Invited Lectures
A1-L5
Mitotic chromosome condensation and segration
C. D’Ambrosio
1
, Y. Katou
2
, K. Shirahige
2
,F. Uhlmann
1
1
Chromosome Segregation Laboratory, London, UNITED KINGDOM,
2
Division of Gene Research, Tokyo Institute of Technology, Yoko-
hama, JAPAN.
Mitotic chromosome structure depends on the chromosomal condensin complex. Without condensin, metaphase chromosomes remain
undercondensed and lack structural stability. We have asked where along budding yeast chromosomes the condensin complex associates,
and what we can learn from its binding pattern about the mechanism of chromosome condensation. Our results suggest that condensin,
like its relative the cohesin complex, is loaded onto chromosomes by a loading factor, the Scc2/4 complex. Unlike cohesin, that moves
away from its loading sites after the loading reaction, condensin remains at the loading sites. We discuss the implications of these pat-
terns for interphase and mitotic chromosome structure.
Furthermore, condensin is required during anaphase to promote sister chromatid resolution. In the absence of condensin, anaphase brid-
ges and segregation defects are observed. How condensin promotes sister chromatid resolution is unknown. We have used the budding
yeast rDNA as a model locus, whose segregation depends on condensin during anaphase. We show that anaphase bridges in a condensin
mutant are resolved by ectopic expression of a foreign (Chlorella virus) but not endogenous yeast topoisomerase II (topo II). This sug-
gests that catenation prevents sister rDNA segregation, and that yeast topo II is ineffective in decatenating the rDNA, and maybe other
chromosomal regions, in the absence of condensin.
B5-L1
The Meningococcal Transformation Machine
T. Tønjum
Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, NORWAY
Neisseria meningitidis is a leading cause of bacterial meningitis and septicaemia worldwide. This bacterium is constitutively competent
for transformation throughout its entire life-cycle. Transformation in neisserial species is particularly important for genetic exchange and
diversity and is coupled to the expression of type IV pili. Meningococcal type IV pili are present on the cell surface as bundled filamen-
tous appendages and are assembled, extruded and retracted by pilus biogenesis components. These proteins are homologous to type II
secretion components in Gram-negative species.
The binding and uptake of transforming DNA into the meningococcal cell can be divided into four stages: entry through an outer mem-
brane pore, transit of the periplasm, transport across the inner membrane and genome integration. We propose that the early stage of
meningococcal transformation is coupled to pilus retraction and that transforming DNA is non-specifically attached to retracting pili.
Previously, we have shown that pili directly interact with the secretin PilQ in the outer membrane. We have evidence that DNA is intro-
duced into the cell through the positively charged pore formed by the PilQ complex and that other DNA binding components process
DNA inside the periplasm and inner membrane.
To address the multi-step nature of DNA binding and uptake during transformation, we have identified DNA binding components in
meningococcal cellular fractions. By using a combination of molecular and imaging approaches, DNA binding candidates are being
assessed for their structure-function relationships to define how they act and interact, with each other and with DNA. The goal is to
define how these DNA binding components provide dynamic multi-site targeting and entry of DNA.
Invited Lectures Late Abstracts
341
E1-L2
Global Mapping of the Yeast Genetic Interaction Network
C. Boone,III
University of Toronto, Toronto, ON, CANADA.
Synthetic Genetic Array (SGA) analysis automates yeast genetics, enabling a number of different large-scale/systematic studies. In partic-
ular, we are attempting to generate the complete synthetic lethal genetic interaction map for yeast cells. This map can be used to define
complexes and pathways in the cell, but perhaps more importantly, it adds functional information to the protein-protein interaction
map, identifying complexes and pathways that buffer one another and somehow work together as backup systems. One of our major
challenges has been to generate a quantitative model for scoring genetic interactions based upon plate images and the predicted fitness
of the double mutant relative to each single mutant. Another challenge has been to develop robotic platforms for our own high-through-
put analysis and individual users in other labs. I will present the results of our latest network compilation and describe our plans for
tackling the yeast genome. In addition, I will describe how the yeast genetic interaction network can be used to interpret chemical-genetic
interactions and link bioactive compounds to their target pathways.
