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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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Normal chromosome morphology over insert regions—correlative light and electron microscopy. In native chromosomes of clone Con1, vector inserts appear as a band going over the entire width of the chromosome. Sections are 0.2 μm thick. (A–C) Fluorescent light microscopy of a single section; lac repressor immunostaining signal, staining for total DNA with DAPI, combined A and B, respectively. (D) Two-fold expanded image from C. (E) An EM image of the same section. Arrows indicate insert region labeled with immunofluorescence probes (D) and corresponding regions on EM sections (E). Bars, 1 μm.
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fig7: Normal chromosome morphology over insert regions—correlative light and electron microscopy. In native chromosomes of clone Con1, vector inserts appear as a band going over the entire width of the chromosome. Sections are 0.2 μm thick. (A–C) Fluorescent light microscopy of a single section; lac repressor immunostaining signal, staining for total DNA with DAPI, combined A and B, respectively. (D) Two-fold expanded image from C. (E) An EM image of the same section. Arrows indicate insert region labeled with immunofluorescence probes (D) and corresponding regions on EM sections (E). Bars, 1 μm.

Mentions: These EM results unambiguously demonstrate normal chromosome width at the chromosome insert sites. By light microscopy, though, some insert regions appear as light staining DAPI or propidium iodide bands (Fig. 5). These are comparable to other light bands found elsewhere in the mitotic chromosomes. Despite these variations in staining density, wild-type chromosomes show uniform density by EM, leading to the conclusion that these staining variations over the wild-type chromosomes reflect variations in sequence content as opposed to variations in chromosome compaction. The heavy immunogold staining over insert regions (Fig. 6) obscures visualization of the uranyl and lead chromatin staining. To verify that the lighter staining over our insert regions is also a result of differential staining, as opposed to a lower local chromosome density, we used correlative light and electron microscopy. This approach showed that by EM, the insert regions were indistinguishable from the surrounding chromosomes (Fig. 7)Figure 7.


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)

Normal chromosome morphology over insert regions—correlative light and electron microscopy. In native chromosomes of clone Con1, vector inserts appear as a band going over the entire width of the chromosome. Sections are 0.2 μm thick. (A–C) Fluorescent light microscopy of a single section; lac repressor immunostaining signal, staining for total DNA with DAPI, combined A and B, respectively. (D) Two-fold expanded image from C. (E) An EM image of the same section. Arrows indicate insert region labeled with immunofluorescence probes (D) and corresponding regions on EM sections (E). Bars, 1 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172725&req=5

fig7: Normal chromosome morphology over insert regions—correlative light and electron microscopy. In native chromosomes of clone Con1, vector inserts appear as a band going over the entire width of the chromosome. Sections are 0.2 μm thick. (A–C) Fluorescent light microscopy of a single section; lac repressor immunostaining signal, staining for total DNA with DAPI, combined A and B, respectively. (D) Two-fold expanded image from C. (E) An EM image of the same section. Arrows indicate insert region labeled with immunofluorescence probes (D) and corresponding regions on EM sections (E). Bars, 1 μm.
Mentions: These EM results unambiguously demonstrate normal chromosome width at the chromosome insert sites. By light microscopy, though, some insert regions appear as light staining DAPI or propidium iodide bands (Fig. 5). These are comparable to other light bands found elsewhere in the mitotic chromosomes. Despite these variations in staining density, wild-type chromosomes show uniform density by EM, leading to the conclusion that these staining variations over the wild-type chromosomes reflect variations in sequence content as opposed to variations in chromosome compaction. The heavy immunogold staining over insert regions (Fig. 6) obscures visualization of the uranyl and lead chromatin staining. To verify that the lighter staining over our insert regions is also a result of differential staining, as opposed to a lower local chromosome density, we used correlative light and electron microscopy. This approach showed that by EM, the insert regions were indistinguishable from the surrounding chromosomes (Fig. 7)Figure 7.

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