1. Introduction to yeast genetics Michelle Attner July 24, 2012

2. What is budding yeast, S. cerevisiae? Electron micrographDIC (light microscopy)

3. Advantages to budding yeast as a model organism Simple, eukaryotic cell (~10μm diameter) Compact genome (genome is sequenced) Cells grow on plates and in culture Short generation time (~90 minutes) Live happily as haploids and diploids Easy to manipulate genes (swap promoters, delete genes) Easy to conduct genetic screens Many yeast genes have evolutionarily conserved homologs in humans

4. Outline and Learning Objectives: Intro to Yeast Genetics 1.Understand the life cycle of budding yeast 2.Describe how yeast cells mate, and understand how the BAR1 gene contributes to the regulation of this process 3.Understand the mitotic cell cycle of budding yeast a.Explain how budding yeast was used as a model system to isolate genes required for cell cycle regulation b.Understand the basics of doing a genetic screen in yeast 4.Understand the meiotic cell divisions of budding yeast a.Explain how sporulation and tetrad formation aids scientists studying yeast

5. The life cycle of budding yeast mating Image: Wikipedia Yeast have 2 matings types: a and alpha a haploids and alpha haploids divide An a and alpha cell can fuse to form an a/alpha diploid. a/alpha diploids can divide asexually or they can undergo meiosis to form four haploid gametes called spores

6. Brief genetics review What is a gene? What is an allele? What is a mutation? What is a genotype? What is a phenotype? What is the difference between mutations conferring recessive and dominant phenotypes? – Why is yeast great for studying mutations conferring recessive phenotypes? – How can you use yeast to determine if your mutation confers a recessive or dominant phenotype?

7. Yeast mating is a fusion event Image: Wikipedia 1.What is the signal for mating? a cells secrete a factor α cells secrete α factor a cells have receptors for α factor, and vice versa 2.When α factor binds to receptors on a cell, a MAP kinase pathway is activated. 3.The output of this pathway is cell cycle arrest and shmoo formation 4.A shmoo is a mating projection that is necessary for cell fusion * Not shown in this diagram are nuclei, but they fuse too.

8. Light microscopy image of yeast shmoos

9. Control of yeast mating by BAR1 The BAR1 gene is expressed in MATa cells The Bar1 protein is a secreted protease that degrades α factor Why might the cell have a mechanism for degrading α factor? What do you predict would happen to bar1Δ mutants?(BAR1 gene is deleted)

10. Outline and Learning Objectives: Intro to Yeast Genetics 1.Understand the life cycle of budding yeast 2.Describe how yeast cells mate, and understand how the BAR1 gene contributes to the regulation of this process 3.Understand the mitotic cell cycle of budding yeast a.Explain how budding yeast was used as a model system to isolate genes required for cell cycle regulation b.Understand the basics of doing a genetic screen in yeast 4.Understand the meiotic cell divisions of budding yeast a.Explain how sporulation and tetrad formation aids scientists studying yeast

11. Overview of the cell cycle The goal of mitosis is to produce two daughter cells genetically identical to the mother

12. Zoom in on budding (vegetative growth) Phases of the cell cycle G1: Gap 1 S: DNA replication G2: Gap 2 M: Mitosis What happens during G1 and G2? Note that bud size gives you an indication of where the cell is in the cell cycle

13. Imaging the cytoskeleton during the cell cycle in budding yeast Journal of Cell Biology Actin stained with phalloidin Tubulin immunofluorence of an anaphase cell

14. How can we design a screen to find genes required for cell cycle progression?

15. Designing a screen to find cell cycle genes 1.Mutagenize yeast cells using a chemical that induces mutations in DNA 2.What phenotype will we screen for? 3.If these genes are essential for cell cycle progression, how will we pick mutants if they are all dead? 4.How do we know which genes have the mutations?

16. This screen was done! Lee Hartwell and colleagues screened mutants for temperature-sensitive arrest in a cell cycle stage For example, all cells with mutation 1 arrest as large- budded cells. Therefore, a wild-type copy of that gene is required for progression past the large-budded stage. The scientists then figured out which genes the mutations were in. These genes were named cell division cycle or cdc In this way, genes that control the different phases of the cell cycle were discovered. Similar screen was done in another yeast species, S. pombe

17. Outline and Learning Objectives: Intro to Yeast Genetics 1.Understand the life cycle of budding yeast 2.Describe how yeast cells mate, and understand how the BAR1 gene contributes to the regulation of this process 3.Understand the mitotic cell cycle of budding yeast a.Explain how budding yeast was used as a model system to isolate genes required for cell cycle regulation b.Understand the basics of doing a genetic screen in yeast 4.Understand the meiotic cell divisions of budding yeast a.Explain how sporulation and tetrad formation aids scientists studying yeast

18. Budding yeast undergo meiosis to produce four haploid gametes in a process called sporulation Marston et al., 2004 In yeast, scientists can manipulate all four spores that are the products of one diploid cell undergoing meiosis Starvation induces sporulation in yeast

19. Using sporulation in genetic analysis of mutants How can we ensure that our mutants have a mutation in only one gene? What would happen if our mutant has a mutation in two genes?

20. Some processes that have been elucidated using budding yeast Regulation of the cell cycle Components of secretory pathway Signaling pathways (ex. mating pathway) Gametogenesis Central dogma (transcription, translation) Cytoskeleton Many, many more examples!

21. Conclusions Today we discussed: – Life cycle of budding yeast: haploid, mating, diploid, sporulation – The basics of setting up a genetic screen Questions?

22. Acknowledgements Mandana Sassanfar Angelika Amon Members of the Amon Lab Questions or comments: Michelle Attner mattner@mit.edu Thanks!