carlo v. bruschi senior scientist & group leader icgeb microbiology group area science park...
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Carlo V. Bruschi
Senior Scientist & Group Leader
ICGEB Microbiology Group
AREA Science Park
Trieste, ITALY
bruschi@icgeb.org
The yeast Sacchromyces cerevisiae as a model microorganism for molecular radiopharmacology research
SISSA - ISAS
Miramare, Trieste - 23/11/04
Respiration (O2 + dispensable mitochondria, ) and Fermentation (anaerobic) convert sugars (hexoses): sucrose>fructose>glucose>maltose CO2 + ethanol
MITOSIS: presence of fermentable C-sources + N2
MEIOSIS: presence of non-fermentable C-sources - N2
Chromosomes of Sacccharomyces cerevisiae strains
280 Kb
1.5 Mb
12.25 Mb
6,200 ORFs
750 ARS
16 CENs
32 TELs
3.3 Gb30 - 40 K ORF
46 CENs
Approximately 50% of yeast (~3,000) genes are estimated to have a structural and/or functional homologue in human
With the systematic genome analysis of the human DNA, new genes are constantly found, which have homologues in yeast
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 ofthe yeast and human sequence. For more details, see Bassett, D.E. Jr., et al., (XREFdb) ------------------------------------------------------------------------Steve A. Chervitz and the SGD teamhttp://genome-www.stanford.edu/Saccharomyces/mammal/
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”CELL-BASED DRUG DISCOVERY”
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 compounds at very high
speed and to identify the cellular target of their biological
action with the technology of the
THE YEASTTHE YEAST Saccharomyces cerevisiae Saccharomyces cerevisiae
� THIS TECHNOLOGY IS BASED UPON THE UTILIZATION OF SIMPLE MODEL CELL MICROORGANISMS LIKE
““CELL-BASED DRUG DISCOVERY”CELL-BASED DRUG DISCOVERY”
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.
CELL-BASED DRUG DISCOVERYCELL-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
Pharmacological Target
Plants
Those cells that are affected at the physiological level may grow more or less and thereforethey can be identified by titrating their DNA in chip microarrays. The deleted gene lackingin those cells is identified through the “barcode” and the corresponding protein that is encoded by it can be considered as a potential pharmacological target.
Mutant cell populationExposure of the yeastcell population to the compounds
10B-Phe
SELECTION
Further modifications:
Combination with NLS (Nuclear Localization Factor) peptide
N
NLS-10B-Phe
B
Kan MX4 NLS-tetR-GFP
A
Sal I
Nde I
Eco RV
Spe I
Kpn I
Xho I
Nde I
Pvu I
Bam HI
Sal I
PvuI
Tc
URA3
CEN4
ARS1
ORI
AMP
FRT
KanMX4
pHKHE
9722.00 Kb
SnaB I, Sac II, Not I, Eco RI
9722 bp
FRT
Primer tail 40-45 bp homologous to the target gene
Primer head 20 bp homologous to the plasmid
FRTFRT
Waghmare S. et al. Biotechniques (2003)
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