CHMI 4226 - W20091 Recombinant DNA Technology CHMI 4226 Week of March 16, 2009 Isolating and characterizing genes.

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CHMI W20091 Recombinant DNA Technology CHMI 4226 Week of March 16, 2009 Isolating and characterizing genes

CHMI W20092 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 W20093 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 W20094 Genomic library

CHMI W20095 Genomic DNA MM MboII Cells (e.g. fibroblasts) Proteinase K + EDTA + SDS DNAse-free RNAse A Extraction 1. phenol 2. chloroform Ethanol precipitation

CHMI W20096 Genomic library – cosmid vectors

CHMI W20097 Walking on the chromosome

CHMI W20098 Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem [2]:

CHMI W20099 mRNA intron ORF

CHMI W Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem [2]:

CHMI W Example of gene isolation: Identifying intron-exon boundaries S1 nuclease mapping

CHMI W Using S1 nuclease mapping to locate the transcription start site

CHMI W Mapping the transcription start site using “primer extension”

CHMI W Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem [2]:

CHMI W mRNA intron ORF Characterization of a promoter

CHMI W Promoter bashing

CHMI W Linker Scanning Mutagenesis

CHMI W Characterization of a promoter

CHMI W Characterization of a promoter using reporter genes From: BD-Bioscience Clone promoter fragments here! Reporter gene

CHMI W 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 W Reporter gene In vitro assayIn vivo assay StrengthWeaknessesLimit 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 10 7 Luciferase 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 10 5  -gal 1.Colorimetric 2. Fluorescence 3. Chemilumin. 1. Histochemistry 2. Biolumin. -Non radioactive -Large dynamic range (5-6 OM) - High endogenous activity - Expensive instrumentation (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 (chemilumin.) GFP 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 W Characterization of a promoter using reporter genes

CHMI W Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem [2]:

CHMI W Characterization of a promoter using reporter genes

CHMI W Characterization of a promoter using reporter genes

CHMI W Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem [2]: