Temperature Sensitive Yeast Library Poised to Uncover Gene Function
University of Toronto ♦ Published in Nature Biotechnology, Mar. 27, 2011
In efforts to document the roles of essential eukaryotic genes, a group of researchers at The Terrence Donnelly Centre for Cellular and Biomolecular Research have constructed an expansive library of yeast mutants that can be manipulated with temperature to provide insight into gene function. A set of 787 temperature-sensitive strains was produced by amplifying temperature-sensitive alleles from yeast mutants and then integrating these back into a common genetic background at their native loci. To allow for the use of high-throughput screening methods a selectable marker (kanMX) was linked to each allele.
Temperature sensitive alleles allow for fine tuning of gene function with three conditions or states: permissive, semi-permissive, and restrictive. In this study, led by Dr. Charles Boone, chemical-genetic suppression analyses were carried out on the temperature-sensitive mutant collection using small molecule compounds at both permissive and non-permissive states. If a yeast mutant grows under non-permissive conditions, it can be concluded that it contains a mutation that is suppressed by a given small molecule. By creating a library populated with mutant strains with a common genetic background, researchers were able to use high-throughput strain manipulation and then carry out high-content screens at the single cell level. Gene function was determined by visualization and quantitative measurement of specific morphological features. In order to score the entire library Dr. Boone’s group created customized software that carried out automated image analysis using features such as cell shape, budding index, organelle density and a panel of 85 more reporter-specific parameters. Impressively, all computationally derived phenotypes were confirmed manually with no discrepancy in findings. Researchers took the library for a test run and performed quantitative analysis of a GFP-tubulin marker revealing that condensin and cohesin have roles in spindle disassembly.
Researchers believe the mutant library will be amenable to exploration with high-throughput methods such as high-resolution growth profiling, chemical-genetic suppression, and high content screening to elucidate the role of highly conserved signaling pathways in the model organism. Yeast has 1,101 essential genes; the mutant collection created here represents 45% of these (497 alleles). A non-overlapping set of mutants created recently covers another 250 alleles. Taken together these collections cover roughly 65% of the essential genes in yeast and are a powerful means to begin annotating the functions of all of yeast’s essential genes.
Normalizing Src-Kinase Enhancement of NMDAR, A New Paradigm for Treating Schizophrenia
The Hospital for Sick Children ♦ University of Toronto ♦ Tufts University School of Medicine
Published in Nature Medicine, Mar. 27, 2011
Excessive NRG1β-ErbB4 signaling in the brain is a hallmark of individuals suffering from schizophrenia. This aberrant signaling is believed to contribute to the hypofunction of a specific glutamate receptor, NMDAR, that is crucial for synaptic plasticity and long-term potentiation at Schaffer collateral-CA1 synapses in the hippocampus and prefrontal cortex. A popular hypothesis for the cognitive deficits and hallucinations associated with schizophrenia has been that they are the result of the general hypofunction of NMDAR.
Dr. Michael Salter and his team at The Hospital for Sick Children proposed something a little different. They hypothesized that the underlying cause of schizophrenic symptoms was actually the result of interference in a cellular mechanism that enhances NMDAR function. The tyrosine kinase Src is the primary agent mediating NMDAR phosphorylation and enhancement, and is also involved in promoting NMDAR-dependent long-term potentiation. For these reasons Dr. Salter and his team sought to investigate whether NRG1β-ErbB4 signaling has an effect on Src-mediated phosphorylation of NMDAR. It turns out it does.
Analysis of whole-cell recordings of neurons in the CA1 layer of acute slices of hippocampus removed from adult animals revealed that NRG1β-ErbB4 signaling does indeed affect Src-mediated enhancement of NMDAR function. The same was found in slices of prefrontal cortex. Researchers believe that NRB1β signaling, via its cognate receptor ErbB4, compromises the catalytic activity of Src kinase which in turn interferes with downstream events that require Src-mediated enhancement, including long-term potentiation at Schaffer collateral synapses. NRB1β-ErbB4 signaling could exert its effects by suppressing activators or facilitating inhibitors of kinase function. The work by Dr. Salter and his team provides a vital piece to the mysterious puzzle that is schizophrenia, and identifies a novel therapeutic regime to tackle cognitive dysfunction associated with the disorder.