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Introduction to yeast genetics. Michelle Attner July 24, 2012. What is budding yeast, S. cerevisiae ?. Electron micrograph. DIC (light microscopy). Advantages to budding yeast as a model organism. Simple, eukaryotic cell (~10μm diameter) Compact genome (genome is sequenced)

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introduction to yeast genetics

Introduction to yeast genetics

Michelle Attner

July 24, 2012

what is budding yeast s cerevisiae
What is budding yeast, S. cerevisiae?

Electron micrograph

DIC (light microscopy)

advantages to budding yeast as a model organism
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
outline and learning objectives intro to yeast genetics
Outline and Learning Objectives: Intro to Yeast Genetics
  • Understand the life cycle of budding yeast
  • Describe how yeast cells mate, and understand how the BAR1 gene contributes to the regulation of this process
  • Understand the mitotic cell cycle of budding yeast
    • Explain how budding yeast was used as a model system to isolate genes required for cell cycle regulation
    • Understand the basics of doing a genetic screen in yeast
  • Understand the meiotic cell divisions of budding yeast
    • Explain how sporulation and tetrad formation aids scientists studying yeast
the life cycle of budding yeast
The life cycle of budding yeast

An a and alpha cell can fuse to form an a/alpha diploid.

Yeast have 2 matings types: a and alpha

a haploids and alpha haploids divide

mating

a/alpha diploids can divide asexually or they can undergo meiosis to form four haploid gametes called spores

Image: Wikipedia

brief genetics review
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?
yeast mating is a fusion event
Yeast mating is a fusion event
  • 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
  • When α factor binds to receptors on a cell, a MAP kinase pathway is activated.
  • The output of this pathway is cell cycle arrest and shmoo formation
  • A shmoo is a mating projection that is necessary for cell fusion
  • * Not shown in this diagram are nuclei, but they fuse too.

Image: Wikipedia

control of yeast mating by bar1
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)
outline and learning objectives intro to yeast genetics1
Outline and Learning Objectives: Intro to Yeast Genetics
  • Understand the life cycle of budding yeast
  • Describe how yeast cells mate, and understand how the BAR1 gene contributes to the regulation of this process
  • Understand the mitotic cell cycle of budding yeast
    • Explain how budding yeast was used as a model system to isolate genes required for cell cycle regulation
    • Understand the basics of doing a genetic screen in yeast
  • Understand the meiotic cell divisions of budding yeast
    • Explain how sporulation and tetrad formation aids scientists studying yeast
overview of the cell cycle
Overview of the cell cycle

The goal of mitosis is to produce two daughter cells genetically identical to the mother

zoom in on budding vegetative growth
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

imaging the cytoskeleton during the cell cycle in budding yeast
Imaging the cytoskeleton during the cell cycle in budding yeast

Tubulin immunofluorence of an anaphase cell

Actin stained with phalloidin

Journal of Cell Biology

designing a screen to find cell cycle genes
Designing a screen to find cell cycle genes
  • Mutagenize yeast cells using a chemical that induces mutations in DNA
  • What phenotype will we screen for?
  • If these genes are essential for cell cycle progression, how will we pick mutants if they are all dead?
  • How do we know which genes have the mutations?
this screen was done
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
slide17

Outline and Learning Objectives: Intro to Yeast Genetics

  • Understand the life cycle of budding yeast
  • Describe how yeast cells mate, and understand how the BAR1 gene contributes to the regulation of this process
  • Understand the mitotic cell cycle of budding yeast
    • Explain how budding yeast was used as a model system to isolate genes required for cell cycle regulation
    • Understand the basics of doing a genetic screen in yeast
  • Understand the meiotic cell divisions of budding yeast
    • Explain how sporulation and tetrad formation aids scientists studying yeast
budding yeast undergo meiosis to produce four haploid gametes in a process called sporulation
Budding yeast undergo meiosis to produce four haploid gametes in a process called sporulation

Starvation induces sporulation in yeast

In yeast, scientists can manipulate all four spores that are the products of one diploid cell undergoing meiosis

Marston et al., 2004

using sporulation in genetic analysis of mutants
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?
some processes that have been elucidated using budding yeast
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
Conclusions
  • Today we discussed:
    • Life cycle of budding yeast: haploid, mating, diploid, sporulation
    • The basics of setting up a genetic screen
  • Questions?
acknowledgements
Acknowledgements
  • MandanaSassanfar
  • Angelika Amon
  • Members of the Amon Lab

Questions or comments:

Michelle Attner

mattner@mit.edu

Thanks!