Isolation of chromosome clusters from metaphase-arrested HeLa cells

We have developed a simplified approach for the isolation of metaphase chromosomes from HeLa cells. In this method, all the chromosomes from a cell remain together in a bundle which we call a “metaphase chromosome cluster”. Cells are arrested to 90–95% in metaphase, collected by centrifugation, extracted with non-ionic detergent in a low ionic strength buffer at neutral pH, and homogenised to strip away the cytoskeleton. The chromosome clusters which are released can then be isolated in a crude state by pelleting or they can be purified away from nearly all the interphase nuclei and cytoplasmic debris by banding in a Percoll TM density gradient. — This procedure has the advantages that it is quick and easy, metaphase chromatin is recovered in high yield, and Ca ++ is not needed to stabilise the chromosomes. Although the method does not yield individual chromosomes, it is nevertheless very useful for both structural and biochemical studies of mitotic chromatin. The chromosome clusters also make possible biochemical and structural studies of what holds the different chromosomes together. Such information could be useful in improving chromosome isolation procedures and for understanding suprachromosomal organisation of the nucleus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47359/1/412_2004_Article_BF00327351.pd

Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy

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