Transcript Introduction to yeast genetics

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
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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
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
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
mating
a haploids and
alpha haploids
divide
a/alpha diploids can
divide asexually or they
can undergo meiosis to
form four haploid
gametes called spores
Image: Wikipedia
Brief genetics review
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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
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.
Image: Wikipedia
Light microscopy image of yeast
shmoos
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 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
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)
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
Actin 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 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?
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 largebudded 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
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
Budding yeast undergo meiosis to produce four
haploid gametes in a process called sporulation
Starvation
induces
sporulation
in yeast
Marston et al., 2004
In yeast, scientists can manipulate all
four spores that are the products of
one diploid cell undergoing meiosis
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
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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, mating,
diploid, sporulation
– The basics of setting up a genetic screen
• Questions?
Acknowledgements
• Mandana Sassanfar
• Angelika Amon
• Members of the Amon Lab
Questions or comments:
Michelle Attner
[email protected]
Thanks!