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

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 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 AAAAA mRNA Radioactive antisense RNA Hybridize RNase treat 1.Heat, PAGE 2.Expose to film RNA quantitation

Primer extension analysis Prevent reinitiation TATAA RNA pol II holoenzyme Histone/DNA ratios Primer Also used to identify locations where transcription starts c. Primer extension RNA quantitation mRNA +dNTPs Heat, PAGE, probe

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

Genetically Modified Foods Includes frost-resistant tomatoes Disease-resistant sweet potatoes Muscle-rich cattle …..and many others 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 ,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) Last month-

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

How can we measure promoter activity? CAT  1AT Answer- Link it to a gene that is easy to monitor Luciferase B-galactosidase (Chloramphenicol acetyl transferase)

CAT  1AT + HNF1 CAT Control- TK-CAT  1AT Acetylated Un-acetylated CAT assay Lyse cells, mix with 14C- acetyl CoA, extract and apply to thin layer chromatography migration Chloramphenicol

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

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

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

Fig Fig To identify proteins which bind DNA Photocrosslinking)

Fig TAF 250 and 150 bind promoter DNA TFIID + P-32 labeled promoter DNA which contains bromodoexyuridine (BrdU) UV irradiate (causes BrdU to be crosslinked to proteins it contacts) nuclease SDS-PAGE DNA ******** UV, nuclease * * Photocrosslinking)

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. To make a lie into a truth- “Say it loud, say it often” G. Gordon Liddy All are largely accepted based upon “wart phenomenon”

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

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

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

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

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) Fig Evidence for ternary complex involving HDAC2. Sin3A and Mad1 9E, slide 46 Mad Sin3A HDAC Flag Ac Epitope tagging experiment results

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

Assay- 1. separate nuclear protein on SDS-PAGE impregnated with histones 2. incubate gel with tritium -labeled AcCoA, wash away Fig Activity gel assay for HAT activity H4 Ac H3 H2BH2A * * * * How do we determine whether a protein is a histone acetyltransferase (HAT)? All nuclear proteins

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

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

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

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

Figure B, slide 18

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