RecombinantArticleQuiz

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victimsofadown
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290078
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RecombinantArticleQuiz
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2014-11-26 05:18:56
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RecombinantArticleQuiz
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  1. What are the implicated diseases?
    • Amylotrophic lateral sclerosis (ALS)
    • Frontotemporal dimentia (C9FTD)
  2. What is the name of the gene and what is the recognizable mutation?  What is the normal state?
    • C9orf72
    • contains 400-4400 GGGGCC repeats
    • healthy is <33 repeats
  3. What are the 3 proposed mechanisms for disease state caused by the mutation?
    • 1. Toxic sense/antisense RNA species that sequester key RNA-binding proteins
    • 2. Toxic dipeptide-repeat (DPR) proteins (non-ATG translation AKA RAN translation)
    • 3. Reduced expression of C9orf72
    • *NOTE - lack of severe phenotype in homozygous mutants and coding mutations indicate loss of function is not responsible
  4. What are RNA foci?
    Neuronal RNA aggregates
  5. What is SH-SY5Y?  What is DPR?
    • SHY-SY5Y: human cell line used as in vitro model for neuronal function
    • DPR: dipeptide repeat
  6. What are the potential products from the mutated segment?
    • Can be translated in all sense and anti-sense frame (6 total)
    • 2/6 frames result in identical DPR proteins, so just 5 unique DPR proteins are possible
  7. Describe the difference between pure repeats and RNA-only repeats created in the first stage of the experiment
    • *NOTE - large repeats are unstable
    • Pure repeats: uninterrupted repeats (GGGGCC) which will form RNA and protein
    • 3 sizes - 3, 36, and 103
    • RNA-only repeats: 1 stop codon per 12 repeats for every frame
    • Will not form protein
    • 3 sizes - 36, 108, and ~288
  8. What is G-quadraplex?
    • Repeating RNA forms a stable tertiary structure called the G-quadraplex
    • This is formed in both pure-repeat and RNA-only repeat sequences
    • (foci?)
  9. What is UAS?
    • Upstream activation system: increases expression of neighboring gene
    • Separate from promoter
    • Used to increase expression of the mutated gene
  10. What were the two initial lines of fruit flies engineered for this experiment?  What was unexpected about their expression?
    • Pure repeat and RNA-only repeat
    • Antisense transcripts were entirely absent
  11. Describe the similarities and difference in results for the first phase of the experiment involving fruit flies (expression in eye).  How is this significant?
    • Same: RNA foci formed
    • Pure repeat: DPR proteins (GR and GP confirmed with immunoblotting)
    • expression of 36 and 103 in eye = degeneration
    • *NOTE - increased temp = increased protein expression = lethality
    • RNA-only repeat: no DPR proteins
    • No effect on eye regardless of temp
    • Significance: DPR proteins must be involved in degeneration
  12. Describe the similarities and difference in results for the second phase of the experiment involving fruit flies (expression in adult neurons).  How is this significant?
    • Why?: ALS and FTD are adult-onset diseases
    • Pure repeat: 36 and 108 lethal <30 days after expression
    • sublethal does of cycloheximide (reduce protein synthesis) extended life span
    • RNA-only repeat: no effect for repeats
    • Significance: confirmation that DPR proteins involved in degeneration
  13. Describe the results for the final phase of the experiment involving fruit flies (in eyes).  How is this significant?
    • What?: Protein only constructs using alternate codons (not GGGGCC)
    • compared GR, PR, PA, and GA DPR proteins
    • 36 DPR constructs (emulates repeats) with arginine caused degeneration, lethality
    • ~103 DPR constructs had similar results with an increase in lethality
    • PR, GR, and GA resulted in decreased survivability (from egg)
    • Significance: Arginine-containing DPR proteins are toxic and must be involved in the mechanism
    • *NOTE - GA100 implicated in late-onset decreased viability, but is not correlated to degeneration
  14. What is the final conclusion of the study?
    • GR and PR DPR proteins are the toxic species even though all 5 DPR proteins can form inclusions
    • This does not mean that RNA toxicity or high focal levels of other DPR proteins are not involved, but this study indicates a strong correlation to GR and PR DPR proteins
  15. What potential explanation is given for the selective degeneration of neuronal tissue?
    • Restricted C9orf72 expression in specific neurons OR
    • inability to clear DPR
    • (potential explanations, not proven)
  16. Describe Fig 1A, B, C, and D
    • A: agarose gel showing pure repeats and RNA-only repeats next to a DNA ladder
    • B: Circular dichroism spectra of 24 pure and 24 RO repeats in G-quadraplex structure
    • C: Microscope images of nuclei (blue) in SH-SY5Y cells showing that both pure and RO repeats formed RNA foci (red)
    • D: Number of SH-SY5Y cells containing RNA foci after transfection (note- #cells not #foci per cell)
  17. Describe Fig 2A, B, C, D, E
    • A: dot blot showing poly-GR proteins in pure but not RO repeats
    • B: showing that 36 pure repeats showed mild toxicity, 103 pure repeats showed increased toxicity, and all others had no effect
    • C: egg to adult viability at varying temps
    • D: survival of female flies expressing repeats in adult neurons
    • E: survival of flies w/ 36, 103 pure repeats in presence/absense of cyclohexamine
  18. Describe Fig 3A, B, C, D
    • *NOTE- protein-only constructs, A-C in eye, D in adult nervous system
    • A: eye degeneration based on DPR protein produced (36)
    • B: eye degeneration based on DPR protein produced (100)
    • C: egg-to-adult viability at varying temperatures (in eye)
    • D: egg-to-adult viability (in adult neurons)

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