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Carlo V. Bruschi
Senior Scientist & Group Leader
ICGEB Microbiology Group
AREA Science Park
Trieste, ITALY
[email protected]
The yeast
Sacchromyces
cerevisiae as a model
microorganism for
molecular
radiopharmacology
research
SISSA - ISAS
Miramare, Trieste - 23/11/04
Respiration (O2 + dispensable mitochondria, r+ , r-, r0) 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
1.5 Mb
280 Kb
12.25 Mb
6,200 ORFs
750 ARS
16 CENs
32 TELs
3.3 Gb
30 - 40 K ORF
46 CENs
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
Functional Homologs: Sample Alignments (BLASTN)
Yeast Gene
Human Gene
Percent
P-value * Identity
ACT1 1.4e-243
89
CDC28 5.0e-130
60
CMD1 1.3e-55
60
CDC8 5.6e-48
44
RPB8 4.5e-21
37
ADE8 1.0e-13
36
Percent
Similarity
94
78
77
61
65
56
Cytoskeletal gamma actin
Cell cycle control (CDC2)
Calmodulin
Thymidylate kinase
RNA polymerase II subunit (RPB17)
Purine biosynthesis (PGFT)
Disease Genes: Sample Alignments (BLASTN)
Yeast Gene
Human Gene
P-value *
MSH2
YCF1
GEF1
TEL1
YNL161W
SOD1
SGS1
IRA2
3.8e-255
2.4e-157
3.4e-95
8.8e-84
8.5e-82
8.9e-56
3.1e-50
1.0e-28
Percent Percent
Identity Similarity
43
31
33
49
41
55
24
21
65
57
58
36
65
69
34
45
Mutator gene (MSH2, colon cancer)
Cystic fibrosis conductance regulator (CFTR)
Voltage-gated chloride ion channel
Ataxia telangiectasia gene
Myotonic dystrophy associated protein kinase
Superoxide dismutase (SOD-1)
Werner's Syndrome gene
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/
SEARCH FOR NEW BIOLOGICALLY ACTIVE COMPOUNDS
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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
THE YEAST Saccharomyces cerevisiae
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 “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 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.
10
Plants
B-Phe
Exposure of the yeast
cell population to the
compounds
Mutant cell population
SELECTION
Pharmacological
Target
Further modifications:
Combination with NLS (Nuclear Localization Factor) peptide
N
NLS-10B-Phe
A
B
SnaB I, Sac II, Not I, Eco RI
FRT
Kan MX4
FRT
NLS-tetR-GFP
Sal I
PvuI
FRT
Sal I
Tc
Eco RV
KanMX4
Bam HI
FRT
Pvu I
Nde I
AMP
URA3
9722 Kb
bp
9722.00
Primer tail
40-45 bp homologous to the target gene
pHKHE
ORI
CEN4
ARS1
Spe I
Nde I
Xho I
Primer head
20 bp homologous to the plasmid
Kpn I
Waghmare S. et al. Biotechniques (2003)