Prometaphase chromosomes

Slides may be examined under phase contrast to select areas of well-spread metaphase or prometaphase chromosomes. 

The nucleus is bound by a double-membrane, which is contiguous through the nuclear pores, known as the nuclear envelope. The pores are required to allow RNA out and membrane lipids in (which is needed for growth during S phase). The inner face of the inner nuclear envelope (INE) is coated by the nuclear lamina, which contains intermediate fibres called lamins A, B and C (atleast in mammals). Phosphorylation of lamins by kinases cause nuclear envelope breakdown during prometaphase. Chromosomes occupy definite positions within the nucleus because of the interaction between lamins and telomeres, for example the Rabl conformation in yeast. 

The analysis of the localization of GC-poor DNA on metaphase and prometaphase chromosomes will be illustrated in some detail in order to provide an example of the procedure followed. Fig. 7.10 displays the CsCl profiles, the relative DNA level, and the proportion of the isochore families in the DNA fractions (Saccone et al., 1996) used as probes for the in situ hybridizations of GC-poor DNA. LI isochores are only present in the pellet DNA L2 isochores are distributed in the pellet and in fractions 1-3 fractions 4 and 5 contain almost exclusively DNA from FIl isochores. In the case of fraction 5 (as well as in the following fractions shown in Fig. 1 of Saccone et al., 1996), a light satellite peak corresponding to DNA from centromeric heterochromatin is present. The pellet DNA, essentially formed by LI isochores, hybridized on a subset of G bands (Fig. 7.11), which were called LI bands, and corresponded to the G1 and G2 bands, as we will name the two most intensely staining subsets of Francke s R bands (1994). In contrast, the pellet DNA is almost absent in the large majority of the FI3 4oo and H3 4oo bands. This is especially evident in chromosomes 15, 17, 19, and 22 (see Fig. 7.11). 

Bernardi G., Federico C., Saccone S. (2000). The human prometaphase chromosomal bands compositional features and gene distribution. In Chromosomes today Vol. 13 (Olmo E. and Redi C.A., eds.) Birkhauser Verlag AG, Germany. 

Saccone S., Federico C., Solovei L, Croquette M.F., Della Valle G., Bernardi G. (1999). Identification of the gene-richest bands in human prometaphase chromosomes. Chromosome Res. 7 379-386. 

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). 

Centromere protein A (CENP-A), one of several variants of histone H3, is phosphorylated on Ser 7 by Aurora B kinase which is equivalent to Ser 10 of histone H3 (Zeitlin et al, 2001). Recent studies demonstrate that Aurora A kinase also phosphorylates CENP-A (S7) (Kunitoku et al, 2003) (Table 1). The presence of CENP-A in centromeric nucleosomes is required for kinetochore organization and function (Choo 2001). Loss of CENP-A phosphorylation function at Ser 7 caused a mislocalisation of Aurora B, a putative partner phosphatase (PPl-yl) and inner centromere protein (INCENP). H3.3, another variant of histone H3 is phosphorylated on Ser 31 in vivo (Table 1). H3.3 (S31) is a mitosis-specific modification that is present only in late prometaphase and metaphase. Furthermore, H3.3 (S31) is excluded from centromeres. However it is enriched in distinct chromosomal areas immediately adjacent to centromeres (Hake et al, 2005). 

In higher eukaryotes, at the onset of S phase cyclin A accumulates which stimulates DNA synthesis. The amount of cyclin A continues to be high after the S phase because of its role in chromosome condensation. Cyclin A is degraded when cells enter prometaphase. The level of another cyclin called cyclin B rises during G2 phase, which helps to complete the chromosome condensation and spindle assembly, which allow transition to metaphase. Cyclin B is degraded by APC during metaphase. ... 

During mitosis, aU the DNA is highly condensed to allow separation of the sister chromatids. This is the only time in the ceE cycle when the chromosome structure is visible. Chromosome abnormalities may be assessed on mitotic chromosomes by karyotype analysis (metaphase chromosomes) and by banding techniques (prophase or prometaphase), which identify aneu-ploidy, translocations, deletions, inversions, and duplications. 

Bolzer A, Kreth G, Solovei I, Koehler D, Saracoglu K, Fauth C, Muller S, Eils R, Cremer C, Speicher M R, CremerT. Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes. PLoS Biology 3, 826-842. 2005. 

Crespo, N.C., Ohkanda, J., Yen, T.J., Hamilton, A.D., and Sebti, S.M. (2001). The farnesyltransferase inhibitor, FTI-2153, blocks bipolar spindle formation and chromosome alignment and causes prometaphase accumulation during mitosis of human lung cancer cells. J Biol Chem 276 16161-16167. 

