39th Meeting of the

Polish Biochemical Society

Gdañsk 16–20 September 2003

SESSION 5

Structure and function of DNA

Organized by G. Wêgrzyn, R. Paw³owski

154 Session 5. Structure and function of DNA 2003

271 Lecture
Unusual thermal stability of RNA/[All-RP-PS]-DNA/RNA triplexes containing a
homopurine DNA strand

Ma³gorzata Boczkowska, Piotr Guga, Magdalena Janicka, Anna Maciaszek, Wojciech Stec
Department of Bioorganic Chemistry, Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies,
ul. Sienkiewicza 112, £ódŸ

Triple-helical forms of nucleic acids have been known
since 19571. In triplexes, the third strand binds either
in a parallel or an antiparallel orientation with respect
to the purine strand in a duplex, due to formation of
Hoogsteen or reverse Hoogsteen hydrogen bonds, re-
spectively [2]. In principle, triplexes can be formed
from different combinations of RNA and DNA strands,
and the composition affects their stability [3–5]. Since
natural oligodeoxyribonucleotides are easily degraded
by the nucleases, several modifications of the
sugar-phosphate backbone have been proposed [6].
Among them phosphorothioate analogues of DNA
(PS-Oligos) appeared to be very important due to close
similarity of their properties to natural DNA and en-
hanced stability against nucleolytic degradation. An ap-
proach developed in this laboratory allows for prepara-
tion of PS-oligos with a predetermined sense of
P-chirality. It has been found that homopurine
dodeca(deoxyribonucleoside phosphorothioate)s pos-
sessing all internucleotide linkages of RP configuration
form with complementary RNA or 2’-OMe-RNA strands
a triple helix in the ratio 1:2, in which the third strand
is parallel to the homopurine oligomer. They are ther-
mally more stable than complexes formed by unmodi-
fied DNA molecules of the same sequence. The tri-
plexes formed by phosphorothioate DNA dodecamers
containing 4–6 dG residues are thermally stable in pH
7.4, although their stability increases significantly at
pH 5.3. To our best knowledge, a triplex structure
RNA/[PS]-DNA/RNA has not yet been described in the
literature.
Referenes:
1. Fensenfeld G, Davies V, Rich A (1957) J Am Chem Soc, 79:
2023–2024.
2. Helene C (1993) in Antisense Research and Application
(Crook ST, Lebleu B, eds) pp 375–385, CRC Press Inc.
Boca Raton, Ann Arbor, London, Tokyo.
3. Roberts RW, Crothers DM (1992) Science, 258: 1463–1466.
4. Han H, Dervan PB (1993) Proc Natl Acad Sci USA, 90:
3806–3810.
5. Escude C, Francois J-C, Sun J-S, Ott G, Sprinzl M,
Garestier T, Helene C (1993) Nucleic Acids Res, 21:
5547–5553.
6. Verma S, Eckstein F (1998) Annu Rev Biochem, 67:
99–134.

272 Lecture
Complete nucleotide sequences of two large plasmids isolated from clinical strains
of Enterobacteriacae

Piotr Ceg³owski
Instytut Biochemii i Biofizyki, Polska Akademia Nauk, ul. Pawiñskiego 5A, 02-106 Warszawa

Plasmids constitute from 1% to greater than 10% of
the genome of many bacterial species. They represent
the most fluid part of bacterial genomes and they en-
dow the host bacteria with high genetic variability and
flexibility in response to environmental stimuli.
Plasmids are mobile genetic elements and as such are
one of the most efficient vehicles in the horizontal gene
transfer (HGT). Analysis of complete bacterial
genomes has revolutionized not only the way we think
about the HGT but also that it is far more prevalent
than previously thought.
The genomic methodology can be applied to newly iso-
lated plasmids as a first and powerful step of their char-
acterization. We used the whole plasmid sequencing ap-
proach to characterize two large plasmids, p1658/97
(125 kb) and pCTX-M-3 (90 kb) that were isolated from
clinical strains of Enterobacteriaceae. We wanted to
gain more information about those plasmids because of
their intriguing properties: the p1658/97 contains the
beta-lactamase gene that undergoes amplification within
a plasmid molecule [1] and the pCTX-M-3 very quickly
disseminated among many species of enteric bacteria
allover Poland [2].
Complete nucleotide sequences of p1658/97 and
pCTX-M-3 were determined, annotated and deposited
in the GenBank database under accession numbers
2003 39th Meeting of the Polish Biochemical Society 155

AF550679 and AF550415, respectively. The most inter-
esting features of both plasmids as well as their organi-
zation and evolution will be presented.
References:
1. Pa³ucha A, Mikiewicz B, Hryniewicz W, Gniadkowski M
(1999) J Antimicrob Chemother, 44: 489–99.
2. Baraniak A, Fiett J, Sulikowska A, Hryniewicz W,
Gniadkowski M (2002) Antimicrob Agents Chemother,
46: 151–9.
Supported by the Centre of Excellence in Molecular Biotech-
nology, Grant ICA1-CT-2000-70010

273 Lecture
Initiation of replication in bacteria: Essential variations on a theme
Donald Helinski
Division of Biological Sciences and Center for Molecular Genetics, University of California at San Diego, 9500 Gliman Drive, La
Jolla, USA

The interactions responsible for the initiation of chro-
mosomal replication from the E. coli replication origin
and subsequent DNA elongation events have, for the
most part, been defined. The key role of the E. coli
DnaA protein in the formation of an open complex and
the recruitment of the DnaB helicase and the role of the
E. coli DnaC protein as a DnaB loader in the formation
of a pre-priming complex at the E. coli origin are now
well established. An important variation on this series
of events has been shown for plasmid RK2 in an in vitro
system utilizing the DnaA and DnaB proteins of Pseu-
domonas species [1, 2]. These studies demonstrated
that the formation of a pre-priming complex requires
the larger form of a plasmid encoded initiation protein
and the DnaB helicase but not the DnaA protein or the
DnaC helicase loading protein. This is consistent with
the failure to identify a homologue of the E. coli dnaC
gene in P. aeruginosa or P. putida [1]. The chromosomal
origins of P. aeruginosa and P. putida have been isolate
and sequenced [3]. We have examined the activity of
these origins in an in vitro assay that measures the for-
mation of a pre-priming complex. These analyses
showed that the Pseudomonas DnaA protein interacts
with its respective DnaB protein to form the
pre-priming complex in the absence of a DnaC-like
loader protein [4]. Thus, for both the chromosomal ori-
gin and the plasmid RK2 origin, initiation of replica-
tion in Pseudomonas occurs in the absence of the DnaC
protein.
Several cytological studies have shown that the E. coli
chromosomal origin of replication is located near one
end of a newborn cell. Low copy number plasmids F and
P1 are, however, positioned at mid-cell in E. coli and dur-
ing cell growth and elongation plasmid foci move to
one-quarter and three-quarter positions which become
the mid-cell positions of daughter cells after cell division
[5]. Using GFP-tagging and FISH we carried out a simi-
lar study using multi-copy plasmids RK2 (5–8 copies per
chromosome) and pUC19 (> 50 copies per cell). To our
surprise these multi-copy plasmids were not present as
multiple foci corresponding in number to their copy
number in E. coli, but in most cells were clustered as a
single focus at the mid-cell position in shorter cells, or as
two foci located at the 1/4 and 3/4 positions in longer
cells [6]. A similar finding was observed for plasmid RK2
in Pseudomonas aeruginosa and Vibrio cholerae [7]. We
are presently addressing the major question of the na-
ture of the E. coli proteins or structures and/or plasmid
encoded proteins responsible for the clustering of multi-
ple copies at fixed locations in the bacterial cell.

274 Lecture
Mechanisms for helicase recruitment and loading at broad host range plasmid ori-
gin. DnaA-boxes, iterons and 13-mers as sites for helicase delivery and loading
1 1 1 1 2 3
Igor Konieczny , Marcin Pacek , £ukasz Kowalczyk , Katarzyna Herman , Anna Janaszak , Donald Helinski ,
3 3
Yong Jiang , Aresa Toukdarian
1 — Katedra Biologii Molekularnej i Komórkowej, Miêdzyuczelniany Wydzia³ Biotechnologii, Uniwersytet Gdañski, ul. K³adki 24,
80-822 Gdañsk, 2 — Katedra Biologii Molekularnej, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk, 3 — Division of Biological
Sciences and Center for Molecular Genetics, University of California at San Diego, 9500 Gliman Drive, La Jolla, USA

The origins of prokaryotic and some eukaryotic cerevisiae possess characteristic functional elements,
replicons such as DNA viruses and Saccharomyces including specific binding sites for the appropriate ini-
156 Session 5. Structure and function of DNA 2003

tiation protein and an AT-rich region where DNA du-
plex destabilization occurs. Plasmid origins usually
contain multiple binding sites (iterons) for the
plasmid-specific replication initiation protein as well as
a binding site(s) for the host replication initiation pro-
tein, DnaA (DnaA boxes). Several lines of evidence sug-
gest that these origin structural elements are employed
for broad-host-range plasmid replication and mainte-
nance in different host bacteria species. We found that
broad-host-range plasmid RK2 can utilize different ori-
gin structural elements to facilitate host-specific path-
ways for bacterial helicase recruitment at plasmid ori-
gin. Our studies revealed the role of DnaA boxes,
iterons and 13-mers of AT-rich region in helicase re-
cruitment and loading on to ssDNA. Another important
factor effecting helicase recruitment and loading mech-
anism is the nature of the protein interactions between
the two forms of the RK2 replication initiation protein
and the host replication machinery. Recruitment of the
helicase through the N-terminal domain uniquely pres-
ent in the larger form of the plasmid replication initia-
tion protein is critical in P. aeruginosa, but plays no role
in helicase recruitment in E. coli. Similarly, the neces-
sity of host specific DnaA-DnaB interactions utilized
for helicase recruitment at the plasmid origin depends
on the bacterial species.

275 Lecture
DnaA, an ATPase switch controlling bacterial replication initiation
Walter Messer
Molecular Genetics, Max-Planck-Institute for Molecular Genetics, Berlin, Germany

DnaA is a member of the AAA+ (ATPases associated
with a variety of cellular activities) superfamily of
ATPases. All family members contain matches to the
Walker A motif for ATP binding and to the Walker B
motif that is important for ATP hydrolysis. The family
includes many proteins involved in DNA replication in
prokaryotes, archaea and eukaryotes, that therefore
provide a unifying link in the biochemistry of DNA rep-
lication throughout the living world. The presence of
ATP or ADP in the binding site results in a significant
change in protein structure and function and provides
a molecular switch that couples key events during initi-
ation of replication with cell cycle progression.
Several monomers of E. coli DnaA form a complex
with the replication origin, oriC, resulting in the local
unwinding of an AT-rich region in oriC. Only
ATP-DnaA is active in unwinding and initiation. The
binding properties of DnaA as well as the ability of
monomers to cooperate are profoundly influenced by
its ATP/ADP status. Both forms bind to an asymmetric
9-mer consensus sequence, the DnaA box
5’-TTA/TTNCACA. But only ATP-DnaA is able to
oligomerize and bind to a relaxed DnaA box with one or
two mismatches, as well as to a 6-mer sequence, the
ATP-DnaA box with the consensus sequence 5’-AGatct.
Sequential binding of DnaA to these sites with graded
affinities is responsible for the unwinding. The unwind-
ing and initiation reaction of bacteria other than E. coli
is similar, but with variations in the details of the reac-
tion.
All organisms have developed mechanisms that en-
sure that chromosomal replication occurs once and
only once per generation. The regulatory inactivation
of DnaA is the prominent mechanism in bacteria. The
intrinsic ATPase of DnaA is activated at the end of the
initiation cycle due to the loading of the sliding clamp,
the b subunit of DNA polymerase III holoenzyme. This
prevents further initiation.

276 Lecture
Molecular mechanisms of activation transcription in Escherichia coli
Zygmunt Wasylewski
Zak³ad Biochemii Fizycznej, Wydzia³ Biotechnologii, Uniwersytet Jagielloñski, ul. Gronostajowa 7, 30-387 Kraków

cAMP receptor protein, CRP, allosterically activated
by cAMP, regulates the several genes in Escherichia
coli. The protein is a homodimer and each monomer is
folded into two distinct domain — C-terminal domain re-
sponsible for DNA binding and N-terminal one respon-
sible for cAMP binding. The fast kinetic measurements
of CRPwt and several single amino acid substituted mu-
tants, which play a crucial role in inter-domain and
inter-subunit communication, have been used to study
the kinetic of allosteric conformational changes of the
2003 39th Meeting of the Polish Biochemical Society 157

protein induced by cAMP binding. The conformational
changes observed upon CRP-(cAMP)2 complex forma-
tion can be described by sequential model of allostery.
The amino acid substitutions influence the allosteric
changes. The formation of CRP-(cAMP)4 complex re-
sults from displacement of equilibrium between two
forms of CRP-(cAMP)2. Intermolecular signal transmis-
sion, triggered by cAMP binding, indicates that the pro-
tein can exist in various states, the distribution of
which can be influenced by these mutations. This
cAMP dependent distribution of conformational states
of CRP, which differ in affinity for DNA and RNA poly-
merase, may play a crucial role in the fine regulation of
transcription. Using steady-state and time resolved flu-
orescence measurements such as FRET we have shown
that the binding of cAMP results in 8Å movement of
the CRP domains and the protein can interact in solu-
tion with alpha subunit as well as with the sigma sub-
unit of RNA polymerase.

