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 substratedetection gusA  -glucuronidase (E. coli) MUGfluorescence X-gluc histochemical luc luciferase (fire fly)luciferinluminiscence gfp green fluorescent protein xxx fluorescence (gelly fish)

Fluorescent proteins: GFP, DsRed, mCherry, EosFP Aequoria victoria Barevné varianty GFP a 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)–

GUS Qualitative 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 level Induction of RNA interference (dsRNA formation): 1) antisense RNA 2) 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 intramolecular pairing complementary strand synthesis

Promoter analysis Fusion 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