E2-L2
Multi-factorial disease and robustness: Where Systems Biology makes a difference
H. V. Westerhoff
Manchester Center for Integrative Systems Biology and Netherlands Institute for Systems Biology, Manchester, UNITED KINGDOM.
As we now know what Systems Biology is, it may be worthwhile to examine the difference it will make. In particular it may be useful to
examine where such a difference is needed and how it may be brought about by those who are interested.
There are a number of diseases that can be related to a single faulty gene product and or a single invading microorganism. Quite a few
of these ‘monofactorial’ diseases can be cured, or structural molecular biology has defined a strategy to identify or design the corres-
ponding drugs. However, there is a more substantial number of diseases for which a vast amount of biomedical research has been moun-
ted and that cannot be cured. The research has led to substantial scientific success and biochemical understanding, but to little if any
progress in defining cures. The examples are on the long list of ‘multifactorial diseases’ and include type-2 diabetes, obesity, heart dis-
ease, cancer and arthritis.
The approach that has been successful for monofactorial diseases is not optimal vis-a
`-vis these diseases. Finding cures for these diseases
may require a shift in strategy. These diseases are ‘network’ or ‘Systems Biology diseases’ and to manage them one needs to address
faults in the network rather than in just a molecule.
I shall discuss some of the ways the Manchester-Amsterdam Systems Biology axis tries to devise such network-based strategies. These
include differential network based drug design against Trypanosoma brucei, modular kinetics of a connection between type-2 diabetes
and obesity, and new paradigms for searching new anti-tumor drugs. Fragility and robustness are Leitmotive, but differentially so.
Late Abstracts Invited Lectures
342
E5-L5
Insight into alternative-splicing mechanisms with the solution structures of several RRM-RNA
complexes
F. Allain
Institute of Molecular Biology and Biophysics, Zu
¨rich, SWITZERLAND.
Alternative-splicing is probably the most effective post-transcriptional gene regulatory event, as more than 60% of the human genes are
alternatively spliced. Defects in alternative-splicing are the cause of many genetic diseases. We recently determined the structure of sev-
eral important human alternative-splicing factors in complex with RNA, namely the Poly-pyrimidine Tract Binding Protein (PTB1),
Fox-12, SRp203 and of a potential splicing factor RBMY4. The structures of PTB and Fox-1 showed that these alternative-splicing fac-
tors bind RNA sequence-specifically and might control the fate of an alternative-exon in remodeling the RNA. The structures of SRp20
and of RBMY in complex with RNA revealed unusual RNA recognition modes. SRp20 binds RNA in a semi-sequence specific manner
3
and the human RBMY binds a stem-loop RNA whereas its mouse homolog doesn’t
4
. Implications of these structural findings for under-
standing the mechanism of action of these alternative-splicing factors will be discussed.
References:
1. Oberstrass, F.C. et al. Structure of PTB bound to RNA: specific binding and implications for splicing regulation. Science 309, 2054–7
(2005).
2. Auweter, S.D. et al. Molecular basis of RNA recognition by the human alternative splicing factor Fox-1. Embo J 25, 163–73 (2006).
3. Hargous, Y. et al. Molecular basis of RNA recognition and TAP binding by the SR proteins SRp20 and 9G8. Embo J 25, 5126–37
(2006).
4. Skrisovska, L. et al. The testis-specific human protein RBMY recognizes RNA through a novel mode of interaction. EMBO Rep 8,
372–79 (2007).
