structure and function of dna - acta biochimica polonica and function of dna 2003 protein induced by...

SESSION 5

Structure and function of DNA

Organized by G. Wêgrzyn, R. Paw³owski

39th Meeting of thePolish Biochemical SocietyGdañsk 16–20 September 2003

271 Lecture

Unusual thermal stability of RNA/[All-RP-PS]-DNA/RNA triplexes containing ahomopurine 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 knownsince 19571. In triplexes, the third strand binds eitherin a parallel or an antiparallel orientation with respectto the purine strand in a duplex, due to formation ofHoogsteen or reverse Hoogsteen hydrogen bonds, re-spectively [2]. In principle, triplexes can be formedfrom different combinations of RNA and DNA strands,and the composition affects their stability [3–5]. Sincenatural oligodeoxyribonucleotides are easily degradedby the nucleases, several modifications of thesugar-phosphate backbone have been proposed [6].Among them phosphorothioate analogues of DNA(PS-Oligos) appeared to be very important due to closesimilarity 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 ofP-chirality. It has been found that homopurinedodeca(deoxyribonucleoside phosphorothioate)s pos-sessing all internucleotide linkages of RP configurationform with complementary RNA or 2’-OMe-RNA strandsa 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 dodecamerscontaining 4–6 dG residues are thermally stable in pH7.4, although their stability increases significantly atpH 5.3. To our best knowledge, a triplex structureRNA/[PS]-DNA/RNA has not yet been described in theliterature.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 strainsof Enterobacteriacae

Piotr Ceg³owski

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

Plasmids constitute from 1% to greater than 10% ofthe genome of many bacterial species. They representthe most fluid part of bacterial genomes and they en-dow the host bacteria with high genetic variability andflexibility in response to environmental stimuli.Plasmids are mobile genetic elements and as such areone of the most efficient vehicles in the horizontal genetransfer (HGT). Analysis of complete bacterialgenomes has revolutionized not only the way we thinkabout the HGT but also that it is far more prevalentthan 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 fromclinical strains of Enterobacteriaceae. We wanted togain more information about those plasmids because oftheir intriguing properties: the p1658/97 contains thebeta-lactamase gene that undergoes amplification withina plasmid molecule [1] and the pCTX-M-3 very quicklydisseminated among many species of enteric bacteriaallover Poland [2].

Complete nucleotide sequences of p1658/97 andpCTX-M-3 were determined, annotated and depositedin the GenBank database under accession numbers

154 Session 5. Structure and function of DNA 2003

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, LaJolla, USA

The interactions responsible for the initiation of chro-mosomal replication from the E. coli replication originand subsequent DNA elongation events have, for themost part, been defined. The key role of the E. coliDnaA protein in the formation of an open complex andthe recruitment of the DnaB helicase and the role of theE. coli DnaC protein as a DnaB loader in the formationof a pre-priming complex at the E. coli origin are nowwell established. An important variation on this seriesof events has been shown for plasmid RK2 in an in vitrosystem utilizing the DnaA and DnaB proteins of Pseu-domonas species [1, 2]. These studies demonstratedthat the formation of a pre-priming complex requiresthe larger form of a plasmid encoded initiation proteinand the DnaB helicase but not the DnaA protein or theDnaC helicase loading protein. This is consistent withthe failure to identify a homologue of the E. coli dnaCgene in P. aeruginosa or P. putida [1]. The chromosomalorigins of P. aeruginosa and P. putida have been isolateand sequenced [3]. We have examined the activity ofthese origins in an in vitro assay that measures the for-mation of a pre-priming complex. These analysesshowed that the Pseudomonas DnaA protein interactswith its respective DnaB protein to form thepre-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 DnaCprotein.

Several cytological studies have shown that the E. colichromosomal origin of replication is located near oneend of a newborn cell. Low copy number plasmids F andP1 are, however, positioned at mid-cell in E. coli and dur-ing cell growth and elongation plasmid foci move toone-quarter and three-quarter positions which becomethe 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 perchromosome) and pUC19 (> 50 copies per cell). To oursurprise these multi-copy plasmids were not present asmultiple foci corresponding in number to their copynumber in E. coli, but in most cells were clustered as asingle focus at the mid-cell position in shorter cells, or astwo foci located at the 1/4 and 3/4 positions in longercells [6]. A similar finding was observed for plasmid RK2in Pseudomonas aeruginosa and Vibrio cholerae [7]. Weare presently addressing the major question of the na-ture of the E. coli proteins or structures and/or plasmidencoded 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