277 Lecture
DnaA protein and oriC region — key elements of initiation of bacterial chromosome
replication
Jolanta Zakrzewska-Czerwiñska
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroc³aw

The initiation of DNA replication is a complex process
involving multiple regulated steps: (i) binding of the ini-
tiator protein(s) to sites localized within the ori region;
(ii) unwinding of the ori region to provide the entry site
for the DNA helicase; (iii) loading of helicase and addi-
tional proteins. In bacteria, initiation of chromosomal
replication starts from a single replication origin oriC
and proceeds bi-directionally until the replication forks
reach termination site (terC). Initiation of bacterial
chromosome replication is mediated by the initiator
DnaA protein which interacts with repetitive
nonpalindromic nonamer sequences, the DnaA boxes
that are localised within the oriC region.
The structure of the oriC region has been analyzed
within Gram-negative and Gram-positive bacteria. The
sequences of oriC regions are conserved only among
closely related organisms. Replication origins of differ-
ent bacteria have varying sizes (from approx. 200 up to
1000 bp) but (nearly) all contain several DnaA boxes,
and an AT-rich region. A cluster of four or more DnaA
boxes is an indication for a functional chromosomal ori-
gin. The functional oriC region is always located within
the intergenic region; frequently within the cluster of
genes rnpA-rmpH-dnaA-dnaN-recF-gyrB rnpA, usually
next to the dnaA gene encoding initiator protein DnaA.
DnaA homologs have been found in all eubacterial
species studied so far, with the exception of the
Wigglesworthia glossinidia genome. Based on structural
similarity and specific functions, four domains have
been identified in DnaA. The N-terminal domain I me-
diates protein-protein interactions, DnaA oligo-
merization and interaction with DnaB helicase. Do-
main II appears to be less evolutionary conserved and
is probably a flexible linker that connects the
N-terminal domain with the highly conserved domains
III and IV. The length of the domain II varies from 48
amino acids in H. pylori to 247 amino acids in Strepto-
myces coelicolor A3(2). The ATP binding domain III in-
cludes canonical Walker A/B motifs and contains an
additional oligomerization site. The C-terminal domain
IV interacts specifically
A comparison of replication initiation in different or-
ganisms particularly in E. coli, H. pylori, M. tuberculosis
and S. coelicolor allows to define those aspects of the
process that are universally used in the eubacterial
kingdom.

278 Oral Presentation

Potential functions of a new member of the mouse SIR-gene family, Sir7, in cellular
proliferation, differentiation and stress response
Eva Bober, Olesya Vakrusheva
Institut für Physiologische Chemie, Martin Luther Universität Halle-Wittenberg, Hollystr. 1, Halle, Germany

+
Sir (silent information regulator) encode NAD de- DNA-repair, stress resistance and regulation of the life
pendent histon-deacethylases and are involved in sev- span. Especially in two lower eucaryots, S. cerevisie and
eral essential processes as transcriptional silencing, C. elegans, where Sir function has been most inten-
158 Session 5. Structure and function of DNA 2003

sively studied, the life span seems to be directly depend-
ent on the number of active Sir gene copies. Sir
homologs were identified in many other species includ-
+
ing mouse and man. Although the NAD -dependent
histone deacetylase activity has been demonstrated for
mammalian Sir proteins, their physiological role in
higher vertebrates remains elusive. Recently it has
been shown, that human SIR2 can deacetylate other
proteins in addition to histones with p53 as one of the
possible specific targets.
We have identified a new member of the mouse Sir
family, that shows the highest homology to the human
Sirt7. To this end we studied the function of Sir7 by
overexpression in mammalian cell tissue cultures and
using in vitro protein interaction assays. In addition,
stable cell clones overexpressing Sir7 were established
and screened for differences in gene expression as re-
lated to the reference cells. Upon overexpression, Sir7
prevents differentiation of myogenic cells. This func-
tion may be explained by the capability of Sir7 to acti-
vate promoters of genes involved in cell cycle control
and apoptosis as myc and p53 and to downregulate the
MyoD promoter. Using GST-based pull down assays a
direct physical interactions of Sir7 with p53 and sev-
eral transcription factors of the bHLH-family were
demonstrated.

279 Oral Presentation

Protein-protein interactions at simple and complex bacterial promoters
Steve Busby
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom

At many simple bacterial promoters, transcription
initiation is dependent on a single activator that func-
tions by interacting directly with a target in RNA poly-
merase. Some recent advances in understanding these
activator-RNA polymerase interactions will be out-
lined. Many bacterial promoters are more complex, and
transcription is co-dependent on two or more activa-
tors; this ensures the appropriate expression from
these promoters in response to particular environmen-
tal triggers. Recent research concerned with under-
standing the different mechanisms of co-dependence
will be presented.
References:
Busby S, Ebright R (1999) J Mol Biol, 293: 199–213.
Browning D, Cole J, Busby S (2000) Mol Microbiol, 37:
1258–1269.
Lloyd G, Landini P, Busby S (2001) Essays in Biochemistry,
37: 17–32.
Wade J, Belyaeva T, Hyde E, Busby S (2001) EMBO J, 20:
7160–7167.

280 Oral Presentation

Analysis of the EcoVIII endonuclease active site using site directed mutagenesis
1 1 2 1
Magdalena Cichowicz , Iwona Mruk , Janusz Bujnicki , Tadeusz Kaczorowski
1 — Katedra Mikrobiologii, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk, 2 — Laboratorium Bioinformatyki,
Miêdzynarodowy Instytut Biologii Molekularnej i Komórkowej, ul. Ks. Trojdena 4, 02-109 Warszawa

Our research focuses on investigating the nature of
the isospecificity phenomenon among type II restric-
tion-modification (R-M) systems. This phenomenon is
interesting in many ways. Firstly, because it poses the
intriguing question — how do genes that encode func-
tional homologs evolved in bacteria that are often not
phylogenetically related. As well as this, it is interest-
ing because structural analysis of these enzymes can
help in the localization of particular motifs responsible
for catalytic reaction and for target recognition, and in
consequence could be used in designing enzymes with
novel specificities.
As a model in our study we decided to use a group of
systems isospecific to HindIII (Haemophilus influenzae
Rd), a R-M system which recognizes the palindromic se-
quence 5’-AAGCTT-3’. This group consists of over 30
R-M systems isolated from different bacteria. To date,
except for HindIII only two other systems, LlaCI
(Lactococcus lactis subsp. cremoris W15) and EcoVIII
(Escherichia coli E1585-68), have been cloned and se-
quenced. In our studies we address the following ques-
tions: (i) how similar are the genes encoding isospecific
enzymes? (ii) is it possible to map their functional do-
mains? (iii) do they recognize cognate sequence in the
same way? (iv) what is their mode of action?
In the present study we decided to investigate the
structure of the catalytic center of the EcoVIII
endonuclease (R.EcoVIII). The putative catalytic DNA
cleavage/Mg(II) binding motif of restriction
endonucleases (PD/EXnD/EXK) is essential for their
function. The predicted amino acid sequence of
R’EcoVIII allowed a putative catalytic/Mg(II) binding
sequence motif characteristic for restriction endonucle-
ases, PE66X54DXK123 to be located in N-terminal por-
2003 39th Meeting of the Polish Biochemical Society 159

tion of the enzyme. This motif is also present in
R’HindIII (PE52X56DXK111) and R.LlaCI (PD49X54-
DXK106). The site-directed mutagenesis of the gene en-
coding HindIII enzyme produced evidence that any mu-
tation within P51X56DXK111 catalytic motif abolishes
enzyme activity (Dahai et al. 1999; Biosci Biotechnol
Biochem, 63: 1703–1707). However, analysis of the
R.HindIII molecular model revealed that residues
PE52 are located a significant spatial distance from the
second part of the catalytic motif (DXK111). Therefore,
we have concluded that most probably the catalytic mo-
tif of this enzyme is different from that suggested
above, and comprises of charged residues located in
close proximity. The spatial architecture of the
R.HindIII molecular model prompted us to propose
that the D94X12DXK111 motif is involved in the forma-
tion of this enzyme active site. The role of the PE52 mo-
tif remains obscure. Thus suggested catalytic motifs for
R.EcoVIII and R.LlaCI are D108X12DXK123 and
D91X12DXK106, respectively. Site directed mutagene-
sis within putative catalytic domain produced two
R.EcoVIII null mutants (D108A and D121A) providing
evidence that changed amino acids are involved in the
process of catalysis.

281 Oral Presentation

Application of DNA sequencing by indexer walking in analysis of plasmids isolated
from enteropathogenic strains of Escherichia coli
Katarzyna Gromek, Tadeusz Kaczorowski
Katedra Mikrobiologii, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

The strategy of DNA sequencing by primer walking
enables direct and systematic analysis of large DNA
fragments with low redundacy and without subcloning.
This approach is based on the use of individual primers
specifically designed for each sequencing step. Al-
though potentially the primer walking strategy seems
to be atractive for large scale projects, recent progress
in sequencing of many genomes was achieved by using
the random approach. Among the disadvantages of the
primer walking technology are high cost of primers and
delays associated with their design and synthesis. How-
ever, these drawbacks could be bypassed by the use of
(i) presynthesized short primers (e.g. 8–10-mers); (ii)
methods for primers assembly from libraries of 5- or
6-mers; (iii) primers produced by the high-throughput
multichanel oligosynthesizers.
In our laboratory we have developed another ap-
proach to DNA sequencing by primer walking which
relies on the usage of the presynthesized library of
oligonucleotide adaptors refered to as indexers and
class IIS restriction endonucleases. These dou-
ble-stranded adaptors consist of an individually syn-
thesised indexing strand (24 nt) annealed to a comple-
mentary common shorter oligomer (20 nt). The
shorter oligonucleotide serves as primer for DNA am-
plification and sequencing whereas the longer one is
responsible for the specificity of ligation to the ana-
lyzed DNA. The use of the presynthesized library of in-
dexers enables amplification and subsequent direct
sequential analysis of any DNA fragment. Such a li-
brary eliminates the necessity for custom synthesis of
oligonucleotides.
The protocol of DNA sequencing by indexer walking
incorporates efficient ligation of double-stranded syn-
thetic oligonucleotides (indexers) to DNA fragments,
produced by class IIS restriction endonucleases which
generate four nucleotide long 5’ overhangs, and their
subsequent amplification which provides enough tem-
plate for automated DNA sequencing. Data gathered in
the first sequencing reaction permits further move-
ment into the unknown DNA sequence by digestion
with class IIS restriction endonuclease followed by liga-
tion of next indexer. The presynthesized library of in-
dexers (256 oligonucleotides) enables bi-directional
analysis of any DNA molecule and provides universal
primers for sequencing. This approach was success-
fully applied to sequence several natural plasmids iso-
lated from entreopathogenic strains of Escherichia coli.

282 Oral Presentation

Binding of regulatory proteins to the putative sigma54 promoter region of the Esch-
erichia coli rpoH gene
Anna Janaszak, Wiktor Majczak, Jacek Jasiecki, Beata Nadratowska, Gra¿yna Konopa, Alina Taylor
Katedra Biologii Molekularnej, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

The sigma54-promoter consensus sequence was proteins for this promoter are unknown, but potential
found in the regulatory region of the rpoH gene coding consensus sequences for IHF binding and enhancers
for the main heat-shock factor sigma32. Regulatory for PspF and NtrC were found. These two activators act
160 Session 5. Structure and function of DNA 2003

by different mechanisms. Since two other heat-shock
inducible and sigma54 controlled units were described:
pspA-E operon and ibpB gene, a third-sigma54 con-
trolled heat-shock regulon emerges besides the two
known, controlled by sigma32 and sigma24 RNA poly-
merase subunits.
The sigma54-RNA polymerase holoenzyme (sigma54-
RNAP) is responsible for transcription of genes whose
products are involved in utilization of various nitrogen
and carbon sources, energy metabolism, development
and many other activities. Hence one may suppose that
the stress response encompasses also gene regulation
adequate for these conditions.
Initiation of transcription at a sigma54 promoter is
tightly regulated at the step of promoter melting. The
sigma54-RNAP binds to the promoter to form a stable
closed complex. Its isomerisation to open complex de-
pends on the interaction with enhancer-bound activa-
tor protein in an ATP dependent manner. The
enhancer sequences can be distant from sigma54 pro-
moter, so the interaction requires looping-out of the in-
tervening DNA, facilitated by the DNA bending IHF
protein.
Sigma54, PspF and NtrC proteins have been purified
for in vitro studies. Gel mobility shift assays have re-
vealed that sigma54-RNAP, but not sigma54 subunit
alone, binds to the regulatory region of the rpoH gene.
It is consistent with the data that sigma54 unlike other
sigma factors can bind to its promoter in the absence of
the core RNAP but only if the ‘T-tract’ element within
promoter sequence is present. The promoter in ques-
tion does not contain the ‘T-tract’. Electron microscopy
experiments confirmed this result and showed that
sigma54-RNAP binds specifically to its consensus se-
quence within the rpoH promoter region. PspF, the ac-
tivator of the pspA-E operon, binds to the regulatory re-
gion, as demonstrated by gel mobility shift assay. NtrC
interaction with this site is under investigation. IHF
protein also has been purified for these and in vitro
transcription studies.
The project was supported by State Committee for Scientific
Research (KBN, Poland) grant nr 3 P04A 001 23).

283 Oral Presentation

The gene expression profiles in patients with abdominal aortic aneurysm (AAA)
1 2 1 2 2
Aleksandra Korcz , Krzysztof Waliszewski , Daniel Lipinski , Marcin Gabriel , Stanislaw Zapalski ,
3
Ryszard Slomski
1 — Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszyñska 32, 60-479 Poznañ, 2 — Clinics of General and
Vascular Surgery, University of Medical Sciences, Poznañ, 3 — Department of Biochemistry and Biotechnology, Agricultural
University, ul. Wo³ynska 35, 60-637 Poznañ, Poland

Abdominal aortic aneurysm (AAA), a localized abnor-
mal dilatation of aorta, is a life-threatening condition
affecting 4–9% of population with a risk increasing
with age. Other risk factors include hypertension, ath-
erosclerosis and smoking. Familial occurrence of ab-
dominal aortic aneurysm indicates involvement of ge-
netic factors in development of AAA however no single
gene was shown to be responsible for it. Based on the
results of the studies done so far, it is believed that
pathogenesis of abdominal aortic aneurysm is a com-
plex and probably multifactorial process. Since gene ex-
pression profiling of abdominal aortic aneurysm tis-
sues in comparison with clinically unchanged aorta
may help to understand complex biological processes
responsible for pathogenesis of AAA we applied DNA
array technique in our studies. The expression profil-
ing experiments were performed on cDNA arrays sup-
plied by Clontech covering 588 genes from AtlasTm Hu-
man Cardiovascular Array and 234 genes from
AtlasTm Human Stress Array. We have identified sev-
eral up-regulated and down-regulated gene expression
changes in abdominal aortic aneurysm tissues when
compared to control unchanged aortas.