Invited Lectures Late Abstracts
343
Posters
A1-13
Characterization of a subtelomeric satellite DNA
in the mollusc Donax trunculus
V. Petrovic
1
,C.Pe
´rez-Garcı
´a
2
, J. J. Pasantes
2
, E. Prats
3
, M. Plohl
1
1
Rudjer Boskovic Institute, Zagreb, CROATIA,
2
University of Vigo,
Vigo, SPAIN,
3
Institut de Biologia Molecular de Barcelona – CSIC,
Barcelona, SPAIN.
Satellite DNAs are non-coding, tandemly repeated DNA sequences
that comprise long arrays in the genome and are usually located
within heterochromatic regions of chromosomes. Among marine
invertebrates, satellite DNAs have so far been studied in detail
only in a couple of taxonomic groups. We detected a novel satellite
DNA present in the genome of the bivalve mollusc Donax trunculus.
The monomer repeat length of this satellite is 169 bp, while the
sequence analysis reveals high sequence conservation maintained
throughout the entire monomer length. In contrast to the other
satellites detected previously in the genome of D. trunculus
(Petrovic and Plohl, Gene 2005, 362:37, and references therein),
this is a GC rich satellite that has also been shown to exhibit CpG
site methylation. In addition, this is the most abundant (5 %)
among all detected satellites. Fluorescence in situ hybridization
revealed that this satellite is located in subtelomeric regions on
more than half of D. trunculus chromosome pairs. Its location
on a subset of chromosomes and homogeneity of randomly cloned
variants indicate different homogenization mechanisms acting
between chromosomes, and/or different evolutionary history of
chromosomes in the D. trunculus genome.
A1-14
The evolutionary role of the LCR16A element in
Mendelian disorders
O. Symmons, H. de Boussac, A. Va
´radi, T. Aranyi
Institute of Enzymology, Hungarian Academy of Sciences,
Budapest, HUNGARY.
With the completion of the human genome, a major challenge is to
determine correlation between genome evolution and phenotypic
changes. The best studied human phenotypes are Mendelian disor-
ders. Two such examples are Polycystic Kidney Disease (PKD)
and Pseudoxanthoma Elasticum (PXE) caused by PKD1 and
ABCC6, respectively. Both genes were mapped to the short arm of
Chr 16. However, their study is complicated by the presence of
multiple pseudogenes of high degree of similarity and also located
on Chr 16. Our aim was to characterize these pseudogenes and
understand the mechanism and potential impact of their duplica-
tion. We found that every pseudogene is situated in close proximity
to an LCR16a element, which is a very proliferative duplicon on
Chr 16. LCR16a was proposed to be the driving force for the for-
mation of intrachromosomal duplication blocks. We analyzed the
phylogenetic relationship of the human LCR16a elements, which
form three distinct clusters. The duplicated copies of the ABCC6
and PKD1 genes are all associated with the same cluster. We dem-
onstrated that some of the duplication events were hominin-specific
and showed that this can lead to hominin-specific chimeric tran-
scripts. We have also investigated the possible consequences of the
presence of pseudogene sequences in the aetiology of PXE and
PKD. Our results demonstrate that non-allelic homologous recom-
bination has occurred and is manifested in these regions and gene
conversion is still an on-going process.
A2-27
The BAH domain of Sir3 is the primary
nucleosome binding domain in the SIR
silencing complex
M. Onishi
1
, G. Liou
1,2
, D. Moazed
1
1
Harvard Medical School, Boston, MA,
2
Division of Molecular and
Genomic Medicine, National Health Research Institutes, Miao-Li
County, TAIWAN.
