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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

Insert DNA is noncontiguous in clones with large inserts. Genomic DNA of clones Con-610, Con-1, dSAR-g12, and dSAR-d11 was cut with MluI, NheI, and EcoRV, endonucleases not cutting the vectors. (A) Hybridization pattern of the EcoRV digest. (B) Hybridization pattern of the MluI digest. (C) Hybridization pattern of the NheI digest. For all panels, lane M is phage λ DNA 48.5-kbp ladder.
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fig2: Insert DNA is noncontiguous in clones with large inserts. Genomic DNA of clones Con-610, Con-1, dSAR-g12, and dSAR-d11 was cut with MluI, NheI, and EcoRV, endonucleases not cutting the vectors. (A) Hybridization pattern of the EcoRV digest. (B) Hybridization pattern of the MluI digest. (C) Hybridization pattern of the NheI digest. For all panels, lane M is phage λ DNA 48.5-kbp ladder.

Mentions: One cell line, Con-610, produced clear evidence for large, contiguous vector concatemers (Fig. 2)Figure 2.


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)

Insert DNA is noncontiguous in clones with large inserts. Genomic DNA of clones Con-610, Con-1, dSAR-g12, and dSAR-d11 was cut with MluI, NheI, and EcoRV, endonucleases not cutting the vectors. (A) Hybridization pattern of the EcoRV digest. (B) Hybridization pattern of the MluI digest. (C) Hybridization pattern of the NheI digest. For all panels, lane M is phage λ DNA 48.5-kbp ladder.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Insert DNA is noncontiguous in clones with large inserts. Genomic DNA of clones Con-610, Con-1, dSAR-g12, and dSAR-d11 was cut with MluI, NheI, and EcoRV, endonucleases not cutting the vectors. (A) Hybridization pattern of the EcoRV digest. (B) Hybridization pattern of the MluI digest. (C) Hybridization pattern of the NheI digest. For all panels, lane M is phage λ DNA 48.5-kbp ladder.
Mentions: One cell line, Con-610, produced clear evidence for large, contiguous vector concatemers (Fig. 2)Figure 2.

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