Understanding the Role of Sterile5 in Modulating Quantitative Responses in the Yeast Alpha Mating Re

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Understanding the Role of Sterile5 in
Modulating Quantitative Responses in the Yeast
Alpha Mating Response Pathway

• Tori Anderson
• Jessica Bersonda
• Rachelle Hampton
• Camille Stanton
• Yang Yang

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Outline

• The Goal
• Introduction to Saccharomyces cerevisiae
• Yeast Alpha Pheromone Mating Response Pathway
• Assays for Pheromone Response
• Experimental Design
• Results
• Whats Next
• Conclusion

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The Goal of MSI

• To understand and predict the quantitative
  behavior of the yeast cell in response to mating
  pheromone.
• Progress toward this goal will increase our
  understanding of biological systems and may lead
  to improvements in the treatment of diseases.

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The Goal of the Interns

• To understand and analyze the contribution of
  Sterile5 to the quantitative behavior of the
  yeast alpha pheromone mating response pathway
• Find mutations that alter the function of
  Sterile5 and change its quantitative behavior in
  response to alpha factor

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Outline

• The Goal
• Introduction to Saccharomyces cerevisiae
• Yeast Alpha Pheromone Mating Response Pathway
• Assays for Pheromone Response
• Experimental Design
• Results
• Whats Next
• Conclusion

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SACCHAROMYCES CEREVISIAE

• Commonly known as bakers yeast
• Popularly used for baking and brewing
• Can be a haploid or diploid
• Reproduces sexually by mating- two mating types
  or sexes a and alpha (a)
• Reproduces asexually by budding- mother cells
  make daughter cells that are genetically
  identical (clones)

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Yeast As A Model Organism

• A model organism is an experimental system used
  to study processes that takes place in humans and
  other living organisms
• Yeast is a simple eukaryote that grows fast and
  can be genetically modified easily
• Entire genome contains approximately 6,000 genes
• Genetically tractable- we can precisely change
  single nucleotides to entire sets of genes
• Reproduces by budding- mother cells make daughter
  cells that are genetically identical (clones)

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Budding
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Yeast Mating
Haploid alpha (a) cell secretes a factor
Haploid a cell secretes a factor

• Cell Cycle Arrest
• Cellular Morphogenesis
• Induction of Genes Required for Mating

Fusion
Diploid a/ a cell
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Sterile5

• Sterile5 is a 917 amino acid protein involved in
  the yeast alpha pheromone mating response pathway
• Sterile5 plays a central role in signal
  transduction
• Sterile5 localizes to the cell membrane in
  response to alpha factor and acts as a scaffold
  (binding site) for other proteins Ste11, Ste7,
  and Fus3

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Outline

• The Goal
• Introduction to Saccharomyces cerevisiae
• Yeast Alpha Pheromone Mating Response Pathway
• Assays for Pheromone Response
• Experimental Design
• Results
• Whats Next
• Conclusion

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Binds to Ste 2
Ste 2 a receptor
a F binds to Ste 2 receptor
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Ste 2 a receptor
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Ste 2 a receptor
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Ste 2 a receptor
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Ste 2 a receptor
Note When Fus3 is absent, Kss1 can enter
the nucleus and phosphorylate Dig 1 and Dig 1 and
Ste12. However, the results are not as effective.
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Ste 2 a receptor
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Responses to Pheromone

• Responses
• Cell Cycle Arrest
• Morphological Changes (e.g. Shmooing)
• Gene Expression

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Outline

• The Goal
• Introduction to Saccharomyces cerevisiae
• Yeast Alpha Pheromone Mating Response Pathway
• Assays for Pheromone Response
• Experimental Design
• Results
• Whats Next
• Conclusion

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A Visual Assay For Cell Cycle Arrest
wild type
?Ste5G418

• Conclusion
• The wild type responded to alpha factor.
• Sterile 5 is needed for cell cycle arrest.

