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Biomedical subjects

Roger Brent

Publications and source records attributed to Roger Brent.

9 recordsLinked to original sources
PubMed2005

Regulated cell-to-cell variation in a cell-fate decision system.

Here we studied the quantitative behaviour and cell-to-cell variability of a prototypical eukaryotic cell-fate decision system, the mating pheromone response pathway in yeast. We dissected and measured sources of variation in system output, analysing thousands of individual, genetically identical cells. Only a small proportion of total cell-to-cell variation is caused by random fluctuations in gene transcription and translation during the response ('expression noise'). Instead, variation is dominated by differences in the capacity of individual cells to transmit signals through the pathway ('pathway capacity') and to express proteins from genes ('expression capacity'). Cells with high expression capacity express proteins at a higher rate and increase in volume more rapidly. Our results identify two mechanisms that regulate cell-to-cell variation in pathway capacity. First, the MAP kinase Fus3 suppresses variation at high pheromone levels, while the MAP kinase Kss1 enhances variation at low pheromone levels. Second, pathway capacity and expression capacity are negatively correlated, suggesting a compensatory mechanism that allows cells to respond more precisely to pheromone in the presence of a large variation in expression capacity.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2005

Automatic generation of cellular reaction networks with Moleculizer 1.0.

Accurate simulation of intracellular biochemical networks is essential to furthering our understanding of biological system behavior. The number of protein complexes and of chemical interactions among them has traditionally posed significant problems for simulation algorithms. Here we describe an approach to the exact stochastic simulation of biochemical networks that emphasizes the contribution of protein complexes to these systems. This simulation approach starts from a description of monomeric proteins and specifications for binding, unbinding and other reactions. This manageable specification is reasonably intuitive for biologists. Rather than requiring the inclusion of all possible complexes and reactions from the outset, our approach incorporates new complexes and reactions only when needed as the simulation proceeds. As a result, the simulation generates much smaller reaction networks, which can be exported to other simulators for further analysis. We apply this approach to the automatic generation of reaction systems for the study of signal transduction networks.

Algorithms↗
PubMed2004

Using protein-DNA chimeras to detect and count small numbers of molecules.

We describe general methods to detect and quantify small numbers of specific molecules. We redirected self-splicing protein inteins to create 'tadpoles', chimeric molecules comprised of a protein head covalently coupled to an oligonucleotide tail. We made different classes of tadpoles that bind specific targets, including Bacillus anthracis protective antigen and the enzyme cofactor biotin. We measured the amount of bound target by quantifying DNA tails by T7 RNA polymerase runoff transcription and real-time polymerase chain reaction (PCR) evaluated by rigorous statistical methods. These assays had a dynamic range of detection of more than 11 orders of magnitude and distinguished numbers of molecules that differed by as little as 10%. At their low limit, these assays were used to detect as few as 6,400 protective antigen molecules, 600 biotin molecules and 150 biotinylated protein molecules. In crudely fractionated human serum, the assays were used to detect as few as 32,000 protective antigen molecules. Tadpoles thus enable sensitive detection and precise quantification of molecules other than DNA and RNA.

Antigens, Bacterial↗
PubMed2002

Cyclin D3 activates Caspase 2, connecting cell proliferation with cell death.

Precancerous cells that enter S phase without appropriate growth and viability factors undergo programmed cell death, suggesting that apoptosis may help guarantee organismic integrity [Evan, G. & Littlewood, T. (1998) Science 281, 1317-1322]. However, the connection between proliferation and cell death has remained unclear. Here, we show that the positive cell cycle regulator cyclin D3 [Matsushime H., Roussel M. F., Ashmun, R. A. & Sherr, C. J. (1991) Cell 65, 701-713] interacts with the death enzyme Caspase 2 [Wang, L., Miura, M., Bergeron, L., Zhu, H. & Yuan, J. (1994) Cell 78, 739-750]. Directed expression of cyclin D3 and Caspase 2 in human cells potentiated apoptosis compared with expression of Caspase 2 alone. Cyclin D3 expression increased the amount of cleaved (active) Caspase 2. We describe a PCR mutagenesis/ligation/two-hybrid/green fluorescent protein approach that facilitates the isolation of missense mutant proteins defective in interaction with particular partners absent other phenotypes or knowledge of the system. We used this approach to isolate Caspase 2 mutants that did not bind cyclin D3 (noninteractors). Noninteractors were sensitive to apoptosis-dependent proteolysis, but did not potentiate apoptosis. Noninteractors did not block apoptosis caused by wild-type Caspase 2. Our results are consistent with the idea that an interaction with cyclin D3 may stabilize Caspase 2, and suggest that a physical interaction between cyclin D3 and Caspase 2 connects the genetic networks that govern cell-cycle progression with those that govern cell death.

Apoptosis↗