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Experimental approaches

Experimental approaches. qPCR microarrays. VISUALISATION OF EXPRESSION DATA. CLUSTER ANALYSIS. RESULTS: CLUSTER ANALYSIS. Co-expressed gene clusters Associations not previously reported Functionally related genes co-induced: evidence for induction of specific biological pathways.

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Experimental approaches

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  1. Experimental approaches • qPCR • microarrays

  2. VISUALISATION OF EXPRESSION DATA

  3. CLUSTER ANALYSIS

  4. RESULTS: CLUSTER ANALYSIS • Co-expressed gene clusters • Associations not previously reported • Functionally related genes co-induced: • evidence for induction of specific biological pathways

  5. ALTERNATIVE RESPIRATORY PATHWAY WAS INDUCED

  6. | Entry of substrates into mt Limit availability of substrates for ETC? …limit flux through ETC …limit ROS production/oxidative damage v SUBSTRATE TRANSPORT WAS REPRESSED

  7. Amino acid metabolism AMINO ACID METABOLIC PATHWAYS WERE INDUCED

  8. BCKDC IVD MCCAse HIBDH BRANCHED CHAIN AMINO ACID PATHWAYS INDUCED Branched chain amino acids BCAT * Branched chain amino acid metabolism Branched chain a-keto acids BCKDC Acyl-CoA IVD * MCCAse Enoyl-CoA * E-CoAH 3-methyl-gluteconyl-CoA * E-CoAH Hydroxy-acyl-CoA Val Leu * Ile HIBDH Acetoacetate + Acetyl-CoA Propionyl-CoA Propionyl-CoA + Acetyl-CoA Image adapted from: Taylor et. al. (2004) Plant Physiol 134(2): 838-48.

  9. PROBLEMS WITH CLUSTERING • Good at identifying genes that are co-expressed in response to all/most treatments • Not good at identifying genes that are co-expressed in response to specific subsets of treatments

  10. Treatments Genes PROBLEMS WITH CLUSTERING Treatments Genes

  11. NEW ALGORITHM: ModuleFinder • Choose an initial subset of treatments • Identify subset of genes that are co-expressed in response to these treatments • Cluster into groups

  12. NEW ALGORITHM: ModuleFinder • Look for other treatments under which these gene clusters are co-expressed

  13. NDB2 AOX1a carriers Classical ETC components All associated with: flux through ETC ATP synthase activity ROS RESULTS: ModuleFinder Amino acid synthesis

  14. Research focus - mitochondria protein import replication, transcription & translation biosynthetic pathways amino acids nucleotides vitamins folate lipids heme TCA cycle membrane transport respiratory chain

  15. TIM17 TIM22 TIM23 TIM44 TOM7 TOM9 TIM16 TIM14 TOM20 TOM40 MPP Transcript profiling: import components

  16. Transcript profiling: mRNA stability Cytc(a) t ½ = 11 h Average transcript abundance (relative to pre-treatment) TIM22-3 t ½ = 54 min

  17. Transcript profiling: mRNA stability Transcript abundance Time (hours)

  18. Degradation of Transcripts • Inhibit Transcription • Measure [T] at different times • Rate of degradation

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