284 Oral Presentation

The EcoVIII endonuclease gene expression is modulated by the low-usage arginine
codons
Iwona Mruk, Tadeusz Kaczorowski
Katedra Mikrobiologii, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

Each organism represents its own preference of us- reflected by the particular cell tRNA pool of those. The
age of the 61 available amino-acids (aa) codons. This is aa arginine can be encoded by six codons. In E. coli, the
2003 39th Meeting of the Polish Biochemical Society
most frequent arginine codons are CGC and CGU. The
other four belong to the low-usage codons with AGA
and AGG codons which are the rarest among all codons
in E. coli cells.
The two E. coli E1585-68 genes: EcoVIII endonuclease
(R.EcoVIII) and EcoVIII methyltransferase
(M.EcoVIII) which together build up the EcoVIII re-
striction-modification system (R-M system) have been
investigated. Both genes possess high percent of
low-usage arginine codons: 18 out of 20 for R.EcoVIII
and 13 out of 15 for M.EcoVIII respectively. In addi-
tion, the rarest AGA and AGG codons occur in tandem,
located at 16–17 aa position in respect to N-terminus in
case of R.EcoVIII whereas the same tandem is observed
at 167–168 aa position in case of M.EcoVIII. It has been
shown that presence of such consecutive sequence of
rare codons close to the gene start site can lead to
translational pausing, frameshifting and aa
misincoporating. This effect is not observed when the
same rare codons occure beyond the first 25 triplets.
The insufficient tRNA population for rare codons may
explain our unsuccessful attempts in R.EcoVIII over-
production. This was overcome when the overproduc-
ing strain BL21(DE3) was transformed with the
plasmid pRARE (Novagen) carrying the dnaY gene cod-
285
Cauliflower complex I and complex V mitochondrial genes: structure and expression
Katarzyna Raczyñska, Sabina Janicka, Krzysztof Lesniewicz, Micha³ Rurek, Halina Augustyniak
Instytut Biologii Molekularnej i Biotechnologii, Zak³ad Biologii Molekularnej Roœlin, Uniwersytet im. Adama Mickiewicza,
ul. Miêdzychodzka 5, 60-371 Poznañ
In the present study we characterized the cauliflower
gene of subunits of complex I (nad3, nad6 and nad9)
and genes of complex V subunits (atp6, atp9) as well as
their expression. Although the nad9, nad6 and nad3
genes as well as atp6 and atp9 were analyzed in a few
plant species, no data about the structure and expres-
sion of these genes are known for cauliflower.
The copy number analysis confirmed the presence of
one copy for each analyzed gene in mtDNA. Our se-
quence analysis revealed the open reading frames for
nad9, nad6 and nad3 genes of 573, 618 and 357 bp, re-
spectively. The 5’ and 3’ flanking region of the nad9
varied more in the sequence than the gene. A similar
variability was observed for the 5’ flanking region of
nad6 and 3’ flanking region of nad3.
To examine the expression of analyzed genes, North-
ern blots of mtRNA revealed transcripts of about 1.4,
161
ing for the proper tRNA that recognises AGA/G
codons.
The EcoVIII R-M system is a possible result of horizon-
tal gene transfer. This finding may support not only the
strong codon usage bias, but also the difference in over-
all GC content of genes (34.5% for R.EcoVIII and 37.7%
for R.EcoVIII) in comparison to genomic E. coli DNA
(50.8%). We calculated the value of the codon adaptation
index (CAI) for each EcoVIII gene. Genes with high CAI
(near 1) belong to the family of highly expressed genes.
The obtained CAI values of 0.151 and 0.185 for
R.EcoVIII and M.EcoVIII respectively, indicate that both
genes belong to the E. coli genes that were most probably
acquired by horizontal gene transfer. It is extremely im-
portant for bacterial cell to balance these two activities
of R-M systems: restriction and modification. In any
case, the overrestriction leads to cell suicide.
We show that the abundance of the low-usage codons
and their position within the EcoVIII endonuclease
gene may seriously affect its expression. We hypothes-
ise that the activity of the endonuclease gene can be
modulated depending on the host genetic context.
Thus, we propose another mode of R-M system regula-
tion which allows to control the potential lethal action
of the restriction enzyme.
Oral Presentation
1.2 and 1.1 kb for nad9, 1.6 and 0.8 kb for nad6, 1.6 and
1.43 kb for nad3, 1 kb for atp6, and 0.3 kb for atp9 gene
were detected. All the transcripts were long enough to
cover the entire coding region of all the genes analyzed.
The presence of longer transcripts may indicate that
cotranscription with other genes is possible.
The 5’ termini of the nad9 and nad6 mRNAs were
identified. They were mapped at a distance of about 380
and 160 bp from the start codon of nad9 gene and at
distance of about 90 and 190 bp from the start codon
for nad6 gene.
In order to detect proteins which bind to 5’ flanking
sequence of nad9 gene, the EMSA analysis was carried
out. In the two analyzed fractions of cauliflower mito-
chondrial proteins, some polypeptides showing binding
activity were detected.
162 Session 5. Structure and function of DNA 2003

286 Oral Presentation

Loss of heterozygosity at the p16 locus in human endometrial carcinomas
1 2 3 1
Agnieszka Stenzel-Bembenek , Andrzej Semczuk , Barbara Marzec , Anna Szczygielska , Marta
1
Stryjecka-Zimmer
1 — Katedra i Zak³ad Biochemii i Biologii Molekularnej, Akademia Medyczna im. Prof. Feliksa Skubiszewskiego,
ul. Lubartowska 85, 20-123 Lublin, 2 — II Katedra i Klinika Ginekologii, Akademia Medyczna w Lublinie, ul. Jaczewskiego 8,
20-954 Lublin, 3 — Zak³ad Genetyki Medycznej z Pracowni¹ Diagnostyki Molekularnej i Cytogenetycznej, Akademia Medyczna
w Lublinie, ul. Radziwi³³owska 11, 20-080 Lublin

p16INK4A — tumor suppressor gene, encoding pro-
tein associated with regulation of the cell-cycle is often
altered in human carcinomas. Some of these alter-
ations are caused by point mutations and deletions,
some by modifications of DNA sequence as well as by
loss of heterozygosity (LOH).
Currently, we examined 50 endometrial carcinomas
from women treated at the IInd Department of Gyne-
cology, Lublin University School of Medicine, Lublin,
for the presence and frequency loss of heterozygosity
(LOH) correlated with immunohistochemical expres-
sion of the p16INK4A protein and clinicopathological
features. All the samples were classified according to
the FIGO stage system and hist.-path. examination.
DNA was isolated from frozen tissues using the stan-
dard method. For LOH study of the p16INK4A , the Se-
quence-Tagged-Site (STS) marker c.5.1 (localized be-
tween coding regions of exon 2 and exon 3 was investi-
gated. DNA was amplified by PCR and PCR-products
were separated by electrophoresis through polyacry-
lamide gels cross-linked with piperazindiacrylamide
and visualized by silver staining. The immunoreaction
was performed with a mouse monoclonal anti human
IgG antibody, clone DSC-50.1/H4 and the streptavidin
— biotin-complex method was used for visualization.
Results: An allelic loss of p16INK4A was detected in 12
of 50 (24%) carcinomas with a higher incidence in ad-
vanced endometrial carcinomas than in early-stage
uterine tumors. p16INK4A-LOH was significantly cor-
related with reduced nuclear p16INK4A expression
immunohistochemically.

287 Poster
Relationship of polymorphism in the apolipoprotein E gene with coronary artery
disease or ischemic stroke in Silesian patients
1 1 1 2 3 1
Anna Balcerzyk , Pawe³ Niemiec , Beata Sarecka , Zbigniew Ciemniewski , El¿bieta Marsza³ , Iwona ¯ak
1 — Department of Biochemistry and Medical Genetics, Medical University of Silesia, ul. Medyków 18, 40-752 Katowice, 2 — The
First Department and Clinic of Cardiology, Medical University of Silesia, ul. Zio³owa 45/47, 40-635 Katowice, 3 — Department
and Clinic of Pediatric Neurology, Medical University of Silesia, ul. Medyków 16, 40-752 Katowice

Background: Apolipoprotein E (apoE) is involved in
the cholesterol transport and lipoprotein metabolism.
Human apoE exists in three isoforms encoded by dis-
tinct alleles (epsilon 2, epsilon 3 and epsilon 4). Varia-
tion at the apoE gene locus has been known to affect
plasma cholesterol concentrations, what may be one of
the reasons of its association with coronary artery dis-
ease (CAD). The E4 allele increases the risk of mortal-
ity after myocardial infarction and susceptibility to
cerebrovascular disease.
Aim: The aim of this study was to assess a possible as-
sociation between the epsilon polymorphism of apoE
gene and CAD or ischemic stroke.
Materials and Methods: Sixty seven adult patients
with angiographically documented CAD, 15 ischemic
stroke children and 105 control subjects were studied.
The apoE genotypes were determined using PCR-RFLP
analysis with HhaI restriction enzyme.
Results: The frequency of the E4 allele in the CAD pa-
tients was 0.1, which was 1.4-fold higher than in controls
(0.07), however this difference was not statistically signifi-
cant. The E4 allele frequency in stroke children was signif-
2
icantly higher than in controls (0.27 vs. 0.07, chi =12.6,
p=0.0004). Carriers of the E4 allele were more frequent in
the stroke group (0.33) than in the CAD patients (0.19)
and controls (0.13). The E4E4 genotype was not found in
the CAD patients and controls, while 20% of ischemic
stroke children were E4E4 homozygotes.
Conclusions: Our data suggest a strong association
between carrier-state of the E4 allele in the apoE gene
and ischemic stroke in children, but further analysis of
larger group is needed.
2003 39th Meeting of the Polish Biochemical Society 163

288 Poster
Direct sequencing of plasmids isolated from hospital environment using cassettes
carying antibiotic resistance genes inserted randomly
Agnieszka Dekowska, Ewa Chêæ, Tadeusz Kaczorowski
Katedra Mikrobiologii, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

There are two basic strategies of genomic DNA se-
quencing. First is based on random shotgun methods
employing DNA fragmentation, subcloning of small
fragments, and their sequencing from universal prim-
ers (e.g. M13/pUC). Second strategy employs direct se-
quencing of the DNA fragments either by primer walk-
ing, which requires designing and synthesis of a new
oligonucleotide primer for each run, or by using antibi-
otic resistance gene cassettes containing primer bind-
ing sites. Random insertion of such elements enables
DNA sequencing by producing overlapping sequences
that are used to obtain the total nucleotide structure of
the analyzed DNA. The overall goal of each of these
strategies is to make DNA sequencing faster, more ac-
curate, and less expensive. The approach based on the
use of the chloramphenicol resistance marker has been
successfully applied in our laboratory for direct se-
quencing of bacterial plasmids isolated from entero-
pathogenic strains of Escherichia coli (EPEC). These
bacteria are responsible for infantile diarrhea, espe-
cially in less developed countries. Epidemiological stud-
ies revealed that EPEC strains are especially abundant
in plasmids. Some of them are associated with pathoge-
nicity (e.g. EAF plasmid), while function of others is
still unclear. In our studies we are interested in how
plasmids enable bacteria to adapt in the hospital envi-
ronment where antibiotic pressure and other factors
promote fast evolution of strains with novel properties.
Plasmids analyzed in our laboratory were processed
by partial digestion with restriction endonuclease
Sau3AI (a frequent cutter) in the presence of ethidium
bromide which severly inhibits the enzyme’s activity
promoting creation of linear forms of plasmid DNA. In
the next step, after excision from delivering plasmid
with BamHI enzyme, a chloramphenicol cassette
[pKRP10; Reece and Phillips, Gene 165 (1995)
141–142] was inserted into natural plasmids partially
digested with Sau3AI, as described earlier. Both en-
zymes after digestion produce the same 5’ protruding
ends. Recombinants were introduced into laboratory E.
coli strain (MM294) and selected on plates supple-
mented with chloramphenicol. Individual colonies were
screened for the presence of plasmid DNA. Selected
recombinants were sequenced bidirectionally using a
single set of sequencing primers (cat1 and cat2, respec-
tively). Products were analyzed on ABI310 DNA se-
quencer. The described procedure enabled us to obtain
nucleotide sequences of several medium-size E. coli
plasmids (4-10 kb) at low cost and minimum laboratory
work. However, initial analysis of their structure did
not reveal presence of any open reading frames that
could be associated with virulence.

289 Poster
Cloning and characterization of the genes of the restriction-modification system
from Neisseria cuniculi
Beata Furmanek-Blaszk, Tadeusz Kaczorowski
Katedra Mikrobiologii, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

Restriction-modification (R-M) systems are distrib-
uted in a variety of microorganisms. The simplest sys-
tems are type II R-M systems, which usually consist of
two separate enzymes, a restriction endonuclease and a
methyltransferase. Genes of R-M systems have some-
times been found to be located on transferable ele-
ments such as plasmids and bacteriophages, and in
some cases, genes encoding proteins involved in DNA
mobility, such as transposases, integrases and
invertases, are found in the vicinity of the genes for
R-M systems. These genetic structures may facilitate
the transfer of R-M systems and may contribute to the
wide distribution of the R-M systems. The above find-
ings, together with recent studies of the complete se-
quences of bacterial genomes have led to a proposal
that R-M systems are likely to be mobile genetic ele-
ments. However, a few examples in which genes for
DNA transfer are not colocalized with a gene for DNA
transfer are known, indicating that other mechanisms
are involved in the spread of R-M systems.
Neisseria cuniculi ATCC 14688 is a gram-negative
coccus isolated from the oral mucosa of the rabbit.
Recently we showed that this strain produces the re-
striction enzyme NcuI which is an isoschizomer of
164 Session 5. Structure and function of DNA 2003

MboII. We have cloned the genes of the NcuI R-M sys-
tem, as primary sequence data might help to com-
pare it to the isospecific system MboII from
Moraxella bovis. The search for homologous se-
quences using the computer program CLUSTAL W
showed significant homology to MboII endonuclease.
A comparison of the DNA sequences revealed simi-
larities between NcuI and MboII coding regions
which differ in 189 nucleotides. The overall nt se-
quence similarity is 84%. Homology searches showed
that the deduced protein was 87% identical to MboII
endonuclease with a much higher similarity if conser-
vative substitution is considered.
The second enzyme of the NcuI R-M system is NcuI
methyltransferase (M.NcuI) which recognizes and
methylates the same sequences as M.MboII. The nucle-
otide sequence for M.NcuI was determined and showed
87% identity to M.MboII; the two methyltransferases
M.NcuI and M.MboII are 90% identical at the amino
acid sequence level. We were interested in a compara-
tive analysis of the structural and functional features of
M.MboII and M.NcuI.

