Cell Cycle Control Mechanisms and Protocols Methods in Molecular Biology
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Moreover, transcriptional profiling of cells overexpressing SKO1 revealed that genes involved in the pheromone response pathway are significantly upregulated. However, genes involved in the pheromone response pathway do not appear to be regulated by SKO1 under normal culture conditions, at least as measured by chromatin-immunoprecipitation of SKO1 . Therefore, our results suggest that SKO1 regulates genes in the pheromone response pathway through a gain-of-function mechanism, e.
In this paper, we describe a near-saturating screen for yeast genes whose overexpression causes cell cycle delays and which are thus likely to function in cell cycle progression. Our analysis thus complements previous screens. These results lay the foundation for future experiments to elucidate the precise roles of these genes in cell cycle progression, such as the mechanisms of RFA1 and YPRC.
Overexpression screens such as we have described here provide complementary information to loss-of-function studies and therefore offer new opportunities for discovery of genetic interactions, such as by systematically testing the overexpression plasmids in deletion strains to screen for phenotype suppression or synthetic interactions. Finally, since overexpression is an efficient technique in human cell culture and since regulation of cell proliferation is an important aspect of studying human diseases, we anticipate that a similar effort to this work in human cell lines could accelerate our understanding of cell cycle control in mammalian systems and help to further clarify the many connections between cell cycle control and cancer.
Flow cytometry histograms of ORF overexpression strains causing cell cycle defects upon induction. Protein expression is significantly induced in overexpression strains, even for proteins expressed natively at high levels. Makkuni Jayaram for providing the plasmid, strain and protocol for mitotic instability assay, and Dr.
Angela M. Bardo in the Microscopy and Imaging Facility of the Institute for Cellular and Molecular Biology for her assistance with flow cytometry. We also thank Dr.
Elizabeth J. Grayhack at University of Rochester for providing the vector BG Abstract Regulation of cell cycle progression is fundamental to cell health and reproduction, and failures in this process are associated with many human diseases. Author Summary All cells require proper cell cycle regulation; failure leads to numerous human diseases.
Introduction The budding yeast Saccharomyces cerevisiae undergoes a cell cycle similar to other eukaryotic organisms except for the lack of nuclear envelope dissolution during mitosis and the production of daughter cells via budding, and thus budding yeast has become a model system for studying eukaryotic cell cycle progression  due to its rapid division, the availability of genetic tools, and homology to higher eukaryotic cell cycle processes.
Cell division control – Cabimer
High-Throughput Flow Cytometry Flow cytometry analyses were performed as in . Download: PPT. Nuclear Staining and Bud Size Measurements ORF strains showing reproducible cell cycle arrest were grown and induced as described above. Independent Validation by Bud Size Measurements The size of the bud relative to the size of the mother cell is the most notable morphological landmark of the cell cycle stages in budding yeast.
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Independent support for cell cycle delays from histograms of the percentages of cells with no bud, small bud or large bud. Subcategorizing Genes Newly Implicated in the Cell Cycle using Drug Sensitivities One major expected cause of defective cell cycle progression is chromosome instability, especially chromosome loss and non-disjunction. Functional Analysis of Genes Affecting Cell Cycle Progression when Overexpressed We examined in more detail the functions for the genes that caused cell cycle defects when overexpressed.
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Mechanisms and Protocols
Overexpression of RFA1 causes chromosomal segregation and spindle defects. Figure 8. Overexpression of SKO1 activates the pheromone response pathway. Overexpression Phenotypes Are Generally Distinct from Loss-of-Function Phenotypes Overexpression of a normal gene product can result in gain-of-function, but may also mimic loss-of-function phenotypes  , such as in cases where precise levels of a protein are required, with either too much or too little equally disruptive.
Figure 9. Overexpression phenotypes are generally distinct from loss-of-function phenotypes. Conclusions In this paper, we describe a near-saturating screen for yeast genes whose overexpression causes cell cycle delays and which are thus likely to function in cell cycle progression. Supporting Information.
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Figure S4. Overlap of identified genes with previous large-scale studies. Table S1. Table S2. Summary of strains with cell cycle defects. Table S3.
Comparison between over-expression and loss-of-function phenotypes. Table S4. Genes whose overexpression induces slow growth, drug sensitivity. Table S5. Genes upregulated following overexpression of SKO1. Table S6. Over-expression strains appearing diploid or 3C. References 1.
Hartwell LH Saccharomyces cerevisiae cell cycle. Bacteriol Rev — View Article Google Scholar 2. Microbiol Mol Biol Rev — View Article Google Scholar 3. Schafer KA The cell cycle: a review.
Vet Pathol — View Article Google Scholar 4. Mol Cell 2: 65— View Article Google Scholar 5. Bioinformatics — View Article Google Scholar 6. Mol Biol Cell 9: — View Article Google Scholar 7. Hartwell LH Genetic control of the cell division cycle in yeast. Genes controlling DNA replication and its initiation. J Mol Biol — View Article Google Scholar 8. Genes controlling bud emergence and cytokinesis.
Exp Cell Res — View Article Google Scholar 9. Hartwell LH Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae. J Bacteriol — View Article Google Scholar Detection of mutants. Nature — Nat Cell Biol 9: — Mol Biol Cell — Genes Dev — Biochemical and biophysical research communications, 3.
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