In Saccharomyces cerevisiae, Silent information regulator (Sir) pro-
teins are required for regional gene silencing at the silent mating
type cassettes and telomeres. The initiation of transcriptional silen-
cing at these domains is proposed to involve the recruitment of the
Sir complex, composed of Sir2, Sir3, and Sir4, by DNA-binding
proteins. This recruitment is followed by iterative cycles of NAD-
dependent deacetylation, production of O-acetyl-ADP-ribose, and
binding of Sir3 and Sir4 to the deacetylated nucleosomes, and
leads to the creation of extended silent chromatin domains. These
silenced regions have histones that are hypoacetylated and hypo-
methylated, while disruption of enzymes that acetylate and methy-
late histones leads to mislocalization of Sir3. Thus, histone
modifications play a crucial role in the assembly of silent chroma-
tin. However, while Sir3 has been shown to have histone and nu-
cleosome binding properties in vitro, specific binding of Sir3 to
nucleosomes, as it occurs in vivo, has yet to be observed. We show
that the Bromo-adjacent-homology (BAH) domain of Sir3 is neces-
sary for nucleosome binding and that this binding is regulated by
histone acetylation and methylation. These results suggest that the
BAH domain, found in many eukaryotic chromatin proteins, binds
to specifically modified nucleosomes.
A2-28
Post synthetic acetylation of HMGB1 protein
stimulates DNA end-joining
I. Ugrinova
1
, E. Mitkova
1
, C. Moskalenko
2
, I. Pashev
1
,
E. A. Pasheva
1
1
Institut Molecular biology,Bulgarian.Academy of Sciences, Sofia,
BULGARIA,
2
Laboratoire Joliot-Curie, Ecole Normale Superieure,
Lyon, FRANCE.
The ability of HMGB1 protein to recognize bent DNA and to
induce bending in linear duplex DNA defines HMGB1 as an archi-
tectural factor. We already demonstrated that the binding affinity
of the protein to various bent DNA structures is enhanced upon in
vivo acetylation at Lys 2. Here we investigate how this modifica-
tion of HMGB1 affects its ability to bend DNA. We report that
the modified protein cannot bend short DNA fragments but,
instead, it stimulates joining of the same fragments via their ends.
The same properties demonstrates in vivo acetylated HMGB1 lack-
ing its acidic tail. Further in vitro acetylation with acetyltransferase
CBP of the truncated protein at Lys81 (possible upon tail removal
only), restores the protein’s bending ability, while the stimulation
of DNA end-joining is strongly reduced. We conclude, therefore,
that the ability of HMGB1 to bend DNA or to stimulate end-join-
ing is modulated in vitro by acetylation. In an attempt to explain
the properties of in vivo acetylated HMGB1, its complexes with
DNA have been analysed by both protein-DNA cross linking and
AFM (atomic force microscopy). Unlike the parental protein,
bound mainly within the internal sequences, the acetylated
HMGB1 binds preferentially to DNA ends. We propose that the
loading of acetylated protein on DNA ends accounts for both the
failure to bend DNA and the stimulation of DNA end-joining.
Late Abstracts Posters
344
A3-11
Analysis of restriction fragments distribution in
genomic fungal DNAs.
V. N. Tomilov
1
, D. A. Gonchar
1
, M. A. Abdurashitov
1
,
A. Schueffler
2
, H. Anke
2
, S. K. Degtyarev
1
1
SibEnzyme Ltd., Novosibirsk, RUSSIAN FEDERATION,
2
IBWF,
Kaiserslautern, GERMANY.
Theoretical diagrams of fungal chromosomal DNA (Magnaporthe
grisea,Mycosphaerella graminicola and Schizosaccharomyces po-
mbe) cleavage at more than 125 different 4 - 8 nucleotides
sequences have been simulated based on recently determined DNA
primary structures. All chosen DNA sequences are the recognition
sites of restriction enzymes. In all diagrams obtained we have selec-
ted the fragments presented in peak’s quantities. Each peak has
been characterized by multiplication of the number of fragments
by their length. In the analysis we have considered peaks with a
value of 0.15% or higher compared to the length of the corres-
ponding chromosomes. Calculated Schizosaccharomyces pombe
DNA digestions did not contain such peaks. Theoretical cleavage
of Magnaporthe grisea and Mycosphaerella graminicola DNAs at
the appropriate recognition sites of restriction enzymes resulted in
more than 10 such peaks. A more detailed study has shown that
the presence of peaks in DNA digestions depends on genome com-
plexity. Hydrolysis of the fungal DNA with several restriction
enzymes has confirmed the presence of the predicted fragments.
