Spring 2015 Module 2 Lecture 4 System Engineering and Protein
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1 Spring 2015 Module 2 Lecture 4 System Engineering and Protein Founda@ons Shannon Hughes Noreen Lyell Leslie McLain Nova Pishesha (TA) Leona Samson (Lectures) Zachary Nagel (help with development) Alex Chaim
2 Key Experimental Methods for Module 1 Mammalian tissue cell culture Monitoring protein level by Western blot Generating plasmids with DNA damage Transfecting plasmids into mammalian cells Using fluorescent proteins as reporters of biological processes Flow cytometry to measure DNA repair Statistical analysis of biological data
3 Key Experimental Methods for Module 1 Mammalian tissue cell culture Monitoring protein level by Western blot Generating plasmids with DNA damage Transfecting plasmids into mammalian cells Using fluorescent proteins as reporters of biological processes Flow cytometry to measure DNA repair Statistical analysis of biological data
4 Six Major DNA Repair Pathways BER NER DR MMR HR NHEJ
5 DNA Double Strand Break (DSB) Repair
6 NHEJ HCR in WT and NHEJ defeclve cells at 18 hours post- transfeclon: 80 % Reporter Expression NHEJ Deficient 0 M059K V79 xrs6 M059J (WT) (WT) (Ku80) (DNA Pkcs)
7 Double digest to produce DSBs with ends that are not compalble with ligalon: PflMI PspOMI BstXI 5 3 A C T G C C A G G A G A T G G A C T G G G G C C C A C T G C C A G T T T T C T G G A C T G T G A C G G T C C T C T A C C T G A C C C C G G G T G A C G G T C A A A A G A C C T G A C 3 5 PflMI, PspOMI The restric@on sites are surely different in your constructs; this slide is meant to show one way in which we can illustrate genera@ng various combina@ons of DSB ends 5 3 A C T G C C A G G A G G G C C C A C T G C C A G T T T T C T G G A C T G T G A C G G T C G T G A C G G T C A A A A G A C C T G A C 3 5 Cannot re- circularize directly
8 NHEJ HCR in WT and NHEJ defeclve cells at 18 hours post- transfeclon: 80 % Reporter Expression NHEJ Deficient 0 M059K V79 xrs6 M059J (WT) (WT) (Ku80) (DNA Pkcs)
9 Non- Homologous End Joining (NHEJ) Ku70 Ku80 DNA- PKcs Xrcc4 Ligase IV
10 NHEJ Inhibitor Compound 401 Specifically Inhibits DNA- PKcs and thus NHEJ Compound 401 Chemical Name: 2- (4- Morpholinyl)- 4H- pyrimido[2,1- a]isoquinolin- 4- one
11 NHEJ in Human Lymphoblastoid Cells
12 Reactivation of damaged DNA multiplexed Each Fluorescent Protein gene will harbor a different type of DNA damage DsRed2FP EGFP EOFP ECFP EBFP
13 NHEJ Inhibitor Compound 401 Specifically Inhibits DNA- PK and thus NHEJ
14 Four Different DNA- PKcs Inhibitors that work in Human Cells will they work in CHO cells?
15 Mibefradil dihydrochloride Loperamide Hydrochloride DMNB
16 TransfecLon Experimental Design K1 + K1 + K1 + K1 +
17 How can we get DNA into Mammalian Cells? hhp://
18 How can we get DNA into Mammalian Cells? hhp://
19 What experimental question will you ask in Module 2? How efficiently does DNA repair by the Non Homologous End Joining (NHEJ) pathway act on DNA damage with different topologies? This raises the following questions How does DNA get damaged? What is DNA repair? Why does DNA repair exist? Why do we care about how efficient DNA repair is? How will we actually measure DNA repair efficiency?
20 Six Major DNA Repair Pathways BER NER DR MMR HR NHEJ
21 Six Major DNA Repair Pathways BER NER DR MMR HR NHEJ
22 Six Major DNA Repair Pathways BER NER DR MMR HR NHEJ
23 Six Major DNA Repair Pathways BER NER DR MMR HR NHEJ PANCREATIC CANCER
24 Cancers arise from the accumulalon of heritable changes in gene funclon Normal Epithelium Neoplasia 1st Mutation Invasive Carcinoma 2nd Mutation Metastasis Additional Mutations
25 MulLple MutaLons More and more MutaLons
26 The GeneLc Basis of Cancer and Theodor Boveri Established that chromosomes carry the hereditary informalon by showing that aberrant segregalon of chromosomes leads to certain phenotypes in sea urchin eggs. Suggested that aberrant segregalon of human chromosomes could be responsible for a normal cell becoming a tumor cell Suggested that some chromosomes promote cell growth and others inhibit cell growth Marcella O Grady Boveri ( ) also contributed
27 Marcella O Grady Boveri ( ) also contributed to Boveri s theory She was the first woman student to graduate from MIT with a Biology Major in 1885! J Med Genet. 1985;22(6): Marcella O'Grady Boveri ( ) and the chromosome theory of cancer
28 Chromosomes from a Normal cell Chromosomes from a Tumor cell Spectral Karyotyping (SKY) SKY Painted Chromosomes
29 Chromosomes from a PancreaLc Tumor Cell
30 The GeneLc Basis of Cancer and Theodor Boveri Established that chromosomes carry the hereditary informalon by showing that aberrant segregalon of chromosomes leads to certain phenotypes in sea urchin eggs. Suggested that aberrant segregalon of human chromosomes could be responsible for a normal cell becoming a tumor cell Suggested that some chromosomes promote cell growth and others inhibit cell growth Marcella O Grady Boveri ( ) also contributed
31 AlteraLons (mutalons) in different kinds of Genes cause Cancer Oncogenes genes that ordinarily promote cell proliferalon but when mutated or overexpressed promote uncontrolled growth Tumor suppressor genes genes that ordinarily prevent inappropriate proliferalon but when mutated allow uncontrolled growth Mutator genes genes that ordinarily prevent mutalons; alteralons in these genes allow increased mutalon rates
32 PBS WGBH
33 Mechanisms of Chromosome TranslocaLon
34 Mechanisms of Chromosome TranslocaLon a Balanced reciprocal translocalons from the fusion of two double- strand breaks that arise in the same cell; ligalon of the free DNA ends is mediated by the non- homologous end- joining pathway. Red and blue strands represent different chromosomes. b Telomere uncapping or ahrilon generates a DNA double- strand break response, which potenlally leads to the fusion of telomeres, generalng end- to- end fusions. During anaphase, dicentric fusion chromosomes are pulled apart, leading to the formalon of translocalons and double- strand breaks. Broken chromosomes act as substrates for addilonal rounds of fusion and breakage, generalng increasingly complex translocalons.
35 Chronic Myelogenous Leukemia (CML) breakpoint cluster region protein (BCR)/ C- Abl non- receptor tyrosine
36 Janet Rowley (April 5, 1925 December 17, 2013) American human genelcist and the first scienlst to idenlfy a chromosomal translocalon as the cause of leukemia and other cancers.
37
38 Six Major DNA Repair Pathways BER NER DR MMR HR NHEJ
39 Spring 2015 Module 2 Lecture 4 System Engineering and Protein Founda@ons Shannon Hughes Noreen Lyell Leslie McLain Nova Pishesha (TA) Leona Samson (Lectures) Zachary Nagel (help with development) Alex Chaim
Spring 2015 Module 2 System Engineering and Protein Founda<ons
20.109 Spring 2015 Module 2 System Engineering and Protein Founda
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