Igor Konieczny1, Marcin Pacek1, £ukasz Kowalczyk1, Katarzyna Herman1, Anna Janaszak2, Donald Helinski3,Yong Jiang3, Aresa Toukdarian3

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 BiologicalSciences and Center for Molecular Genetics, University of California at San Diego, 9500 Gliman Drive, La Jolla, USA

The origins of prokaryotic and some eukaryoticreplicons such as DNA viruses and Saccharomyces

cerevisiae possess characteristic functional elements,including specific binding sites for the appropriate ini-

2003 39th Meeting of the Polish Biochemical Society 155

tiation protein and an AT-rich region where DNA du-plex destabilization occurs. Plasmid origins usuallycontain multiple binding sites (iterons) for theplasmid-specific replication initiation protein as well asa 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 employedfor broad-host-range plasmid replication and mainte-nance in different host bacteria species. We found thatbroad-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 importantfactor effecting helicase recruitment and loading mech-anism is the nature of the protein interactions betweenthe two forms of the RK2 replication initiation proteinand the host replication machinery. Recruitment of thehelicase 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 rolein helicase recruitment in E. coli. Similarly, the neces-sity of host specific DnaA-DnaB interactions utilizedfor helicase recruitment at the plasmid origin dependson 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 associatedwith a variety of cellular activities) superfamily ofATPases. All family members contain matches to theWalker A motif for ATP binding and to the Walker Bmotif that is important for ATP hydrolysis. The familyincludes many proteins involved in DNA replication inprokaryotes, archaea and eukaryotes, that thereforeprovide a unifying link in the biochemistry of DNA rep-lication throughout the living world. The presence ofATP or ADP in the binding site results in a significantchange in protein structure and function and providesa molecular switch that couples key events during initi-ation of replication with cell cycle progression.

Several monomers of E. coli DnaA form a complexwith the replication origin, oriC, resulting in the localunwinding of an AT-rich region in oriC. OnlyATP-DnaA is active in unwinding and initiation. Thebinding properties of DnaA as well as the ability ofmonomers to cooperate are profoundly influenced by

its ATP/ADP status. Both forms bind to an asymmetric9-mer consensus sequence, the DnaA box5’-TTA/TTNCACA. But only ATP-DnaA is able tooligomerize and bind to a relaxed DnaA box with one ortwo mismatches, as well as to a 6-mer sequence, theATP-DnaA box with the consensus sequence 5’-AGatct.Sequential binding of DnaA to these sites with gradedaffinities is responsible for the unwinding. The unwind-ing and initiation reaction of bacteria other than E. coliis similar, but with variations in the details of the reac-tion.

All organisms have developed mechanisms that en-sure that chromosomal replication occurs once andonly once per generation. The regulatory inactivationof DnaA is the prominent mechanism in bacteria. Theintrinsic ATPase of DnaA is activated at the end of theinitiation cycle due to the loading of the sliding clamp,the b subunit of DNA polymerase III holoenzyme. Thisprevents 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 activatedby cAMP, regulates the several genes in Escherichiacoli. The protein is a homodimer and each monomer isfolded into two distinct domain — C-terminal domain re-sponsible for DNA binding and N-terminal one respon-

sible for cAMP binding. The fast kinetic measurementsof CRPwt and several single amino acid substituted mu-tants, which play a crucial role in inter-domain andinter-subunit communication, have been used to studythe kinetic of allosteric conformational changes of the


protein induced by cAMP binding. The conformationalchanges observed upon CRP-(cAMP)2 complex forma-tion can be described by sequential model of allostery.The amino acid substitutions influence the allostericchanges. The formation of CRP-(cAMP)4 complex re-sults from displacement of equilibrium between twoforms of CRP-(cAMP)2. Intermolecular signal transmis-sion, triggered by cAMP binding, indicates that the pro-tein can exist in various states, the distribution ofwhich can be influenced by these mutations. This

cAMP dependent distribution of conformational statesof CRP, which differ in affinity for DNA and RNA poly-merase, may play a crucial role in the fine regulation oftranscription. Using steady-state and time resolved flu-orescence measurements such as FRET we have shownthat the binding of cAMP results in 8Å movement ofthe 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 chromosomereplication

Jolanta Zakrzewska-Czerwiñska

Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroc³aw

The initiation of DNA replication is a complex processinvolving 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 sitefor the DNA helicase; (iii) loading of helicase and addi-tional proteins. In bacteria, initiation of chromosomalreplication starts from a single replication origin oriCand proceeds bi-directionally until the replication forksreach termination site (terC). Initiation of bacterialchromosome replication is mediated by the initiatorDnaA protein which interacts with repetitivenonpalindromic nonamer sequences, the DnaA boxesthat are localised within the oriC region.

