Metaphase chromosomes preparation

Chromosomal aberrations. The study of chromosomal aberrations involves examination of individual chromosomes for deletion, duplication or rearrangement of its normal gene array (Swierenga et al., 1991). Studies are initiated by exposing organisms or cell lines to contaminants, followed by the addition of colchicine to capture cells at metaphase. Chromosome preparations are then stained and the incidence of chromosome aberrations is quantified (Figure 8.8). 

Fluorescence In Situ Hybridization is a technique that utilizes fluorescently modified DNA probes to hybridize to specific sequences in metaphase chromosome preparations or interphase nuclei. These probes can be used to localize single-gene copies, teleomeres, centromeres, and specific whole chromosomes... 

For monolayer cultures, the cultures are set up the day before BrdU treatment so that the cells will be in exponential growth before the addition of BrdU or the test compound. After BrdU addition the cells are allowed to undergo the equivalent of two cell cycles before cell harvest. A spindle inhibitor such as colchicine or colcemid is introduced for the final 1-2 h of culture to arrest cells in metaphase, after which the cells are harvested and chromosome preparations are made by routine cytogenetic techniques. 

The simplest and most sensitive assays for detecting clastogenic (i.e. chromosomal breaking) effects involve the use of mammalian cells. Cultures of established cell lines (e.g. Chinese hamster ovary) as well as primary cell cultures (e.g. human l)nnphocyte) may be used. After exposure to a range of chemical concentrations in the presence and absence of an appropriate metabolic activation system, the cell cultures are treated with a spindle inhibitor (e.g. vinblastine) to accumulate cells in a metaphaselike stage of mitosis. Cells are harvested at appropriate times and chromosome preparations are made, stained with DNA-specific dye and the metaphase cells are analysed under the microscope for chromosome abnormalities. 

Prior to sacrifice (3-5 hours), animals are treated with a metaphase-arresting agent (e.g. colchicine). Chromosome preparations are then made from the respective tissues and stained with an appropriate method. For a better discrimination of the chromosomes and to detect translocations, the FISH technology can be used (Natrajan and Boei 2003). Metaphase cells are microscopically analyzed for the occurrence of structural and numerical chromosome aberrations. 

In the chromosome mutation test Chinese hamster lung fibroblast cell line (CHL) can be used. The cells are treated for 24 or 48 hours with a test compound at different dose levels. Both with and without the metabolic activation system, the cells are treated with colcemid (0.2 mg/ml) for 2 h to arrest cell divisions and chromosome preparations are made. The chromosome aberrations are recorded in at least 100 metaphases of each treatment. Five groups of structural chromosome aberrations can be found ... 

For mapping, BrdU is added to enhance chromosome elongation and banding for metaphase chromosomes and to help distinguish G1 from S and G2 phase nuclei in the case of interphase chromosomes. Treatment of the cells for a few minutes with hypotonic solutions to make them swell and exposure to low temperatures, thus interfering with the stability of spindle fibres, can improve the preparations. 

A EXPERIMENTAL FIGURE 10-23 An electron micrograph of a histone-depleted metaphase chromosome reveals the scaffold around which the DNA is organized. The long loops of DNA are visible extending from the nonhistone protein scaffold (the dark structure). The scaffold shape reflects that of the metaphase chromosome itself The chromosome was prepared from HeLa cells by treatment with a mild detergent. [From J. R. Paulson and U. K. Laemmli, 1977, Ce//12 817. Copyright 1977 MIT]... 

Lohka, M. J.. and Masui, Y. (1984). Effects of Ca ions on the formation of metaphase chromosomes and sperm pronuclei in cell-free preparations from unactivated Rana pipiens eggs. Dev. Biol. 103, 434-442. 

S. S. Hoo and C. A. Bowles, An air-drying method for preparing metaphase chromosomes from the spermatogonial cells of rats and mice, Mutat. Res. 13, 85-88 (1971). 

The methanol/acetic acid fixation techniques described above preserve chromosome morphology very well, but they remove a substantial fraction of chromosomal proteins. Thus, preparations obtained by these techniques are not suitable for immunolocalization of proteinaceous components of metaphase chromosomes. We have therefore developed a series of fixation procedures that result in good chromosomal quality with minimal removal of proteins. We stress here that different fixation protocols result in differential extraction of chromosomal proteins. Thus, for each protein under study, develop a fixation procedure that minimizes its removal from the chromosomes. In addition, we have... 

Metaphase Analysis. Metaphase analysis can be performed in any tissue with actively dividing cells, but bone marrow is the tissue most often examined. Cells are treated with a test compound and are arrested in metaphase by the administration of colcemid or colchicine at various sampling times after treatment. Preparations are examined for structural chromosome damage. Because the bone marrow has a good blood supply, the cells should be exposed to the test compound or its metabolites in the peripheral blood supply. Additionally, these cells are sensitive to S-dependent and S-independent mutagens (Topham et al., 1983). 

The preparations most often affixed to silane- (9) or poly-L-lysine-coated slides are Carnoy-fixed suspensions of metaphase cells for chromosome studies (see Chapter 47) cytospins of cultured cells followed by methanol fixation sections of formalin-fixed, paraffin-embedded tissues and frozen sections fixed with acetone or ethanol/acetic acid after cutting. Each of these approaches imparts different intracellular effects that may modify cytological detail and/or hybridization. (See Chapters 8 and 9 for a discussion of fixatives and frozen-section preparations.)... 

Figure 26-13 Synaptonemal complexes. (A) Aligned pairs of homologous chromatids lying 0.4 pm apart in Allium cepa. Arrows indicate "recombination nodules" which may be involved in initiating formation of crossovers. Portions of meiotic chromosomes of lily are shown at successive stages (B) Pachytene. (C) Portion of diplotene nucleus. (D) A bivalent at diplo-tene. (E) Two bivalents at diakinesis. Pairs of sister chromatids are coiled with appropriate handedness. (F) Sister chromatid cores are far apart in preparation for separation. A chiasma is present between the two central strands. (B) through (F) courtesy of Stephen Stack.279,279d (G) Pair of sister chromatids coiled with opposite handedness at metaphase. These are immun-ostained with anti-topoisomerase II antibodies. From Boy de la Tour and Laemmli.280 Courtesy of U. K. Laemmli.

To prepare a karyotype (the chromosome constitution of a cell) a series of metaphase preparations is photographed and the individual chromosomes cut out and arranged in order of decreasing size (an Idiogram — Fig.7.6). In most cases groups of chromosomes are apparent — e.g. in man there are seven groups — but seldom is it possible to identify individual chromosomes solely on the basis of size and position of the centromere. 

There is no reason why chromosome spreads should not be prepared from cells other than PHA-activated lymphocytes (whole blood cultures). In practice, however, unless there is a specific reason for using other sources of cells, such as mapping translocations, deletions, and so on, blood cultures are easy to set up, and provide an ample source of metaphase spreads. 

Marked chromosomal abnormalities and chromosomal breaks were found in metaphases in direct bone marrow preparations from 40 patients undergoing maintenance hemodialysis (SEDA-11, 477) (17). During the period of these cjdogenetic studies, the dialysers were reused after sterilization with formaldehyde, and each patient may have received residual amounts of as much as 127 (sd 51) mg of formaldehyde during each dialysis. 

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