290 Poster
C®T substitution in the promoter region of CYP17 gene and prostate cancer risk
1 2 3 2 1
Monika Gos , Ma³gorzata Sadowska , Marek Grzegrzó³ka , Tomasz Demkow , Przemys³aw Janik
1 — Zak³ad Biologii Komórki, Centrum Onkologii — Instytut im. M. Sk³odowskiej-Curie, ul. Roentgena 5, 02-781 Warszawa,
2 — Klinika Nowotworów Uk³adu Moczowego, Centrum Onkologii — Instytut im. M. Sk³odowskiej-Curie, ul. Roentgena 5,
02-781 Warszawa, 3 — I Oddzia³ Wewnêtrzny, Centralny Szpital Kolejowy, ul. Bursztynowa 2, Warszawa

Prostate cancer, besides lung and colon cancer, is
commonly diagnosed among elder Polish men. Many
factors are involved in prostate cancer etiology, but the
most important from them is age. In the year 2000, the
incidence rate was about 252/100 000 for men after
their seventies and was about 28 times higher as com-
pared to values calculated for younger men. Also the
androgen metabolism influences prostate cancer devel-
opment, therefore any changes in genes correlated with
sex-hormone synthesis can be important for its
etiology. A single-nucleotide change (T®C) in the pro-
moter region of CYP17 gene encoding cytochrome
P450c17a was described as well as its possible role in
prostate cancer development.
The aim of our work was the examination if this poly-
morphism can serve as a marker in assessment of pros-
tate cancer risk. Therefore, we examined T—>C change
in patients with prostate cancer and age-matched con-
trols using PCR technique followed by digestion with
MspAI endonuclease and the electrophoresis in
agarose gel.
The prevalence of TT, TC and CC genotypes among
patients did not differ from values obtained for the con-
trols. When we divided examined population according
to age (<70 and =70) the CC genotype appeared to be
more common in the younger prostate cancer group
(31%) as compared to the adequate control group (17%).
Contrary results were obtained for the elder group how-
ever it can be due to non-homogenous control popula-
tion.
Therefore, we suggest that the CC variant occurrence
in the promoter region of CYP17 gene can be correlated
with higher risk of prostate cancer development among
men before their seventies, although further studies
are necessary to confirm this hypothesis.

291 Poster
Polymorphism analysis of vulpine (Vulpes vulpes) androgen receptor gene
1 2 3 2 4 5
Piotr Gronek , Dariusz Brzezinski , Robert Kalak , Karolina Doba , Piotr Przysiecki , S³awomir Nowicki ,
2 6
Katarzyna Nuc , Ryszard S³omski
1 — Department of Pigs Breeding, Agricultural University of Poznan, ul. Wo³yñska 33, 60-637 Poznañ, 2 — Department of
Biochemistry and Biotechnology, Agricultural University of Poznan, ul. Wo³yñska 35, 60-637 Poznañ, 3 — Katedra Biochemii
i Biotechnologii, Akademia Rolnicza, ul. Wo³yñska 35, 60-637 Poznañ, 4 — RKS £ubnica, Fox Breeding Farm, Œniaty,
5 — Department of Fur Animal Breeding, Agricultural University of Poznan, Poznañ, 6 — Instytut Genetyki Cz³owieka, Polska
Akademia Nauk, ul. Strzeszyñska, 60-479 Poznañ

Androgens are important steroid hormones for ex- The androgen receptor gene is located on the
pression of the male phenotype. The actions of andro- X-chromosome at Xq11–12 and codes for a protein with
gens are mediated by the androgen receptor. a molecular mass of approximately 110 kDa.
2003 39th Meeting of the Polish Biochemical Society 165

The gene consists of eight coding exons. The amino
acid sequence encoded by exon 1 is partly involved in
transactivation of androgen target genes, the region en-
coded by exons 2 and 3 enables DNA-binding, and the
region encoded by exons 4 to 8 is involved in ligand
binding. The first exon of the human androgen receptor
(AR) contains a translated CAG (poly-glutamine) re-
peat. The repeat length is polymorphic in the normal
population ranging from 8 to 35 repeats. Short tracts
are associated with high intrinsic AR activity and in-
creased severity and earlier age of onset of the andro-
gen-regulated tumor prostate cancer, whereas longer
CAG tracts are associated with low AR activity and
oligospermic infertility. There is possibility of involv-
ing AR in aggression behaviour.
We research group of 135 foxes. The aim is searching
of polymorphism distribution in exon 1 and poly-
morhisms in following exons. All exons were amplified
and analysed using HD, PCR-SSCP in different condi-
tions. The polymorphism in exon 1 was identified.

292 Poster
Identification of genes important in modulation of radiosensibility of human
melonoma cells
1 2 3 1
Robert Herok , Krzysztof Fujarewicz , Maria Wide³ , Joanna Rzeszowska-Wolny
1 — Zak³ad Radiobiologii Doœwiadczalnej i Klinicznej, Centrum Onkologii w Gliwicach, ul. Armii Krajowej 15, 44-100 Gliwice,
2 — Institute of Automation Control, Silesian University of Technology, Gliwice, 3 — Zak³ad Radiobiologii Doœwiadczalnej
i Klinicznej, Centrum Onkologii Oddzia³ Gliwice, Wybrze¿e Armii Krajowej 15, 44-101 Gliwice

Melanomas are very malignant cancers capable of
forming distant metastases. Usually, albeit not always,
they are ionizing radiation-resistant. During periods
between therapy sessions as well as during metastatic
spread, selective expansion of neoplastic cell clones oc-
curs. Due to great genetic instability of these neo-
plasms both genotype and phenotype of arising clones
may differ (for example in their radiosensitivity). The
goal of our experiments was to check gene expression
pattern changes during growth of a new cell population
and whether clones obtained differ indeed in their
radiosensitivity. Answer to these questions is essential
for understanding mechanisms of metastasis. The pres-
ent study is the first of the planned series.
Starting with Me45 melanoma line, three subclones
were obtained. Ionizing radiation sensitivity of the
starting line and that of the subclones was compared.
Cell survival tests indicated no statistical differences in
radiosensitivity. Total RNA was then isolated from the
starting cell line as well as from the subclones and ex-
pression levels of some 22 thousand genes were
checked using high-density DNA microarrays from
Affymetrix.
Among genes with significantly altered expression
level were those coding for G antigens and other cell
surface proteins, transcription factors, protein mem-
brane receptors and other signaling proteins.
The study was financed by the State Committee for Scientific
Research (KBN, Poland) grants no. PBZ-040/PO4/2001
and 4PO5015190.

293 Poster
Ionizing radiation-induced changes of gene expression patterns in human melanoma
cells
Robert Herok, Joanna Rzeszowska-Wolny
Zak³ad Radiobiologii Doœwiadczalnej i Klinicznej, Centrum Onkologii w Gliwicach, ul. Armii Krajowej 15, 44-100 Gliwice

Ionizing radiation is a factor both increasing the risk
of malignancy as well as an important antitumor ther-
apy tool. Damages induced by radiation trigger in cells
various signaling pathways, repair mechanisms as well
as cause changes in gene expression patterns. Knowl-
edge of processes induced in cells by radiation should
help in understanding the basis of different radio-
sensitivity of various cell types.
The goal of this study was to compare gene expres-
sion patterns in non-irradiated control cells and cells
exposed to ionizing radiation as well as to monitor the
time course of these changes. Material used in our
study was human melanoma Me45 cell line (obtained at
the Gliwice Center of Oncology). Cells were irradiated
with 4Gy dose and total RNA was then isolated from
the cells (a) immediately; (b) after 12 h and (c) after 24
166 Session 5. Structure and function of DNA 2003

hours following radiation exposure. As a control, sion was substantially increased or decreased in irradi-
non-irradiated cells were used. Expression levels of ated cells were those coding for:
some 22 thousand genes were then assessed using high — transcription factors
density oligonucleotide microarrays, hybridization — cell cycle control proteins
equipment and procedures from Affymetrix. — proteins participating in metabolic processes
Groups of genes were selected for which gene expres- — signal transduction proteins
sion pattern differed in a specific manner following var- — repair proteins
ious exposure times. Among genes for which expres- The study was financed by the State Committee for Scientific
Research (KBN, Poalnd) grants no. PBZ-040/PO4/2001
and 4PO5015190.

294 Poster
Analysis of transcription in the region encoding protein phosphatase PrpE
in Bacillus subtilis
1 2 1 1
Krzysztof Hinc , El¿bieta Ratajczak , Adam Iwanicki , Micha³ Obuchowski
1 — Katedra Biologii Molekularnej, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk, 2 — Miêdzyuczelniany Wydzia³
Biotechnologii, Uniwersytet Gdañski i Akademia Medyczna, ul. K³adki 24, 80-822 Gdañsk

In spite of huge progress in research on protein
phosphorylation, still the new representatives of pro-
teins kinases and phosphatases are being described.
One of these enzymes is protein phosphatase PrpE
from Bacillus subtilis. The existence of characteristic
motifs in the amino-acid sequence of this protein sug-
gests that it belongs to the PPP protein phosphatases
family. Although, PrpE possesses different properties
from other members of this family. This protein has un-
usual substrate specificity. It was able to remove phos-
phate from phosphotyrosine but not from phospho-
threonine or phosphoserine. Its catalytic activity de-
2+
pends on Ni ions, which is not common property.
Also it is not inhibited by typical inhibitors of PPP
phosphatases: sodium orthovanade and ocadic acid.
This phosphatase also shows in vitro activity of asym-
metrical Ap4A hydrolase and ATP-ase.
In this work we present results of transcription analy-
sis in the region of B. subtilis genome encoding protein
phosphatase PrpE.
The PrpE gene is a single open reading frame localised in
the 106 of B. subtilis chromosome. Preliminary computer
analysis showed presence of at least six potential promot-
ers in this region. Analysed fragment was cloned to the vec-
tor harbouring reporter gene coding for thermostable
beta-galactosidase and integrated into the amyE locus of
the B. subtilis chromosome. Obtained results confirmed
presence of the promoter in the PrpE region. The promoter
becomes activated during the osmotic stress. Results of ex-
periments with strains deleted for prpE and sigB genes
provide indication that this phosphatase is involved in re-
sponse to changing environment osmolarity. Elucidation
of the conditions in which expression of the prpE gene be-
comes activated may help in establishing the cellular role of
this protein phosphatase.

295 Poster
Transcription in the prpC-yloQ region with conserved genes layout
“phosphatase-kinase-essential gene” in Bacillus subtilis
Adam Iwanicki, Krzysztof Hinc, Micha³ Obuchowski
Katedra Biologii Molekularnej, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

Project of the genome sequencing of the gram posi-
tive soil bacterium Bacillus subtilis has been finished in
1997. During realisation of this task a putative tran-
scription unit spanning from the yloI to the yloS gene
was identified. Within this region of B. subtilis chromo-
some 11 putative open reading frames were found.
Four of them have been cloned and their products have
been characterised. These are PriA primosomal replica-
tion factor Y, Def polypeptide deformylase, PrpC pro-
tein Ser/Thr phosphatase and PrkC protein Ser/Thr
kinase. The gene prpC overlaps 3 bp with the next gene,
prkC. Product of these genes may regulate one or more
cellular processes, since autophosphorylated form of
the PrkC kinase is a good substrate for PrpC
2003 39th Meeting of the Polish Biochemical Society 167

phosphatase. Genes encoding these enzymes are
co-transcribed and form with adjacent gene yloQ a con-
served genes layout phosphatase-kinase-gene. Product
of the yloQ gene is a protein of unknown function that
does not have any known homologues. It appears to be
the essential gene in some specific conditions (growth
in the minimal medium).
In this work we map the promoter which is responsi-
ble for transcription of the pair of genes prpC and prkC.
We also show, that yloQ gene is transcribed from the
promoter located 7 bp upstream the start codon of the
YloQ protein. This promoter was previously shown to
be induced by the ethanol stress. Presence of the se-
quence similar to the sigma H consensus near the start
site of transcription suggests that this is sigma
H-dependent promoter. We show that transcription of
the yloQ gene depends neither on this sigma factor nor
sigma B, which is involved in transcription of general
stress response genes.

