Identification of cell cycle regulated genes

Proliferating cells have to duplicate their constituents and segregate them equally to their daughter cells. In each cell division cycle a single round of DNA replication during S-phase is followed by an accurate distribution of chromosomes to the two daughter cells in mitosis. This process is controlled by the oscillatory activity of cyclin dependent kinases (CDK) that regulate the transitions between the major phases of the cell cycle, such as entry into S-phase, prevention of rereplication and entry and progression through mitosis. Although the essential role of these kinases is well documented their precise functions are still poorly understood. Errors in this process, such as chromosome segregation defects in mitosis, cause aneuploidy, one of the hallmarks of cancer. This study aims to identify new genes that are regulated during the cell cycle and function either in the regulation or execution of mitosis. These genes might provide new insights into the mechanisms of cell cycle regulation in human cells and might further our understanding of cancerogenesis. To achieve this goal new technologies will be used to identify CDK substrates, proteins that interact with cyclin, as well as proteins marked with green fluorescent protein to make them visible during cell division. Genes that turn up to be interesting using these screens shall be evaluated using RNA interference, a novel technology to suppress gene function efficiently. To identify kinase substrates we will employ small pool expression cloning to look for proteins that can be phosphorylated by recombinant active CDK in vitro and by performing a yeast two-hybrid interaction screen to identify proteins that interact with cyclin. Genome-wide expression profiling of genes expressed cyclically during the cell division cycle has revealed a set of genes with both known and unknown functions during the cell cycle. We propose to exploit this data set to identify novel cell cycle genes by defining a set of 100 uncharacterized genes that are induced in late G2 phase or mitosis, which, in addition to the genes identified in the substrate and protein interaction screens, will be evaluated by using stable RNA interference (RNAi). In addition, we will express GFP-tagged novel full-length cDNA clones in tissue culture cells and look for changes in sub-cellular localization and altered protein stability as cells go through mitosis by using time-lapse fluorescence videomicroscopy. These functional genomic screens should lead to the discovery of CDK substrates and new genes that function during the cell cycle.

Proliferating cells have to duplicate their constituents and segregate them equally to their daughter cells. In each cell division cycle a single round of DNA replication during S-phase is followed by an accurate distribution of chromosomes to the two daughter cells in mitosis. This process is controlled by the oscillatory activity of cyclin dependent kinases (CDK) that regulate the transitions between the major phases of the cell cycle, such as entry into S-phase, prevention of rereplication and entry and progression through mitosis. Although the essential role of these kinases is well documented their precise functions are still poorly understood. Errors in this process, such as chromosome segregation defects in mitosis, cause aneuploidy, one of the hallmarks of cancer. This study aims to identify new genes that are regulated during the cell cycle and function either in the regulation or execution of mitosis. These genes might provide new insights into the mechanisms of cell cycle regulation in human cells and might further our understanding of cancerogenesis. To achieve this goal new technologies will be used to identify CDK substrates, proteins that interact with cyclin, as well as proteins marked with green fluorescent protein to make them visible during cell division. Genes that turn up to be interesting using these screens shall be evaluated using RNA interference, a novel technology to suppress gene function efficiently. To identify kinase substrates we will employ small pool expression cloning to look for proteins that can be phosphorylated by recombinant active CDK in vitro and by performing a yeast two-hybrid interaction screen to identify proteins that interact with cyclin. Genome-wide expression profiling of genes expressed cyclically during the cell division cycle has revealed a set of genes with both known and unknown functions during the cell cycle. We propose to exploit this data set to identify novel cell cycle genes by defining a set of 100 uncharacterized genes that are induced in late G2 phase or mitosis, which, in addition to the genes identified in the substrate and protein interaction screens, will be evaluated by using stable RNA interference (RNAi). In addition, we will express GFP-tagged novel full-length cDNA clones in tissue culture cells and look for changes in sub-cellular localization and altered protein stability as cells go through mitosis by using time-lapse fluorescence videomicroscopy. These functional genomic screens should lead to the discovery of CDK substrates and new genes that function during the cell cycle.