Yeast as a Model Organism

1. Yeast as a Model Organism RaghavRamachandran Ambhi Ganesan September 9,2008

2. A model for life science research Test out various hypotheses on a smaller scale. Saves time and money. Practically impossible to carry out certain research directly on intended targets. Learn from mistakes and go back to the drawing board. Insights into the complex mechanisms of Life, that are conserved over different levels.

3. Yeast based models in basic Science and applied research • Neurodegenerative disorders: • Ease of expt’al manipulation, high conservation of mechanisms, well defined genome • Studying mechanisms in yeast provide insights into human system. • Designing therapeutics/drugs against homologous protein aggregates in yeast. • Aging: • Calorie restriction affects life span in worms, flies, and rodents, apart from yeast (reducing glucose or amino acid concns can increase life spans) • Life span extension from Sir2-overexpression, TOR-inhibition, Sch9/Akt or DR has been observed in yeast, worms, and flies • Nutrient responsive protein TOR speculated to play conserved role.

4. Why use yeast? • The S.cerevisiae yeast has one of the smallest genomes of eukaryotes, being unicellular. • Its genome contains 12, 495,682 base pairs and 5770 genes as opposed to 3.3 *109 base pairs and ~20500 genes in humans. • Also, S.cerevisiae (baker’s yeast) is viable with numerous markers and available in large quantities making it cheap to study.

5. Why use yeast? • Perhaps, the most striking feature of S.cerevisiae is its existence in both haploid and diploid forms. • This makes it easy to isolate recessive mutations in haploids. • Also, DNA transformed in S.cerevisiae can undergo homologous recombination readily, into the S.cerevisiae genome.

6. Why use yeast? • By analyzing the S.cerevisiae mutants observed from homologous recombination of foreign DNA, the functions of several proteins in vivo can be discerned. • The entire genome of S.cerevisiae was sequenced in 1996 and since then, it has been used as a eukaryotic model for the study of protein interactions and infectious diseases.

7. Yeast Cell Cycle • Each Yeast Cell undergoes four phases in its life cycle: G1,S,G2,M (growth, synthesis, mitosis) • In S. cerevisiae, arrangement of microtubules and duplication of spindle pole bodies takes place early in the life cycle to allow for bud formation. • Thus, budding S.cerevisiae lacks clear distinction between S, G2 and M phases.

8. Yeast Cell Cycle

9. Regulation of cell cycle • In the G1 phase of the cycle, the yeast cell has three options • It can complete the cycle and divide • It can leave the cycle, if nutritionally starved, where it is resistant to heat and chemical treatment • It can mate with a cell of opposite sex, if haploid, after a transient arrest in G1. • It can undergo meiosis to produce four haploid cells under nutritional starvation, if diploid

10. Regulatory genes in yeast • A gene is a cell cycle regulator if its mutant causes inappropriate progression through the cycle. • CDC 28-p34 protein kinase in G1/S and G2/M • CLN1, CLN2, WHI1-G1 cyclins in G1/S • MIH1- inducer of mitosis in G2/M • CKS 1, CDC 37, CDC 36 (haploids), CDC 39 (haploids)

11. G1/S phase of yeast life cycle • The START phase occurs during G1 and after this phase, the cell is committed to DNA replication and cell division. • Before passing START, cells must obtain a critical mass. • CDC28 is a critical START p34 protein kinase whose mutants can block cells at G1/S or G2 phases.

12. G2/M phase of yeast life cycle • While START commits the cell to its life cycle, mitosis can only take place after the S phase thus requiring a second checkpoint in the life cycle, namely the G2/M phase. • The MIH1 gene speeds up the entry of cells into mitosis. The role of other genes in regulating G2/M in S.cerevisiae is unclear.

13. Relationship between M and S phases • Initiation of M phase depends upon successful completion of DNA replication in S phase. The RAD9 gene performs this function in S.cerevisiae. If DNA replication is delayed, cells undergo mitosis with lethal effects. • The p34 kinase increases in activity on the onset of mitosis and its activity can be regulated by tyrosine dephosphorylation at the G2/M stage.

14. yeast life cycle-Meiosis • START-specific genes, like CDC 28, act after DNA replication in G1/S meiosis, whereas they have an indirect affect on DNA replication in meiosis. • However, CDC28 is required for the second G2/M meiosis, where the M and S phases are uncoupled from each other.

15. Exit from life cycle • Under nutritional starvation, yeast cells stop growing and exit the life cycle (G0 phase). • Unlike in mammalian cells, growth factors may not play a role in growth control in yeast cells and such cells in the stationary (G0 phase) are metabolically dormant.

16. Yeast as a model organism: Genetic analyses using S.cerevisiae life cycle Chemical mutagen  grow colonies  replica plate  identify isolates Complementation to identify recessive lethal mutants Clone WT gene and sequence it Linkage analysis using Meiotic analysis.

17. Functional characterization of the S. cerevisaeGenome by Gene Deletion and Parallel Analysis • The usual methods: • Random mutagenesis – rapid but matching phenotypes is slower • Genetic footprinting – simultaneous testing but mutant strains can not be recovered • The new method: • Delete entire ORFs using PCRs and homologous recombination • Direct and simultaneous analysis • Rapid and increased sensitivity

18. Deletion of ORFs by homologous recombination

19. Essential Genes • Deletions of ‘essential’ genes: • Essential for viability and lacking human homologues => targets for antifungal drugs • 356 ORFs identified as essential – failed to grow (YEPD, 30 oC) as haploid deletants. • Only 56 % of these previously shown to be essential for viability. • 1620 non-essential genes identified. • Additional homozygous and 2 haploid deletants constructed.

20. Non-essential genes • Non-essential genes: • Relatively more complicated to analyze than essential genes; may require complicated growth conditions to observe the effects for some. • 558 homozygous deletion mutants pooled and grown in Rich (R) and Minimal (M) media. • Aliquots from both pools  Amplify tags  Hybridize to complements on array  Hybrid. Data, measure of growth rate. • Correlation of UPTAG and DOWNTAG growth rates (<0.6 of WT for the growth-impaired strains)

21. Results • New findings: • Genes whose inactivation affects growth are not necessarily the ones induced during growth under the same particular conditions. • Caveats: • Neomycin phosphatase (product of KanMX4) may affect fitness • Composition of pool, culture conditions • Complementation (?)

22. Yeast as a tool:Yeast two hybrid (Y2H) system • Native GAL4 protein (881aa) contains 2 distinct domains: DNA binding and Activation Domains • Fuse DB (1-147) with protein X • Fuse portion of AD (768-881) with protein Y • If X and Y interact with each other in vivo, DB and AD will be brought together sufficient enough to activate the AD. • This recruits the transcription machinery • LacZ product is formed. • Caveats: • Interactions need to occur within yeast nucleus • GAL4 Activation region is accessible to transcription machinery • BD-X hybrid is itself not an activator http://www.biologicalprocedures.com/bpo/arts/1/16/m16f1lg.gif