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Morphological Change

• Shmooing
• (a factor)

• Budding
• (a factor)

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Alpha Pheromone Responsive Gene Expression
What we already know In normal yeast cells, the
PRM1 promoter drives expression of the PRM1
(Pheromone-Regulated Membrane Protein), which is
involved in membrane fusion during mating.

• PRM1 is an example of a Pheromone Responsive
  Gene. It only turns on when pheromone is
  sensed.
• Its expression is regulated by the PRM1 Promoter.

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How We Used the PRM1 Promoter to Monitor
Pheromone Responsive Gene Expression

• We were given a yeast strain that contained a
  reporter gene for His3, which produces the His3
  enzyme in the biochemical pathway that produces
  histidine, and also a reporter for YFP ( yellow
  fluorescent protein).

YFP (ORF)
PRM1 Promoter
YFP (ORF)
What does this mean and its importance?

• Now the yeast cell can synthesize histidine and
  YFP
• His3 and YFP expression is completely dependent
  on pheromone

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Reverse Halo Assay
Objective Demonstrates that the His3 gene
is pheromone responsive.
wild type (-His/aF)
wild type (-His/aF/1NMPP1)

• Why plate on His plates?
• If cells are able to produce their own histidine
  then they should be able to survive on a plate
  that lacks the amino acid.
• 1NMPP1 (inhibits Fus3 and prevents cells from
  arresting).

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1NMPP1 Inhibitor of Fus3-as2 Allele

• Our yeast strain contained a mutated Fus3-as2
  allele (behaves normally, but is inhibitable)
• Involved in mating response (phosphorylated by
  Ste7)
• Fus3 phosphorylates Far1, the cell cycle
  regulator, which mediates cell cycle arrest.

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Results Explained

• Conclusion
• Since His3 expression is regulated by pheromone,
  we can make the survival of cells on His plates
  completely dependent on alpha factor.
• We used Fus3-as2 inhibitor to prevent cells from
  arresting.

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Outline

• The Goal
• Introduction to Saccharomyces cerevisiae
• Yeast Alpha Pheromone Mating Response Pathway
• Assays for Pheromone Response
• Experimental Design
• Results
• Whats Next
• Conclusion

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Approach to Obtain Mutants

• Our experiment was to mutate the Ste5 gene,
  insert it back into yeast (yeast strain has
  endogenous Ste5 deleted), and screen for mutants
  that respond differently to alpha factor (when
  compared to wild type).
• The process went as follows

Mix segments and gapped plasmid, re-insert into
yeast
Select colonies with repaired plasmid
Mutate Ste5 gene
Pick candidates (streak for single colonies)
Select colonies with functional Ste5
Screen for mutants
Quantify differences in PRM1-YFP gene expression
and halo assays
Retest Still look like mutants?
Rescue mutants.
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Ste5 Protein Characteristics
0
917
Cell Membrane/ Nuclear Localization
Ste4 binding domain
Ste11binding domain
Ste7 binding domain
Fus3 binding domain
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The Plasmid
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Restriction Digest

• To cut Ste5 out of the plasmid we did a
  restriction digest
• A restriction digest is cutting DNA molecules
  into smaller pieces using special enzymes called
  site specific restriction enzymes
• The restriction enzymes recognized specific sites
  that define the segments that we wish to
  introduce.
• Segment 1 Yang and Rachelle
• Segment 2 Tori
• Segment 3 Jessica and Camille

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The Gapped Plasmid
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Mutagenesis

• Mutagenesis is the induction of a mutation either
  in nature or experimentally.
• We wanted to create mutations in each segment and
  we did this through error prone PCR.
• PCR, polymerase chain reaction, is the technique
  we used to amplify our segments of DNA through
  enzymatic replication
• The PCR was performed using the kit, Genemorph2.
  This polymerase has been mutated so that during
  DNA synthesis the mutated polymerase
  misincorporates nucleotides and it does not have
  the ability to proofread misincorporated
  nucleotides.

