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Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning
Abstract:
The higher-order assembly of the approximately 30 nm chromatin fibers into the characteristic morphology of HeLa mitotic chromosomes was investigated by electron microscopy. Transmission electron microscopy (TEM) of serial sections was applied to view the distribution of the DNA-histone-nonhistone fibers through the chromatid arms. Scanning electron microscopy (SEM) provided a complementary technique allowing the surface arrangement of the fibers to be observed. The approach with both procedures was to swell the chromosomes slightly, without extracting proteins, so that the densely-packed chromatin fibers were separated. The degree of expansion of the chromosomes was controlled by adjusting the concentration of divalent cations (Mg2+). With TEM, individual fibers could be resolved by decreasing the Mg2+ concentration to 1.0-1.5 mM. The predominant mode of fiber organization was seen to be radial for both longitudinal and transverse sections. Using SEM, surface protuberances with an average diameter of 69 nm became visible after the Mg2+ concentration was reduced to 1.5 mM. The knobby surface appearance was a variable feature, because the average diameter decreased when the divalent cation concentration was further reduced. The surface projections appear to represent the peripheral tips of radial chromatin loops. These TEM and SEM observations support a "radial loop" model for the organization of the chromatin fibers in metaphase chromosomes.
Insights
HeLa mitotic chromosomes are organized by radial loops of chromatin fibers. Electron microscopy revealed these fiber arrangements, supporting a radial loop model for chromosome structure.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Understanding the higher-order structure of chromatin is crucial for comprehending chromosome function and dynamics.
- HeLa mitotic chromosomes exhibit a characteristic morphology that arises from complex chromatin fiber organization.
Purpose of the Study:
- To investigate the higher-order assembly of chromatin fibers within HeLa mitotic chromosomes.
- To elucidate the spatial arrangement of DNA-histone-nonhistone fibers in chromatid arms.
Main Methods:
- Utilized transmission electron microscopy (TEM) on serial sections to visualize internal fiber distribution.
- Employed scanning electron microscopy (SEM) to observe the surface arrangement of chromatin fibers.
- Controlled chromosome swelling using divalent cations (Mg2+) to resolve fiber organization.
Main Results:
- TEM revealed a predominant radial organization of chromatin fibers in both longitudinal and transverse sections.
- SEM showed surface protuberances, interpreted as the peripheral tips of radial chromatin loops, with variable diameters.
- Fiber resolution was achieved by decreasing Mg2+ concentration to 1.0-1.5 mM.
Conclusions:
- The observed fiber organization supports a "radial loop" model for metaphase chromosome structure.
- Chromatin fibers are arranged in radial loops extending from a central scaffold within chromatids.
- This study provides ultrastructural evidence for a specific model of chromosome higher-order organization.