Download The Cell Cycle and other Schmoos.

Introduc)on to yeast gene)cs Michelle A5ner July 24, 2012 What is budding yeast, S. cerevisiae? Electron micrograph DIC (light microscopy) Advantages to budding yeast as a model organism Simple, eukaryo)c cell (~10μm diameter) Compact genome (genome is sequenced) Cells grow on plates and in culture Short genera)on )me (~90 minutes) Live happily as haploids and diploids Easy to manipulate genes (swap promoters, delete genes) •  Easy to conduct gene)c screens •  Many yeast genes have evolu)onarily conserved homologs in humans • 
• 
• 
• 
• 
• 
Outline and Learning Objec)ves: Intro to Yeast Gene)cs 1.  Understand the life cycle of budding yeast 2.  Describe how yeast cells mate, and understand how the BAR1 gene contributes to the regula)on of this process 3.  Understand the mito)c cell cycle of budding yeast a.  Explain how budding yeast was used as a model system to isolate genes required for cell cycle regula)on b.  Understand the basics of doing a gene)c screen in yeast 4.  Understand the meio)c cell divisions of budding yeast a.  Explain how sporula)on and tetrad forma)on aids scien)sts studying yeast The life cycle of budding yeast Yeast have 2 ma)ngs types: a and alpha a haploids and alpha haploids divide Image: Wikipedia An a and alpha cell can fuse to form an a/
alpha diploid. mating
a/alpha diploids can divide asexually or they can undergo meiosis to form four haploid gametes called spores Brief gene)cs review • 
• 
• 
• 
• 
• 
What is a gene? What is an allele? What is a muta)on? What is a genotype? What is a phenotype? What is the difference between muta)ons conferring recessive and dominant phenotypes? –  Why is yeast great for studying muta)ons conferring recessive phenotypes? –  How can you use yeast to determine if your muta)on confers a recessive or dominant phenotype? Yeast ma)ng is a fusion event 1.  What is the signal for ma)ng? •  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 ac)vated. 3.  The output of this pathway is cell cycle arrest and shmoo forma)on 4.  A shmoo is a ma)ng projec)on that is necessary for cell fusion * Not shown in this diagram are nuclei, but they fuse too. Image: Wikipedia Light microscopy image of yeast shmoos Control of yeast ma)ng 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) Outline and Learning Objec)ves: Intro to Yeast Gene)cs 1.  Understand the life cycle of budding yeast 2.  Describe how yeast cells mate, and understand how the BAR1 gene contributes to the regula)on of this process 3.  Understand the mito)c cell cycle of budding yeast a.  Explain how budding yeast was used as a model system to isolate genes required for cell cycle regula)on b.  Understand the basics of doing a gene)c screen in yeast 4.  Understand the meio)c cell divisions of budding yeast a.  Explain how sporula)on and tetrad forma)on aids scien)sts studying yeast Overview of the cell cycle The goal of mitosis is to produce two daughter cells gene)cally iden)cal to the mother Zoom in on budding (vegeta)ve growth) Phases of the cell cycle G1: Gap 1 S: DNA replica)on G2: Gap 2 M: Mitosis What happens during G1 and G2? Note that bud size gives you an indica)on of where the cell is in the cell cycle Imaging the cytoskeleton during the cell cycle in budding yeast Ac)n stained with phalloidin Journal of Cell Biology Tubulin immunofluorence of an anaphase cell How can we design a screen to find genes required for cell cycle progression? Designing a screen to find cell cycle genes 1.  Mutagenize yeast cells using a chemical that induces muta)ons in DNA 2.  What phenotype will we screen for? 3.  If these genes are essen)al for cell cycle progression, how will we pick mutants if they are all dead? 4.  How do we know which genes have the muta)ons? This screen was done! •  Lee Hartwell and colleagues screened mutants for temperature-­‐sensi)ve arrest in a cell cycle stage •  For example, all cells with muta)on 1 arrest as large-­‐
budded cells. Therefore, a wild-­‐type copy of that gene is required for progression past the large-­‐budded stage. •  The scien)sts then figured out which genes the muta)ons 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 Outline and Learning Objec)ves: Intro to Yeast Gene)cs 1.  Understand the life cycle of budding yeast 2.  Describe how yeast cells mate, and understand how the BAR1 gene contributes to the regula)on of this process 3.  Understand the mito)c cell cycle of budding yeast a.  Explain how budding yeast was used as a model system to isolate genes required for cell cycle regula)on b.  Understand the basics of doing a gene)c screen in yeast 4.  Understand the meio)c cell divisions of budding yeast a.  Explain how sporula)on and tetrad forma)on aids scien)sts studying yeast Budding yeast undergo meiosis to produce four haploid gametes in a process called sporula)on Starva)on induces sporula)on in yeast Marston et al., 2004 In yeast, scien)sts can manipulate all four spores that are the products of one diploid cell undergoing meiosis Using sporula)on in gene)c analysis of mutants •  How can we ensure that our mutants have a muta)on in only one gene? •  What would happen if our mutant has a muta)on in two genes? 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!
Conclusions •  Today we discussed: –  Life cycle of budding yeast: haploid, ma)ng, diploid, sporula)on –  The basics of sejng up a gene)c screen •  Ques)ons? Acknowledgements •  Mandana Sassanfar •  Angelika Amon •  Members of the Amon Lab Ques)ons or comments: Michelle A5ner [email protected] Thanks!