The structure of the oriC region has been analyzedwithin Gram-negative and Gram-positive bacteria. Thesequences of oriC regions are conserved only amongclosely related organisms. Replication origins of differ-ent bacteria have varying sizes (from approx. 200 up to1000 bp) but (nearly) all contain several DnaA boxes,and an AT-rich region. A cluster of four or more DnaAboxes is an indication for a functional chromosomal ori-gin. The functional oriC region is always located withinthe intergenic region; frequently within the cluster of

genes rnpA-rmpH-dnaA-dnaN-recF-gyrB rnpA, usuallynext to the dnaA gene encoding initiator protein DnaA.

DnaA homologs have been found in all eubacterialspecies studied so far, with the exception of theWigglesworthia glossinidia genome. Based on structuralsimilarity and specific functions, four domains havebeen 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 andis probably a flexible linker that connects theN-terminal domain with the highly conserved domainsIII and IV. The length of the domain II varies from 48amino 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 anadditional oligomerization site. The C-terminal domainIV interacts specifically

A comparison of replication initiation in different or-ganisms particularly in E. coli, H. pylori, M. tuberculosisand S. coelicolor allows to define those aspects of theprocess that are universally used in the eubacterialkingdom.

278 Oral Presentation

Potential functions of a new member of the mouse SIR-gene family, Sir7, in cellularproliferation, 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-pendent histon-deacethylases and are involved in sev-eral essential processes as transcriptional silencing,

DNA-repair, stress resistance and regulation of the lifespan. Especially in two lower eucaryots, S. cerevisie andC. elegans, where Sir function has been most inten-


sively studied, the life span seems to be directly depend-ent on the number of active Sir gene copies. Sirhomologs were identified in many other species includ-ing mouse and man. Although the NAD+-dependenthistone deacetylase activity has been demonstrated formammalian Sir proteins, their physiological role inhigher vertebrates remains elusive. Recently it hasbeen shown, that human SIR2 can deacetylate otherproteins in addition to histones with p53 as one of thepossible specific targets.

We have identified a new member of the mouse Sirfamily, that shows the highest homology to the humanSirt7. To this end we studied the function of Sir7 by

overexpression in mammalian cell tissue cultures andusing in vitro protein interaction assays. In addition,stable cell clones overexpressing Sir7 were establishedand screened for differences in gene expression as re-lated to the reference cells. Upon overexpression, Sir7prevents 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 controland apoptosis as myc and p53 and to downregulate theMyoD promoter. Using GST-based pull down assays adirect physical interactions of Sir7 with p53 and sev-eral transcription factors of the bHLH-family weredemonstrated.


Protein-protein interactions at simple and complex bacterial promoters

Steve BusbySchool of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom

At many simple bacterial promoters, transcriptioninitiation is dependent on a single activator that func-tions by interacting directly with a target in RNA poly-merase. Some recent advances in understanding theseactivator-RNA polymerase interactions will be out-lined. Many bacterial promoters are more complex, andtranscription is co-dependent on two or more activa-tors; this ensures the appropriate expression fromthese promoters in response to particular environmen-tal triggers. Recent research concerned with under-

standing the different mechanisms of co-dependencewill 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.


Analysis of the EcoVIII endonuclease active site using site directed mutagenesis

Magdalena Cichowicz1, Iwona Mruk1, Janusz Bujnicki2, Tadeusz Kaczorowski1

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 ofthe isospecificity phenomenon among type II restric-tion-modification (R-M) systems. This phenomenon isinteresting in many ways. Firstly, because it poses theintriguing question — how do genes that encode func-tional homologs evolved in bacteria that are often notphylogenetically related. As well as this, it is interest-ing because structural analysis of these enzymes canhelp in the localization of particular motifs responsiblefor catalytic reaction and for target recognition, and inconsequence could be used in designing enzymes withnovel specificities.

As a model in our study we decided to use a group ofsystems isospecific to HindIII (Haemophilus influenzaeRd), a R-M system which recognizes the palindromic se-quence 5’-AAGCTT-3’. This group consists of over 30R-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 isospecificenzymes? (ii) is it possible to map their functional do-mains? (iii) do they recognize cognate sequence in thesame way? (iv) what is their mode of action?