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Missense Mutation

• There are 3 types of mutations missense,
  nonsense, and silent

1. Missense mutation the codon is altered and
encodes for a different amino acid.
A A
A A
C
A
Asparagine
Lysine
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Nonsense Mutation

• 2. Nonsense Mutation causes a change in the
  codon to a stop codon, which ends protein
  translation

T G
G
A
T G
Tryptophan
STOP
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Silent Mutation

• 3. Silent Mutation codon change does not alter
  the encoded amino acid.

C G
C G
A
C
Arginine
Arginine

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Gap Repair

• We did gap repair, which uses homologous
  recombination in order to insert out mutated
  segment into the plasmid.
• Homologous recombination is the process of
  physical rearrangement occurring between 2
  strands of DNA
• This happens inside of the yeast cell

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Repairing the Gapped Plasmid

Mutagenized PCR product (Ste5 segment)
The PCR product has homology to the gapped
vector, which is where the vector and PCR
product will recombine.
Gapped vector
Ste5
This is a repaired gapped plasmid with
introduced mutation
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What Next?
After Ste5 was mutagenized and reinserted back
into yeast we had to select for transformants
Select for transformants
Insert into yeast
Mutagenize Ste5
Pick candidates (streak for single colonies)
Screen for mutants (faster/ slower growth rate)
Select for functional Ste5
Quantify differences in PRM1-YFP gene expression
and halo assays
Retest and Refrog Still look like mutants?
Rescue mutants. Put them back into standard lab
strains and ensure single copy
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Yeast Transformation

• After we have the gapped plasmid and the Ste5
  mutant segment, we heat shocked the cells and
  forced them to take up and recombine the DNA
  through homologous recombination.

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Types of Cells After Transformation
PCR Segment
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Only Yeast w/ the Plasmid Will Grow
Ura3 gene
PCR Segment
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Pheromone Response Selection
-His/ -Ura/10mM 1NM PP1/ 100 nM a factor plate
With functional Ste5
x
With non- functional Ste5
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What do we do next?!?!
Collect cells to make a library and then find
mutants with altered response to pheromone by
screening for faster or slower growth rate.
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How Do We Screen For Mutants?What Are We Looking
For?

• We have a pheromone responsive gene that allows
  cells to grow without added histidine in the
  presence of a factor (PRM1 His3).
• We will use this pheromone response promoter to
  identify mutants that have increased or decreased
  pheromone responsive gene expression.
• We can grow our cells on plates with a factor,
  1NMPP1 and another inhibitor called 3-AT that
  blocks the activity of the His3 enzyme and
  eliminates the background (uninduced) expression.
  In the plates without added histidine or uracil,
  we can look for mutants with increased or
  decreased rate of growth

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Collecting Cells to Make Library
Mutant Library
Plate on 500 colonies on -Ura
Grow them on individual wells
Replica plate onto His/-Ura 100nM a F/100 mM
3AT/1NMPP1
Frog 40-1000 cell onto His/-Ura/ 100nM a
F/100nM 3AT/1NMPP1/
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Frogging
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Pheromone Response Screen
Each dot has 35-45 cells
-His/-Ura/10nM of 1NMPP1/ 100mM 3AT/100 nM a
factor plate
The colonies grow according to the level of His3
gene expression
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Pheromone Response
Wild type (normal) response
Faster Growing
Slower Growing
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Secondary Screening
Pheromone Response Plate
YPD Plate
WT
WT
B
C
D
H2
WT
WT
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How does the mutation affect the pheromone
response?

