1

Drosophila melanogaster

Modifiers, mosaics and cancer genetics

Modifier genes and screens

Site-specific recombinases and mosiac analysis

Genetics of Ras signaling

Modifier lociEffects of double mutants are usually additive

white andsmall

w/w Bar/+

white small

+

w/w ; Bar/+

=

Modifier lociUnless the mutations act in the same pathway

Multi-vulvae

lin3/lin3 let23D/+

no vulva Multi-vulvae

+

lin3/lin3 ; let23D/+

=

Modifier lociSometimes mutations in the same pathway act synergistically

no eye!

so1/+ ; ey/ey

=

so1/+ ey/ey

normal small

+

sine oculis is a dominant modifier of eyeless

Modifiers can enhance or suppress a phenotype

roughest acts upstream of Drosophila cell death genes

Weak alleles of rstconfer a mild rougheye phenotype

rst/rst

Delta mutationsdominantly enhancethe roughness

rst/rst ; Delta/+

Dras mutationsdominantly suppressthe roughness

rst/rst ; Ras/+

2

Screens for dominant modifiers of eyeless

Enhancer/Enhancer or Supressor/Supressor often lethal orshow no visible phenotype

Irradiation induced mitotic recombination

InefficientCrossovers at random locations

Restricted choice of markers

FLP-FRT site-specific recombinase

FLP catalyses recombination between FRT sequences

The DNA between the FRT sites is excised

FLP-FRT site-specific recombinaseFLP also catalyses interchromsomal recombination

Heat shock induction of FLP expression generates mutant cells

Short heat pulses induce recombinationin a small proportion of cells whichgive rise to marked mutant clones

Human cancers result fromsomatic mutations in tumoursuppressor genes creatingclones of mutant cells

Tumourigenesis can bemodelled by FLP inducedrecombination in flies

3

Screening for novel tumour suppressor genes

Mutagenise FRT/FRT males

Cross with HsFLP/HsFLP ;

FRT-myc/FRTmyc females

F1 individuals carrying mutations in potential tumour supressor genes develop tumours

Heat shock HsFLP ;

FRT-myc/FRT* progeny

Ras is an oncogene

Dominant mutations in Ras associated with tumourigenesis

Wild type Ras forms part of a signaling pathway

Controls growth and development in many different systems

Ras is a membrane bound G-protein

Inactive when bound to GDP and active when bound to GTP

Oncogenic forms of Ras bind GTP permanently

Ras activation stimulated by growth factors such as EGF

Ras Receptor tyrosinekinases

RTKs such as the EGF receptor are transmembrane proteins

Extracellular domain binds ligand (EGF or TGFα)

Intracellular domain has tyrosine kinase activity

Ligand binding induces dimerisation and autophosphorylation

What links RTK phosphorylation to Ras activation?

Candidate molecules

Cellular proteins that are tyrosine phosphorylatedfollowing RTK activation

Activated RTK-complex forming proteins

In vivo function and order of activity?

Drosophila R7 cell development

R8 develops first

R7 develops last

R7 is UV sensitive

4

Drosophila R7 cell developmentsevenless and bride of sevenless mutants lack R7

Photoreceptor fate

Sev acts in the R7 cell and is an RTK

Boss is a membrane bound ligandexpressed in R8

Downstream targets of Sev signal notuncovered in standard screens

May take part in multiple signalingpathways and homozygous lethal

Screens for dominant modifiers of eyeless

Enhancer/Enhancer or Supressor/Supressor often lethal orshow no visible phenotype

Screens for dominant modifiers of sevenless

sevts has wild typefunction at 22.7 C butinactive at 24.3 C

Dominant enhancermutations causeinactivity at 22.7 C

Dominant enhancers of sevenless

First E(sev) mutation in aDrosophila homologue of Ras

Dras mutations homozygouslethal

Clones homozygous for Dras

in the eye lack R7 cells

Where do other E(sev) genes fit in the pathway?

SummaryMutations in genes acting in the same pathway canhave synergistic effects enhancing or suppressing phenotypes

Site specific recombinases increase the efficiency withwhich genetic mosaics can be generated

Enhancer/suppressor screens have been used toidentify a Drosophila homologue of the Ras oncogenethat is required for R7 development

ReferencesMolecular Cell Biology 6th Edition

Modifier mutations in yeast pp 173-174

Ras signalling pp 684-688

Insights from model systems: Understanding human cancer ina fly (1997) St John MAR and Xu T AM. J. Hum. Genet vol61pp1006-1010

Dickson B and Hafen E (1994) Invertebrate Signal transductionCurrent Opinion in Genetics and Development 4 64-70