Presentation on theme: "Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes Redundancy? No perfect redundancy => subtle phenotype Gain-of-function."— Presentation transcript:
Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes Redundancy? No perfect redundancy => subtle phenotype Gain-of-function Many give observable phenotypes (e.g. oncogenes) mis-expression enhanced expression ectopic expression constitutive activation
Using Drosophila to analyze human gene function Homologous gene in fly (FlyBase) find fly mutation study fly mutation identify interacting genes check in human for involvement of the interacting genes Human gene family usually have more genes than in fly easier to study in fly can not address functional diversity
-/-+/+ +/- Clonal (mosaic) analysis Analysis of gene with pleiotropic functions (e.g. at later stages) Determine the site of gene function
+ m FRT marker hs-FLP + m FRT marker + +FRTmarker + mFRT m HS Making a mitotic clone Random location
No fly homolog Ectopic expression in fly => phenotype Determine what process/pathway is affected Similar function in human? Search for interacting genes in fly Similar gene interaction in human?
Flowchart for ectopic expression in fly 1. Make UAS-X construct 2. Microinjection to fly embryo 3. Isolate transformants and set up balanced lines 4. Drive expression with GAL4 lines GMR-GAL4 dpp-GAL4 Eq-GAL4 5. Examine phenotype what cell types are affected? what cellular processes are affected? fate transformation? cell death or proliferation?
Using fly to study human gene function No fly homolog Misexpression in fly Phenotype in fly Function Interacting genes Fly homolog Fly mutant
6. Predict interacting genes based on knowledge in fly study obtain relevant mutants test for genetic interactions Modifier mutation (suppressor, enhancer) Second site mutation that modifies the mutant phenotype of the first gene a - /a + b - /b + => mutant phenotype a - /a + b - /b - => stronger phenotype (dominant enhancer) a - /a - b - /b - => synergistic (more than additive) effect
A C B D X Effect of X (ectopic expression) may be suppressed by l-o-f mutation in C
7. Screen for modifier mutations Screen for (EMS) mutations that enhance or suppress the ectopic expression phenotype Map mutations by recombination or deficiency Predict candidate genes from map location Get mutants for candidate genes Confirm genetic interactions Similar relationship in human?
Reverse genetics: From a gene to function 1. Candidate genes by location Map position FISH mapped genomic clones genome sequence Known mutations mapped at same location Molecular defect of the gene in mutants Southern expression
5. Dominant-negative protein e.g. Transcription factor Domains: DNA-binding, Activation, Oligomerization D - A + O + oligomer without DNA-binding D + A - O + seq-specific repressor D + A - O - blocks target sites