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)