Gene function analyses,Reporter genes in direct and

reverse genetics

Reporter genes

• encode proteins that can be directly visualized or enzymes, whose activity can be visualized

• quantitative or qualitative assessment• promoter activity analyses, subcellular localization of

proteins, optimizing transformation procedure, ….

Constrains:- background (autofluorescence, natural enzyme activity

within the tissue) - protein stability (mask changes in promoter activity)- degradation of fusion proteins

Reporter genes

gene product substrate detection

gusA -glucuronidase (E. coli) MUG fluorescence

X-gluc histochemical

luc luciferase (fire fly) luciferin luminiscence

gfp green fluorescent protein xxx fluorescence

(gelly fish)

Fluorescent proteins: GFP, DsRed, mCherry, EosFP

Aequoria victoria

Barevné varianty GFPa DsRed

- unique tools for in vivo labelling

- encoded with small genes

- origin: sea Coelenterata (corals, gelly fish)- modified forms:

- fluorescent features, - codon usage, splicing, stability, …

EosFP – photoactivatable fluorescent protein (green to red FP)

GUSQualitative detection (X-gluc)• oxidized blue precipitate of reaction product• low background• slow diffusion• mostly in fixed material

(X-gluc = 5-bromo-4-chloro-3-indolyl glucuronide)

Quantitative detection (MUG)• GUS enzyme isolation, fluorimetric statement• highly sensitive, low background

(MUG = 4-methylumbelliferyl-beta-D-glucuronide)

Gene function analyses

Modulation of expression:- increased protein level (overexpression) –

introduction of a gene with a strong constitutive promoter

- alt. gain-of-function mutations

- decreased protein level by RNAi

- alt. loss-off-function mutation

Tilling – point mutation in commercial collections

Reporter gene fusions

Decreasing protein levelInduction of RNA interference (dsRNA formation): 1) antisense RNA2) hairpin RNA (e.g. sense-intron-antisense) 3) non-terminated RNA (dsRNA via RdRP)

- dsRNA cleavage by DCL, siRNA formation, sequence specific mRNA degradation or block of transcription due to promoter methylation

+

RdRP

Intermolecular pairing

intramolecularpairing

complementary strandsynthesis

Promoter analysisFusion of analyzed promoter with reporter gene (transcription fusion), with or without the original transcript:

Indicates: - tissue, organ, developmental specificity- responses to external factors

Confirmation with other approaches advisible (risk of artifacts)

P gen T

reportérový gen

- usually introduction of new copy into the genome

Promoter fusion with GFP and GUS

Arabidopsis thaliana

Fusion protein formation

- stop codon removal, fusion in reading frame (= translational fusion)- functional domains, natural interaction(!)

GFP fusion proteins

- Protein localization analyses, protein interactions

- in vivo labelling of cellular structures

Golgy complex chromosomes microtubules