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Molecular Biology Bio4751 Spring 2003 Gary A. Bulla, PhD Methods in Molecular Biology RNA quantitation a. Northern 1. Lyse with detergent, 2. Phenol extract (removes proteins) 3. Precipitate RNA 4. Load onto agarose gel 5. Transfer RNA to nylon membrane 6. Add radioactive DNA or RNA to detect individual species Nylon membrane probed with labeled a1antitrypsin RNA, then tubulin DNA RNA quantitation b. RNase protection 1. Lyse with detergent 2. Phenol extract (removes proteins) 3. Precipitate RNA 4. Incubate with radioactive antisense RNA 5. Degrade single strand RNA with RNases 6. Load on 8% PAGE mRNA AAAAA Radioactive antisense RNA Hybridize RNase treat 1. 2. Heat , PAGE Expose to film RNA quantitation c. Primer extension RNA pol II holoenzyme Histone/DNA ratios TATAA Prevent reinitiation Primer extension analysis mRNA Primer +dNTPs Heat, PAGE, probe Also used to identify locations where transcription starts Molecular Biology Bio4751 Spring 2003 Gary A. Bulla, PhD Western (protein detection) A. Immunoblot Cell extract PAGE (+ SDS) Incubate with anti-Rx Transfer to nylon membrane Alkaline peroxidase Rx Anti-myc AntiAnti-Rx Enhancer B. Radiolabeling Transfect C ells Western 35S amino acid, immunoprecipitate electrophoresis Genetically Modified Foods Includes frost-resistant tomatoes Disease-resistant sweet potatoes Muscle-rich cattle …..and many others Last month• Zambia’s government rejected 1000s of tons of corn from US because it may contain some GM kernels •Approx 2.9 people at risk of starvation from droughtinduced famine since 2001 •35,000 will die by 2003 if food not provided (WHO) •GM corn produces a bacterial toxin that is toxic to insects •GM corn used world-wide for 6 years without adverse effects (FDA) How can we detect measure gene activation? DNA RNA Northern RNase Protection Primer extension Protein Western How do we examine DNA-protein interactions? Mad Electrophoretic Mobility Shift Assay (EMSA) (aka gel shift) DNaseI protection Photo-crosslinking How do we examine protein-protein interactions? GST pull-down EMSA Supershift Co-immunoprecipitation How can we measure promoter activity? Answer- Link it to a gene that is easy to monitor a1AT -261 CAT (Chloramphenicol acetyl transferase) +44 Luciferase B-galactosidase CAT assay a1AT CAT Lyse cells, mix with 14Cacetyl CoA, extract and apply to thin layer chromatography Acetylated Chloramphenicol migration Un-acetylated Chloramphenicol + HNF1 - - + + a1AT -261 CAT +44 - + ControlTK-CAT Protein-DNA interactions EMSA (Gel Shift) DNAseI footprinting Photo-crosslinking EMSA (Gel Shift assay)- 4% PAGE (non-denaturing) -To detect protein-DNA interactions - Usually transcription factor binding Theory 80V, 3hr 15 min. Expose to film PAGE Labeled DNA + protein DNAprotein complexes Unbound DNA fos jun TRE TATA J=jun F=fos M=myc (another bZIP protein) C= bZIP domain only Note- complexes migrate according to protein size Fig. 12.31- Fos and jun binding to a TRE Observe• jun or fos cannot bind alone (lanes 1-3) • Jun+ fos does bind (lane 4) • only bZIP domain (C) is required for binding (lanes5, 10 , 11) • another bZIP factor (myc) fails to allow fos or jun to bind (lanes 14-15) DNAseI footprinting 32P Footprinting by DNaseI and Cu++ Experiment TFIID, A and/or B added to DNA treat with DNaseI or Cu++ polyacrylamide gel electrophoresis (PAGE) Observe•TFIID binds poorly •A + D binds strongly •B doesn’t enhance binding of D+A Fig. 11.4 DNaseI footprinting DNase I digestion products Photocrosslinking) • To identify proteins which bind DNA Fig. 6.25 Fig. 6.24 Photocrosslinking) * * * * * * * *DNA UV, nuclease * * TFIID + P-32 labeled promoter DNA which contains bromodoexyuridine (BrdU) UV irradiate (causes BrdU to be crosslinked to proteins it contacts) nuclease SDS-PAGE Fig. 11.15- TAF 250 and 150 bind promoter DNA Health stupidity reigns supreme • Magnet therapy • electronic ab exercisers- called “pump fiction” by Federal Trade Commission -ftc/gov/opa/2002/05/projectabsurd.htm • Acupuncture-No proven benefit in controlled studies • Chiropractic medicine- only useful for lower back pain. Period. All are largely accepted based upon “wart phenomenon” To make a lie into a truth- “Say it loud, say it often” G. Gordon Liddy How can we detect measure gene activation? DNA RNA Northern RNase Protection Primer extension Protein Western How do we examine DNA-protein interactions? Mad Electrophoretic Mobility Shift Assay (EMSA) (aka gel shift) DNaseI protection Photo-crosslinking How do we examine protein-protein interactions? GST pull-down EMSA Supershift Co-immunoprecipitation Protein-protein interactions How do we determine identify protein-protein interactions? Method- Epitope Tagging Ligate a small peptide onto a protein, introduce that protein into cells, then lyse the cells, and use antibodies raised against the small peptide to bind the protein plus any proteins interacting with that protein. Gene of interest ATG TAA ATG TAA Promoter FLAG epitope (7-9 amino acids) Poly-Adenylation sequence Epitope-tagged protein 3A, slide 5 Method- Epitope Tagging Figure 10.13 RNA polymerase II structure- yeast has 12 subunits How do we determine identify protein-protein interactions? HDAc Co-immunoprecipitation Example- Epitope tagging experiment FLAG Epitope-tagged histone deacetylase (HDAC2) to generate FLAG-HDAC2 Introduce FLAG-HDAC2 + Mad1 plasmids into cells Prepare cell extracts Flag Sin3A Mad HDAC Ac Immunoprecipitate with anti-FLAG Ab PAGE Transfer to membrane Probe with anti- SinA or anti-Mad1 HDAC Sin3A Mad Flag Epitope tagging experiment results Ac Fig. 13.38- Evidence for ternary complex involving HDAC2. Sin3A and Mad1 ObserveFLAG alone doesn’t interact with Sin3A or Mad1 (lanes 1-3) HDAC2 interacts with Sin3A (Lane 4) Mad1, but not mutant Mad1pro, interacts with Sin3A (lanes 5 and 6) 9E, slide 46 Another clever assay for protein-protein interaction M2 High Sensitivity Capture Assay CMV FLAG CBP PolyA CMV C-myc HNF1a PolyA Co-transfect Cos7 cells Alkaline peroxidase Anti-myc HNF1 CBP Anti-FLAG 96-well format How do we determine whether a protein is a histone acetyltransferase (HAT)? Assay1. separate nuclear protein on SDS-PAGE impregnated with histones 2. incubate gel with tritium -labeled AcCoA, wash away All nuclear proteins Ac* Ac* H4 H3 H2A H2B Ac * Ac * Fig. 13.33 Activity gel assay for HAT activity How do we identify methylated DNA? CCGG HpaII •Digest genomic DNA with enzyme pair •Load onto agarose gel •Southern transfer •probe with 32-P DNA CCmGG MspI CCGG DNA probe Methylation analysis: The results of MspI and HpaII cleavage are compared by Southern analysis How does one find “open” vs “Closed” DNA Inactive DNase sensitivity assay DNAseI Active DNAseI Remove proteins Remove proteins Cut with restriction enzyme 6kb 4kb 6kb 4kb 3kb 5kb 5kb 3kb Isolate chromatin Treat with DNaseI Remove protein, Isolate DNA Digest with BamHI Agarose gel Southern blot Probe: a-globin Ovalbumin MSB= non-expressing cells Fig. 13.31 Dnase I hypersensitivity of an active gene Transcription run-off assay • To monitor transcriptional activity of a gene Measure transcription directly. Thus post-transcriptional processing in not a concern Figure 5.27 8B, slide 18 Transcription run-off assay SP1 375 nt transcript TBP TATAA Note- Each lane contains RNA pol II + TFIIA,B,E and F bh= bacterially derived human TBP vh=virus derived human TBP Fig. 11.18- TBP alone can’t respond to Sp1