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Carlo V. Bruschi Senior Scientist & Group Leader

Carlo V. Bruschi Senior Scientist & Group Leader ICGEB Microbiology Group AREA Science Park Trieste, ITALY bruschi@icgeb.org.

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Carlo V. Bruschi Senior Scientist & Group Leader

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  1. Carlo V. Bruschi Senior Scientist & Group Leader ICGEB Microbiology Group AREA Science Park Trieste, ITALY bruschi@icgeb.org

  2. The yeast Sacchromyces cerevisiae as a model microorganism for molecular radiopharmacology research SISSA - ISAS Miramare, Trieste - 23/11/04

  3. Respiration (O2 + dispensable mitochondria, r+ , r-, r0) and Fermentation (anaerobic) convert sugars (hexoses): sucrose>fructose>glucose>maltose =CO2 + ethanol

  4. MITOSIS: presence of fermentable C-sources + N2 MEIOSIS: presence of non-fermentable C-sources -N2

  5. Chromosomes of Sacccharomyces cerevisiae strains 1.5 Mb 280 Kb

  6. 12.25 Mb 6,200 ORFs 750 ARS 16 CENs 32 TELs 3.3 Gb 30 - 40 K ORF 46 CENs

  7. With the systematic genome analysis of the human DNA, new genes are constantly found, which have homologues in yeast Approximately 50% of yeast (~3,000) genes are estimated to have a structural and/or functional homologue in human

  8. Functional Homologs: Sample Alignments(BLASTN) Yeast Gene Human Gene Percent Percent P-value * Identity Similarity ACT1 1.4e-243 89 94 Cytoskeletal gamma actin CDC28 5.0e-130 60 78 Cell cycle control (CDC2) CMD1 1.3e-55 60 77 Calmodulin CDC8 5.6e-48 44 61 Thymidylate kinase RPB8 4.5e-21 37 65 RNA polymerase II subunit (RPB17) ADE8 1.0e-13 36 56 Purine biosynthesis (PGFT) Disease Genes: Sample Alignments(BLASTN) Yeast Gene Human Gene Percent Percent P-value * Identity Similarity MSH2 3.8e-255 43 65 Mutator gene (MSH2, colon cancer) YCF1 2.4e-157 31 57 Cystic fibrosis conductance regulator (CFTR) GEF1 3.4e-95 33 58 Voltage-gated chloride ion channel TEL1 8.8e-84 49 36 Ataxia telangiectasia gene YNL161W 8.5e-82 41 65 Myotonic dystrophy associated protein kinase SOD1 8.9e-56 55 69 Superoxide dismutase (SOD-1) SGS1 3.1e-50 24 34 Werner's Syndrome gene IRA2 1.0e-28 21 45 Neurofibromin (NF1) * The P-value links to a file containing a Needleman and Wunsch alignment of the yeast and human sequence. For more details, see Bassett, D.E. Jr., et al., (XREFdb) ------------------------------------------------------------------------ Steve A. Chervitz and the SGD team http://genome-www.stanford.edu/Saccharomyces/mammal/

  9. THE YEAST Saccharomyces cerevisiae SEARCH FOR NEW BIOLOGICALLY ACTIVE COMPOUNDS � THE SEARCH FOR NEW, BIOLOGICALLY ACTIVE COMPUNDS IS CARRIED OUT BY UTILIZING AN ADVANCED TECHNOLOGY OF MODERN PHARMACOGENOMICS, THE SO-CALLED “CELL-BASED DRUG DISCOVERY” �THIS TECHNOLOGY IS BASED UPON THE UTILIZATION OF SIMPLE MODEL CELL MICROORGANISMS LIKE Yeast has been the first eukaryotic expression system in which several recombinant proteins have been expressed: � insulin (Humulin) � hepatitis B s.a. (Recombinovax HB) � Today, yeast cells represent the workhorse of modern biotechnology. With them it is possible to screen hundreds of chemical compoundsat very high speed and to identify the cellular target of their biological action with the technology of the “CELL-BASED DRUG DISCOVERY”

  10. e (A) Samples from the indicated time points were assayed by Northern analysis. Genes were chosen to be representative of the four previously identified temporal classes. DMC1, SPS1, DIT1, and SPS100 belong to the early, middle, mid-late, and late classes, respectively. (B) Data from the microarray analysis of RNA samples from the same time course are graphically displayed using color to represent the quantitative changes. Increases in mRNA (relative to pre-sporulation levels) are shown as shades of red and decreases in mRNA levels are represented by shades of green.

  11. Exposure of the yeast cell population to the compounds Mutant cell population 10B-Phe SELECTION m1 Plants m2 m2 m2 m2 m3 m6000 CELL-BASED DRUG DISCOVERY A population of yeast cells marked with a system similar to the barcode for each of its 6,000 deleted genes “m” is exposed to the compounds to be screened Those cells that are affected at the physiological level may grow more or less and therefore they can be identified by titrating their DNA in chip microarrays. The deleted gene lacking in those cells is identified through the “barcode” and the corresponding protein that is encoded by it can be considered as a potential pharmacological target. Pharmacological Target

  12. Further modifications: Combination with NLS (Nuclear Localization Factor) peptide N NLS-10B-Phe

  13. A FRT FRT SnaB I, Sac II, Not I, Eco RI Sal I PvuI FRT Tc Nde I Sal I KanMX4 Eco RV Bam HI FRT URA3 9722 bp 9722.00 Kb AMP Pvu I pHKHE ORI CEN4 ARS1 Spe I Nde I Kpn I Xho I B NLS-tetR-GFP Kan MX4 Primer tail 40-45 bp homologous to the target gene Primer head 20 bp homologous to the plasmid Waghmare S. et al. Biotechniques (2003)

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