David Stuart

Cell division and proliferation are driven by the action of Cyclin Dependent protein Kinases (CDKs) in conjunction with their regulatory subunits, cyclins. Inappropriate activation of cyclin/CDK complexes can drive unregulated cell division resulting in cell death or tumor formation. Cyclin/Cdk complexes also play essential roles in cellular development and differentiation processes. For example meiosis and the formation of gametes in all eukaryotic cells is strictly dependent upon Cyclin/CDK activity. Because of their crucial roles in cell proliferation and development cyclin/CDK expression and activity have multiple forms of regulation imposed upon them. Work in my laboratory is focused on understanding the mechanisms that regulate cell division and differentiation. Currently we are involved in three major lines of research:

1. Regulation of progression through the meiotic cell cycle. Meiosis is a form of cellular differentiation in which somatic diploid cells develop into haploid sex cells. I have shown that in budding yeast this process is dependent upon the Clb5/Cdc28 kinase. Genetics, molecular biology and proteomic approaches are being used to characterize the regulation of Clb5 at the levels of gene expression, protein stability, and interaction with specific kinase inhibitors. Additionally we are investigating the function of the meiosis-specific protein kinase Ime2 which appears to perform many of the functions attributed to G1 cyclin/CDK complexes in proliferating cells. These projects also involve investigations of checkpoints that restrain progression through meiosis when DNA has been damaged or is incompletely replicated.

2. The role of Cyclin/CDK complexes in the initiation of DNA replication. The protein complexes that assemble to initiate DNA replication are highly conserved among eukaryotes. Some components of these complexes must be "activated" by phosphorylation in order for the complex to fully assemble and initiate DNA synthesis. Clb/CDK and Dbf4/Cdc7 kinase activities are necessary for these activation events to occur in a timely fashion. We are using genetic and proteomic approaches to identifying substrates of the Clb5/Cdc28 complex and Ime2 in order to determine how these kinases promotes DNA replication. Additionally we are investigating the composition of protein complexes that regulate the initiation of DNA replication during meiosis. Our investigations are revealing a model indicating that the primary role of Clb5/Cdc28 kinase is to recruit several other proteins to the origin of replication and some of these substrates may be uniquely involved in regulating DNA replication during meiosis.

3. Regulation of meiotic gene expression. When yeast cells are deprived of nutrients they abandon mitotic proliferation and embark on a program differentiation that proceeds through meiosis and ultimately produces haploid spores. This process is driven by regulated waves of meiosis-specific gene expression. Meiosis-specific genes are repressed during normal growth by the action of repressor complexes that contain Histone DeACetylases (HDACs). We are investigating the regulation of HDAC containing repressor complexes to determine how they are inactivated to allow the induction of meiosis-specific genes. We have characterized the function of the meiosis-specific transcription factor Ndt80 in activating middle sporulation genes, and how its function is integrated with the repressor complex that contains the HDAC Hst1. Early meiosis-specific genes are repressed by a complex that contains Ume6 and the HDAC Rpd3. We are investigating how the HDAC activity of Rpd3 is regulated during meiosis. We are using molecular biology and biochemical techniques to perform structure function analysis of both repressor complexes to determine how these proteins regulate a diverse set of genes during cellular differentiation.

Ultimately we seek to understand the mechanisms that regulate cell division in hopes of gaining insight into the causes of and therapies for proliferative disorders.

Synthetic biology and metabolic engineering approaches to biofuel and bioproduct synthesis. The increasing cost of fossil fuel production, and concerns about global warming have elevated interest in the development and application of alternative fuel sources. We are applying synthetic biology and metabolic engineering approaches to manipulate the metabolism of yeast and other microbial cells for the production of biofuels and valuable chemicals. These investigations involve the construction of novel metabolic pathways, the ablation of competing metabolic pathways and metabolomic analysis.

1. Engineering yeast for the synthesis of butanol, isobutanol and pentanols. Budding yeasts are being engineered by introducing multi-enzyme complexes for the production of medium chain alcohols that can be used as fuels. To optimize the process, genes are sourced from a variety of organisms and utilized based upon their in vivo activity in yeast. Additionally, targeted and random approaches are being applied to increase the tolerance of cells to alcohols. These studies are also yielding and increased understanding of the regulation of metabolism and metabolic pathways.

2. Engineering yeast for the production of fatty acid molecules with potential for use as transport fuels. Budding yeast are being engineered to synthesize and secrete lipids that can be refined and used as "biodiesel". Additionally, we are interested in engineering yeast for the production of specific lipid molecules that have medical and industrial applications.