In addition to cohesion between sister chromatids established during the S phase of the cell cycle, chromosomes are further condensed to allow for the movement and segregation of sister chromatids to daughter cells during mitosis. The formation of stable, rod-like chromosomes during prometaphase of mitosis is mediated by several scaffolding proteins, such as topoisomerase II and the condensin complex (38, 39). 

Discovery and Biochemical Studies ent-15-oxokaurenoic acid (EKA, 1) was identified as a chemical that causes prolonged mitotic arrest at a stage resembling prometaphase [1]. EKA inhibits the association of the mitotic motor protein centromeric protein E with kinetochores, and inhibits chromosome movement. Unlike most antimitotic agents, EKA does not inhibit the polymerization or depolymerization of tubuhn. 

In an effort to determine the magnitude of the resistance to dinitroaniline herbicides, we (IS) utilized root tip squashes to determine changes in the mitotic indices of the R and S biotypes. Dinitroaniline herbicides disrupt mitosis at prometaphase, due to a loss of microtubules. Chromosomes fail to arrange at the... 

A FIGURE 20-36 Capture of chromosomes by microtubules in prometaphase, (a) In late prophase, spindle microtubules probe randomly for chromosomes by alternately growing and shrinking at their distal (+) ends, (b) Some chromosomes first encounter the side (D), not the end, of a microtubule, interacting with the microtubule through proteins at the kinetochore. Kinetochore-associated (+) end-directed motor proteins (e.g., MCAK) then move the chromosome to the (+) end (B), thereby stabilizing the microtubule. 

When metaphase cells are depleted of ATP by mild detergent treatment, poleward chromosome movement (anaphase A) proceeds but the spindle poles do not separate (anaphase B). This finding Indicates that microtubule motor proteins take part in separating the spindle poles, as they do in centrosome movement In prometaphase (see Figure... 

Figure 17.6 Attachment of kinetochores to the spindle. During prometaphase, kinetochores on chromosomes capture microtubules emanating from the spindle poles. Several types of attachments are possible, however only amphitelic (i.e., bipolar) attachment is compatible with segregation of chromosomes to opposite poles when anaphase begins. Monotelic and syntelic attachments trigger the spindle assembly checkpoint to give more time for them to be converted to amphitelic attachments. Merotelic attachments do not trigger the spindle assembly checkpoint but are corrected by a mechanism that requires Aurora B kinase.

These considerations will center on the usual course of chromosome motion occurring when the spindle is present—prometaphase through anaphase. Three questions suggest the analytical Tormat. First, descriptive cytology What are the phenomena to be explained Second, mechanics What forces and mechanical properties are necessary to account for chromosome motion Third, preliminary attempts at molecular explanation How are mitotic forces produced and controlled This gives a progression from the best to the least understood features of chromosome motion. 

The transition from prometaphase to metaphase is indistinct, but the termination of metaphase is readily recognized by the sudden separation of chromosomes and their anaphase motion toward opposite poles. Anaphase motion, like prometaphase, is parallel to the interpolar spindle axis and similar speeds are observed (reviewed by Mazia, 1961, p. 274). Anaphase differs in that daughter units separate and motion is more uniform. All the chromosomes in any one cell begin motion at about the same time and move at about... 

Spindle Mechanical Properties. How does the spindle respond to the mitotic forces so that force directionality is controlled and orderly chromosome motion results Artificial forces have been used to study chromosome attachment to the spindle. Especially informative are the centrifugation studies of Schrader (1934), Shimamura (1940), and Yamamoto (1964), and the micromanipulation experiments of Carlson (1952). Recent micromanipulation results (Nicklas and Staehly, 1967) add some details to the earlier observations and confirm them during normal prometaphase and anaphase motion. Artificial forces sufficient to stretch the chromosome cause little or no increase in the distance from the pole to the kinetochore, but there is much less resistance to lateral or poleward displacement. The spindle as a whole behaves in micromanipulation and during isolation as a single body it is a mechanical unit independent of the rest of the cell (reviewed by Mazia, 1961). The simplest, and the classic, interpretation attributes these mechanical properties to individual spindle fibers in order to account for the... 

PROMETAPHASE. No entirely satisfactory hypothesis of prometaphase congression is available. The chief issues are the extent to which poleward forces are responsible and, of course, the mechanism of force production or regulation which produces a stable chromosome position midway between the poles. Consideration of the force equilibrium hypothesis of Ostergren (summary 1950 see also Rashevsky, 1941 and Ostergren et al., 1960) illustrates the problems. The hypothesis postulates, first, poleward forces at the kinetochores of each chromosome (or bivalent). If these kinetochores are oriented to... 

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