NE102 Lecture Notes 2

Key point 1 during transfection only a fraction of

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Key point #1: During transfection, only a fraction of cells actually get transfected Key point #2: Transfected cells take up many copies of plasmid DNA Two different plasmids can be  co-transfected  into cells
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Recombinant DNA pt. 2 19:00 To visualize proteins within LIVING cells Expression plasmids can be generated encoding  GFP-fusion protein FINAL POINT: Recombinant DNA can be used to introduce proteins into animals Recombinant DNA molecules can be microinjected into fertilized eggs = Transgenic animals (animals carrying foreign DNA)
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Integrating Concepts 19:00 QUESTION: How can we use these tools to dissect molecular events that govern cell  behavior? Example: p53 Hypothesis: p53 is rapidly degraded in cells.  Cycloheimide =  a drug commonly used to inhibit global translation in cells Hypothesis: p53 degradation is mediated by the proteasome MG132 = drug that inhibits the proteasome Steady levels of p53 How about if we just treat with MG132?  p53 would not be present because the proteasome is not inhibited Hypothesis:   p53 accumulates in cells following DNA damage UV light induces DNA damage Hypothesis: p53 acccumulates in THE NUCLEUS following DNA damage Immunocytochemistry with DAPI QUESTION: Can we examine p53 phosphorylation at serine-9 & serine-15 with  antibodies? Hint 1) We can make p53 protein in vitro (i.e., in a test tube) Hint 2) We can also phosphorylate p53 on Ser-9 and Ser-15 in vitro What if we inoculated an animal with p53 that is phosphorylated at Ser-9 or -15? QUESTION: What’s the experiment we want to run? Create plasmid of Ser-15-Ala p53 mutant and add a transfection reagent and introduce  UV light Does Ser-15-Ala p53 accumulate? CONCLUSION: p53 is phosphorylated on many, MANY serine & threonine residues
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Protein Sorting pt. 1 19:00 Cells spatially “sort” proteins to their functional compartments/locations.
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Protein Sorting pt. 1 19:00 HOW? General Mechanisms: Pre-translational sorting Post-translational sorting Sorting via the “secretory pathway” PRE-TRANSLATIONAL SORTING mRNA localization mRNA can be localized to specific subcellular locations which control WHERE the  protein is made Neuro Ex.) Localization of specific mRNAS to synapses plays a role in synaptogenesis  & synaptic plasticity Ex.) FMRP (Fragile X Mental Retardation Protein) = mRNA localization protein involved  in localization of synaptic mRNAs Mutations in FMRP is a major cause of mental retardation Ex.) Localization of mRNA in  Xenopus  oocytes QUESTION: How do cells localize specific mRNAs? Not extremely well understood Key elements/events: The 3’ UTF of mRNAs that are localized to subcellular locations contain specific RNA  sequences that target them for transport.  3’ UTR sequences are bound by “mRNA localization proteins” that transport them to 
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