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Exploitation de la variabilité naturelle Core-collection

Une autre méthode de génomique inverse : TILLING (targeted induced local lesins in genomes, Comai, Henikoff, 2001). Applicable a tous organismes, produit des séries alléliques, backcross nécessaires (plusieurs mutations / génome). Exploitation de la variabilité naturelle Core-collection.

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Exploitation de la variabilité naturelle Core-collection

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  1. Une autre méthode de génomique inverse : TILLING (targeted induced local lesins in genomes, Comai, Henikoff, 2001) Applicable a tous organismes, produit des séries alléliques, backcross nécessaires (plusieurs mutations / génome)

  2. Exploitation de la variabilité naturelleCore-collection Identification des facteurs génétiques impliqués dans les caractéres complexes par analyse de la variabilité génétique au sein d’une espéce Arabidopsis: plusieurs centaines d’écotypes (isolats géographiques de l’Himalaya au Cap Vert) -> séquencage de quelques régions uniformément réparties dans le génome chez 265 écotypes-> sélection de 48 écotypes représentant la quasi totalité de la variabilité génétique de l’espéce. Applications: séquencage systématique de gènes candidats et identification des alléles associés a des adaptations environnementale (génétique d’association du génotype au phénotype) Froid , sécheresse

  3. 1A 4E eIF2 PABP 4G 2 4A 3 1 40S 5 Life after transcription: Genome wide analysis of translational regulations • Multiple mechanisms • Multiple roles(viral expression,metabolism,development, cell growth and division). • Require appropriate methods for analysis Access to translation factors Interaction with small RNA Translation inhibition Degradation (RNA silencing) uORFs/reinitiation

  4. AAAAAAA monosomes AAAAAAA AAAAAAA AAAAAAA polysomes AAAAAAA Large scale analysis of mRNA translation during stress Sucrose gradient Total RNA 80S Polysomal RNA -stress 260nm 60S +stress 40S Comparison of polysomal RNA to total RNA ratio for each mRNA in different physiological conditions Sédimentation Microarrays: Sugar: Rhobio/Biogemma company; Sarda Freyssinet et al, Evry, 25607 oligos (Operon) Cadmium: URGV Evry; Renou et al, 24576 GeneSpecificTags (CATMA Consortium)

  5. A 60 Control 50 200uM 300uM 40 Cadmium addition PCV (%) 30 20 3,5 10 0 12 24 48 72 2,5 0 0 24 48 72 96 120 144 168 192 216 240 264 1,5 Time (hours) 0,5 0 • phytochelatin production C control Cadmium Cadmium intoxication Arabidopsis cells Polysome purification RNA extraction Microarray analysis Glutathion Phytochelatins

  6. Homodirectional regulation ARN totaux TTemTCad PTemPCad TCadPCad TTemPTem ARN polysomaux -Cd +Cd Decrease in total RNA Decrease in polysomal RNA

  7. Homodirectional regulation TTemTCad PTemPCad TCadPCad TTemPTem Total RNA Polysomal RNA -Cd +Cd Increase total RNA Increase polysomal RNA

  8. TTemTCad PTemPCad TCadPCad TTemPTem Translational regulation Total RNA Polysomal RNA -Cd +Cd No variation in total RNA Polysomal RNA decrease

  9. TTemTCad PTemPCad TCadPCad TTemPTem Translational regulation ARN totaux ARN polysomaux -Cd +Cd No variation in total RNA Polysomal RNA increase

  10. Uncoupling Total RNA Polysomal RNA -Cd +Cd TTemTCad PTemPCad TCadPCad TTemPTem Total RNA decrease No variation polysomal RNA

  11. TTemTCad PTemPCad TCadPCad TTemPTem Total RNA Polysomal RNA -Cd +Cd Uncoupling No variation polysomal RNA total RNA increase

  12. Different stresses leads to different types of translational responses Total RNA Total RNA Polysomal RNA Polysomal RNA - stress 1 + stress - stress 2 + stress - stress 3 + stress UP DOWN Class 1: Homodirectional variation: Cadmium: 23% , Sucrose 17% Class 2: Translational regulation: Cadmium 7%, Sucrose 76% Class 3: Uncoupling: Cadmium 70%, Sucrose 7% Class 2 and 3 define subcellular sites for accumulation of mRNA

  13. les ARNm sont localisés et traduits dans des régions cellulaires particuliéres->gradients

  14. Génomique comparative Comparative Genomics Identifies a Flagellar and Basal Body Proteome that Includes the BBS5 Human Disease Gene (Cell, 117 p541 2004) Flagelles: structures complexes (100-250 proteines) Plantes: cellules non-flagellées Algues, animaux: cell. flagellées Proteome humain + proteome chlamydomonas - proteome arabidopsis= ensemble des composants du flagelle!!!!

  15. Protein synthesis: initiation 1A AUG 4E PABP 4G 2 4A 3 1 40S 5 AAAAA 4E+4G+4A=eucaryotic initiation complex 4F (eIF4F)

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