• 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 Genetics1. 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• 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 50kbFOR 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 sevenlessCauses a “rough” phenotype

A screen for dominant suppressors ofSev receptor identified a loss-of-functionAllele 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…

SH2SH3

*GTP*GDP

“sevenless in absentia”

Well-used Pathways

•GPCR-linked signaling

•RTK-linked signaling

•JAK-STAT pathway

•Others…

Model of G-protein-Coupled

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…

DeltaNotch

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

• Chimeras

• Transfection

• GFP as a marker of transfection

• Co-IP

• Yeast 2 hybrid

Yeast two-hybridWhat 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

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

What it looks like

Co-Immunoprecipitation (co-IP)

How it works:

Why do we use it?

To capture our protein of interest and look for protein-protein interactions

The Fc region of the antibody sticks to the bead.

Incubate the antibody beads with cell lysate to pull down the protein of interest (and anything else that stuck to it).

1.

2.

G 1.

G

2.

G

3.

Co-Immunoprecipitation (co-IP)

Note: This does not prove a direct interaction but it does suggest that the proteins interact in vivo.

Protein B

Protein A

Immunoprecipitate withAntibody to A and see if itBrings down protein B

Ant

i-A a

ntib

ody

Con

t ro l

ant

ibod

y

i.e. is it A-C-B??

Question: Does Protein BBind to Protein A??

G

3.

Protein A

Protein B

Use an antibody to B to seeif it’s there…

Antibodies stuck to the beads

Use an antibody to A to confirm it’s there

Antibodies for visualizing protein

What do you do if there are no antibodies specific to your protein?

Protein of interest HA

anti-HA antibody

HA-tagMyc-tagFlag