A3-12
Nitric oxide levels and eNOS gene
polymorphism in patients with coronary artery
disease documented by angiography
G. Yilmaz
1
,I. Mehmetoglu
1
, S. Kurban
1
, H. Aacar
2
1
Meram Faculty of Medicine, Department of Biochemistry, Univer-
sity of Selcuk, Konya, TURKEY,
2
Meram Faculty of Medicine,
Department of Medical Genetics, University of Selcuk, Konya,
TURKEY.
The aim of this study was to investigate 4a/4b VNTR polymorph-
ism in intron 4, the G10-T polymorphism in intron 23 and plasma
NO levels in patients with coronary artery disease (CAD) docu-
mented by angiorgaphy and healty controls.
The study was performed on 106 patients (30 F, 76 M) aged 40–70
years and 89 healty controls (40 males, 49 females) aged 41–73
years. All patients had more than 50% stenosis in at least one cor-
onary artery documented by angiography.
Blood samples were obtained from all subjects after fasting. NO
was measured as NOx by Griess reaction on EDTA samples and
eNOS gene polymorphism was investigated by PCR-RFLP tech-
nique.
NO levels of the patients and the controls were 47,78 ± 27 lmol/
L and 41,44 ± 25,9 lmol/L respectively the difference of which
was significant.
There was no significant difference between allelic frequencies of
eNOS gene intron 4 a/b VNTR and intron 23 polymorphisms of
the groups.
Therefore, our results show that eNOS gene intron 4 a/b VNTR
and intron 23 polymorphisms are not independent risk faktors for
CAD in a central area (Konya) of TURKEY.
A4-11
The role of miRNAs in DNA replication and
damage repair
E. Zlotorynski, J. van Duijse, R. Agami
The Netherlands Cancer Institute, Amsterdam, THE NETHER-
LANDS.
The miRNA genes encode short RNAs, which suppress the expres-
sion of mRNAs bearing complementary target sequences. Each mi-
RNA is predicted to target up to hundreds of genes, but the
function of only a handful miRNAs has been determined, under-
ling the need for a systematic screening approach to identify their
roles in development and disease. Primary human cells grown with
low levels of DNA replication inhibitors undergo irreversible
growth arrest due to the accumulation of DNA damage. In order
to identify miRNAs that can modulate DNA replication and DNA
damage repair, we performed a screen for miRNAs that can affect
replication inhibition-induced growth arrest. Human primary cells
were transduced with a human miRNA expression library, and
grown for 3 weeks with low levels of the replication inhibitors
Aphidicolin, Hydroxyurea, Etoposide or Doxorubicin. The relative
abundance of miRNA expressing cells was then measured by
hybridization of the DNA of the different cell populations on a
miRNA micro-array. We subsequently validated the screen results
for each miRNA individually. Several miRNAs were found to
override the replication inhibition-induced arrest, whereas others
were found to enhance the effect of replication inhibition. We are
currently studying the mechanism of action of these miRNAs, in
order to understand their role in DNA replication and DNA dam-
age repair.
A4-12
Identification of novel regulators of microRNA
function
M. Kedde, R. Agami
Netherlands Cancer Institute, Amsterdam,
THE NETHERLANDS.
The 3¢-untranslated regions (UTRs) of many mRNAs are subject
to posttrancriptional regulation by microRNAs (miRNAs), which
function as repressors of their target mRNAs. Little is known
about the regulation of miRNA targeting and the reversibility of
the process. Here I will present evidence that certain RNA binding
proteins can repress miRNA activity both in vitro and in vivo.
Posters Late Abstracts
345
