Studies on the Isolation of Metaphase Chromosomes

Abstract

A method for the isolation of metaphase chromosomes from mouse L1210 leukemia cells has been developed. Cells, arrested at metaphase with colchicine, were exposed to hypotonic solution and the pH was then adjusted to 5.6 to stabilize the chromosomes. The metaphase figures were subsequently disrupted and the chromosomes isolated by a series of differential ccntrifugations in sucrose. The isolated chromosomes were well preserved, as judged by morphological criteria. The effect of various enzymes and chemical agents on the isolated chromosomes was studied. Chymotrypsin, trypsin, and deoxyribonuclcase caused a marked disintegration of the chromosomes, whereas treatment with pepsin and ribonuclease induced no significant morphological alterations.

Structure of Metaphase Chromosomes: A Role for Effects of Macromolecular Crowding

“…In general, these images resemble those of chromosomes prepared by other methods [8]–[12]. The diameter of chromosomes measured on longitudinal sections was 1370±85 nm (mean ±SEM, n = 14), larger than that of chromosomes isolated in cation- or polyamine-containing buffers (700–800 nm) [10].…”

“…The conservation of chromosome structure in crowded media in which the concentrations of K + , Na + , Ca 2+ , and Mg 2+ ions were 100–1000-fold lower than those usually employed for their isolation, often together with polyamines [8]–[12], is consistent with the elimination of a requirement for ions for stabilisation of other macromolecular assemblies in crowded conditions [25]–[27], [44]. The extent to which ionic conditions in the cell are reproduced by media commonly used to isolate chromosomes is difficult to evaluate; concentrations of diffusible (osmotically active) ions in vivo cannot be derived from measurements of their total quantities because significant fractions of K + and Na + appear to be bound to macromolecules [51]–[55] and of Mg 2+ to ATP, mitochondria, and the sarcoplasmic reticulum [56], and it has been argued that the cytoplasm contains essentially no free ions [57].…”

A Study of Newt Mitotic Chromosomes by Negative Staining

“…It may be noted in this connection that Cantor and Hearst (7) found that isolated chromosomes from mouse ascites tumor cells contained as much RNA as DNA, namely, 13.5%. However Chorazy, Bendich, Borenfreund and Hutchinson (8) found that in an acid medium which prevented chromosome disintegration, the chromosomes were liable to be contaminated by ribosomes and other cytoplasmic components. b.…”

Micro-interferometry of Insect Polyploid Nuclei

“…The amount of histone, however, is lesser than that expected on the basis of total mass of the sheath. The bulk of the sheath protein is thought to be acidic, which is supported by the fact that isolated metaphase chromosomes are stabilized in acid solution (Chorazy et al 1963, DuPraw 1965. Therefore, one can assume that most of the remaining nuclear dry mass of nuclei from Melipona quadrifasciata Malpighian tubes is due to acidic proteins.…”