Download Genetic screens, sevenless revisited, pathways and paper techniques

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Genetic Screens
Sevenless revisited
Pathways you’ll want to know
Quick review of techniques
Genetic Screens
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
Forward Genetics
1. 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
Making mutations
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EMS can be fed to flies…
Typically causes point mutations
Ave. mutation rate for a gene is 1:1000
Drawback is mosaicism (some cells carry
mutation while others do not)
• X-ray irradiation induces double-stranded DNA
breaks that don’t cause mosaicism
• Often large chromosomal rearrangements or
deletions
• About an order of magnitude less efficient than
EMS
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
don’t affect the process of interest
•Sometimes a more laborious and specific screen
saves you time in the end (less sequencing of
irrelevant genes)
Some obvious phenotypes one
can screen for…
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
Sevenless revisited
An omatidium is made up of 8 photoreceptors
(R1-R8) & accessory cells
The sevenless mutation is
relatively easy to screen for…
All 8 present
Sevenless!
Too many sevens Almost back to normal
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 on…no longer regulated)
Supressor and enhancer screens can give one an idea about
downstream effectors of the protein of interest
Suppressor & Enhancer Screens con’t
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
And the final result…
*GTP*
GDP
SH2
SH3
“sevenless in absentia”
Well-used Pathways
•GPCR-linked signaling
•RTK-linked signaling
•JAK-STAT pathway
•Others…
Model of
G-proteinCoupled
Activation
CREB
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
RTK Signaling Complex con’t…
JAK/STAT: the TF is its own
second messenger!
Some signaling pathways are less
conventional…
Notch
Delta
Axon Guidance Signaling
• Eph receptors are traditional RTKs
• Semaphorins, netrins and slits signal
through novel receptors
What themes do you notice???
Quick review of techniques
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Chimeras
Transfection
GFP as a marker of transfection
Co-IP
Yeast 2 hybrid
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.
Yeast two-hybrid
What it looks like
Positive control with colonies
expressing beta-gal
Negative control.
Colonies do not
express beta-gal
Test protein. Amount of beta-gal
production indicates strength of
interaction
Co-Immunoprecipitation (co-IP)
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Why do we use it?
To capture our protein of interest and look
for protein-protein interactions
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How it works:
1. The Fc region of the antibody sticks to the bead.
2. Incubate the antibody beads with cell lysate to
pull down the protein of interest (and
anything else that stuck to it).
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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
Protein B
Antibodies stuck to the beads
Protein A
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Protein A
Use an antibody to A to confirm
it’s there
Protein B
Use an antibody to B to see
if it’s there…
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??
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