Recombinant DNA Technology CHMI 4226 Week of March 16, 2009 Isolating and characterizing genes CHMI 4226 - W2009 1 Genes structure • Promoter: – DNA sequence located upstream of the gene; – Bind transcription factors and RNA polymerase; – indicates where transcription should begin (TSS: transcription start site). • Intron splicing sequences: – Introns always (well, almost…) begin with GT and end with AG. CHMI 4226 - W2009 2 Why clone genes • Identifying and characterizing promoter sequences which participate in regulating transcription; • Identification and characterization of the introns and exons (size, number, involvement in regulation of gene expression). CHMI 4226 - W2009 3 Genomic library CHMI 4226 - W2009 4 Genomic DNA MboII M Cells (e.g. fibroblasts) Proteinase K + EDTA + SDS M 1 353 1 078 872 DNAse-free RNAse A 603 310 Extraction 1. phenol 2. chloroform Ethanol precipitation CHMI 4226 - W2009 5 Genomic library – cosmid vectors CHMI 4226 - W2009 6 Walking on the chromosome CHMI 4226 - W2009 7 Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem. 1994. 269[2]: 1001-1009 CHMI 4226 - W2009 8 mRNA intron CHMI 4226 - W2009 ORF 9 Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem. 1994. 269[2]: 1001-1009 CHMI 4226 - W2009 10 Example of gene isolation: Identifying intron-exon boundaries S1 nuclease mapping CHMI 4226 - W2009 11 Using S1 nuclease mapping to locate the transcription start site CHMI 4226 - W2009 12 Mapping the transcription start site using “primer extension” CHMI 4226 - W2009 13 Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem. 1994. 269[2]: 1001-1009 CHMI 4226 - W2009 14 Characterization of a promoter mRNA CHMI 4226 - W2009 intron ORF 15 Promoter bashing CHMI 4226 - W2009 16 Linker Scanning Mutagenesis CHMI 4226 - W2009 17 Characterization of a promoter CHMI 4226 - W2009 18 Characterization of a promoter using reporter genes Clone promoter fragments here! Reporter gene From: BD-Bioscience CHMI 4226 - W2009 19 Characterization of a promoter using reporter genes • Chemiluminescence: – Generation of light through an enzymatic reaction– « artificial system» – E.g. B-galactosidase, horseradish peroxidase • Bioluminescence: – Generation of light through an enzymatic reaction– « natural system» – E.g. luciferase • Autofluorescence: – E.g. Green fluorescent protein • Fluorescence • Radioactivity • Generation of a chromophore (colored compound) CHMI 4226 - W2009 20 Reporter gene In vitro assay In vivo assay Strength Weaknesses Limit of detection (molecules) CAT 1.Chromatography. 2.Fluorescence 3. ELISA None -Stable - minimal endogenous activity -Labor intensive -Radioactivity -Low sensitivity -Low Dynamic range (2 OM) - Short half life of mRNA 5-10 x 107 Luciferase Bioluminescence Bioluminescence -Non radioactive -Large dynamic range (4 orders of magnitude) - minimal endogenous activity - Short half life of protein - Expensive instrumentation - Low reproducibility 1-2,5 x 105 b-gal 1.Colorimetric 2. Fluorescence 3. Chemilumin. 1. Histochemistry 2. Biolumin. -Non radioactive -Large dynamic range (5-6 OM) - High endogenous activity - Expensive instrumentation 104 -105 (chemilumin.) SEAP 1. Colorimetric 2. Bioluminescence. 3. Chemiluminescence None -Non radioactive -Large dynamic range (4 OM) - High endogenous activity - dependent on the secretory activity of the cell line used 104 -105 (chemilumin.) GFP Fluorescence Fluorescence -Works in living cells - non-toxic -No photobleaching -Signal too faint for some applications n/a Source: Current Protocols in Molecular Biology. Table 9.6.1 CHMI 4226 - W2009 21 Characterization of a promoter using reporter genes http://www.promega.com/pnotes/70/7618_25/7618_25.html CHMI 4226 - W2009 22 Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem. 1994. 269[2]: 1001-1009 CHMI 4226 - W2009 23 Characterization of a promoter using reporter genes CHMI 4226 - W2009 24 Characterization of a promoter using reporter genes CHMI 4226 - W2009 25 Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem. 1994. 269[2]: 1001-1009 CHMI 4226 - W2009 26