Janos Szabad Department of Biology University of Szeged 6720 Szeged, Somogyi str

Transcription

1 Janos Szabad Department of Biology University of Szeged 6720 Szeged, Somogyi str

2 - Through the use of antibodies - against the protein - against a fusion partner - Through lighting up them - The Shimomura, Chalfie and Tsien story - Luminescence (cold body radiation) - Fluorescence (the emission of light by a substance that has absorbed light) - GFP (green fluorescent protein) - FRET - Features of the fluorescent proteins - Reporter transgenes; types and uses - Cell biology examples from home

3 Osamu Shimomura Martin Chalfie Roger Y. Tsien

4 Aequorea victoria Osamu Shimomura isolated the AEQUORIN protein in AEQUORIN emits blue luminescence upon receiving energy from a chemical reaction

5 GFP (green fluorescent protein) was isolated as a byproduct along the isolation of AEQUORIN.

6 Principles of the origin of fluorescence Conversion to heat eg

7 Structure of GFP - GFP is composed from 238 amino acids - Its molecular mass is 26.9 kda - Its diameter is 4 nm - The 65 th - 67 th amino acids (Ser-Tyr-Gly) compose the fluorophore part

8 The basis of fluorescence The chromophore itself is a p-hydroxybenzylidene-imidazolidone (green background). It consists of residues (Ser - dehydrotyr - Gly) of the protein. The cyclized backbone of these residues forms the imidazolidone ring. The peptide backbone trace is shown in red. Although the amino acid sequence Ser-Tyr-Gly is present in a number of other proteins, it is neither cyclized in any of these, nor is the tyrosine oxidized and thus these proteins are not fluorescent. This implies that the tendency to form such a chromophore is no intrinsic property of this tripeptide.

9 The excitation spectrum (blue) and the fluorescence emission spectrum (green) of GFP

10 Features of the fluorescent proteins 1,0 BFP GFP CFP EGFP YFP DsRed Absorption spectra 0,5 Wavelength (nm) 1,0 GFP: Ser-Thr-Gly EGFP: Thr-Thr-Gly BFP CFP YFP DsRed Florescence spectra 0,5 Wavelength (nm)

11 ATP Luminescence Aequorin ADP + P i

12 ATP Luminescence ADP + P i Aequorin ATP Aequorea victoria Fluorescence Aequorin GFP ADP + P i Transfer of electronic excitation energy (FRET, fluorescence resonance energy transfer)

13 Can GFP be used as a reporter? The GFP encoding so-called GFP gene was cloned by Douglas Prasher in 1992

14 - Through the use of antibodies - against the protein - against a fusion partner - Through lighting up them - The Shimomura, Chalfie and Tsien story - Luminescence (cold body radiation) - Fluorescence (the emission of light by a substance that has absorbed light) - GFP (green fluorescent protein) - FRET - Features of the fluorescent proteins - Reporter transgenes; types and uses - Cell biology examples from home

15 Reporter gene types DNA Promoter mrna Protein Gene Transcribed part DNA Reporter gene Promoter Reporter DNA mrna Reporter protein Promoter DNA mrna Fusion protein Reporter gene Transcribed part

16 Reporter gene expression 1. Transient, following trasfection

17 Reporter gene expression 1. Transient, following trasfection 2. Permanent, once in transgene Way to generate transgene in Drosophila Nucleus DNA solution Polar plasm Germ-line founder cell Cell with a transgene

18 Ketel The LacZ gene of E. coli as reporter and X-gal Villányi et al., Mech Dev 125, 822, 2008.

19 Luciferase as a reporter gene Transgenic Arabidopsis thaliana with a Luciferase reporter transgene

20 Luciferin The mode of luciferase action: Luciferin + ATP luciferyl-adenylate + PP i luciferyl adenylate + O 2 Luciferase oxyluciferin + AMP + LGHT The reaction is energetically very efficient: nearly all of the energy input into the reaction is transformed into light (560 nm). As a comparison, the incandescent light bulb only converts about 10% of its energy into light.

21 Firefrly, Photuris pennsylvanica Common glow-worm of Europe (Lampyris noctiluca)

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23 Making use of GFP in reporter genes DNA Promoter mrna Protein Gene Transcribed part DNA Reporter gene Promoter Reporter DNA mrna Reporter protein Martin Chalfie (1994) Transgene expressed in the nervous system of the nematode Caenorhabditis elegans Promoter DNA mrna Fusion protein Reporter gene Transcribed part Tulle Hazelrigg (1994) Transgene reporting the expression of the exuparentia gene of Drosophila melanogaster

24 Expression of the EGFP reporter gene as ensured by the promoter of the engrailed gene of Drosophila. The engrailed gene is expressed in the posterior compartments of the body segments. ( To show the efficiency of stem cell theraphy, father of the green-glowing mice was first sterilized and was made fertile by impalnting green-glowing tissue cultured stem cells into his testis. The stem cells as well as the offspring mice produce GFP. (

25 Features of the fluorescent proteins 1,0 BFP GFP CFP EGFP YFP DsRed Absorption spectra 0,5 Wavelength (nm) 1,0 GFP: Ser-Tyr-Gly EGFP: Thr-Tyr-Gly BFP CFP YFP DsRed Florescence spectra 0,5 Wavelength (nm)

26 Discosoma (mushroom anemones) the Ds-Red source

27 - Through the use of antibodies - against the protein - against a fusion partner - Through lighting up them - The Shimomura, Chalfie and Tsien story - Luminescence (cold body radiation) - Fluorescence (the emission of light by a substance that has absorbed light) - GFP (green fluorescent protein) - FRET - Features of the fluorescent proteins - Reporter transgenes; types and uses - Cell biology examples from home

28 Light pathways in confocal microscopy

29 LSM information flow schematic diagram

30 Ours is an Olympus FV1000 LSM, laser scanning microscope

31 Microtubules

32 Chromatin

33 Microtubules Chromatin

34 Histone Tubulin Lodestar Merged telophase metapahse prophase anaphase 5 m

35 Egg primordia in a Drosophila ovariole Stg 7 Stg 6 Stg 2 Stg 4 Stg 9 50 m Stg 6 Stg 7 Stg 8 Stg 9 5 m

36 Colchicine stops nuclear wiggling Colchicine-treated 5 m

37 Wiggling is the succession of random and sudden minor turnings of the nuclei Importinchromatin and

38 Microtubules poking the nuclei

39 Wriggling of the ER

40 EB1 highlighted microtubules in follicle cells

41 EB1 highlighted microtubules in neuroblasts

42 EB1 highlighted microtubules in nurse cells

43 Osamu Shimomura Martin Chalfie Roger Y. Tsien