296 Poster
Molecular diagnostics of medullary thyroid cancer in Polish patients
1 2 3 4 2
Marta Kaczmarek , Katarzyna Ziemnicka , Justyna Hoppe-Golebiewska , Karolina Wielgus , Jerzy Sowinski ,
4
Ryszard Slomski
1 — Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszynska 32, 60-479 Poznañ, 2 — Clinics of Endocrinology,
University of Medical Sciences, Poznañ, 3 — Delta Pharma BV, Delta Pharma BV, Poznañ, 4 — Department of Biochemistry and
Biotechnology, Agricultural University, ul. Wo³yñska 35, 60-637 Poznañ, Poland

Medullary thyroid carcinoma (MTC) occurs in famil-
ial and sporadic forms and is the major feature of the
multiple endocrine neoplasia type 2 syndromes (MEN
2). Hereditary form of MTC may occur as familiar
medullary thyroid cancer (FMTC) or more commonly is
associated with phaeochromocytoma and hyper-
parathyroidism in multiple endocrine neoplasia type
2A (MEN 2A) and also with mucosal neuromas,
ganglioneuromatosis of the gastrointestinal tract in
multiple endocrine neoplasia type 2B (MEN 2B). It is
an autosomal dominant cancer syndrome, caused by
mutation in RET protooncogene, mapped to the
centromeric region of chromosome 10q11.2. Proto-
oncogene RET encodes a member of the receptor tyro-
sine kinase family of transmembrane receptors and is
expressed in various tissues as thyroid, adrenal, devel-
oping kidney, nerve tissue and in some human
neoectodermal tumors as well. It is possible that RET
play also a role in the differentiation and proliferation
of neural cells.
In case when the medullary thyroid cancer is recog-
nized, genetic tests are performed, independently from
data obtained on basis of family interview and physical
recognition, indicating hereditary form of cancer. It is
estimated that individuals with negative interview in
direction of hereditary form show 10% probability of ge-
netic predisposition.
Genetic tests include analysis of mutation in RET
protooncogene in DNA obtained from whole blood lym-
phocytes. Positive results prove directions to testing
patient’s families. In our laboratory at Institute of Hu-
man Genetic in Poznañ we perform screening analysis
of six exons of RET gene (10, 11, 13, 14, 15, 16) by
PCR-SSCP analysis with using fluorescent-labeled
primers as it enabled analysis of many samples in rela-
tively short time. Every detected change in SSCP band
pattern predisposed to sequencing.
Group of 168 individuals was analyzed, including pa-
tients diagnosed and hospitalized in Clinics of Endocri-
nology at University of Medical Sciences in Poznañ and
their families. The group included families with FMTC,
MEN 2A syndrome. The most frequently identified
changes occurred in exon 11 (26 changes) and in exon
10 (21 changes). Occasionally we detected irregulari-
ties in exon 13, 14 and 16.
Virtually all patients with FMTC, MEN 2A and MEN
2B develop MTC, what is a clear rationale for perform-
ing thyreoidectomy as soon, as RET mutation has been
identified. Statistically in families of patients with he-
reditary form of MTC, 50% of 1st degree relatives are
carriers of mutations, and genetic analysis should be
performed for all 1st and 2nd degree relatives. Conse-
quently full medical characteristics describing develop-
ment of the disease should be performed immediately
to consider necessity of surgery. In case when clinical
symptoms are not present prophylactic thyroidectomy
should be considered, as much better solution than fre-
quent monitoring of blood calcitonin level.
2003 39th Meeting of the Polish Biochemical Society 168

297 Poster
Detection of the mutations in the methylenetetrahydrofolate reductase (MTHFR) and
cystathionine b-synthase (CBS) genes
1 2 3 3 4
Leszek Kadziñski , Grzegorz Wêgrzyn , Zyta Banecka-Majkutewicz , Walenty Nyka , Wojciech Sawu³a ,
4 5
Bogdan Banecki , Joanna Jakóbkiewicz-Banecka
1 — Department of Molecular Biology, University of Gdañsk, ul. K³adki 24, Gdañsk, 2 — Katedra Biologii Molekularnej,
Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk, 3 — Department of Neurology, Medical University of Gdañsk, Gdañsk, 4 —
Department of Molecular and Cellular Biology, University of Gdañsk, Gdañsk, 5 — Genetics and Marine Biotechnology
Department, Institute of Oceanology PAS, Gdynia

Methylenetetrahydrofolate reductase (MTHFR) is an pitalised in the Department of Neurology of the Medi-

enzyme involved in the metabolism of homocysteine.
The homocysteine is formed by demethylation of the es-
sential amino acid methionine, and it can be further
metabolized by cystathionine beta-synthase (CBS).
Homocysteine may be remethylated to methionine in
the presence of methyl-cobalamin (Me-Cbl) and
5-methyl-tetrahydrofolate (Me-THF) as a cosubstrate.
The production of Me-THF requires proper function of
MTHFR and the presence of an adequate supply of re-
duced folate. Some mutations in the MTHFR and CBS
genes lead to reduction of the activity of MTHFR and
CBS enzymes and as a result to increase total homo-
cysteine level in serum or plasma.
It has been shown that an elevated level of homo-
cysteine (hyperhomocysteinemia) is a risk factor for ar-
terial and venous thrombosis.
We isolated DNA from patients’ EDTA anticoagu-
lated whole blood samples. All of the subjects were hos-
cal Academy in Gdañsk, with patients being the sub-
jects in the acute phase of ischemic stroke.
Patient DNA is assayed for the C677T and A1298C
mutations in the MTHFR gene as well as for mutations
in the CBS gene by PCR and restriction enzyme analy-
sis. Obtained PCR products and restriction fragments
are easily detected and separated with a 2.5% agarose
gel electrophoresis. These results are compared with
the homocysteine level for each patient.
Heterozygotic genotype C677T of MTHFR was de-
tected in 24% of patients, A1298T in 52%. However only
1% of homozygotic C677C and 15% of T1298T were de-
tected. In 60% of subjects at least one heterozygotic mu-
tation was detected. Hyperhomocysteinemia
(>14 mmol/l) was correlated with mutations in either
gene (p=0.04). Additionally, significantly higher homo-
cysteine level in homozygotes than in heterozygotes
was detected (p=0.01).

298 Poster
Reconstituted mononucleosome as a model to study proteins binding to DNA dam-
aged by cis-platinum
1 2 1
Magdalena Kalinowska , Monika Pietrowska , Piotr Wid³ak
1 — Center of Oncology, Department of Experimental and Clinical Radiobiology, Wybrze¿e Armii Krajowej 15, 44-100 Gliwice, 2
— Zak³ad Radiobiologii Doœwiadczalnej i Klinicznej, Centrum Onkologii w Gliwicach, ul. Armii Krajowej 15, 44-100 Gliwice

Proteins that recognize and bind to damaged DNA
participate in all repair pathways. Another group of
damaged DNA-binding proteins (DDB-proteins) are
chromatin proteins (e.g. HMG-1/2), that do not take
part in the repair directly yet modulate its efficiency. In
contrast to interactions between DDB proteins and na-
ked DNA interactions between such proteins and
chromatin are poorly understood. The aim of presented
work was to evaluate the feasibility of using in vitro re-
constituted mononucleosomes to detect DDB-proteins.
Mononucleosomes were reconstituted in vitro using
core histones purified from rat hepatocytes and DNA of
the Lytechinus variegatus 5S rRNA gene. A 195bp re-
32
striction fragment was end-labeled with P-g ATP, pu-
rified from acrylamide gel and damaged with
cis-platinum (cis-DDP) before chromatin reconstitu-
tion. DNA was damaged by 24 hours incubation with 3
and 30 mM cis-DDP. Estimated level of the damage was
about 1 and 20 of platinum-adducted nucleotides per
DNA molecule, respectively. The ratio between his-
tones and DNA was established experimentally to get
optimal mononucleosome recovery. Either naked DNA
or reconstituted mononucleosomes were complexed
with nuclear extracts from rat hepatocytes in the pres-
ence of ATP. DNA-protein complexes were analyzed us-
ing Electrophoretic Mobility Shift Assay (EMSA) on 4%
native polyacrylamide gels.
2003 39th Meeting of the Polish Biochemical Society 169

DNA damaged by cis-DDP was bound with high strin-
gency by proteins from nuclear extracts. These
DDB-DDP proteins were identified as HMG-1/2 by
co-migration with complexes between damaged DNA
and purified HMG. The presence of platinum-adducted
nucleotides affected stability of the mononucleosomes
— at higher level of the damage mononucleosomes were
disrupted during the storage. When mononucleosomes
formed on DNA with lower level of the damage were in-
cubated with nuclear extracts this changed the nature
of protein-DNA complexes detected on native gels.
Bands specific for mononucleosomes were replaced
with bands specific for complexes between DNA and
DDP-DDB proteins. This suggests that binding of HMG
proteins to cis-DDB-damaged DNA resulted in disrup-
tion of a nucleosome formed on such damaged tem-
plate.
The project was supported by the State Committee for Scien-
tific Research (KBN, Poland) grant no. 3P05A 10524.

299 Poster
Properties of methylenetetrahydrofolate reductase (MetF) from Escherichia coli
— in vivo assay
1 2 3 4
Anna Kloska , Joanna Jakóbkiewicz-Banecka , Bogdan Banecki , Grzegorz Wêgrzyn
1 — Department of Molecular Biology, University of Gdañsk, ul. K³adki 24, Gdañsk, 2 — Genetics and Marine Biotechnology
Department, Institute of Oceanology PAS, Gdynia, 3 — Department of Molecular and Cellular Biology, University of Gdañsk,
Gdañsk, 4 — Katedra Biologii Molekularnej, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

Methylenetetrahydrofolate reductase (MTHFR) is an
essential enzyme involved in methionine metabolic
pathway. Two common polymorphisms of the human
MTHFR have been identified. One of them is the C677T
(Ala222Val) polymorphism that is associated with in-
creased thermolability of the enzyme and with 60% loss
of its activity. This polymorphism is the cause of
hyperhomocysteinemia — a risk factor for cardiovascu-
lar disease.
The methylenetetrahydrofolate reductase from Esch-
erichia coli (MetF) is highly homologous with the hu-
man MTHFR. Also the C530T (Ala177Val) mutation in
E. coli MetF is analogous to the C677T mutation in hu-
man MTHFR.
In our studies we used the bacterial MetF reductase
as a model to investigate the C530T mutation influence
on the enzyme activity and the effect of the cofactors
presence (folic acid, vitamin B6 and B12) to suppress
the effect of mutation in in vivo assay.
We constructed the mutant strain (E. coli delMetF2)
with the disrupted chromosomal copy of metF gene. To
investigate the function of the MetF reductase we de-
signed and constructed series of plasmids containing
the wild-type metF gene and the metF gene with the
C530T mutation (metF177) under the control of differ-
ent inducible promoters (plac, pTc, para).
The E. coli delMetF2 strain was transformed by these
plasmids and we are investigating the temperature ef-
o o o o
fect (25 C, 30 C, 37 C, 43 C) and cofactor suppression
on the bacterial growth in strains containing the wild
type metF, the mutant metF177 and in the heterozy-
gous strain with both wild-type metF and mutant
metF177.
Deletion of the chromosomal copy of metF results in E.
coli delMetF2 strain becoming a methionine auxotroph,
so it is unable to grow in minimal medium lacking
methionine. The presence of plasmids containing the
wild-type metF supports the growth in medium lacking
methionine in every temperature. The strain with
plasmid carrying the mutant metF177 gene is unable to
o o
grow in 37 C and 43 C. The growth is also poorly sup-
o o
ported in lower temperatures (25 C and 30 C).

300 Poster
Radiation-induced bystander effects in human leukemic K562 cells in vitro
1 1 1 2 3
Maria Konopacka , Jacek Rogoliñski , Robert Herok , Andrzej Orlef , Krzysztof Fujarewicz , Joanna
3 3 1
Polañska , Andrzej Œwierniak , Joanna Rzeszowska-Wolny
1 — Zak³ad Radiobiologii Doœwiadczalnej i Klinicznej, Centrum Onkologii w Gliwicach, ul. Armii Krajowej 15, 44-100 Gliwice,
2 — Department of Medical Physics, Center of Oncology, Maria Sk³odowska-Curie Memorial Institute, Gliwice, 3 — Institute of
Automation Control, Silesian University of Technology, Gliwice

One of the biological effects of radiation is phenome- netic damage occuring in cells that were not directly ir-
non that was termed “bystander effect”. It involves ge- radiated but responded to signals transmitted from ir-
170 Session 5. Structure and function of DNA
radiated cells. A number of studies suggest that cell-cell
communication plays a role in mediating this phenome-
non.
In the present study we tested the medium-mediated
bystander effects in non-irradiated K562 cells. The ge-
netic changes were estimated as a chromosomal dam-
age as well as a alterations of gene expression.
Chromosomal damage. The cultures of human leukemic
K562 cells were exposed to X-radiation (dose of 4 Gy). Af-
o
ter one-hour incubation at 37 C the medium (ICM) was
remowed, filtered and transferred to non-irradiated flasks
containing cells from the same line.
Medium transfered from irradiated cells to non-irra-
diated ones reduced the number of viable cells after 3
days of incubation. The increase of apoptotic and
micronucleated cells was observed after conditioned
medium (ICM) exposure.
Alterations of gene expression. In order to solve the
problem whether cells grown in conditioned medium
301
Abnormal nucleoid distribution and cell division in cells overproducing partition
proteins of P1 plasmid
Krystyna Krajewska-Grynkiewicz, Ma³gorzata £obocka
Instytut Biochemii i Biofizyki, PAN, ul. Pawiñskiego 5a, 02-106 Warszawa
Partition of low copy number plasmids resembles mi-
tosis in that prior to cell division sibling DNA molecules
are actively moved apart from the center of the mother
cell into positions corresponding to the centers of fu-
ture daughters. In the case of P1 it depends on plasmid
proteins, ParA and ParB, and a centromeric site in
plasmid DNA. ParA and ParB belong to families of con-
served plasmid and chromosome partition proteins.
Proteins of ParB family interact with their target
centromeric sites in DNA. Proteins of ParA family are
Walker-type ATPases involved in plasmid trans-
location within a cell. They bind to centromeric sites via
their partner ParB proteins. To find out whether the P1
partition proteins can compete with bacterial proteins
for a common target in a host cell we looked for effects
of their overproduction on the cell cycle. Overproduc-
tion of ParA caused cell filamentation. Nucleoid DNA
in the majority of cells was decondensed and uniformly
distributed. Surprisingly, cells overproducing ParA
2003
(so called “bystander cells”) have a genome-wide ge-
netic expression profile similar or distinct with respect
to directly irradiated cells, we examined the genetic ex-
pression profiles of these cells using oligonucleotide ar-
rays that contained probes representing 22 283 human
genes. Using hierarchical clustering analysis we sepa-
rated from all analyzed genes some groups of genes
with up- (or down-) regulated expression.
The results suggest that for genotoxic damages seen in
cells growing in conditioned medium responsible are pro-
teins participating in the folowing processes, for example:
apoptosis, cation transport, DNA repair, autocrine medi-
ating IL1 and TNF cytokines, protein-protein interac-
tions, regulation chromatin structure and gene transcrip-
tion, mRNA synthesis and maturation, glycolysis.
This work was partially supported by the State Committee for
Scientific Research (KBN, Poland) Grants: No. 4 PO5A 015
190, No. 4T11F 01824, and PBZ-KBN-040/PO4/2001, in
2003 year.
Poster
and ParB differed from those overproducing ParA.
Their nucleoids were only slightly decondensed, but
nucleoid DNA was distributed unevenly, often concen-
trated in two or more regions of different size sepa-
rated by unequal “empty” regions. Many prolonged
cells contained single or multiple cell division septa,
which were positioned abnormally, usually at regions
of lower DNA concentration. Sometimes, guillotining
of DNA by cell division septa occurred. Cells overpro-
ducing ParB alone did not differ from normal cells, in-
dicating that ParB influenced cell divisions and
nucleoid distribution via interaction with ParA. Nei-
ther the effect of ParA alone nor ParA and ParB on dis-
tribution of nucleoid DNA was reversed by cephalexin,
an inhibitor of cell division. Thus it is likely that the pri-
mary target of P1 ParA, or ParA and ParB in cells over-
producing those proteins may be a component of
nucleoid condensation or partitioning machinery.
2003 39th Meeting of the Polish Biochemical Society 171

