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

2. CHMI 4226 - 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.

3. CHMI 4226 - 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).

4. CHMI 4226 - W20094 Genomic library

5. CHMI 4226 - W20095 Genomic DNA MM MboII 1 353 1 078 872 603 310 Cells (e.g. fibroblasts) Proteinase K + EDTA + SDS DNAse-free RNAse A Extraction 1. phenol 2. chloroform Ethanol precipitation

6. CHMI 4226 - W20096 Genomic library – cosmid vectors

7. CHMI 4226 - W20097 Walking on the chromosome

8. CHMI 4226 - W20098 Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem. 1994. 269[2]: 1001-1009

9. CHMI 4226 - W20099 mRNA intron ORF

10. CHMI 4226 - W200910 Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem. 1994. 269[2]: 1001-1009

11. CHMI 4226 - W200911 Example of gene isolation: Identifying intron-exon boundaries S1 nuclease mapping

12. CHMI 4226 - W200912 Using S1 nuclease mapping to locate the transcription start site

13. CHMI 4226 - W200913 Mapping the transcription start site using “primer extension”

14. CHMI 4226 - W200914 Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem. 1994. 269[2]: 1001-1009

15. CHMI 4226 - W200915 mRNA intron ORF Characterization of a promoter

16. CHMI 4226 - W200916 Promoter bashing

17. CHMI 4226 - W200917 Linker Scanning Mutagenesis

18. CHMI 4226 - W200918 Characterization of a promoter

19. CHMI 4226 - W200919 Characterization of a promoter using reporter genes From: BD-Bioscience Clone promoter fragments here! Reporter gene

20. CHMI 4226 - W200920 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)

21. CHMI 4226 - W200921 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 10 4 -10 5 (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 10 4 -10 5 (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

22. CHMI 4226 - W200922 Characterization of a promoter using reporter genes http://www.promega.com/pnotes/70/7618_25/7618_25.html

23. CHMI 4226 - W200923 Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem. 1994. 269[2]: 1001-1009

24. CHMI 4226 - W200924 Characterization of a promoter using reporter genes

25. CHMI 4226 - W200925 Characterization of a promoter using reporter genes

26. CHMI 4226 - W200926 Example of gene isolation: Mouse Heme oxygenase J. Biol. Chem. 1994. 269[2]: 1001-1009