Tetrads with n genes
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Tetrads with n genes. A B C D x a b c d. A/a B/b C/c D/d. ABCD AbCd aBcD abcd. ABCD AbCD aBcd abcd. ABCD Abcd aBCD abcd. How many types???. 2:2 segregation for each locus If no linkage: 1/(2 n ) spores. 3-5 x oversampling to ensure obtaining strain.

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Tetrads with n genes

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Tetrads with n genes

Tetrads with n genes

A B C D x a b c d

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

ABCD

AbCd

aBcD

abcd

ABCD

AbCD

aBcd

abcd

ABCD

Abcd

aBCD

abcd

How many types???

2:2 segregation for each locus

If no linkage: 1/(2n) spores

3-5 x oversampling to ensure obtaining strain


Tetrads with n genes

Suppressor screens, examples

Suppressor of Multivulva in C. elegans

Activated Go-alpha in C. elegans

Enhancer screens, examples

Rough eye in Drosophila


Tetrads with n genes

LET-23 EGFR

C. elegans

KINASE

rasGAP

LET-60 RAS

KINASE

LIN-45 RAF

GNEF

SEM-5 Grb2

KINASE

SH2

Y~P

MEK-2

LET-341 SOS

RING +

SH3

pro

SH2

Y~P

SH3

pro

pro

KINASE

SLI-1 Cbl

KINASE

Y~P

MPK-1

ARK-1

Ack-related kinase

Vulval differentiation


Tetrads with n genes

Sevenless RTK

KINASE

GAP

RAS1

KINASE

MAP-KKK

GNEF

Drk Grb2

KINASE

Y~P

MAP-

KK

SOS

GNEF

SH3

pro

SH2

Y~P

SH3

KINASE

Y~P

MAP K

R7


Tetrads with n genes

Drosophila Photoreceptor Development

R8 induces R7


Tetrads with n genes

Multiple Ommatida

in each eye:

a population assay


Tetrads with n genes

An enhancer screen for essential genes required

for R7 development

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 the

R8 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 specify

R7'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 are

likely 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 a

temperature-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


Tetrads with n genes

X

R7 present

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

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

R7 absent

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

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

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

R7 absent

Look for mutation (*) that confers

dominant enhancement of sev phenotype

sev/sev; +/+; P[sev-ts]/balancer

sev/Y; +/+; +/+male

Screen for absence of R7 in individual flies.

Isolate these chromosomes by balancing.


Tetrads with n genes

Sevenless RTK

KINASE

GAP

RAS1

KINASE

MAP-KKK

GNEF

Drk Grb2

KINASE

Y~P

MAP-

KK

SOS

GNEF

SH3

pro

SH2

Y~P

SH3

KINASE

Y~P

MAP K

R7


Tetrads with n genes

Receptor is

“exchange factor”

GPCR

g

b

Effector

GDP

GTP

b

GTP

a

Effector

a

g

GDP

Pi

RGS

RGS is the GTPase Activating Protein


Tetrads with n genes

GPCR

g

b

Effector

GDP

GTP

b

GTP

a

Effector

a

g

GDP

Pi

a GTPase-

or

RGS-

RGS


Tetrads with n genes

G proteins Gq and Go control movement

C. elegans

GenotypePhenotype

Wild typewild-type

egl-30(lf)paralyzed

egl-30(gf)hyperactive

goa-1(lf)hyperactive

goa-1(gf)paralyzed

egl-30(lf) goa-1(lf)paralyzed

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


Tetrads with n genes

Mutations that Suppress activated Goa

syIs17

syIs17; sag-4(sy433)

Before

Heat Shock

After

Heat Shock

Jane Mendel, Yvonne Hajdu-Cronin, Wen Chen


Tetrads with n genes

Suppressors of Activated Go (Sag)

CLASSI

hyperactive

(14 alleles) encodes diacylgycerol kinase

dgk-1/sag-1

eat-16(sy348)

(p.k.a.

sag-2) encodes RGS7 homologue

CLASSII

wild type

sag-4, 8sag-4 encodes cyclin L homologue

CLASSIII

Egg-laying defective

sag-3, 5 sag-3 encodes Heat Shock Factor

CLASSIV wild type

•sag-6

CLASSV Egg-laying defective

•sag-7

Yvonne Hajdu-Cronin & Wen Chen


Tetrads with n genes

G Protein Coupled Receptors (GPCRs)

EAT-16 RGS

EGL-10 RGS

GOA-1 Go

EGL-30 Gq

?

[IP3]

EGL-8 PLCb

[PIP2]

DGK-1

[DAG]

[PA]

UNC-13 [DAG-binding] etc.

Synaptic transmission: movement


Extragenic suppression

Extragenic suppression

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


Suppression by compensatory change in direct interactor

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

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

suppression by formation of new protein-protein interactions

ACT

SAC

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


Gene non specific allele specific

gene non-specific, allele specific

  • suppression at level of gene expression: ‘informational’

  • Nonsense suppression

  • Frameshift suppression

  • Splicing machinery

  • stabilization of unstable mRNA or protein

  • suppression of transposon insertion alleles


Nonsense suppression

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

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

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


Mrnas with premature stop codons produce truncated proteins

mRNAs with premature stop codons produce truncated proteins.

stop

AUG

AAAA

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

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

stop

AUG

AAAA

SMG factors

stop

AAAA

decapping and exonucleolytic cleavage


Screens for suppressors of nonsense mutations revealed smg genes

Screens for suppressors of nonsense mutations revealed smg genes

  • smg-1phosphatidylinositol-3 kinase homolog

  • smg-2Upf1 helicase homolog, phosphoprotein

  • smg-3Upf2 homolog

  • smg-4Upf3 homolog

  • smg-5novel, binds SMG-7

  • smg-6--

  • smg-7novel, binds SMG-5

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

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.


In the absence of smg proteins mrnas with premature stop codons will persist

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

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

stop

AUG

AAAA

Short protein fragment is not functional or antimorphic


Suppression by stabilization of protein

suppression by stabilization of protein

  • E. colilon protease degrades aberrant proteins

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


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