Multivariate flow cytometry using specific cyclin proteins and DNA content can identify cell populations at different points within the cell cycle. Quantification of cyclin B1 and DNA content reveals that cells with high levels of cyclin B1 predominantly have a 4C DNA content and are therefore in G2 or mitosis. We have examined whether separation of cells by levels of cyclin B1 could be used to discriminate cells at discrete times within these phases. Post‐replicative cells progressively enter into fractions with higher levels of cyclin B1, indicating that this protein can be used as a marker of time in G2. Furthermore, cells in particular phases of mitosis can be greatly enriched by separation based on cyclin B1 levels. This method can thus be used to isolate cells at specific times within G2 and mitosis, periods of the cell cycle that have been difficult to study by cell fractionation. Cytometry 27:250–254, 1997. © 1997 Wiley‐Liss, Inc.

G 2 was defined originally as the temporal gap between the termination of DNA replication and the beginning of mitosis. In human cells, the G 2 period was estimated to be 3-4 h. However, the absence of replicative DNA synthesis during this period designated G 2 has never been shown conclusively. In this report, we show that, at some autosomal and X linked loci, programmed DNA replication continues within 90 min of mitosis. Furthermore, the major accumulation of cyclin B1, a cell-cycle marker that is usually ascribed to G 2 , overlaps extensively with very late DNA replication. We conclude that the G 2 period is much shorter than previously thought and may, in some cells, be nonexistent.The eukaryotic cell cycle consists of an orderly series of events in which cells grow, replicate their DNA, and segregate the duplicated genome to the daughter cells. Our current textbook view of the cell cycle was first put forth by Howard and Pelc (1) as encompassing four phases: S phase, the time in which replicative DNA synthesis occurs; M phase, the time in which mitotic chromosome segregation and cell division proceed; and two gap phases, G 1 and G 2 , that temporally separate S phase from mitosis.The G 2 phase was inferred from the length of time between the addition of radiolabeled DNA precursors and the appearance of labeled, condensed mitotic chromosomes (1). Autoradiography, however, may not be sufficiently sensitive to detect low levels of replicative synthesis in the G 2 phase. The absence of detectable incorporation of radiolabel could thus be taken to indicate erroneously the absence of DNA replication. Furthermore, DNA repair synthesis can occur throughout the cell cycle (2, 3); replicative and repair DNA synthesis are not easily distinguishable by the autoradiographic method. An additional problem is that no cellular marker signaling the termination of replicative DNA synthesis has been identified. These features contribute to an inherent lack of both sensitivity and specificity for accurately defining the terminal border of S phase.Our studies on fragile sites and the replication timing of FMR1, the gene responsible for the fragile X syndrome, first brought our attention to the possibility of replication very close to mitosis (4). In cell lines derived from patients with fragile X syndrome, the replication of a large region surrounding the FMR1 gene is abnormally late, occurring within the G 2 ͞M compartment defined by DNA content (5, 6). Surprisingly, equally late replication was observed for several loci on the inactive X chromosome in normal female fibroblasts (7,8), as well as on the inactive X chromosome in human-hamster hybrids (8). In this report, we have determined more precisely the timing of late DNA replication in the human cell cycle, by using a new, sensitive method for fractionating cells late in the cell cycle (9). We address two questions. Does this very late replication occur during what is usually termed G 2 ? What proportion of the genome replicates in this late compartment? MATERIALS A...

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BACKGROUND One of the most dramatic events during the course of the mammalian cell cycle is mitosis, when chromosomes condense and segregate, the nuclear envelope breaks down, and the cell divides into two daughter cells. Although cells undergoing mitosis are cytologically distinguishable from nonmitotic cells, few molecular markers are available to specifically identify mitotic cells, especially cells within different stages of mitosis. METHODS We applied the flow cytometric method of Juan et al. (Cytometry 32:71–77, 1998) to obtain cells with various levels of the molecular markers cyclin B1 and phosphorylated histone H3; fluorescence microscopy was then used to identify sorted cells in different stages of mitosis. RESULTS We observed the substantial enrichment of submitotic cell populations. CONCLUSIONS This method represents an effective approach to obtain an enriched population of submitotic cells without the use of drug treatments or prior synchronization. Cytometry 39:126–130, 2000 © 2000 Wiley‐Liss, Inc.