Growth control of the eukaryote cell: a systems biology study in yeast.

BACKGROUND: Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. RESULTS: Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. CONCLUSION: This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome-scale systems biology models of the eukaryotic cell.

Identification of changes in gene expression in dorsal root ganglia in diabetic neuropathy: correlation with functional deficits.

This study aimed to correlate the onset of functional deficits in diabetic neuropathy with changes in gene expression in rat dorsal root ganglia (DRG). After 1, 4, or 8 weeks of streptozotocin-induced diabetes, sensory and motor nerve conduction velocities (NCV) were measured as an indicator of neuropathy and changes in gene expression were measured using Affymetrix oligonucleotide microarrays. No significant changes in NCV were found after 1 week of diabetes, but after 4 and 8 weeks, there was a significant reduction in both sensory and motor NCV. Global gene expression changes in diabetic rat DRG were evident from principal component analysis of microarray data after 1, 4, and 8 weeks. Expression changes in individual genes were relatively small in line with a gradual degenerative neuropathy indirectly resulting from diabetes. Sets of differentially expressed genes have been identified and quantitative reverse transcriptase-polymerase chain reaction has been used to confirm the microarray data for several genes. Gene ontology overrepresentation analysis was performed on the microarray data to identify biologic processes altered in diabetic DRG. The genes identified in this study may be responsible for causing the functional deficits and suggest pathways/processes that require further investigation as possible targets for therapeutic intervention.

Doxycycline, the drug used to control the tet-regulatable promoter system, has no effect on global gene expression in Saccharomyces cerevisiae.

The tet-regulatable promoter system is commonly used for genetic studies in many eukaryotic organisms. The promoter is regulated using doxycycline. There are no obvious phenotypic effects observed when doxycycline is added to the growth medium of yeast to control expression from the promoter. It is widely accepted that doxycycline is innocuous to yeast. Global genetic studies are now commonplace and the tetO-system is being used in transcriptome studies. Hence, we wanted to ensure that the absence of phenotypic effects, on addition of doxycycline to the growth medium, is mirrored in transcriptome data. We have demonstrated that doxycycline has no significant effect on global transcription levels and will continue to use the tetO-regulatable promoter system for genetic studies.

Functional TIM10 chaperone assembly is redox-regulated in vivo.

The TIM10 chaperone facilitates the insertion of hydrophobic proteins at the mitochondrial inner membrane. Here we report the novel molecular mechanism of TIM10 assembly. This process crucially depends on oxidative folding in mitochondria and involves: (i) import of the subunits in a Cys-reduced and unfolded state; (ii) folding to an assembly-competent structure maintained by intramolecular disulfide bonding of their four conserved cysteines; and (iii) assembly of the oxidized zinc-devoid subunits to the functional complex. We show that intramolecular disulfide bonding occurs in vivo, whereas intermolecular disulfides observed in vitro are abortive intermediates in the assembly pathway. This novel mechanism of compartment-specific redox-regulated assembly is crucial for the formation of a functional TIM10 chaperone.