Tetrads with n genesTetrads with n genes

A B C D x a b c dA B C D x a b c d

A/a B/b C/c D/dA/a B/b C/c D/d

2:2 segregation for each locus2:2 segregation for each locusIf no linkage: 1/(2If no linkage: 1/(2nn) spores) spores

3-5 x oversampling to ensure obtaining strain3-5 x oversampling to ensure obtaining strain

ABCDABCDAbCdAbCdaBcDaBcDabcdabcd

ABCDABCDAbCDAbCDaBcdaBcdabcdabcd

ABCDABCDAbcdAbcdaBCDaBCDabcdabcd

How many How many types???types???

Suppressor screens, examplesSuppressor screens, examples

Suppressor of Multivulva in Suppressor of Multivulva in C. elegansC. elegans

Activated Go-alpha in Activated Go-alpha in C. elegansC. elegans

Enhancer screens, examplesEnhancer screens, examples

Rough eye in Rough eye in DrosophilaDrosophila

pro

RING +SH2

SH3

LET-23 EGFR

SEM-5 Grb2

LET-341 SOS

LET-60 RAS

Vulval differentiation

ARK-1

KINASE

SH2

SH3

pro

GNEF

pro

KINASE

SLI-1 Cbl

Y~P

Y~P

rasGAP

Y~P

Ack-related kinase

KINASE

KINASE

KINASE

LIN-45 RAF

MEK-2

MPK-1

C. elegansC. elegans

SH3

Sevenless RTK

Drk Grb2

SOSGNEF

RAS1

R7

KINASE

SH2

SH3

pro

GNEF

Y~P

Y~P

GAP

Y~P

KINASE

KINASE

KINASE

MAP-KKK

MAP-KK

MAP K

Drosophila Photoreceptor DevelopmentDrosophila Photoreceptor Development

R8 induces R7R8 induces R7

Multiple OmmatidaMultiple Ommatidain each eye:in each eye:a population assaya population assay

The fly eye consists of approximately 800 20-cell repeating units known as ommatidia.  Each ommatidium consists of eight photoreceptor neurons (R1-R8), four lens secreting cone cells and eight additional accessory cells.  The ommatidia arise from an undifferentiated epithelium by a series of cell interactions.  We will only consider an interaction between theR8 and presumptive R7 cells that determines the fate of R7.  The R7 photoreceptor detects light in the UV range.  Screens for mutants with ommatidia that lack R7 cells identified three genes:  sevenless (sev), bride of sevenless (Boss) and seven-in-abstentia (sina).  Adult flies homozygous for mutations in any of these genes have ommatidia that lack an R7 cell and contain an additional cone cell.  In the absence of R7 differentiation, the presumptive R7 cell becomes a cone cell.  sev and sina are a receptor tyrosine kinase and a nuclear protein, respectively, and both genes act in R7 to specifyR7's fate.  boss appears to encode the ligand for the Sev receptor tyrosine kinase, and in contrast to sev and sina, acts in R8 cell to specify R7's fate.

Now consider the problem that many genes functioning downstream of receptor tyrosine kinse receptor activation arelikely to be required for other tyrosine kinase signaling pathways that are required for the viability of the organism. How can one use the fly eye to identify such mutations in such genes.

Make a partially active mutant version of sev and introduce it into a sev mutant background. These flies have atemperature-sensitive phenotype. A fly carrying one copy of this transgene is wildtype at 22.7oC (R7 is present).However, at 24.3oC R7 is absent

An enhancer screen for essential genes required for R7 development

sev/Y; +/+; +/+male sev/sev; +/+; P[sev-ts]/balancerX

sev/sev; */+; P[sev-ts]/+ sev/Y; */+; P[sev-ts]/+

Screen for absence of R7 in individual flies.Isolate these chromosomes by balancing.

sev/sev; +/+; P[sev-ts]/Y at 22.7oC

sev/sev; +/+; P[sev-ts]/Y at 24.3oC

sev/sev; */+; P[sev-ts]/Y at 22.7oC

Look for mutation (*) that confersdominant enhancement of sev phenotype

R7 present

R7 absent

R7 absent

SH3

Sevenless RTK

Drk Grb2

SOSGNEF

RAS1

R7

KINASE

SH2

SH3

pro

GNEF

Y~P

Y~P

GAP

Y~P

KINASE

KINASE

KINASE

MAP-KKK

MAP-KK

MAP K

Effector

RGS

GPCR

GDP

GTP

GDPGTP

Effector

Pi

Receptor is“exchange factor”

RGS is the GTPase Activating Protein

Effector

RGS

GPCR

GDP

GTP

GDPGTP

Effector

Pi GTPase-orRGS-

G proteins Gq and Go control movement

Genotype Phenotype

Wild type wild-typeegl-30(lf) paralyzedegl-30(gf) hyperactivegoa-1(lf) hyperactivegoa-1(gf) paralyzedegl-30(lf) goa-1(lf) paralyzed

lf, loss-of-function; gf, gain-of-function

C. elegansC. elegans

Mutations that Suppress activated Go

syIs17 syIs17; sag-4(sy433)

