Genetic Screens

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• Genetic Screens
• Sevenless revisited
• Pathways youll want to know
• Quick review of techniques

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Genetic Screens
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Benefits of Drosophila

• Only 4 chromosomes
• Short generation time (10 days)
• Lots of external features with visible mutations
  (bristles, wings, eyes, etc.)
• A large number of human homologs
• Ability to carry out large-scale genetic screens
  for mutations

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Forward Genetics

• Create Random Mutations
• EMS (ethyl methane sulphonate) introduces point
  mutations (Protocol by Lewis and Bacher 1968)
• 2. Screen for a phenotype of interest
• Be sure to design a simple screen that can be
  done in bulk
• 3. Clone gene from mutants of interest
• Involves lots of sequencing

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Making mutations

• EMS can be fed to flies
• Typically causes point mutations
• Ave. mutation rate for a gene is 11000
• Drawback is mosaicism (some cells carry mutation
  while others do not)
• X-ray irradiation induces double-stranded DNA
  breaks that dont cause mosaicism
• Often large chromosomal rearrangements or
  deletions
• About an order of magnitude less efficient than
  EMS

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Screening for phenotypes

• Want a phenotype that is easy to identify, but
  specific enough to your question of interest
• Need to minimize the background of mutants that
  dont affect the process of interest
• Sometimes a more laborious and specific screen
  saves you time in the end (less sequencing of
  irrelevant genes)

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Some obvious phenotypes one can screen for
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Cloning the gene

• FOR EMS-BASED MUTATIONS
• Single nucleotide polymorphisms (SNPs) are passed
  down from parents to offspring
• A map of SNPs for flies exists
• Mapping the inheritance of a phenotype to the
  inheritance of SNPs allows the rapid mapping of
  mutations to regions less than 50kb
• FOR X-RAY BASED MUTATIONS
• Because these are large scale chromosomal
  rearrangements or deletions, can often be
  detected cytologically in larval polytene
  chromosomes
• Allows mutation to be mapped rapidly to a region
  and then IDed on Southern blots

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Sevenless revisited
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An omatidium is made up of 8 photoreceptors
(R1-R8) accessory cells
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The sevenless mutation is relatively easy to
screen for
All 8 present
Sevenless!
Too many sevens
Almost back to normal
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Suppressor Enhancer Screens

• Forward genetic screens can generate a variety of
  alleles of a gene
• Amorphs (null mutations)
• Weak hypomorphs (partial loss-of-function
  mutations)
• Constitutively active (always onno longer
  regulated)
• Supressor and enhancer screens can give one an
  idea about downstream effectors of the protein of
  interest

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Suppressor Enhancer Screens cont
Constitutively active form of sevenless Causes a
rough phenotype A screen for dominant
suppressors of Sev receptor identified a
loss-of-function Allele of drk
Hypomorphic mutation in sevenless causes decrease
in the number of R7 cells A screen for dominant
suppressors that increase the number of R7 cells
turned up a gain of function muant in sos
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And the final result
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GDP
GTP
SH2
SH3
sevenless in absentia
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Well-used Pathways

• GPCR-linked signaling
• RTK-linked signaling
• JAK-STAT pathway
• Others

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Model of G-protein-Coupled Activation
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CREB
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Receptor Tyrosine Kinases

• Receptors dimerize in response to ligand binding
• Cross-phosphorylation fully activates the
  receptors
• They phosphorylate other residues
• Recruit other proteins to these binding sites
• These other proteins can then be activated by
  phosphorylation

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RTK Signaling Complex cont
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JAK/STAT the TF is its own second messenger!
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Some signaling pathways are less conventional
Delta
Notch
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Axon Guidance Signaling

• Eph receptors are traditional RTKs
• Semaphorins, netrins and slits signal through
  novel receptors

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What themes do you notice???
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Quick review of techniques

• Chimeras
• Transfection
• GFP as a marker of transfection
• Co-IP
• Yeast 2 hybrid

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Yeast two-hybrid
What it tells you Screen for interacting proteins
How do you do it? Transfect yeast with designed
plasmids 1. Gene for protein A upstream of gene
encoding GAL4 DNA binding domain, creating a
fusion protein. 2. Genes from a library
upstream of gene encoding GAL4 activation
domain If the two proteins interact, the GAL4-AD
will be brought into close proximity with the
binding domain and will be able to initiate
transcription of a reporter gene.
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Yeast two-hybrid
What it looks like
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Co-Immunoprecipitation (co-IP)
Why do we use it?
To capture our protein of interest and look for
protein-protein interactions
How it works
The Fc region of the antibody sticks to the bead.
1.
Incubate the antibody beads with cell lysate to
pull down the protein of interest (and anything
else that stuck to it).
2.
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Co-Immunoprecipitation (co-IP)
Question Does Protein B Bind to Protein A??
Immunoprecipitate with Antibody to A and see if
it Brings down protein B
Anti-A antibody
Protein B
Control antibody
Antibodies stuck to the beads
Protein A
Protein A
Use an antibody to A to confirm its there
Use an antibody to B to see if its there
Protein B
Antibodies for visualizing protein
Note This does not prove a direct interaction
but it does suggest that the proteins interact
in vivo.
i.e. is it A-C-B??
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What do you do if there are no antibodies
specific to your protein?
HA-tag Myc-tag Flag
anti-HA antibody
Protein of interest
HA