Giles 23 April 09
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Giles 23 April 09

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Giles 23 April 09 Giles 23 April 09 Presentation Transcript

  • Analysis of transcription related H3/H4 displacement at the individual gene level
  • Following to be done for ArdC, ProP and AltB at mid and late G2
    Quantification of transcription
    Quantification of PolII occupancy
    Quantification of H3/H4 replacement
    Perform high resolution mapping at mid and late G2 for AltB
    Experiments
  • Showed change in AltB expression as expected
    Showed possible enrichment of PolIIon AltB in late G2
    Previously...
    Late G2
    Mid G2
    Imp UnIP
    Imp Un IP
  • Following to be done for ArdC, ProP and AltB at mid and late G2
    Quantification of transcription
    Quantification of PolII occupancy
    Quantification of H3/H4 replacement
    Perform high resolution mapping at mid and late G2 for AltB
    Experiments


  • Mapping of AltB
    -230
    -156
  • Comparison of mononucleosomes from control and flag incorporated cells
    Testing effect of Flag histone incorporation
  • Determine relative amplification from DNA cut 5 min and 15 min compared to uncut
    Testing Mnase sensitivity of AltB
  • AltBnucleosomepostition in Mid (off) and Late (on) G2
    -230
    -156
    100bp
  • Confirmation of quantitative mapping
    -230
    -156
    Primer extension assay
  • Development of a novel method for high resolution mapping
    -230
    -156
    Tried to quantify/amplify of different products in primer extension reaction
    Late G2
    Mid G2
    PE Q1 Q2 Q3 Q4 Q5
    PE Q1 Q2 Q3 Q4 Q5
  • Single primer tests
    -230
    -156
    Neat 1:100
    Neat 1:100
    Mid G2
    Late G2
    P1/P2 P1 P2
    P1/P2 P1 P2
    P1/P2 P1 P2
    P1/P2 P1 P2
  • Use reconstituted 5s nucleosomes
    Confirm position
    restriction digest
    New method
    Apply new method to map completely AltB
    Validation of new mapping method