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LT15 Drosophila Tumourigenesis
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Drosophila Tumourigenesis
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Drosophila: Transgenics and TumorigenesisFly’s eye view of biology (Thomas, 1999)
Determining how genes function in multicellular organisms is complex and difficult
- Inactivation of most genes results in subtle and undetectable phenotypic
alterations
- Phenotypes are observed are difficult to interpret as most genes play multiple
roles in development
Drosophila eye addresses processes: cell cycling regulation, cell proliferation and
differentiation, neuronal connectivity, programmed cell death, protein degradation
and trafficking, tissue patterning, polarity
Eyes develop in a series of precise developmental events – from larval, pupal, adult
stages – coordination of cell cycle regulation and differentiation in 3 rd larval stage
eye disc generates neuronal photoreceptor cells (R cells), lens-secreting cone cells,
pigment cells, mechanosensory bristles
- Differentiated cells assembled into patterned unit eyes or ommatidia in turn
organised into a perfect lattice
Good features of using eye development in molecular, genetic and cell biological
studies: stages can be analysed at level of single cells, eye is dispensable for
viability and fertility of the fly – flies can be maintained for further analysis, 2/3
of 3200 vital genes for Drosophila – required for assembly and neuronal
connectivity of the eye
Genetics of Ras Signalling
Ras proto-oncogene overexpression leads to cancer, usually last to be mutated
(eg. in gut cancers) transform benign cancer to metastasis
Dominant mutations in Ras associated with tumourigenesis
Part of signalling pathway controls growth and development in many different
systems
Ras is a membrane bound G-protein
- inactive when bound to GDP, active when bound to GTP
- oncogenic forms of Ras bind GTP permanently
- Cell culture studies – ras activation stimulated by growth factors such as EGF
- Growth factors that activate Ras bind to Receptor tyrosine kinases
transmembrane proteins
- Extracellular domain binds ligands – intracellular domain has tyrosine kinase
activity ligand binding induces dimerization and autophosphorylation
What links RTK phosphorylation to Ras activation?
, Find candidate molecules – cellular proteins that are tyrosine phosphorylated
following RTK activation/activated RTK-complex forming proteins
- Problem: hundreds of proteins that fit this profile which ones function in vivo
and which context, order of activity – address with genetics!
Mis-expression of wild-type Drosophila regulate cell cycle progression such as
cyclins E and A, decapo, roughex
Mis-expression of wild-type genes does not always disturb eye development –
encoded protein is normally constitutively expressed but is non-functional in the
absence of induced alterations such as nucleotide binding, post-translational
modification, structural arrangement or cellular localisation
- Wild-type RAS1 mis-expression using sev-vector does not affect eye
development while both constitutively active RAS1 (reduced GTPase) activity
and dominant negative RAS1 (interferes with the function of endogenous RAS1)
affect cell fate decisions when expressed in the same vector
- Expression of analogous altered forms of RAB and RHO GTPase family members
has been shown to affect cell shape changes and vesicular transport
Using mosaic analysis – Drosophila R7 cell development
Sevenless and bride of sevenless mutants lack R7
Sev acts in the R7 cell and is an RTK
Boss is a membrane bound ligand expressed in R8 but stays tethered
to it so it can only activate adjacent cells so R7 cells are not
expressed everywhere
Downstream Ras-like targets of Sev signal not uncovered in standard
screens
- Possible explanation: downstream targets of this system take place
in other signalling pathways or induce homozygous lethals, so they are never
uncovered
Screens for dominant modifiers of sevenless – pick up mutations that are
homozygous lethal in their heterozygous form – in the heterozygous form they act
as dominant modifiers of the phenotype)
- Used temperature sensitive mutant of sevenless – system is weakened, but a
dominant modifier will restore wild-type function enable system to work in
restrictive conditions
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