C-metaphase

Observations are made in metaphase cells arrested with a spindle inhibitor such as colchicine or colcemid to accumulate cells in a metaphase-like stage of mitosis (c-metaphase) before hypotonic treatment to enlarge cells and fixation with alcohol-acetic acid solution. Cells are then dispersed on to microscope slides and stained and slides are randomized, coded and analyzed for chromosome aberrations with high-power light microscopy. Details of the procedure are given in Dean and Danford (1984) and Preston et al. (1981, 1987). The UKEMS guidelines (Scott et al., 1990) recommend that all tests be repeated regardless of the outcome of the first test and... 

Mammalian cells in vitro are exposed to the test chemical with and without an exogenous mammalian metaboUc activation system and cultured for two rounds of replication in bromodeox3Uiridine (BrdU) containing medium. After treatment with a spindle inhibitor (e.g., colchicine) to accumulate cells in a metaphase-Uke stage of mitosis (c-metaphase), cells are harvested, stained, and metaphase cells analyzed for SCEs. Primary cultures (e.g., human lymphocytes) or established cell lines (e.g., Chinese hamster ovary or lung cells) may be used in the assay. At least three adequately spaced concentrations of the test substance should be used. 

A series of experiments using corn root tips, Zea mays cv Norfolk Market White, was established using the free acid from 10 3 to 10 4 M. Results definitively showed that mitosis was inhibited at the C-metaphase plate at both concentrations... 

Fig. 2. Quantification of the increase in t PJNAD labeling of nuclear and chromosomal proteins resulting from incubation with DMS. Autoradiograms, such as those in Fig. 1, were scanned with a Cary spectrophotometer equipped with a gel scanning stage. The relative weights of the tracings were measured for incorporation by total proteins ( ), nonhistones (O), and the 116,000 dalton protein (x). Panel A, nuclear proteins from cells treated in growth medium panel B, cells treated in isolation buffer, panel C, metaphase chromosome proteins from cells incubated with mutagen in chromosome isolation buffer.

FIGURE 2 Nuclear transplantation. (A) Mature oocyte with first polar body (arrowhead). (B) Aspiration of chromosomes in a membrane-bounded cytoplasmic vesicle. (C) Metaphase II plate (small arrowhead) and first polar body (large arrowhead) in the enucleation pipette. (D) Aspiration of an isolated 32-cell stage blastomere. (E) Insertion of the blastomere into the perivitelline space of the oocyte. (F) Enucleated recipient oocyte and adjacent donor blastomere. 400X, phase contrast. 

Figure 5.3. Second meiotic division in D. melanogaster males, a) Prophase secondary spermatocyte nucleus, (b) Two prometaphase secondary spermatocyte nuclei (top) plus a prometaphase cell (bottom) without nuclear-cytoplasmic demarcation, (c) Metaphase (d) early anaphase (e) mid-anaphase (/) late anaphase (g,h) telophases. Bar, 10 itm. (Reprinted, with permission, from Cenci et al. 1994.)...

Notice the presence of several submetacentrics and many small biarmed chromosomes. (c) Metaphase of hybrid DC. Notice the presence of chromosomes characteristic of both parents, including the D chromosome (arrow). 

Lewis, C.D. and Laemmli, U.K. (1982). Higher order metaphase chromosome structure evidence for metal-loprotein interactions. Cell 29, 171-181. 

Labbe, J-C., Capony, J-P., Caput, D., Cavadore, J-C., Derancourt, J., Kaghad, M., Lelias, J-M., Picard, A., and Doree, M. (1989a). MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule of cyclin B. EMBO J. 8 3053-3058. 

Also in need of further study is the function of Mos in male germ cells. It appears that Mos is expressed prior to meiosis of spermatocytes, consistent with the possibility that it acts in the initiation of meiosis and/or during progression from meiosis I to meiosis II. However, c-mos expression continues in postmeiotic spermatids, where it does not induce metaphase II arrest. This may be due to the absence of other components of cytostatic factor, but the role of c-mos expression in postmeiotic male germ cells remains unclear. 

Burton PBJ, Raff MC, Kerr P, Yacoub MH, Barton PJR 1999 An intrinsic timer that controls cell-cycle withdrawal in cultured cardiac myocytes. Dev Biol 216 659—670 Chen X, Ko LJ, Jayaraman L, Prives C 1996 p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells. Genes Dev 10 2438—2451 Duesbery NS, Choi T, Brown KD et al 1997 CENP-E is an essential kinetochore motor in maturing oocytes and is masked during mos-dependent cell cycle arrest at metaphase II. Proc Natl Acad Sci USA 94 9165-9170... 

FIG. 3. Chromosome arms begin to separate in pro metaphase. Scanning electron micrographs of human chromosomes isolated from cells in prophase (A), prometaphase (B), metaphase (C) and early anaphase (insert in C). Size bar, 1 /tm. Reprinted with permission from Sumner (1991). 

Ciosk R, Zachariae W, Michaelis C, Shevchenko A, Mann M, Nasmyth K 1998 An ESP1 /PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in... 

Surana U, Amon A, Dowzer C, McGrew J, Byers B, Nasmyth K 1993 Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast. EMBO J 12 1969-1978... 

L3. Lichter, P., Cremer, T., Borden, J., Manuelidis, L., and Ward, D. C., Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80, 224-234 (1988). 

Townsley, F. M., Aristarkhov, A., Beck, S., Hershko, A., and Ruderman, J. V. Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase. Proc. Natl. Acad. Sei. USA 1997, 94, 2362-67. 

J. B. Rattner, and C. C. Lin, Radial loops and helical coils coexist in metaphase chromosomes. Cell 42, 291-296 (1985). 

Van Prooijen-Knegt, A. C. and Raap, A. K. (1983) Spreading and staining human metaphase chromosomes on aminoalkylsilane-treated glass slides. Histochem. J. 14, 333,334. 

Ubiquitinated H2A and H2B disappear at metaphase and reappear in anaphase [251-253]. Histone ubiquitination may also be involved in cell cycle progression through S phase [254]. In dividing and non-dividing cells, the ubiquitin moiety of the ubiquitinated histones is in rapid equilibrium with a pool of free ubiquitin [238,239]. The turnover of the ubiquitinated histones is presumably catalyzed by ubiquitin-C-terminal hydrolases. Uni- and multi-cellular eukaryotes contain... 

One of the major difficulties in the synthesis of these binary indole-indoline alkaloids is the necessity of generating the natural PARF (priority antireflective) (12) relative stereochemistry between C-14 and C-16, as well as the requirement for controlling the absolute stereochemistry at C-16, which must be (5). Other epimers at these positions lack the high cytotoxicity, with mitotic arrest at metaphase, that is the basis of the anticancer activity of these compounds (13,14). 

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