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