drosophila melanogaster - university college londonucbhhks/biol2005/fly3ho.pdf1 drosophila...
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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/+
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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
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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
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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