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Approaches

Transcript data from detailed lab studies Transcript data from past experiments reveals co-expression. Large transcript data-sets Clustering of array data over development, biotic/abiotic stresses in order to discover co-regulation through co-expression.

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Approaches

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Presentation Transcript

  1. Transcript data from detailed lab studies Transcript data from past experiments reveals co-expression. Large transcript data-sets Clustering of array data over development, biotic/abiotic stresses in order to discover co-regulation through co-expression. Comparative genomics: Inter-species promoter comparison Comparison of promoter sequences of a gene between different species can identify elements highly conserved since divergence. Approaches Different resources used to identify regulatory promoter motifs:

  2. Identify tightly co-expressed mitochondrial genes and predict regulatory motifs Cluster genes with a motif of interest based on motif organisation and expression Test activity of motifs Test motif activity and importance of organisation Network discovery driven by large-scale transcriptional profiling Vast publicly-available Arabidopsis transcript data sets

  3. ADP + Pi ATP NAD+ O2 H2O NAD(P)+ fumarate O2 H2O NADH H+ H+ H+ succinate NAD(P)H int II AOX I III IV V ext c NAD(P)H H+ NAD(P)+ Respiratory chain MATRIX UQ IMS Number of genes in Arabidopsis: • cytochrome pathway – approx. 150 • AOX – 5 • alternative NAD(P)H DHs - 7

  4. The alternative respiratory pathway was induced

  5. ModuleFinder - Biclustering Salicylic acid (100mM) Salicylic acid (10mM) Rotenone (3hr) Rotenone (12hr) Chloramphenicol (3 hr) Chloramphenicol (12 hr) Cysteine H2O2 Oligomycin (1.25mM) Oligomycin (0.125mM) Anoxia (N2)

  6. 0% 50% 100% Identification of regulatory motifs SR2 SR1 SR3 SR4 SR5 SR6 SR6 SR8

  7. Models of regulation - testable Model of module promoters:

  8. Leaf development transcript profiles QRT-PCR to measure transcripts over 2˚ rosette leaf development 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6

  9. Identification of Import Component Regulatory Elements Tom20-2 / Tom20-3 Normalised Transcript Abundance Leaf age (Weeks) 0 1 2 3 4 5 6 Tom9-1 / Tom20-4 Leaf Development 0 1 2 3 4 5 6

  10. Identification of Import Component Regulatory Elements Tom20-2 / Tom20-3 0 1 2 3 4 5 6 Tom9-1 / Tom20-4 0 1 2 3 4 5 6

  11. Test predictions

  12. Testing functionality Measure reporter gene

  13. Phylogenetic analysisAtAox1c and GmAox2b motif

  14. Aox1a - motif stress specific

  15. Functional analysis of motifs in leaves Aox 2kb Aox 2kb  1683-1677 Aox 1kb

  16. Identification of common active elements Aox1a NDB2

  17. Model of gene expression for Aox1a and NDB2 upon treatment with rotenone

  18. Identification of Import Component Regulatory Elements Tom20-2 / Tom20-3 0 1 2 3 4 5 6 Tom9-1 / Tom20-4 0 1 2 3 4 5 6

  19. Identification of Import Component Regulatory Elements Tom20-2 / Tom20-3 0 1 2 3 4 5 6 Site II Elements Tom9-1 / Tom20-4 0 1 2 3 4 5 6

  20. + + Enhancer function Promoter deletion/biolistics Electrophoretic mobility shift assays + + +

  21. Motif identification Oligo + nuc extract Oligo + comp + nuc extrct Oligo + comp + nuc extract Oligo + nuc extract Oligo Oligo Reporter gene activity 20-4 60-mer 20-4 60-mer ∆TGGGCT

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