302 Poster
Application of polymerase chain reaction (PCR) amplification in estimation of
methylation status of selected DNA fragments
Barbara Krawczyk, Dorota Wyczechowska, Krystyna Fabianowska-Majewska
Zak³ad Chemii Medycznej IFiB, Uniwersytet Medyczny, ul. Mazowiecka 6/8, 92 215 £ódŸ

DNA methylation as epigenetic modification is one of the
combined mechanisms of gene expression regulation, a fac-
tor of chromatin structure stability and is a requisite of nor-
mal cell development. For the last 10 years, numerous
studies of cancer cells were focused on hypermethylation of
promoter regions of tumour-suppressor genes or tis-
sue-specific genes. Promoters of tumour-suppressor genes
are characterized by extended density of CpG sequences
(i.e. CpG rich islands) or CCGG sequences. Examination of
differences between DNA methylation level in normal and
cancer cells can concern both genomic DNA (for example,
estimation of C-5 methyltransferase activity or genomic
ability to accept methyl groups) and any fragment of a se-
lected gene.
Current studies are designed to adopt a simple
Iwase’s method (MSP, methylation-specific polymerase
chain reaction) (Iwase et al. Brit J Cancer, 1999; 80:
1982–1986) to estimate the level of methylated CpG se-
quences in, for example, a fragment (347 pb) of estro-
gen receptor alpha (ER alpha) gene. The analysis con-
sists of three steps: 1) digestion of DNA (isolated from
K562 cells which were cultured for 48 hr) with restric-
tion enzymes, HpaII and BstU1, which recognize
unmethylated CCGG and CGCG sequences, respec-
tively; 2) amplification (PCR) of digested fragment of
ER alpha gene; 3) electrophoretic analysis of amplified
DNA fragments on polyacrylamide gel dyed with
ethidium bromide.
The results demonstrate that when the fragment of
the gene contains methylated CpG sequences, DNA
samples are not recognized and digested by the restric-
tion enzymes (HpaII and BstU1), the fragment of the
gene is amplified and bands on the gel corresponding to
347 pb are detected. In contrast, the unmethylated frag-
ment of the gene was digested and no fragments was
amplified.
The adopted MSP analysis of the level of methylated
CpG sequences is a simple method which, firstly, elimi-
nates laborious bisulfite DNA modification and sec-
ondly, may be a useful diagnostic tool to estimate
methylation status of any fragment of tumour-sup-
pressor genes in cancer cells.

303 Poster
The contribution of the promoter –35 and –10 elements to RNA polymerase binding
Katarzyna Lewandowska, Ma³gorzata Marcyjaniak, W³adys³aw Werel
Katedra i Zak³ad Mikrobiologii Farmaceutycznej, Akademia Medyczna w Gdañsku, ul. Gen. Józefa Hallera 107, 80-416 Gdañsk

Transcription initiation is the first step in one of the
most important processes taking place in a living cell.
At this stage the RNA polymerase holoenzyme recog-
nizes specific DNA sequences, called promoter se-
quences, which contain two conserved regions
(hexamers) –35 and –10. Although a wealth of work
has been done in this field we still do not fully under-
stand the mechanism of promoter DNA-RNA polymer-
ase interaction, function and contribution of each
hexamer to enzyme binding.
In order to determine the importance and influence
of the promoter –35 and –10 regions on RNA polymer-
ase binding, we constructed various promoter DNA
fragments which lack the –35 element or contain –10
hexamer modified entirely or in part. Functional char-
acteristic of the obtained promoter fragments were de-
termined by analysis of the effectiveness of promoter
DNA — RNA polymerase complexes formation. For this
analysis we adopted gel mobillty shift assay on a native
polyacrylamide gel.

304 Poster
Gene constructs for xenotransplantation
1 2 2 1 2 2
Daniel Lipinski , Wojciech Juzwa , Joanna Zeyland , Andrzej Plawski , Karolina Wielgus , Ryszard Slomski
1 — Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszyñska 32, 60-479 Poznañ, 2 — Department of
Biochemistry and Biotechnology, Agricultural University, ul. Wo³yñska 35, 60-637 Poznañ, Poland

The presence in humans of xenoreactive antibodies face of xenograft donor cells, leads to the complement
directed against swine Gal antigen present on the sur- activation and immediate xenograft rejection — as a
2003 39th Meeting of the Polish Biochemical Society 172

consequence of hyperacute immunological reaction.
The Gal antigen (Gala1,3Gal) is synthesized by the
a1,3-galactosyltransferase. In aim to prevent hyper-
acute rejection it is possible to modify swine genome by
transfection of human genes — controlling enzymatic
cascade of complement or modifying set of the cell sur-
face proteins of the graft donor. For this purpose ge-
netic constructs containing human CD59, CD55 and
CD46 genes and human gene encoding a1,2-fuco-
syltransferase were prepared. CD59 blocks formation
of the membrane attack complex. CD46 blocks forma-
tion of C3 convertases in the classical and alternative
pathways of complement activation. CD46 has Factor I
cofactor activity, which cleaves and inactivates C4b and
C3b compounds. CD55 accelerates decay of C3 con-
vertases in the classical and alternative pathways. It
dissociates C2 and C2a from C4b and dissociates B and
Bb from C3b. Introduction of additional copies of hu-
man gene encoding a1,2-fucosyltransferase into the
swine genome leads to reduction of the Gal antigen
level on the surface of donor cells. This results in re-
duced binding of xenoreactive natural antibodies to en-
dothelial cells of xenograft and protection from comple-
ment mediated lysis.

305 Poster
Factors influencing the regulation of change of bacteriophage lambda DNA replica-
tion mode
1 1 2 1
Magdalena Narajczyk , Sylwia Barañska , Magdalena Gabig-Cimiñska , Grzegorz Wêgrzyn
1 — Katedra Biologii Molekularnej, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk, 2 — Laboratorium Biologii Molekularnej
(afiliowane przy Uniwersytecie Gdañskim), Instytut Biochemii i Biofizyki PAN, Gdañsk

During the live cycle of the bacteriophage lambda,
replication of its DNA occurs according to two different
modes: (i) early replication or theta mode followed by
(ii) late or sigma mode. Thanks to the theta mode of rep-
lication, lambda DNA can be synthesized and serve as a
template to produce phage proteins. Late mode of
lambda DNA replication produces lambda genomes
necessary to form complete phage progeny.
According to commonly accepted hypothesis, sigma
mode of replication may be preceded by one round of
theta unidirectional replication. Switch from the theta
to the sigma mode of replication requires a change
from bidirectional mode of theta replication to the uni-
directional replication. There are many hypotheses to
explain the role of different proteins and regulation of
the change from bidirectional to the unidirectional
theta replication mode. Most of them are based on in vi-
tro experiments.
We show in in vivo experiments the effect of some
proteins (ClpXP, lambdaP, lambdaCro) and oopRNA
on the switch from bidirectional to the unidirectional
theta mode of lambda plasmid DNA. Thus we conclude
that those factors influence the initiation of sigma rep-
lication.

306 Poster
DNAzyme to mRNA of the beta1 integrin subunit as an efficient tool to reduce
invasiveness of human carcinoma cells
1 1 1 2 3
Magdalena Nawrot , Jolanta Niewiarowska , Izabela Papiewska , Andrzej Okruszek , Maciej Ugorski , Czes³aw
4
Cierniewski
1 — Zak³ad Biofizyki Molekularnej i Medycznej, Uniwersytet Medyczny, ul. Mazowiecka 6/8, 92-215 £ódŸ, 2 — Centrum Badañ
Molekularnych i Makromolekularnych, Polska Akademia Nauk, £ódŸ, 3 — Instytut Immunologii i Terapii Doœwiadczalnej
im. Ludwika Hirszfelda, Polska Akademia Nauk, Wroc³aw, 4 — Centrum Mikrobiologii i Wirusologii, Polska Akademia Nauk,
ul. Lodowa 106, 92-215 £ódŸ

Integrins are known to mediate a variety of cellular
events including adhesion, migration, proliferation,
invasiveness, and cellular survival. These cellular
events play important roles in biological processes such
as angiogenesis, tumor growth and metastasis. It is be-
coming increasingly clear that particularly members of
the beta1 subfamily of integrins may play a crucial role
in all these processes. Therefore, we developed a novel
approach to downregulate beta1 integrins in several
cell lines and provided evidence that DNA enzymes can
be useful gene-inactivating agents to control expression
of membrane proteins. We designed the DNA enzyme
2003 39th Meeting of the Polish Biochemical Society 173

to beta1 mRNA containing a 15 deoxyribonucleotide
catalytic domain that was flanked by two substrate-re-
cognition segments of eight deoxyribonucleotides. To
engineer specific cleavage site in mRNA, the flanking
domains were derived from the most inhibitory oligo-
deoxynucleotide antisense to beta1, designated beta1
1053.
In preliminary experiments we found that such
DNAzyme significantly inhibited expression of the
beta1 integrin subunit in endothelial cells at the level of
mRNA and protein synthesis. They also reduced cell
surface expression of subunit in endothelial cells mea-
sured by flow cytometry and significantly affected ad-
hesion of the cells to fibronectin and vitronectin. Simi-
lar to initial antisense oligonucleotides, the DNAzyme
abolished microvascular endothelial cell capillary tube
formation in fibrin and matrigel. In several tests, the
DNAzyme and its analogues appeared to be strong in-
hibitors of adhesion and migration of malignant pros-
tate carcinoma cells (PC3) and various human colon
adenocarcinoma cells (HT29, CX1.1, SW620, LS180).
Furthermore, they blocked the invasiveness of malig-
nant cells through the barrier of matrigel matrix in in
vitro assay.

307 Poster
Synergistic effect of the p22 (phox) C242T polymorphism and ACE insertion/dele-
tion polymorphism in Silesian CAD patients
1 1 1 2 1
Pawe³ Niemiec , Beata Sarecka , Anna Balcerzyk , Zbigniew Ciemniewski , Iwona ¯ak
1 — Department of Biochemistry and Medical Genetics, Medical University of Silesia, ul. Medyków 18, 40-752 Katowice, 2 — The
First Department and Clinic of Cardiology, Medical University of Silesia, ul. Zio³owa 45/47, 40-635 Katowice

Background: Increased reactive oxygen species (ROS)
production is implicated in the pathogenesis of athero-
sclerosis and coronary artery disease (CAD). The
p22(phox) subunit is a critical component of NAD(P)H
oxidase, which plays an essential role in superoxide pro-
duction. However the NAD(P)H oxidase is a constitutive
enzyme, it can be also regulated by humoral factors,
such as angiotensin II. Angiotensin converting enzyme
(ACE) hydrolyses ang I into ang II and ACE plasma ac-
tivity is related to the insertion/deletion (I/D) polymor-
phism in the ACE gene. Therefore the ACE DD genotype
increases p22(phox) production and ROS generation.
Our previously published data indicate that carrier-state
of D allele in ACE gene I/D polymorphism is strongly as-
sociated with CAD in Silesians (p=0.0029).
Aim: In the present study we investigated allele fre-
quencies of p22(phox) polymorphism in the same pa-
tients and tried to find relationship between genotype
distribution of ACE gene I/D polymorphism and
p22(phox) gene C242T polymorphism in CAD patients
and controls.
Materials and Methods: Sixty six patients with
angiographically proven CAD, and 98 healthy controls
were studied. ACE and p22(phox) polymorphisms were
evaluated by PCR and PCR-RFLP (RsaI) respectively.
Results: The frequencies of p22(phox) genotypes in
cases and controls were respectively: CC 40.9% vs.
46.9%, CT 45.5% vs. 42.9% and TT 13.6% vs. 8.2%, but
these differences were not significant. The DD geno-
type of ACE gene I/D polymorphism was significantly
more frequent in CAD patients (p=0,027). Additionally,
we found differences in a number of subjects with both
homozygous genotypes (DD and TT). The frequencies
of double-homozygous genotypes in cases and controls
were respectively: 9.09 vs. 1.02 (p=0.017, OR=9.70; CI
1.10-222.84).
Conclusion: The coexistence of ACE DD genotype and
p22(phox) TT genotype is more common in patients
with CAD, than in controls and considerably increases
CAD risk. There is a synergistic effect of the ACE I/D
and p22(phox) C242T gene polymorphisms in Silesian
patients with coronary artery disease.

