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-expressionenhanced expressionectopic 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

FRT

marker

hs-FLP

+

m

FRT

FRT

marker

+ marker

+FRT marker

+ marker

mFRT

m

HS

Making a mitotic clone

Random location

loxP loxP

Enhancer-Cre recombinase

=> tissue-specific knock-out

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?

GAL4 X

UAS

GAL4

specific enhancer targeted expression of X

CD2Actin promoter

FRT FRT

STOP GAL4

UAS

UAS

X

GFP

hs-FLP random location; can sample all cells

Enhancer-FLP

Clonal induction of expression

enhancers Endogenous gene

UAS

UAS

Drives anti-sense expression

Drives expression

Cross with GAL4 lines => targeted expression

Targeted expression screen

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

The Drosophilacompound eye

利用果蠅研究人類疾病

人類MJD基因表現於果蠅造成類似的神經退化壞死現象

正常的MJD基因 致病的MJD基因

Eye-antenna

Leg

Wing

dpp-GAL4 + UAS-GFP (dpp>GFP)

dpp>eyg dpp>ey dpp>eyg+ey

Coexpression of eyg and ey showSynergistic effect in eye induction

wt gv (eyg) Eq>hth

Dorsal midline fusion of the thorax

脊索閉鎖不全

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 phenotypeof 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 positionFISHmapped genomic clonesgenome sequence

Known mutations mapped at same location

Molecular defect of the gene in mutantsSouthernexpression

2. Gene knock-out by homologous recombination

3. Insertional mutaenesisscreen by PCR

4. Post-transcriptional inactivation Antisense RNA Ribozyme RNA interference (RNAi, dsRNA)

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