No 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 ∼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.

Mitotic chromosome structure and pathways of mitotic condensation remain unknown. The limited amount of structural data on mitotic chromosome structure makes it impossible to distinguish between several mutually conflicting models. Here we used a Chinese hamster ovary cell line with three different lac operator-tagged vector insertions distributed over an approximately 1 microm chromosome arm region to determine positioning reproducibility, long-range correlation in large-scale chromatin folding, and sister chromatid symmetry in minimally perturbed, metaphase chromosomes. The three-dimensional positions of these lac operator-tagged spots, stained with lac repressor, were measured in isolated metaphase chromosomes relative to the central chromatid axes labeled with antibodies to topoisomerase II. Longitudinal, but not axial, positioning of spots was reproducible but showed intrinsic variability, up to approximately 300 nm, between sister chromatids. Spot positions on the same chromatid were uncorrelated, and no correlation or symmetry between the positions of corresponding spots on sister chromatids was detectable, showing the absence of highly ordered, long-range chromatin folding over tens of mega-basepairs. Our observations are in agreement with the absence of any regular, reproducible helical, last level of chromosome folding, but remain consistent with any hierarchical folding model in which irregularity in folding exists at one or multiple levels.

A fluorescently labeled protein specifically binding to genes was reproducibly found at the periphery of condensed mitotic fruit fly chromosomes, illustrating preservation of a radial structure between consecutive divisions.

CSH Protocols Ampicillin (add at 100 µg/mL to both LB medium and LB agar plates) Bacteria (e.g., Stbl2 competent cells; Invitrogen/GIBCO) CaCl 2 (250 mM) Cells for transfection: CHO DG44 DNA preparation kits (e.g., QIAGEN miniprep and maxiprep kits) Ethanol (70%; ice-cold 100%) Fetal bovine serum (FBS), defined and dialyzed (HyClone Lab) Fluorescein-labeled methotrexate (FMTX) (Molecular Probes) Glycine (100 µM) Hypoxanthine (30 µM) LB medium (liquid and agar plates) Media for cells used for transfection F-12 (HAM) medium (Invitrogen/GIBCO) supplemented with 10% defined FBS: for growth of DG44 cells F-12 medium, lacking thymidine and hypoxanthine, with 10% dialyzed FBS: for selection and further passaging of stably transfected cells Phosphate-buffered saline, calcium-and magnesium-free (CMF-PBS) Restriction enzymes SalI and XhoI Sodium acetate (3 M, pH 5.5) TE buffer (optional; see Step 4.vii) Thymine (30 µM) TR buffer Trypsin Vector (e.g., psv2-DHFR-8.32) and appropriate insert (e.g., 256-copy lac operator repeat) (see Steps 1 and 2) Equipment Dry ice Equipment for agarose gel electrophoresis Filter paper Flasks (25-cm 2 tissue culture; 75-cm 2) Fluorescence-activated cell sorter Forceps (sharp-tipped) Incubator preset to 30°C Inoculation loops Marker (permanent) Microcentrifuge Microcentrifuge tubes (1.5 mL) Micropipette tips (sterile 200-µL) (1 mL) (optional; see Step 8.viii) Microscope Petri dishes (150-mm) Pipette Plastic hose (optional; see Step 8.viii) Plates for growing bacterial cultures Plates (96-well) Shaking platform Tubes for transfection METHOD Preparing Vector DNA Containing Large Direct Repeats 1. Clone the lac operator repeat into a vector.