) dSAR-d11 clone. C shows smaller insert, D shows larger insert. (E and F) dSAR-g12 clone. E shows the smaller insert and F shows the larger insert. Bars: 1 μm for images, 0.5 μm for inserts.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172725&req=5

fig6: Mitotic chromosomes have normal large-scale structure at the resolution of EM. Mitotic chromosomes of all four clones showed no change in the chromosome diameter at the sites of vector insert. Arrows indicate immunogold-stained insert sites. (A) Con-610 clone. (B) Con-1 clone. (C and D) dSAR-d11 clone. C shows smaller insert, D shows larger insert. (E and F) dSAR-g12 clone. E shows the smaller insert and F shows the larger insert. Bars: 1 μm for images, 0.5 μm for inserts.
Mentions: The above observations were based on light microscopy. To examine the labeled chromosome regions at higher resolution, cell lines not expressing lac repressor were studied with EM. Mitotic cells were collected by shakeoff after a 3-h nocodazole block and were deposited on coverslips before fixation with formaldehyde. Precise localization of the inserts was provided by lac repressor staining followed by pre-embedding immunogold staining and silver amplification of the gold-labeled secondary antibody. For all insert regions in all cell clones, chromosome diameters were similar to surrounding chromosome regions (Fig. 6)Figure 6.

Bottom Line: 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.Specifically, an approximately 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.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell and Structural Biology, University of Illinois Urbana-Champaign, B107 CLSL, 601 S. Goodwin Ave., Urbana, IL 61801, USA.

ABSTRACT
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 approximately 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.

Show MeSH
Related in: MedlinePlus