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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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Related in: MedlinePlus

Experimental approach. (A) A pSVII-dhfr vector derivative was used to create control and dSAR constructs with 32 copies of a lac operator 8-mer repeat. (B) DHFR (−) CHO cells (DG44) were stably transfected with either control or dSAR supercoiled DHFR expression vectors. fluorescein-labeled MTX–stained cells with larger inserts were selected with FACS® and cloned. (C) Mitotic chromosomes were isolated and used for extraction and staining by different methods; lac repressor immunostaining or in vivo expression of an EGFP–lac repressor–NLS fusion protein and FISH. (D) Cell flow cytometry (abscissa, logarithm of relative intensity; ordinate, cell number) after staining with fluorescein-labeled MTX. Average DHFR expression is ∼20× higher in cells transformed with the dSAR versus control vector.
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fig1: Experimental approach. (A) A pSVII-dhfr vector derivative was used to create control and dSAR constructs with 32 copies of a lac operator 8-mer repeat. (B) DHFR (−) CHO cells (DG44) were stably transfected with either control or dSAR supercoiled DHFR expression vectors. fluorescein-labeled MTX–stained cells with larger inserts were selected with FACS® and cloned. (C) Mitotic chromosomes were isolated and used for extraction and staining by different methods; lac repressor immunostaining or in vivo expression of an EGFP–lac repressor–NLS fusion protein and FISH. (D) Cell flow cytometry (abscissa, logarithm of relative intensity; ordinate, cell number) after staining with fluorescein-labeled MTX. Average DHFR expression is ∼20× higher in cells transformed with the dSAR versus control vector.

Mentions: Fig. 1Figure 1.


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)

Experimental approach. (A) A pSVII-dhfr vector derivative was used to create control and dSAR constructs with 32 copies of a lac operator 8-mer repeat. (B) DHFR (−) CHO cells (DG44) were stably transfected with either control or dSAR supercoiled DHFR expression vectors. fluorescein-labeled MTX–stained cells with larger inserts were selected with FACS® and cloned. (C) Mitotic chromosomes were isolated and used for extraction and staining by different methods; lac repressor immunostaining or in vivo expression of an EGFP–lac repressor–NLS fusion protein and FISH. (D) Cell flow cytometry (abscissa, logarithm of relative intensity; ordinate, cell number) after staining with fluorescein-labeled MTX. Average DHFR expression is ∼20× higher in cells transformed with the dSAR versus control vector.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Experimental approach. (A) A pSVII-dhfr vector derivative was used to create control and dSAR constructs with 32 copies of a lac operator 8-mer repeat. (B) DHFR (−) CHO cells (DG44) were stably transfected with either control or dSAR supercoiled DHFR expression vectors. fluorescein-labeled MTX–stained cells with larger inserts were selected with FACS® and cloned. (C) Mitotic chromosomes were isolated and used for extraction and staining by different methods; lac repressor immunostaining or in vivo expression of an EGFP–lac repressor–NLS fusion protein and FISH. (D) Cell flow cytometry (abscissa, logarithm of relative intensity; ordinate, cell number) after staining with fluorescein-labeled MTX. Average DHFR expression is ∼20× higher in cells transformed with the dSAR versus control vector.
Mentions: Fig. 1Figure 1.

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