ATP analog-sensitive Pat1 protein kinase for synchronous fission yeast meiosis at physiological temperature

To study meiosis, synchronous cultures are often indispensable, especially for physical analyses of DNA and proteins. A temperature-sensitive allele of the Pat1 protein kinase (pat1-114) has been widely used to induce synchronous meiosis in the fission yeast Schizosaccharomyces pombe, but pat1-114-induced meiosis differs from wild-type meiosis, and some of these abnormalities might be due to higher temperature needed to inactivate the Pat1 kinase. Here, we report an ATP analog-sensitive allele of Pat1 [Pat1(L95A), designated pat1-as2] that can be used to generate synchronous meiotic cultures at physiological temperature. In pat1-as2 meiosis, chromosomes segregate with higher fidelity, and spore viability is higher than in pat1-114 meiosis, although recombination is lower by a factor of 2–3 in these mutants than in starvation-induced pat1⁺ meiosis. Addition of the mat-Pc gene improved chromosome segregation and spore viability to nearly the level of starvation-induced meiosis. We conclude that pat1-as2 mat-Pc cells offer synchronous meiosis with most tested properties similar to those of wild-type meiosis.     

Why minimal is not optimal: Driving the mammalian cell cycle—and drug discovery—with a physiologic CDK control network

Progression through the eukaryotic cell division cycle is governed by the activity of cyclin-dependent kinases (CDKs). For a CDK to become active it must (1) bind a positive regulatory subunit (cyclin) and (2) be phosphorylated on its activation (T) loop. In metazoans, multiple CDK catalytic subunits, each with a distinct set of preferred cyclin partners, regulate the cell cycle, but it has been difficult to assign functions to individual CDKs in vivo. Biochemical analyses and experiments with dominant-negative alleles suggested that specific CDK/cyclin complexes regulate different events, but genetic loss of interphase CDKs (Cdk2, -4 and -6), alone or in combination, did not block proliferation of cells in culture. These knockout and knockdown studies suggested redundancy or plasticity built into the CDK network but did not address whether there was true redundancy in normal cells with a full complement of CDKs. Here, we discuss recent work that took a chemical-genetic approach to reveal that the activity of a genetically non-essential CDK, Cdk2, is required for cell proliferation when normal cyclin pairing is maintained. These results have implications for the systems-level organization of the cell cycle, for regulation of the restriction point and G?/S transition and for efforts to target Cdk2 therapeutically in human cancers.     

ATP analog-sensitive Pat1 protein kinase for synchronous fission yeast meiosis at physiological temperature

To study meiosis, synchronous cultures are often indispensable, especially for physical analyses of DNA and proteins. A temperature-sensitive allele of the Pat1 protein kinase (pat1-114) has been widely used to induce synchronous meiosis in the fission yeast Schizosaccharomyces pombe, but pat1-114-induced meiosis differs from wild-type meiosis, and some of these abnormalities might be due to higher temperature needed to inactivate the Pat1 kinase. Here, we report an ATP analog-sensitive allele of Pat1 [Pat1(L95A), designated pat1-as2] that can be used to generate synchronous meiotic cultures at physiological temperature. In pat1-as2 meiosis, chromosomes segregate with higher fidelity, and spore viability is higher than in pat1-114 meiosis, although recombination is lower by a factor of 2–3 in these mutants than in starvation-induced pat1⁺ meiosis. Addition of the mat-Pc gene improved chromosome segregation and spore viability to nearly the level of starvation-induced meiosis. We conclude that pat1-as2 mat-Pc cells offer synchronous meiosis with most tested properties similar to those of wild-type meiosis.     

Why minimal is not optimal: Driving the mammalian cell cycle—and drug discovery—with a physiologic CDK control network

Progression through the eukaryotic cell division cycle is governed by the activity of cyclin-dependent kinases (CDKs). For a CDK to become active it must (1) bind a positive regulatory subunit (cyclin) and (2) be phosphorylated on its activation (T) loop. In metazoans, multiple CDK catalytic subunits, each with a distinct set of preferred cyclin partners, regulate the cell cycle, but it has been difficult to assign functions to individual CDKs in vivo. Biochemical analyses and experiments with dominant-negative alleles suggested that specific CDK/cyclin complexes regulate different events, but genetic loss of interphase CDKs (Cdk2, -4 and -6), alone or in combination, did not block proliferation of cells in culture. These knockout and knockdown studies suggested redundancy or plasticity built into the CDK network but did not address whether there was true redundancy in normal cells with a full complement of CDKs. Here, we discuss recent work that took a chemical-genetic approach to reveal that the activity of a genetically non-essential CDK, Cdk2, is required for cell proliferation when normal cyclin pairing is maintained. These results have implications for the systems-level organization of the cell cycle, for regulation of the restriction point and G₁/S transition and for efforts to target Cdk2 therapeutically in human cancers.     

Generation of a set of conditional analog-sensitive alleles of essential protein kinases in the fission yeast Schizosaccharomyces pombe

The genome of the fission yeast Schizosaccharomyces pombe encodes for 17 protein kinases that are essential for viability. Studies of the essential kinases often require the use of mutant strains carrying conditional alleles. To inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. The mutation of a single residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. Using this approach, we constructed conditional analog-sensitive alleles of 13 essential protein kinases in the fission yeast S. pombe.