The Pathways over Time Project A one-semester research project in comparative functional genomics Cysteine and methionine are superimposed over a portion of “The Tree of Life” by Gustav Klimt (1909)

The metabolic pathways for synthesizing methionine and cysteine have changed during evolution Methionine and cysteine are sulfur-containing amino acids found in the proteins of all living organisms

Genome annotation projects predict enzyme function based on sequence similarities to known enzymes. Functional testing is usually missing. Over 40,000 completed genomes as of July 15, 2014 Primarily microbial sequences Genome sequencing projects offer opportunities for undergraduate research

This semester-long research project uses Saccharomyces cerevisiae, the budding yeast, to analyze the conservation of enzyme function Nonpathogenic model organism in its natural environment under the microscope Spanish "cerveza" Swahili "pombe" in our kitchens Yeast!

Yeast research has a long history Empirical research dominated for millenia Louis Pasteur shows that fermentation is due to the activities of microorganisms 1866 – “Etudes sur le Vin” Eduard Buchner shows that a cell-free extract of yeast is able to ferment sugar “enzyme = from yeast” 1907 – wins Nobel Prize

Ability to alternate between haploid and diploid forms made yeast an favored organism for geneticists Haploids and some diploids can propagate by division (1) When stressed, haploids of opposite mating types conjugate (2) to form diploids Diploids generally enter meiosis and form hardy, haploid spores (3) Spores are contained within an ascus

Model organisms have many of the same processes as more complex life forms Model organisms have many experimental advantages Why are yeast considered model organisms?

~6000 genes Haploid and diploid forms Small (~4 µm) and unicellular New generation every 1.5 hr Simple growth requirements Few genes have introns Many mutant phenotypes Genetic manipulation possible ~20,000-25,000 genes Diploid Large and multi-cellular New generation every 20 yr Complex growth requirements Many introns –complex splicing Phenotypes are complex Ethical issues preclude experimentation

Yeast are able to synthesize methionine de novo Also MET 1, 8, 7 and 13 Over 20 S. cerevisiae genes encode proteins involved in methionine and cysteine synthesis (not all appear here) We will use S. cerevisiae strains with targeted deletions in MET and CYS genes to analyze foreign gene function More later…….. Genes were identified in genetic screens and confirmed with molecular methods

1. Identify potential genes by their similarity to known yeast genes using bioinformatics databases 2. Clone the foreign genes into plasmids that drive overexpression in S. cerevisiae 3. Express the foreign genes in S. cerevisiae and see if they can compensate for missing MET genes (credits: Tree of Life Web project)Tree of Life Web project Can genes from distant organisms replace genes that are inactivated in budding yeast? The strategy

Ascomycota: spores are contained within an ascus S. cerevisiae and S. pombe are yeast species that separated from a common ancestor from billion years ago Unicellular eukaryotes Members of the ascomycota phylum of fungi Fission yeast Schizosaccharomyces pombe Budding yeast Saccharomyces cerevisiae (sugar fungus found in beer) Difference in lifestyle: One buds, the other divides to produce two equal offspring Shared a common ancestor ~1 billion years ago

Considerable genome remodeling has occurred since the species diverged whole genome duplication 14 Mbp 28 Mbp 9.4 Mbp 39 Mbp 32 Mbp 30 Mbp 12.6 Mbp Genome size (Mbp)

Let’s hope for some great results! You will be building on work from previous years Poster winners participate in the Biology Dept.’s Undergraduate Research Symposium (first study day in May) Students have demonstrated conservation of several MET gene between S. pombe and S. cerevisiae