308 Poster
Characteristics of a second gene 27 product — 27 bis of bacteriophage T4
Józef Nieradko
Katedra Mikrobiologii, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

Gene 27 is a member of the main cluster of six genes that these six genes are cotranscribed from a common
coding for bacteriophage T4 baseplate constituents; 51, promoter located between the gene 26 and 51. These
27,28,29,48,54. Transcription experiments have shown genes were cloned into pT7-5 vector under control of T7
174 Session 5. Structure and function of DNA 2003

bacteriophage f10 promoter. The level of synthesis of
particular products was estimated in the strain E. coli
BL21 (DE3)pLysS, which differs from its original coun-
terpart BL21(DE3) by the presence of a plasmid coding
for T7 lysozyme and by being nonpermissive for the T4
amber mutants. These properties make possible exami-
nation of expression and estimation of complemen-
tation efficiency in the same culture. The examination
of expression of this hybrid and its deletion derivatives
was the base for the following conclusions: the 27 gene
of bacteriophage T4 encode two proteins of 44 and 39
kDa (designated 27-44 and 27-39bis, respectively) as a
result of independent translation initiation at two dif-
ferent start codons with the same reading frame. The
latter with molecular weight 39 kDa probably plays sig-
nificant role in regulation of expression of gene 51. The
44 kDa protein is the structural component of
bacteriophage T4 baseplate.

309 Poster
Microsatellite instability at RAD51 locus in breast cancer
1 1 2 1
Maria Nowacka , Magdalena Bryœ , Hanna Romanowicz-Makowska , Wanda Krajewska
1 — Department of Cytobiochemistry, University of Lodz, ul. Banacha 12/16, 90-237 Lodz, 2 — Department of Pathology, Institute
of Polish Mother’s Memorial Hospital, ul. Rzgowska 281/289, 93-338 Lodz

Identification of chromosomal regions with allelic
losses is a method for screening genes implicated in the
pathogenesis of human tumors. Loss of heterozygosity
(LOH) in the region 15q15.1 (microsatellite region with
tandem repeats CA) where gene RAD51 is localized,
may suggest that this gene can play a role in the devel-
opment of breast cancer and in evaluation of the patho-
logic features of the tumor.
LOH was determined in the region 15q15.1 of the
RAD51 gene in 30 primary breast cancers. Material for
analysis was obtained from paraffin-embedded tissues
and peripheral blood as control. After extraction of
DNA by the phenol-chloroform/proteinase K method
fragments of the RAD51 gene were amplified using
three microsatellite markers: D15S118, D15S214,
D15S1006. Fluorescent PCR products were analysed in
®
ABI PRISM 377 DNA Sequencer. Size and quantity of
®
allele markers were evaluated using GeneScan soft-
ware version 3.1.2. The nuclotide sequences of frag-
ments showing peak shifts were determined using the
®
ABI PRISM BigDye™ Terminator Cycle Sequencing
®
Kit and ABI PRISM 377 DNA Sequencer.
Allelic loss was detected in 11 of the 22 informative
cases for microsatellite marker D15S118, in 7 of the 15
informative cases for microsatellite marker D15S214,
in 4 of the 15 informative cases for microsatellite
marker D15S1006. LOH was observed in the 15q15.1
region for at least one microsatellite marker in 27% of
breast cancer studied cases.

310 Poster
Polimorphism of XPD gene coding for nucleotide excision repair protein and the
risk of head and neck cancers
1 2 2 3 2
Joanna Polañska , Joanna Jaworska , Monika Pietrowska , Dorota Butkiewicz , Joanna Rzeszowska-Wolny
1 — Institute of Automation Control, Silesian University of Technology, Gliwice, 2 — Zak³ad Radiobiologii Doœwiadczalnej i
Klinicznej, Centrum Onkologii w Gliwicach, ul. Armii Krajowej 15, 44-100 Gliwice, 3 — Department of Tumor Biology, Center of
Oncology, Maria Sk³odowska-Curie Memorial Institute, ul. Wybrzeze AK, Gliwice

We investigated the frequency of single nucleotide
substitutions changing Asp to Asn in codon 312 or Lys
to Gln in codon 751 of the protein XPD in the group of
127 donors, 84 healthy and 43 patients with head and
neck cancers. To detect differences in polymorphic
variant distribution between healthy donors and pa-
tient groups we used the Fisher-Freeman-Halton per-
mutation test. This test did not show significant differ-
ences in distribution of codon 312 variants between the
analyzed subgroups (exact p=0.9554). Similar analysis
performed for codon 751 showed significant differ-
ences between them (exact p=0.0450). We observed sig-
nificant departures from Hardy-Weinberg equlibrium
(HWE) (p<0.0001) at that site among cancer patients
and healthy donors. A significant excess of hetero-
zygosity was observed in cancer patients (observed
value 0.8140, expected value 0.4884). The estimated
value of odds ratio OR was equal to 2.692 (exact
p=0.0328) with 95% CI from 1.047 to 7.525. The results
suggest that heterozygotic genotype at XPD 751 codon
could be the cancer risk factor for head and neck can-
cers.
The project was supported by the State Committee for Scien-
tific Research (KBN, Poland) grant no. 4P05A 01519
2003 39th Meeting of the Polish Biochemical Society 175

311 Poster
Genetic instability of (CT/AG) motifs isolated from V. bithynica
1 2
Tomasz Sakowicz , Pawe³ Parniewski
1 — Zak³ad Cytogenetyki i Biloogii Molekularnej Roœlin, Uniwersytet £ódzki, ul. Banacha 12/16, £ódŸ, 2 — Cent. Mikro. i Wirus.
PAN, Akademia Œwiêtokrzyska, Kielce

Plant genomes are extremely rich in DNA repetitive
sequences. Of these repeats microsatellites, stretches
of short, tandemly repeated motifs of 1–6 bp are very
unstable and display very high polymorphism. We
found such sequences are also present in Vicia bithynica
genome. Based on the screening of DNA library, clones
harbouring long tracts of CT/AG motifs have been se-
lected. Sequence analyses precisely revealed the length
and class of the repeating unit, number of repeats, as
well sequence composition flanking repeating motifs.
Representative plasmids were used to investigate their
genetic instability. In the study presented herein, we
employed very well identified and tractable bacterial
genetic system. Wild type and methyl-directed mis-
match repair defective strains have been used to dem-
onstrate that the genetic instability of such micro-
satellites depends primarily on their length and some
factors influencing transcriptions through the repeti-
tive motifs. These observations are in agreement with
similar studies performed on human repetitive se-
quences.

312 Poster
Mutations G98T and A561C of the E-selectin gene are not associated with coronary
artery disease in Silesian patients
1 1 1 2 3 3
Beata Sarecka , Pawe³ Niemiec , Anna Balcerzyk , Zbigniew Ciemniewski , Ewa Rudowska , Stanis³aw Dyl¹g ,
1
Iwona ¯ak
1 — Department of Biochemistry and Medical Genetics, Medical University of Silesia, ul. Medyków 18, 40-752 Katowice, 2 — The
First Department and Clinic of Cardiology, Medical University of Silesia, ul. Zio³owa 45/47, 40-635 Katowice, 3 — Section of
Hematology, Regional Blood Centre, ul. Raciborska 15, 40-074 Katowice

Background: E-selectin mediates in the adhesion of
leukocytes into endothelium. An increased expression
of E-selectin has been observed in activated arterial en-
dothelium and appeares to be involved in the
pathogenesis of atherosclerosis. Several studies have
demonstrated that the G98T and the A561C mutations
in the E-selectin gene are a strong risk factors for early
severe atherosclerosis. Some authors suggest also high
correlation between the G98T and A561C mutations.
Aim: The aim of our study was to investigate relation-
ships between the E-selectin G98T or A561C mutations
and coronary artery disease (CAD) in the Silesian pa-
tients.
Materials and Methods: Sixty four patients with
angiografically documented CAD, and ninety nine
blood donors as control group were studied. The G98T
and A561C mutations were analyzed by PCR-RLFP
(HphI and PstI respectively).
Results: The allele frequencies of G98T in cases and
controls were respectively: G 85.9% vs. 86.4%, T 14.1%
vs. 13.6%. The frequencies of genotypes of the G98T
polymorphism were 75% GG, 21.9% GT and 3.1% TT in
the patients and 75.8% GG, 21.2% GT and 3% TT in the
controls. The allele frequencies of A561C in cases and
controls were respectively: A 85.2% vs. 84.8%, C 14.8%
vs. 15.2%. The distribution of A561C genotypes were
73.4% AA, 23.4% AC and 3.1% CC in the patients and
72.7% AA, 24.2% AC and 3% CC in the control group.
Differences in the frequencies of alleles and genotypes
between CAD patients and controls were not statisti-
cally significant. The frequencies of both C and T muta-
tive alleles are higher in control of blood donors than in
other previously published data. Despite the observa-
tion that there was no significant relationship between
both mutative genotypes and the disease we found that
the G98T mutation is highly correlated with the A561C
mutation (p<0.001; correlation coefficient 0.9553) as
well in patients as in control group.
Conclusion: Our data demonstrate that there is no as-
sociation between E-selectin G98T or A561C mutations
and coronary artery disease in the Silesian patients.
176 Session 5. Structure and function of DNA 2003

313 Poster
Limited use of the Cre/loxP recombination system in efficient production of
loxP-containing minicircles in vivo
Marian Sêktas, Maciej Specht
Katedra Mikrobiologii, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

The Cre/loxP recombination system of bacteriophage
P1 is one of the most powerful tools in genome engi-
neering. We report, however, that activity of Cre/loxP
system interferes with stability of the multicopy
loxP-bearing plasmids. Intermolecular recombination
reduces copy number of plasmids and eventually in-
creases their segregational instability. Due to predomi-
nantly unidirectional Cre-mediated high-order
multimer formation of those plasmids, the number of
their copy (overall yield) gradually decreases. We have
found that in the presence of even the slightest amount
of Cre activity, loxP-bearing plasmids continuously un-
dergo multimerization, which leads to loxP-plasmid
free cells very rapidly. Our results are compatible with
the hypothesis of the multimer catastrophe (Summers
and Sherratt, 1984, Cell 36: 1097–1103).

314 Poster
Sequencing of juvenile hormone binding protein (jhbp) gene from Galleria mellonella
Agnieszka Sok, Kamila Czajewska, Andrzej O¿yhar, Marian Kochman
Zak³ad Biochemii, Politechnika Wroc³awska, ul. Gdañska 7/9, 50 344 Wroc³aw

The juvenile hormone (JH) controls insect growth, de-
velopment and reproduction. In insect larvae JH blocs
expression of subset of genes responsible for larvae
metamorphosis, whereas in adult insects activates ex-
pression of genes that are necessary for reproductive
maturation. A term JH refers to six closely related
sesquiterpenes containing an ester bond and epoxy
bond required for the hormone regulatory function. JH
is produced in corpora allata and transferred to target
cells by juvenile hormone binding protein (JHBP). In
the hemolymph more than 99.9% of hormone(s) ap-
pears in a complex with JHBP. This protein protects
the hormone molecule against action of non-specific hy-
drolyses and prevents its non-specific binding to hydro-
phobic surfaces lining a hemolymph circulatory sys-
tem. JHBP(s) from Lepidoptera are low molecular
weight (25–30 kDa) monomeric proteins with one
JH-specific binding site.
In our laboratory it has been previously demon-
strated that molecule of JHBP from G. mellonella
hemolymph is a glycoprotein containing two disulfide
bonds and that JH binding induces a profound
conformational change of the protein molecule which
might have a significance for signal transmission. Re-
cently we have sequenced cDNA JHBP from G.
mellonella (Gene Bank accession number AF4107772).
Crystallization and preliminary crystallographic data
were obtained in cooperation with Dr M. Jaskolski and
dr G. Bujacz laboratories. Although JHBP(s) have been
isolated and characterized from several insect species,
the amino acid sequences are known for four proteins
and a gene sequence has been reported only for
Manduca sexta jhbp, just a few months ago.
In this report we present the studies on jhbp gene se-
quence from G. mellonella. It appears that the gene
transcript of G. mellonella jhbp contains a characteris-
tic sequence of the initiator (Inr), the downstream pro-
moter element (DPE) and four introns, similarly to
M. sexta jhbp. However, the introns positioning and
their length differ between the two genes. The donor
and acceptor sequences of all exon-intron boundaries
conform to the GT/AG rule. Assuming the first nucleo-
tide coding JHBP from G. mellonella as number one, po-
sitions of introns I, II, III, and IV appear between 55
and 56 nt, 296 and 297 nt, 420 and 421 nt, and 573 and
574 nt, respectively. Their lengths, calculated from
electrophoretic mobility, are close to 5 kbp, 3 kbp, 1.2
kbp, and 0.8 kbp for I, II, III, and IV intron, respec-
tively. At the moment the whole sequence of IV intron
(860 nt) and about 500 nt sequences from each site (5’
and 3’) of remaining introns were elucidated. The se-
quences of all introns will be presented.
This work was supported by the State Committee for Scien-
tific Research (KBN, Poland) grant 3 P04A 003 23)
2003 39th Meeting of the Polish Biochemical Society 177

315 Poster
Searching for Arabidopsis thaliana mutants resistant to Agrobacterium-mediated
transformation
1 2 1
Wojciech Strza³ka , Stanton Gelvin , Alicja Ziemienowicz
1 — Zak³ad Genetyki Molekularnej, Wydzia³ Biotechnologii, Uniwersytet Jagielloñski, ul. Gronostajowa 7, 30-387 Kraków,
2 — Dept. of Biological Sciences, Purdue University, West Lafayette, USA

Development of the advanced techniques of molecu-
lar genetics resulted in the new methods for studying
genomes of various complex organisms. Such a prog-
ress gave researchers powerful tools like forward ge-
netic screening for studying function of many unknown
genes. Recently, it has been widely engaged for an iden-
tification of the plant factors responsible for
Agrobacterium-mediated transformation.
Agrobacterium tumefaciens is a plant pathogen charac-
terized by an unique feature of interkingdom DNA
transfer. This bacterium possesses the ability to trans-
fer a large fragment of the resident Ti-plasmid (T-DNA
— transferred DNA) to the plant cell, where T-DNA is in-
tegrated into the host genome. Such integration may
result in a induction of crown gall disease on
dicotyledonus plants due to the expression of T-DNA
genes encoding enzymes involved in opines and plant
hormones synthesis. Although the Agrobacterium fac-
tors involved in plant transformation are well charac-
terized, our understanding of the T-DNA transport and
integration process into the plant genome still remains
incomplete.
The aim of this study is identification of A. thaliana
RAT (Resistant to Agrobacterium-mediated Transfor-
mation) mutants in order to give more light on the pro-
cess of T- DNA transfer from the bacteria to the plant
cell.
In this project we use a pool of A. thaliana T-DNA
tagged lines (Bressian collection). Sterilized seeds are
germinated in a growth chamber followed by cultiva-
tion of seedlings for next 10 days. After this period of
time roots are cut, infected with A. tumefaciens At10,
cocultivated on MS basal plate, and then scraped off on
a surface of MS basal plate containing timentin. After 6
weeks the grown tumors are scored. From a pool of 400
tested independent lines 120 candidates were chosen
for second screening and it resulted in 5 potential RAT
mutants that are likely to show defects in transforma-
tion process. These defects might be at different stages
of plant-bacterium interaction including: attachment of
the bacteria to the plant cell, T-DNA transfer, T-DNA
nuclear import and integration. The selected candi-
dates will be tested by Southern blot analysis, and the
plant DNA sequence interrupted by the T-DNA insert
will be identified.
Identification of RAT mutants will wide our knowl-
edge about plant factors that are essential for the pro-
cess of Agrobacterium-mediated transformation.

