Cell Cycle Studies on a Temperature-sensitive Cell Division Mutant of Mammalian Cells

A temperature-sensitive Syrian hamster mutant cell line, ts-745, exhibiting novel mitotic events has been isolated. The cells show normal growth and mitosis at 33 degrees C, the permissive temperature. At the nonpermissive temperature of 39 degrees C, mitotic progression becomes aberrant. Metaphase cells and those cells still able to form a metaphase configuration continue through and complete normal cell division. However, cells exposed to 39 degrees C for longer than 15 min can not form a normal metaphase spindle. Instead, the chromosomes are distributed in a spherical shell, with microtubules (MT) radiating to the chromosomes from four closely associated centrioles near the center of the cell. The cells progress from the spherical monopolar state to other monopolar orientations conical in appearance with four centrioles in the apex region. Organized chromosome movement is present, from the spherical shell state to the asymmetrical orientations. Chromosomes remain in the metaphase configuration without chromatid separation. Prometaphase chromosome congression appears normal, as the chromosomes and MT form a stable monopolar spindle, but bipolar spindle formation is apparently blocked in a premetaphase state. When returned from 39 degrees to 33 degrees C, the defective phenotype is readily reversible. At 39 degrees C, the mitotic abnormality lasts 3-5 h, followed by reformation of a single nucleus and cell flattening in an interphase-like state. Subsequent cell cycle events appear to occur, as the cells duplicate chromosomes and initiate a second round of abnormal mitosis. Cell cycle traversion continues for at least 5 d in some cells despite abnormal mitosis resulting in cells accumulating several hundred chromosomes.

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Inhibition of herpes simplex virus type 1 replication in temperature-sensitive cell cycle mutants.

Journal of Virology ◽

10.1128/jvi.25.1.42-50.1978 ◽

1978 ◽

Vol 25 (1) ◽

pp. 42-50 ◽

Cited By ~ 18

Author(s):

K Yanagi ◽

A Talavera ◽

T Nishimoto ◽

M G Rush

Keyword(s):

Cell Cycle ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Temperature Sensitive ◽

Sensitive Cell ◽

Simplex Virus

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Overexpression Phenotypes of Plk1 and Ndrg2 in Schizosaccharomyces Pombe: Fission Yeast System for Mammalian Gene Study

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.277-279.1 ◽

2005 ◽

Vol 277-279 ◽

pp. 1-6 ◽

Cited By ~ 1

Author(s):

Young Joo Jang ◽

Young Sook Kil ◽

Jee Hee Ahn ◽

Jae Hoon Ji ◽

Jong Seok Lim ◽

...

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Rna Splicing ◽

Mammalian Cells ◽

Protein Modification ◽

Genetic System ◽

Functional Study ◽

Mammalian Genes

The fission yeast, Schizosaccharomyces pombe is a single-celled free-living fungus that shares many features with cells of more complicated eukaryotes. Many of the genes required for the cell-cycle control, proteolysis, protein modification, and RNA splicing are highly conserved with those of higher eukaryotes. Moreover, fission yeast has the merit of genetics and its genetic system is already well characterized. As such, the current study evaluated the use of a fission yeast system as a tool for the functional study of mammalian genes and attempted to set up an assay system for novel genes. Since the phenotypes of a deletion mutant and the overexpression of a gene are generally analyzed for a functional study of specific genes in yeast, the present study used overexpression phenotypes to study the functions of mammalian genes. Therefore, based on using a thiamine-repressive promoter, two mammalian genes were expressed in fission yeast, and their overexpressed phenotypes compared with those in mammalian cells. The phenotypes resulting from overexpression were analyzed using a FACS, which analyzes the DNA contents, and a microscope. One of the selected genes was the mammalian Polo-like kinase 1 (Plk1), which is activated and plays a role in the mitotic phase of the cell division cycle. The overexpression of various constructs of Plk1 in the HeLa cells caused cell cycle defects, suggesting that the ectopic Plk1s blocked the endogenous Plk1 in the cells. As expected, when the constructs were overexpressed in the fission yeast system, the cells were arrested in mitosis and defected at the end of mitosis. As such, this data suggests that the Plk1-overexpressed phenotypes were similar in the mammalian cells and the fission yeast, thereby enabling the mammalian Plk1 functions to be approximated in the fission yeast. The other selected gene was the N-Myc downstream-regulated gene 2 (ndrg2), which is upregulated during cell differentiation, yet still not well characterized. When the ndrg2 gene was overexpressed in the fission yeast, the cells contained multi-septa. The septa were positioned well, yet their number increased per cell. Therefore, this gene was speculated to block cell division in the last stage of the cell cycle, making the phenotype potentially useful for explaining cell growth and differentiation in mammalian cells. Accordingly, fission yeast is demonstrated to be an appropriate species for the functional study of mammalian genes.

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Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint.

Genetics ◽

10.1093/genetics/134.1.63 ◽

1993 ◽

Vol 134 (1) ◽

pp. 63-80 ◽

Cited By ~ 9

Author(s):

T A Weinert ◽

L H Hartwell

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Dna Replication ◽

Cell Cycle Control ◽

Dna Lesions ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

A Cell ◽

G2 Phase ◽

Rad Mutants

Abstract In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G2 phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase.

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Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

10.1101/470013 ◽

2018 ◽

Cited By ~ 5

Author(s):

Evgeny Zatulovskiy ◽

Daniel F. Berenson ◽

Benjamin R. Topacio ◽

Jan M. Skotheim

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Cell Growth ◽

Cell Size ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Inverse Correlation ◽

Cell Types ◽

Similar Amount ◽

Cell Cycle Inhibitor

Cell size is fundamental to function in different cell types across the human body because it sets the scale of organelle structures, biosynthesis, and surface transport1,2. Tiny erythrocytes squeeze through capillaries to transport oxygen, while the million-fold larger oocyte divides without growth to form the ~100 cell pre-implantation embryo. Despite the vast size range across cell types, cells of a given type are typically uniform in size likely because cells are able to accurately couple cell growth to division3–6. While some genes whose disruption in mammalian cells affects cell size have been identified, the molecular mechanisms through which cell growth drives cell division have remained elusive7–12. Here, we show that cell growth acts to dilute the cell cycle inhibitor Rb to drive cell cycle progression from G1 to S phase in human cells. In contrast, other G1/S regulators remained at nearly constant concentration. Rb is a stable protein that is synthesized during S and G2 phases in an amount that is independent of cell size. Equal partitioning to daughter cells of chromatin bound Rb then ensures that all cells at birth inherit a similar amount of Rb protein. RB overexpression increased cell size in tissue culture and a mouse cancer model, while RB deletion decreased cell size and removed the inverse correlation between cell size at birth and the duration of G1 phase. Thus, Rb-dilution by cell growth in G1 provides a long-sought cell autonomous molecular mechanism for cell size homeostasis.

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