• What we can do
• Quantify pheromone response gene expression.
• Use the Halo Assay (check size of halo)

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PRM1-YFP Expression (reporter gene)
YFP (yellow fluorescent protein) is controlled by
pheromone responsive promoter (PRM), measurement
of YFP level in mutants compare to wild type
allows us to quantify the difference in pheromone
pathway output.
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Halo Assay
By analyzing the size of the halo, we can decide
how sensitive particular mutant is to a factor.
wild type halo
more sensitive larger halo
less sensitive smaller halo
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Outline

• The Goal
• Introduction to Saccharomyces cerevisiae
• Yeast Alpha Pheromone Mating Response Pathway
• Assays for Pheromone Response
• Experimental Design
• Results
• Whats Next
• Conclusion

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Ste5 Protein Characteristics
Segment 1
Segment 2
Segment 3
Segment 4
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Yangs Results

• Transformation
• Segment 1 nuclear and membrane localization
  binding domains
• I found that 24 of my cells did not have a
  functional Ste5 by checking their response to a
  factor under microscope

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Yangs Results
Transformation I picked 288 colonies from my
primary transformants
I noted the faster and slower growing colonies. I
focused on the faster growing colonies and then
streaked 9 of the fastest growing colonies.
After doing so, I re-confirmed those potential
mutants.
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Yangs Reconfirmation (Halo Assay)

• 2 cm
• 2.4 cm
• 2.6 cm
• 1.8 cm
• 2.3 cm

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Toris Results

• Transformation
• Segment 2 Fus 3 and Ste4 binding domains
• I found that 12 of my cells did not have a
  functional Ste5

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Toris Results
Transformation I picked 384 colonies from my
primary transformants
I noted the faster and slower growing colonies.
I focused on the faster growing colonies and then
streaked 14 of the fastest growing colonies.
After doing so, I re-confirmed those potential
mutants.
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Toris Reconfirmation (Halo Assay)

• 1.5cm
• 1.35 cm
• 1 cm
• 1.25 cm
• 1.4 cm

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Rachelles Results

• Transformation
• Segment 1 nuclear and membrane localization
  binding domains
• I found that 30 of my cells did not have a
  functional Ste5

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Rachelles Results
Transformation I picked 384 colonies from my
primary transformants
I made note of the colonies that appeared to grow
faster and slower. From the frogged plates, I
streaked out 14 of the fastest growing colonies
and re-confirmed using single colonies.
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Rachelles Reconfirmation (Halo Assay)

• 3cm
• 3cm
• 3.2cm
• 3cm
• 2.8cm

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Jessicas Results

• Transformation
• Segment 3 Ste11 binding domain
• I found that 80 of my cells did not have a
  functional Ste5

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Jessicas Results
Transformation I picked 384 colonies from my
primary transformants
I made note of the colonies that appeared to grow
faster and slower. From the frogged plates, I
streaked out 12 of the fastest growing colonies
and re-confirmed using single colonies.
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Jessicas Reconfirmation (Halo Assay)
Positive Control 3 cm
Seg.3 Plate 3 H2 2.7 cm
Seg.3 Plate 1 D1 2.7 cm
Seg.3 Plate 2 D1 2.5 cm
Seg.3 Plate 3 D5 2.5 cm
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Camilles Results

• Transformation
• Segment 3 Ste11 binding domain
• I found that 65 of my cells did not have a
  functional Ste5

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Camilles Results
Transformation I picked 384 colonies from my
primary transformants
I noted the faster and slower growing colonies.
I focused on the faster growing colonies and then
streaked 19 of the fastest growing colonies.
After doing so I re-confirmed those potential
mutants.
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Camilles Halo Assays
Positive Control 1.5 cm
A4 1.6 cm
A10 2 cm
B6 1.5 cm
E9 1.5 cm
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Outline

• The Goal
• Introduction to Saccharomyces cerevisiae
• Yeast Alpha Pheromone Mating Response Pathway
• Assays for Pheromone Response
• Experimental Design
• Results
• Whats Next
• Conclusion

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Whats Next?

• Use microscopy and software to quantify pheromone
  responsive YFP expression
• Use PCR to rescue the mutants
• Send the mutant for sequencing
• Put the mutant into standard lab strains for
  further analysis

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Thank You
A Special Thanks To Leonore Tonya Gustavo Pia Fr
ank Susana Eric The Rest of MSI Our Parents FROM
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Yang
Tonya
Camille
THANK YOU!
Tori
Jessica
Rachelle