316 Poster
Regulation of transcription by the SeqA protein and distribution of GATC sequences
in a promoter region
1 1 1 2
Barbara Strzelczyk , Monika S³omiñska , Grzegorz Wêgrzyn , Alicja Wêgrzyn
1 — Katedra Biologii Molekularnej, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk, 2 — Pracownia Biologii Molekularnej
(afiliowana przy UG), Instytut Biochemii i Biofizyki PAN, ul. K³adki 24, 80-822 Gdañsk

The SeqA protein of Escherichia coli is not only the
main negative regulator of DNA replication initiation
but also a specific transcription factor. It binds to
hemimethylated GATC sequences and, with somewhat
different specificity, to fully methylated GATC regions.
Recently, a microarray analysis was reported, in which
transcriptomes of wild-type and delta seqA strains were
compared. Although in the seqA mutant the levels of
some transcripts were significantly decreased while
certain transcripts were evidently more abundant rela-
tive to wild-type bacteria, no correlation between the
presence of GATC motifs in promoter sequences and
transcription activity was found. However, here we
show that when larger DNA fragments, encompassing
positions from –250 to +250 relative to transcription
start site, are analyzed, some common features of
GATC distribution near promoters activated by SeqA
can be demonstrated. Nevertheless, it seems that
GATC pattern is not the only determinant of
SeqA-dependence of promoter activity.
178 Session 5. Structure and function of DNA 2003

317 Poster
Biological activity of purified hGH, TNFa and FeldI recombinant proteins expressed
in bacterial and eukaryotic systems
1 2 3 4 5 1
Marlena Szalata , Daniel Lipinski , Robert Kalak , Paulina Tobola , Marek Pienkowski , Ryszard Slomski
1 — Department of Biochemistry and Biotechnology, Agricultural University, ul. Wo³yñska 35, 60-637 Poznañ, Poland,
2 — Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszyñska 32, 60-479 Poznañ, 3 — Katedra Biochemii
i Biotechnologii, Akademia Rolnicza, ul. Wo³yñska 35, 60-637 Poznañ, 4 — Delta Pharma BV, Delta Pharma BV, Hengelo,
The Netherlands, 5 — PienGen Biomedical Corporation, PienGen Biomedical Corporation, Knoxville, USA

In the last years proteomics is rapidly developing as a
new field connected with protein structure and func-
tion. Now each protein predicted by genome analysis
can be obtained as recombinant protein. Biologically
active recombinant proteins can be expressed in bacte-
rial or eukaryotic systems. We used for expression of
recombinant proteins E. coli cells and mammary glands
of transgenic animals. Human tumor necrosis factor
TNFa and two chains of major cat allergen FeldI were
purified from bacteria. Human growth hormone hGH
was overexpressed in homozygotic female rabbit. All re-
combinant proteins have 6xHis tag for purification on
metal affinity chromatography column (Talon).
Histidine tags were removed from hGH and FeldI chain
1 and 2 by cleavage of sequence recognized by
thrombin or enterokinase introduced between tag and
protein sequence. Biological and cytotoxic activity of
recombinant proteins was estimated. Additionally, for
both FeldI chains immunological analysis was accom-
plished by surface plasmon resonance technology. Hu-
man growth hormone activity was analyzed towards
growth promoting activity using growth hormone de-
pendent cells Nb2-11. For all recombinant proteins
amino acid sequencing was performed.

318 Poster
Variation of the ribosomal operon rrn within of the strains of Desulfovibrio
desulfuricans species
Joanna Szczerba, Zofia Dzier¿ewicz, Beata Wiœniowska, Ludmi³a Wêglarz, Tadeusz Wilczok
Katedra Biologii Molekularnej, Biochemii i Biofarmacji, Œl¹ska Akademia Medyczna, ul. Narcyzów 1, 41-200 Sosnowiec

Hydrogen sulphide may be important in the
pathogenesis of ulcerative colitis, because of inhibition
of butyrate oxidation within the colonocyte. The sul-
phate reducing bacteria (SRB) have been shown to be
directly responsible for the generation of hydrogen sul-
phide in the colon. Pitcher et al. (2000) have identified
on the basis of 16S rRNA sequence the Desulfovibrio
desulfuricans as a representative strain in patients with
ulcerative colitis with clinically active disease. Al-
though SRB are not considered as pathogenic bacteria
Desulfovibrio spp. have been isolated from pyogenic
liver abscesses, and found to cause septicemia and
bacteremia. Langedijk et al. (2001) stated the occur-
rence of these bacteries in sites affected by periodontal
disease. The possibility of pathological influences of
Desulfovibrio genus on a human body suggests advis-
ability of applying the most appropriate diagnostic
methods with the purpose of identification of this ge-
nus strains.
Over the last years techniques involving the analysis
of the genes encoding rRNA (rDNA) have revolution-
ised prokaryotic taxonomy.
The present study demonstrates the restriction analy-
ses encompassing the 16S-23S rRNA genes and the
spacer region in-between within 15 strains (soil and in-
testinal) of D. desulfuricans species. Seven enzymes were
evaluated for their discriminatory power in ribotyping
16S-23S fragment. The discriminatory power was the
greatest with MboI and HaeIII. Furthermore, these two
cases the most complicated banding pattern was re-
ceived. The enzymes AluIII, HindIII, HinfIII and EcoR1
were not very useful for genotyping examined strains,
and the enzyme PstI was definitively useless for this pur-
pose. These data suggest that PCR ribotyping provides a
reproducible and simple alternative to conventional mo-
lecular approaches for typing strains of D. desulfuricans,
but the careful choice of appropriate restriction enzymes
is indispensable requirement.
2003 39th Meeting of the Polish Biochemical Society 179

319 Poster

SeqA-mediated stimulation of a promoter activity by facilitating functions of a tran-

scription activator
1 2 2 2 2
Alicja Wêgrzyn , Monika S³omiñska , Gra¿yna Konopa , Barbara Kêdzierska , Grzegorz Wêgrzyn
1 — Pracownia Biologii Molekularnej afiliowana przy UG, Instytut Biochemii i Biofizyki PAN, ul. K³adki 24, 80-822 Gdañsk,
2 — Katedra Biologii Molekularnej, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

It was demonstrated recently that the SeqA protein, a
main negative regulator of Escherichia coli chromo-
some replication initiation, is also a specific transcrip-
tion factor. SeqA specifically activates bacteriophage
lambda pR promoter while revealing no significant ef-
fect on the activity of another lambda promoter, pL.
We demonstrate that lysogenization by bacteriophage
lambda is impaired in E. coli seqA mutants. Genetic
analysis demonstrated that CII-mediated activation of
the phage pI and paQ promoters, which are required
for efficient lysogenization, is less efficient in the ab-
sence of seqA function. This was confirmed in in vitro
transcription assays. Interestingly, SeqA stimulated
CII-dependent transcription from pI and paQ when it
was added to the reaction mixture before CII, while
having little effect if added after a pre-incubation of CII
with the DNA template. This SeqA-mediated stimula-
tion was absolutely dependent on DNA methylation, as
no effects of this protein were observed when using
unmethylated DNA templates. Also, no effects of SeqA
on transcription from pI and paQ were observed in the
absence of CII. Binding of SeqA to templates contain-
ing the tested promoters occurs at GATC sequences lo-
cated downstream of promoters, as revealed by elec-
tron microscopic studies. Contrary to pI and paQ, activ-
ity of the third CII-dependent promoter, pE, devoid of
neighboring downstream GATC sequences, was not af-
fected by SeqA

320 Poster

Cloning of the retron structure from clinical E. coli strains

Renata Wolinowska, Krystyna Strze¿ek, Aleksander Masny, Katarzyna ¯ebrowska, Andrzej P³ucienniczak
Zak³ad Bioin¿ynierii, Instytut Biotechnologii i Antybiotyków, ul. Starosciñska 5, 02-516 Warszawa

Retrons are bacterial retroelements encoding the re-
verse transcriptase as well as msd and msr genes. It is
organised as an operon and is responsible for msDNA
production. Multicopy single-stranded DNA (msDNA)
is a structurally unique molecule composed of a sin-
gle-stranded DNA (encoded by msd) branched out from
a single-stranded RNA (encoded by msr) by 2’-5’ phos-
phodiester linkage. msDNAs are common in cells of soil
bacteria (Myxococcus, Stigmatella) but occur also in bac-
teria from Enterobacteriaceae family. Function of
msDNA in bacterial cell is unknown.
Single-stranded DNA purified from polyacrylamide
gel was sequenced by Maxam-Gilbert method. The se-
quence obtained for two strains (W8 and CZD1527) is
identical. It is rather short, i.e. 63 bp, and contains in-
verted repeats of 25 bp. The next aim of research was
cloning retrons present in strains W8 and CZD1527.
Due to this molecule’s structure two problems occur:
this molecule hardly can be used as a probe for hybrid-
ization and designing good primers for PCR is difficult.
Strategy with use of attached adaptors was used. Total
chromosomal DNA was prepared by partial Sau3AI di-
gestion, then 35-bp long adaptor containing GATC pro-
truding end was ligated. This material was used as a
template for PCR with one primer complementary to li-
gated adaptor and the other complementary to the pre-
viously sequenced fragment of msd gene. In two PCRs,
two primers homological to msd of diverse orientation
were used. For CZD1527 strain, two products have
been obtained of 350 and 210 bp containing promotor,
msd and msr genes.
180 Session 5. Structure and function of DNA 2003

321 Poster
The optimal eukaryotic signal for translation initiation from non-aug codons, pres-
ent upstream of bacteriophage l P cistron, is inactive in Escherichia coli
1 2 2
Borys Wróbel , Bartosz S³omiñski , Grzegorz Wêgrzyn
1 — Instytut Oceanologii, Polska Akademia Nauk, ul. Œw. Wojciecha 5, 81-347 Gdynia, 2 — Katedra Biologii Molekularnej,
Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

The expression of replication genes of bacteriophage
l, O and P, is believed to be translationally coupled.
However, it was previously noted that under the condi-
tions of amino acid starvation, when O is not synthe-
sized , P continues to be expressed at a relatively high
level. The results presented in this report, suggest con-
trary to the previously presented hypothesis, that a
AGACUGGAU sequence, an optimal context for trans-
lation initiation from non-AUG codons in eukaryots,
and present upstream the P cistron, is inactive in Esch-
erichia coli. Comparative sequence analysis confirms
that such a signal is unlikely to be important for P syn-
thesis. Instead, a weak Shine-Dalgarno sequence may
be present upstream the P cistron. This weak signal
might be active in the absence of O gene expression.

322 Poster
Demonstration of kidney-specific activity of human uromodulin gene promoter in
transgenic mice
1 2 2 3 4 2
Halina ¯bikowska , Nadia Soukhareva , Reza Behnam , Rosemary Chang , Roman Drews , Henryk Luboñ ,
2 2
David Hammond , Serguei Soukharev
1 — Katedra Biochemii Ogólnej, Uniwersytet £ódzki, ul. Banacha 12/16, 90-237 £ódŸ, 2 — Plasma Derivative Department, American
Red Cross, Rockville, USA, 3 — Clearant, Incorporation, Rockville, USA, 4 — Division of Hematology, FDA, Rockville, USA

The ability to target expression of therapeutically use-
ful proteins into the urine of animals may greatly ex-
pand the potential application of transgenic technology
in medicine and biotechnology. If successful, this alter-
native may appear significantly more time and cost ef-
fective in comparison to the mammary gland-based sys-
tems. The major shortcoming of recombinant protein
production in the urinary tract is the lack of a reliable
and tissue-specific expression vector. The uromodulin
gene seemed to be a good candidate for this purpose
since uromodulin (known as the Tamm-Horsfall pro-
tein; THP) is recognized as the most abundant protein
in human urine. The expression of uromodulin is spe-
cific to the cells of the ascending limbs of Henle’s loop
in the kidney.
In this study, we have isolated and sequenced the
6.75-kb fragment of human uromodulin promoter. In
addition, we have generated transgenic mice for ex-
pression of the lacZ marker gene. Analysis of the
Uro-lacZ transgenic mice indicated that the 5.6-kb frag-
ment of the uromodulin gene, containing the 3.7-kb pro-
moter area, the first exon and part of the second exon
could provide for a highly tissue-specific expression of
the lacZ gene. All tested transgenic lines showed a
steady increase in kidney-specific beta-galactosidase ac-
tivity measured spectrofluorometrically. Histological
evaluation of the beta-galactosidase expression con-
firmed high tissue-specificity of the promoter. Signifi-
cant activity after staining with X-gal as a substrate for
beta-galactosidase was detected in a medulla of the kid-
ney which correlates with the anatomical location of
Henle’s loop.