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Herpesviruses Experiments

Post and Roizman Experiment

What is the significance of the TAATGARAT motif in immediate-early promoters?

  1. Thymidine-kinase gene + Early promoter-TK gene.
  2. Add to early promoter + TAATGARAT + early promoter-TK gene.
  3. TAATGARAT-TK Virus: synthesizes TK mRNA temporally like an IE gene. Synthesizes TK mRNA in the presence of cycloheximide.
  4. When the TAATGARAT-TK is cloned into a plasmid, TK- cells can be transformed into TK+ cells.
  5. When these transformed cells are infected by TK- virions, TK RNA synthesis is induced.
  6. Perform same experiment after the TK- virion has been exposed to UV to form thymidine dimers on the DNA & block transcription.
  7. TK RNA synthesis is still induced.
  8. Perform same experiment in presence of cycloheximde, which blocks translation. This means no ICP0 nor ICP4 will be produced.
  9. TK RNA synthesis is still induced.
  10. Since no viral protein synthesis is required for induction of the TAATGARAT-TK gene, a virion protein must be the inducing agent. That proteinn is called VP16.

Transient Assay
Test to see transcription factors for IE genes

  1. Make a plasmid of the ICP4promoter-luciferase.
  2. Transform cells with this plasmid (use lipofectin to do so).
  3. Incubate for 24-48 hours, harvest and assay for luciferase activity.

Deletion Assay
delete various segments, attach to luciferase, assay for activity to identify for promoters.

deletion assay genetics technique

Peter O'Hare

schematic tree breakdown of the rna virusesschematic tree breakdown of the dna viruses

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