Methods in Molecular Biology

1. Molecular Biology Bio4751 Spring 2003 Gary A. Bulla, PhD Methods in Molecular Biology

2. 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 a1-antitrypsin RNA, then tubulin DNA

3. 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 • Heat , PAGE • Expose to film

4. 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

5. Western (protein detection) Molecular Biology Bio4751 Spring 2003 Gary A. Bulla, PhD Transfect C ells 35S amino acid, immunoprecipitate electrophoresis Alkaline Alkaline peroxidase peroxidase Anti Anti Anti - - - myc A. Immunoblot Cell extract PAGE (+ SDS) Transfer to nylon membrane Incubate with anti-Rx Anti-Rx Rx - - - Enhancer B. Radiolabeling Western

6. 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 drought-induced 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)

7. Mad How can we detect measure gene activation? DNA RNA Northern RNase Protection Primer extension Protein Western How do we examine DNA-protein interactions? Electrophoretic Mobility Shift Assay (EMSA) (aka gel shift) DNaseI protection Photo-crosslinking How do we examine protein-protein interactions? GST pull-down Co-immunoprecipitation EMSA Supershift

8. How can we measure promoter activity? Answer- Link it to a gene that is easy to monitor (Chloramphenicol acetyl transferase) a1AT CAT -261 +44 Luciferase B-galactosidase

9. CAT assay a1AT CAT Lyse cells, mix with 14C-acetyl CoA, extract and apply to thin layer chromatography Acetylated Chloramphenicol migration Un-acetylated Chloramphenicol - + - - + + + HNF1 Control- TK-CAT a1AT CAT -261 +44

10. Protein-DNA interactions EMSA (Gel Shift) DNAseI footprinting Photo-crosslinking

11. EMSA (Gel Shift assay)- 4% PAGE (non-denaturing) • -To detect protein-DNA interactions • - Usually transcription factor binding fos jun TATA TRE Fig. 12.31- Fos and jun binding to a TRE Theory 80V, 3hr 15 min. Expose to film PAGE Labeled DNA + protein J=jun F=fos M=myc (another bZIP protein) C= bZIP domain only DNA-protein complexes Note- complexes migrate according to protein size Unbound DNA • 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)

12. DNAseI footprinting 32P DNase I digestion products Experiment TFIID, A and/or B added to DNA treat with DNaseI or Cu++ polyacrylamide gel electrophoresis (PAGE) Fig. 11.4 DNaseI footprinting Footprinting by DNaseI and Cu++ • Observe- • TFIID binds poorly • A + D binds strongly • B doesn’t enhance binding of D+A

13. Photocrosslinking) • To identify proteins which bind DNA Fig. 6.25 Fig. 6.24

14. * * * * * * * * 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 Photocrosslinking)

15. 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

16. Mad How can we detect measure gene activation? DNA RNA Northern RNase Protection Primer extension Protein Western How do we examine DNA-protein interactions? Electrophoretic Mobility Shift Assay (EMSA) (aka gel shift) DNaseI protection Photo-crosslinking How do we examine protein-protein interactions? GST pull-down Co-immunoprecipitation EMSA Supershift

17. Protein-protein interactions

18. How do we determine identify protein-protein interactions? Gene of interest ATG TAA ATG TAA FLAG epitope (7-9 amino acids) Promoter Poly-Adenylation sequence Epitope-tagged protein 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. 3A, slide 5

19. Figure 10.13 Method- Epitope Tagging RNA polymerase II structure- yeast has 12 subunits

20. How do we determine identify protein-protein interactions? HDAc FLAG Epitope-tagged histone deacetylase (HDAC2) to generate FLAG-HDAC2 Introduce FLAG-HDAC2 + Mad1 plasmids into cells Prepare cell extracts Flag HDAC Immunoprecipitate with anti-FLAG Ab Sin3A Ac Mad PAGE Transfer to membrane Probe with anti- SinA or anti-Mad1 Co-immunoprecipitation Example- Epitope tagging experiment

21. Flag HDAC Sin3A Ac Mad Epitope tagging experiment results Fig. 13.38- Evidence for ternary complex involving HDAC2. Sin3A and Mad1 Observe- FLAG 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

22. Another clever assay for protein-protein interaction CMV Co-transfect Cos7 cells FLAG CBP PolyA CMV C-myc HNF1a PolyA Alkaline peroxidase Anti-myc myc HNF1 CBP FLAG Anti-FLAG M2 High Sensitivity Capture Assay 96-well format

23. * * Ac Ac H4 H3 H2A * H2B * Ac Ac How do we determine whether a protein is a histone acetyltransferase (HAT)? Assay- 1. separate nuclear protein on SDS-PAGE impregnated with histones 2. incubate gel with tritium -labeled AcCoA, wash away All nuclear proteins Fig. 13.33 Activity gel assay for HAT activity

24. How do we identify methylated DNA? • Digest genomic DNA with enzyme pair • Load onto agarose gel • Southern transfer • probe with 32-P DNA Methylation analysis: The results of MspI and HpaII cleavage are compared by Southern analysis

25. How does one find “open” vs “Closed” DNA DNase sensitivity assay Inactive Active DNAseI DNAseI Remove proteins Remove proteins Cut with restriction enzyme 4kb 3kb 6kb 5kb 5kb 6kb 3kb 4kb

26. Isolate chromatin Treat with DNaseI Remove protein, Isolate DNA Digest with BamHI Agarose gel Probe: a-globin Ovalbumin Southern blot MSB= non-expressing cells Fig. 13.31 Dnase I hypersensitivity of an active gene

27. Transcription run-off assay • To monitor transcriptional activity of a gene Measure transcription directly. Thus post-transcriptional processing in not a concern

28. Figure 5.27 8B, slide 18

29. SP1 TATAA Fig. 11.18- TBP alone can’t respond to Sp1 TBP Transcription run-off assay 375 nt transcript Note- Each lane contains RNA pol II + TFIIA,B,E and F bh= bacterially derived human TBP vh=virus derived human TBP