The Measday Lab’s current research is the study of chromosome segregation in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) using molecular biology and genomic tools. We am interested in understanding how chromosomes attach to spindle microtubules and segregate equally in mitosis. Spindle microtubules attach to the kinetochore which is composed of centromere DNA and associated proteins. There are over 40 kinetochore proteins identified in budding yeast that are grouped into inner, central and outer kinetochore categories. We are specifically interested in understanding the function of the outer kinetochore complexes and how they are regulated.
Most yeast researchers are currently studying basic cell processes using only a few strains of yeast. We will be expanding our research program to study chromosome biology in wine yeast. Interestingly, there are fundamental differences between laboratory yeast and industrial wine yeast. For example, wine yeast are able to grow in very high concentrations of sugar and ethanol which would kill a laboratory strain of yeast. Wine yeast also contain abnormal numbers of chromsomes (aneuploidy) whereas laboratory yeast have equal numbers of chromosomes. We will combine molecular biology and genomics knowledge from laboratory S. cerevisiae to identify genes important for survival in an industrial growth environment and to study chromosome segregation in wine yeast. Studying aneuploid wine yeast will provide important insight into diseases hallmarked by abnormal chromosome numbers such as Down’s syndrome and cancer.
Dr. Vivien Measday, PhD, Canada Research Chair and MSFHR Scholar; Associate Professor, Faculty of Land and Food Systems; Associate member of Michael Smith Laboratories and Department of Biochemistry and Molecular Biology
Krystina Ho, Ph.D. candidate
Our lab studies chromosome segregation in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) using molecular biology, genetic and genomic tools. Studying chromosome segregation using yeast as a model system will provide important insights into diseases hallmarked by abnormal chromosome numbers such as Down’s syndrome and cancer. We are specifically interested in understanding how chromosomes attach to spindle microtubules and segregate equally during mitosis. Spindle microtubules attach to the multiprotein kinetochore complex which is composed of centromere DNA and associated proteins. Well over 50 kinetochore proteins have been identified in budding yeast that are grouped into inner, central and outer kinetochore protein complexes depending on their proximity to centromere DNA. Our research focuses on understanding the function of the highly conserved central kinetochore Ndc80 complex and is currently funded by the CIHR (Canadian Institutes of Health Research) and the MSFHR (Michael Smith Foundation for Health Research).
There are approximately 5,000 nonessential genes in the budding yeast genome that have been systematically deleted by the yeast community. A method termed Synthetic Genetic Array (SGA) analysis, which was pioneered at the University of Toronto, has been developed which enables yeast researchers to perform genetic screens using the yeast deletion array. In collaboration with Dr. Brenda Andrews and Dr. Charlie Boone at the University of Toronto, Dr. Kristin Baetz at the University of Ottawa and Dr. Phil Hieter at the University of British Columbia, we have performed genome-wide SGA screens to identify yeast mutants that are defective in kinetochore function. We have an exciting list of genes that have a role in chromosome segregation and the biological tools necessary to decipher their role in this process.
The long term objective of this research program is to identify and characterize genes in yeast that mediate stress responses to survive conditions during fermentation. Functional characterization of the budding yeast Saccharomyces cerevisiae (S. cerevisiae) genome by gene deletion has shown that 20% of the ~6200 genes are essential for growth in standard laboratory medium (2% glucose). However S. cerevisiae is exposed to 20% sugars (equimolar of glucose and fructose) during industrial fermentations in grape juice. Hence many of the genes required for survival under these conditions will not have been identified using standard laboratory media. Profiling of the yeast deletion mutants in their industrial environment will not only identify genes required for industrial fermentation but will also reveal roles for genes that could not otherwise be characterized using conventional laboratory conditions.
We are taking a genomics approach to understand the molecular network of specific fermentation related stress responses in S. cerevisiae. In collaboration with Dr. Mike Tyers’ lab at the University of Toronto, we are using the yeast deletion set barcode method to perform yeast fitness profiling experiments. To enable identification of a single deletion from a pool of deletion mutants, two unique 20mer nucleotide barcodes – called the uptag and downtag – were introduced upstream and downstream, respectively of the kanMX4 marker gene (Figure 1A). The uptags and downtags are flanked by a common pair of PCR primers which are used to amplify the tags from a pool of deletion mutants. The barcodes enable all deletion mutants to be screened for a particular phenotype by growing the deletion pool under certain conditions, isolating genomic DNA, PCR amplification of the tags and hybridization to an oligonucleotide array carrying the tag complements (Figure 1B). The abundance of each deletion strain is then determined by quantifying the associated molecular bar codes. If a gene is important for growth, the corresponding deletion strain diminishes in the culture. Phenotypic profiling studies with the deletion mutant collection using standard laboratory conditions have successfully attributed phenotypes to ~5000 S. cerevisiae genes, however the remaining ~1000 genes still have no known function. Analyzing growth of the yeast deletion set under conditions in which yeast survive both in nature and in the winery will likely identify functions for many of the uncharacterized genes in S. cerevisiae.
Yeast has evolved to survive constant fluctuations in their surroundings by rapidly adapting to meet challenges of new environments. During alcoholic fermentation (conversion of sugars into alcohol and CO2), yeast are exposed to a variety of stresses including high osmolarity, organic acid stress and ethanol toxicity. The yeast deletion set has not yet been profiled under most fermentation related stress conditions. Our research combines the genomic technology of lab yeast with the environmental conditions of wine yeast to uncover key pathways required for surviving the stresses imposed by fermentation and to functionally annotate uncharacterized S. cerevisiae genes. Understanding stress resistance pathways in yeast creates potential to increase stress resistance in wine yeast, improve industrial performance and reduce the cost to Canadian wineries of incomplete (sluggish or stuck) fermentations.