Before Heat Shock

AfterHeat Shock

Jane Mendel, Yvonne Hajdu-Cronin, Wen ChenJane Mendel, Yvonne Hajdu-Cronin, Wen Chen

Suppressors of Activated Go (Sag)

• sag-4, 8 sag-4 encodes cyclin L homologueCLASS II wild type

CLASS I hyperactive• dgk-1/sag-1 (14 alleles) encodes diacylgycerol kinase• eat-16(sy348) (p.k.a. sag-2) encodes RGS7 homologue

CLASS III Egg-laying defective• sag-3, 5 sag-3 encodes Heat Shock Factor

CLASS IV wild type• sag-6

CLASS V Egg-laying defective• sag-7

Yvonne Hajdu-Cronin & Wen Chen

EGL-30 Gq

EGL-8 PLC [IP3]

[DAG]

Synaptic transmission: movement

[PIP2] DGK-1[PA]

GOA-1 Go

EGL-10 RGSEAT-16 RGS

?

UNC-13 [DAG-binding] etc.

G Protein Coupled Receptors (GPCRs)

Extragenic suppression

• many mechanisms--key issue is the genetic specificity of the suppressor

gene-specific

allele-nonspecificepistasis (bypass

suppression)

gene-specific

allele-specificdirect interaction?

gene-nonspecific

allele-specific‘informational’

suppression

suppression by compensatory change in direct interactor?

• ‘Lock and Key’ model: binding site is restored• in general a very rare event as target size is 1(or a few)

bp--need screens of >106 genomes• RNA-RNA interactions:

– restoration of base pairing (nonsense suppression)– splice site suppression e.g. Lesser + Guthrie 1993 Science 262:

1982

• protein-DNA interactions– lac operon: oC mutations suppressed by mutations in repressor that

bind more tightly to operator (Pfahl 1981, J. Mol. Biol. 147: 1-10)

• protein-protein interactions?

allele-specific suppression

• null mutants are not suppressed, so not bypass suppressor

• stabilization or altered processing of mutant gene product

suppression by formation of new protein-protein interactions

Adams + Botstein 1989. suppressors of ts actin mutants– get sac mutants. sac6 is

fimbrin, actin-binding

– sac6 mutations are missense in actin binding domain, increase affinity for mutant actin

– But the affinity for wild type actin is also increased

ACT SAC

act SAC

act sac

ACT sac

gene non-specific, allele specific

• suppression at level of gene expression: ‘informational’

1. Nonsense suppression

2. Frameshift suppression

3. Splicing machinery

4. stabilization of unstable mRNA or protein

5. suppression of transposon insertion alleles

nonsense suppression• conditional ‘amber’ mutations in many T4 genes

(Epstein et al)– grow on one E coli strain (CR63) but not on B– cause premature termination– suppression due to mutant tRNA that can recognize

amber codon UAG and insert amino acid (usually Trp; codon is UGG)

– amber suppressor strains are a bit sick because of readthrough

frameshift suppression

• extragenic suppression of frameshifts by two mechanisms– limitation of Trp-tRNA– other tRNAs loosely bind to codon (mismatch)

and allow frameshifting

– also mutant tRNA with 4-base anticodon now ‘reads’ frameshift as a 3-base codon…

suppression by stabilization of message

• mRNAs with ‘premature’ stop codons are recognized and degraded– nonsense mediated decay/ ‘mRNA surveillance’

– Upf pathway (yeast), SMG pathway (worms)

– get rid of aberrant mRNAs before they get to ribosome

• some nonsense mutations can be suppressed if partially functional protein can be made

AAAA

stopAUG

mRNAs with premature stop codons produce truncated proteins.

Expression of these from many loci can be detrimental to the animal.Cells have mechanisms of removing aberrant mRNAs

mRNAs with premature stop codons are recognized and destroyed by nonsense mediated

decay

AAAA

SMG factors

stopAUG

AAAA

decapping and exonucleolytic cleavage

stop

Screens for suppressors of nonsense mutations revealed smg genes

• smg-1 phosphatidylinositol-3 kinase homolog• smg-2 Upf1 helicase homolog, phosphoprotein• smg-3 Upf2 homolog• smg-4 Upf3 homolog • smg-5 novel, binds SMG-7• smg-6 --• smg-7 novel, binds SMG-5

Hodgkin J, Papp A, Pulak R, Ambros V, Anderson P. A new kind of informational suppression in the nematode Caenorhabditis elegans. Genetics. 1989 Oct;123(2):301-13.

Mutations in the proteins required for nonsense mediated decay suppress nonsense mutations by allowing stabilizing mRNAs with premature stop codons. Functional proteins are made since low levels of readthrough make some normal protein or because expression of the truncated protein can suppress the phenotype

AAAA

stopAUG

In the absence of SMG proteins mRNAs with premature stop codons will persist

Expression of these from many loci can be detrimental to the animal

mRNAs with premature stop codons have a low level of readthrough, these levels may be enough to rescue the mutant phenotype

Short protein fragment is not functional or antimorphic

suppression by stabilization of protein

• E. coli lon protease degrades aberrant proteins

• mutations in lon suppress thermolabile mutations in many genes (RNA polymerase etc)