Chromosomes from mitotic cells

Although the DNA-binding properties of several procaryotic topoisomerases have been well characterized, little information is currently available concerning eucaryotic enzymes. Some eucaryotic topoisomerases may be intimately associated with other nuclear proteins HeLa topoisomerases I and II have been found to be associated with chromatin (Javaherian and Liu, 1983 Liu et al., 1983b). HeLa topoisomerase I has been shown to bind to the nonhistone protein HMG17, which also stimulates DNA catenation by the enzyme (Javaherian and Liu, 1983 Tse et al., 1984). It has been suggested that type II topoisomerase is an important component of the chromosomal scaffold in interphase nuclei and mitotic chromosomes from chicken cell lines (Earnshaw et al., 1985). In addition, an ATP-dependent topoisomerase has been found associated with several other enzymes of DNA metabolism in a complex (termed the replitase complex) isolated from the nuclei of Chinese hamster embryo fibroblast cells (Noguchi et al., 1983). 

The third observation that supported a central role for DNA in cancer came from the study of chromosomes in human leukemia cells. Karyotyping is a cytological technique used to characterize the gross structure of chromosomes in mitotic cells. Researchers noticed that in cells from one type of leukemia, chronic myelogenous leukemia, there was almost always a chromosome rearrangement between chromosomes 9 and 22. This reciprocal translocation led to identification of a small 22 9 chromosome which came to be called the Philadelphia chromosome because it was discovered at the Wistar Institute in Philadelphia. Importantly, the karyotype of nonleukemic cells from patients with this form of leukemia is normal. 

Sunkara, P. S., Wright, D. A., and Rao, P. N. (1979). Mitotic factors from mammalian cells induce germinal vesicle breakdown and chromosome condensation in amphibian oocytes. Proc. Natl. Acad. Sci. USA 76 2799-2802. 

A positive result from a chromosome aberration assay (MNT, cytogenetic analysis or small colonies in MLA) suggests a second in vivo assay for clastogenesis or aneugenesis. Centromere staining of mitotic cells allows the distinction of aneugens and clastogens. 

Micronucleus Test The micronucleus test is an in vivo test usually carried out in mice. The animals are treated in vivo, and the erythrocyte stem cells from the bone marrow are stained and examined for micronuclei. Micronuclei represent chromosome fragments or chromosomes left behind at anaphase. It is basically a test for compounds that cause chromosome breaks (clastogenic agents) and compounds that interfere with normal mitotic cell division, including compounds that affect spindle fiber function. 

The first evidence that diffusible factors regulate the cell cycle came from cell-fusion experiments with cultured mammalian cells. When interphase cells in the Gi, S, or G2 phase of the cell cycle were fused to cells in mitosis, their nuclear envelopes retracted and their chromosomes condensed (Figure 21-3). This finding indicates that some diffusible component or components in the cytoplasm of the mitotic cells forced interphase nuclei to undergo many of the processes associated with early mitosis. We now know that these factors are the mitotic cyclin-CDK complexes. 

During mitosis, the mitotic spindle is responsible for the physical movement of chromosomes. The spindle is composed of hundreds of microtubules. During prophase, numerous microtubules form at the kinetochores and then move the chromosomes first to a central plane (metaphase) and then to the poles during anaphase. At telophase, microtubules are present at the site of cell plate formation. At interphase, microtubules are located in the cytoplasm near the cell wall. Microtubules do not move from one location to another during the cell cycle (e.g., from the cell wall at interphase to the kinetochore of the chromosome during prophase), but rather are depolymerized at one location into a common pool of protein subunits called tubulin. At their new location, the microtubules repolymerize from the tubulin pool. 

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