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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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Visualization of vector sequence distribution within chromosome halos. (A–C) FISH on smaller insert of clone dSAR-g12, isolated, fixed, and extracted with 2 M salt buffer. (D–F) Isolated smaller insert of dSAR-g12 extracted with LIS buffer and stained with GFP–lacI fusion protein. (G–R) Isolated chromosomes extracted with 2 M salt buffer and stained with lac repressor. (G–I) dSAR-g12, larger insert; (J–L) dSAR-d11, larger insert; (M–O) Con-610; (P-R) Con-1. (A, D, G, J, M, and P) total DNA staining (DAPI); (B) FISH signal; (C) combined DAPI and FISH signal; (E) GFP signal; (F) combined DAPI and GFP signal; (H, K, N, and Q) lac repressor immunostaining; (I, L, O, and R) combined DAPI and immunofluorescence signal. Bars, 2 μm.
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fig3: Visualization of vector sequence distribution within chromosome halos. (A–C) FISH on smaller insert of clone dSAR-g12, isolated, fixed, and extracted with 2 M salt buffer. (D–F) Isolated smaller insert of dSAR-g12 extracted with LIS buffer and stained with GFP–lacI fusion protein. (G–R) Isolated chromosomes extracted with 2 M salt buffer and stained with lac repressor. (G–I) dSAR-g12, larger insert; (J–L) dSAR-d11, larger insert; (M–O) Con-610; (P-R) Con-1. (A, D, G, J, M, and P) total DNA staining (DAPI); (B) FISH signal; (C) combined DAPI and FISH signal; (E) GFP signal; (F) combined DAPI and GFP signal; (H, K, N, and Q) lac repressor immunostaining; (I, L, O, and R) combined DAPI and immunofluorescence signal. Bars, 2 μm.

Mentions: Mitotic halos were prepared from isolated mitotic chromosomes using either high salt or LIS extraction. The scaffold fraction of vector DNA was defined as the high intensity DAPI staining located in the central halo area, which retained the general shape of the unextracted chromosome. The measured width of this DAPI-rich core is approximately one half to two thirds that expected for the native chromatid, and compares well with the observed width of axial condensin immunofluorescence staining observed within native chromosomes and mitotic halos (Maeshima and Laemmli, 2003). Consistent with previous papers (Bickmore and Oghene, 1996), traditional FISH conditions led to the loss of the central scaffold structure (Fig. 3Figure 3.


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)

Visualization of vector sequence distribution within chromosome halos. (A–C) FISH on smaller insert of clone dSAR-g12, isolated, fixed, and extracted with 2 M salt buffer. (D–F) Isolated smaller insert of dSAR-g12 extracted with LIS buffer and stained with GFP–lacI fusion protein. (G–R) Isolated chromosomes extracted with 2 M salt buffer and stained with lac repressor. (G–I) dSAR-g12, larger insert; (J–L) dSAR-d11, larger insert; (M–O) Con-610; (P-R) Con-1. (A, D, G, J, M, and P) total DNA staining (DAPI); (B) FISH signal; (C) combined DAPI and FISH signal; (E) GFP signal; (F) combined DAPI and GFP signal; (H, K, N, and Q) lac repressor immunostaining; (I, L, O, and R) combined DAPI and immunofluorescence signal. Bars, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172725&req=5

fig3: Visualization of vector sequence distribution within chromosome halos. (A–C) FISH on smaller insert of clone dSAR-g12, isolated, fixed, and extracted with 2 M salt buffer. (D–F) Isolated smaller insert of dSAR-g12 extracted with LIS buffer and stained with GFP–lacI fusion protein. (G–R) Isolated chromosomes extracted with 2 M salt buffer and stained with lac repressor. (G–I) dSAR-g12, larger insert; (J–L) dSAR-d11, larger insert; (M–O) Con-610; (P-R) Con-1. (A, D, G, J, M, and P) total DNA staining (DAPI); (B) FISH signal; (C) combined DAPI and FISH signal; (E) GFP signal; (F) combined DAPI and GFP signal; (H, K, N, and Q) lac repressor immunostaining; (I, L, O, and R) combined DAPI and immunofluorescence signal. Bars, 2 μm.
Mentions: Mitotic halos were prepared from isolated mitotic chromosomes using either high salt or LIS extraction. The scaffold fraction of vector DNA was defined as the high intensity DAPI staining located in the central halo area, which retained the general shape of the unextracted chromosome. The measured width of this DAPI-rich core is approximately one half to two thirds that expected for the native chromatid, and compares well with the observed width of axial condensin immunofluorescence staining observed within native chromosomes and mitotic halos (Maeshima and Laemmli, 2003). Consistent with previous papers (Bickmore and Oghene, 1996), traditional FISH conditions led to the loss of the central scaffold structure (Fig. 3Figure 3.

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