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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)
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 0.3-1 billion years ago
Unicellular eukaryotesMembers 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