Enrichment for submitotic cell populations using flow cytometry.

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.

Very late DNA replication in the human cell cycle.

G2 was defined originally as the temporal gap between the termination of DNA replication and the beginning of mitosis. In human cells, the G2 period was estimated to be 3-4 h. However, the absence of replicative DNA synthesis during this period designated G2 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 G2, overlaps extensively with very late DNA replication. We conclude that the G2 period is much shorter than previously thought and may, in some cells, be nonexistent.

Fragile-X syndrome and myotonic dystrophy: parallels and paradoxes.

Fragile-X syndrome and myotonic dystrophy are caused by triplet repeat expansions embedded in CpG islands in the transcribed non-coding regions of the FMR1 and the DMPK genes, respectively. Although initial reports emphasized differences in the mechanisms by which the expanded triplet repeats caused these diseases, results published in the past year highlight remarkable parallels in the likely molecular etiologies. At both loci, expansion is associated with altered chromatin, aberrant methylation, and suppressed expression of the adjacent FMR1 and DMAHP genes, implicating epigenetic mediation of these genetic diseases.

Separation of cells at different times within G2 and mitosis by cyclin B1 flow cytometry.

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.

Testis-specific expression of a metallothionein I-driven transgene correlates with undermethylation of the locus in testicular DNA.

Mice carrying a chimeric transgene of the human testis-specific lactate dehydrogenase cDNA driven by mouse metallothionein I promoter have been reported to express the transgene in a testis-specific manner in six founder lines. To study the mechanism by which this testis-specific expression is mediated, we have examined genomic placement, expression pattern, and methylation status of the transgene. Our results indicate that transgene expression is repressed in all somatic tissues examined even when heavy metals are administered. Nuclear run-on assays indicate that failure of expression in the liver (in which the metallothionein I promoter is highly active) occurs at the transcriptional level. In contrast, the transgene mRNA is transcribed in male germ cells and is developmentally regulated during spermatogenesis. Examination of the transgene methylation status reveals that expression is inversely correlated with hypermethylation of the locus; all CpG dinucleotides examined in the promoter region were found to be fully methylated in kidney and liver but were undermethylated in testis. Since methylation of the murine metallothionein I promoter is sufficient to inhibit its activity, it is likely that suppression of the transgene in somatic tissues is mediated by methylation.

Rat urate oxidase produced by recombinant baculovirus expression: formation of peroxisome crystalloid core-like structures.

Urate oxidase (EC 1.7.3.3), which catalyzes the oxidation of uric acid to allantoin, is present in most mammals but absent in humans and hominoid primates. In rats and most other mammals that catabolize uric acid to allantoin, this enzyme is localized within the crystalloid cores of peroxisomes present in liver parenchymal cells. To determine whether urate oxidase forms these crystalloid cores or whether core-forming protein(s) exist in association with urate oxidase, a baculovirus expression vector system was used to overproduce the full-length rat urate oxidase in Spodoptera frugiperda cells. Urate oxidase was expressed to a level of approximately 30% of the total protein in this system. Immunoblot analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunologic properties similar to those of urate oxidase expressed in rat liver. Immunofluorescence and electron microscopic examination revealed that the overexpressed recombinant urate oxidase is present in both the cytoplasm and the nucleus of infected insect cells as numerous 1- to 3-microns discrete particles. These insoluble protein aggregates, which were positively stained for urate oxidase by protein A-gold immunocytochemical approach, did not appear to be delimited by a single membrane. They revealed a crystalloid structure reminiscent of rat peroxisomal core consisting of bundles of tubules with an inner diameter of approximately 50 A. The recombinant urate oxidase particles, isolated by a single-step procedure, were composed entirely of 35-kDa urate oxidase subunit. These studies indicate that rat urate oxidase is capable of forming insoluble crystalloid core-like structures.