In the present study we decided to investigate thestructure of the catalytic center of the EcoVIIIendonuclease (R.EcoVIII). The putative catalytic DNAcleavage/Mg(II) binding motif of restrictionendonucleases (PD/EXnD/EXK) is essential for theirfunction. The predicted amino acid sequence ofR’EcoVIII allowed a putative catalytic/Mg(II) bindingsequence motif characteristic for restriction endonucle-ases, PE66X54DXK123 to be located in N-terminal por-


tion of the enzyme. This motif is also present inR’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 abolishesenzyme activity (Dahai et al. 1999; Biosci BiotechnolBiochem, 63: 1703–1707). However, analysis of theR.HindIII molecular model revealed that residuesPE52 are located a significant spatial distance from thesecond 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 inclose proximity. The spatial architecture of theR.HindIII molecular model prompted us to proposethat 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 forR.EcoVIII and R.LlaCI are D108X12DXK123 andD91X12DXK106, respectively. Site directed mutagene-sis within putative catalytic domain produced twoR.EcoVIII null mutants (D108A and D121A) providingevidence that changed amino acids are involved in theprocess of catalysis.


Application of DNA sequencing by indexer walking in analysis of plasmids isolatedfrom enteropathogenic strains of Escherichia coli

Katarzyna Gromek, Tadeusz KaczorowskiKatedra Mikrobiologii, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

The strategy of DNA sequencing by primer walkingenables direct and systematic analysis of large DNAfragments with low redundacy and without subcloning.This approach is based on the use of individual primersspecifically designed for each sequencing step. Al-though potentially the primer walking strategy seemsto be atractive for large scale projects, recent progressin sequencing of many genomes was achieved by usingthe random approach. Among the disadvantages of theprimer walking technology are high cost of primers anddelays 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- or6-mers; (iii) primers produced by the high-throughputmultichanel oligosynthesizers.

In our laboratory we have developed another ap-proach to DNA sequencing by primer walking whichrelies on the usage of the presynthesized library ofoligonucleotide adaptors refered to as indexers andclass 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 isresponsible for the specificity of ligation to the ana-lyzed DNA. The use of the presynthesized library of in-dexers enables amplification and subsequent directsequential analysis of any DNA fragment. Such a li-brary eliminates the necessity for custom synthesis ofoligonucleotides.

The protocol of DNA sequencing by indexer walkingincorporates efficient ligation of double-stranded syn-thetic oligonucleotides (indexers) to DNA fragments,produced by class IIS restriction endonucleases whichgenerate four nucleotide long 5’ overhangs, and theirsubsequent amplification which provides enough tem-plate for automated DNA sequencing. Data gathered inthe first sequencing reaction permits further move-ment into the unknown DNA sequence by digestionwith class IIS restriction endonuclease followed by liga-tion of next indexer. The presynthesized library of in-dexers (256 oligonucleotides) enables bi-directionalanalysis of any DNA molecule and provides universalprimers for sequencing. This approach was success-fully applied to sequence several natural plasmids iso-lated from entreopathogenic strains of Escherichia coli.


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 TaylorKatedra Biologii Molekularnej, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

The sigma54-promoter consensus sequence wasfound in the regulatory region of the rpoH gene codingfor the main heat-shock factor sigma32. Regulatory

proteins for this promoter are unknown, but potentialconsensus sequences for IHF binding and enhancersfor PspF and NtrC were found. These two activators act


by different mechanisms. Since two other heat-shockinducible and sigma54 controlled units were described:pspA-E operon and ibpB gene, a third-sigma54 con-trolled heat-shock regulon emerges besides the twoknown, controlled by sigma32 and sigma24 RNA poly-merase subunits.

The sigma54-RNA polymerase holoenzyme (sigma54-RNAP) is responsible for transcription of genes whoseproducts are involved in utilization of various nitrogenand carbon sources, energy metabolism, developmentand many other activities. Hence one may suppose thatthe stress response encompasses also gene regulationadequate for these conditions.

Initiation of transcription at a sigma54 promoter istightly regulated at the step of promoter melting. Thesigma54-RNAP binds to the promoter to form a stableclosed complex. Its isomerisation to open complex de-pends on the interaction with enhancer-bound activa-tor protein in an ATP dependent manner. Theenhancer sequences can be distant from sigma54 pro-

moter, so the interaction requires looping-out of the in-tervening DNA, facilitated by the DNA bending IHFprotein.

Sigma54, PspF and NtrC proteins have been purifiedfor in vitro studies. Gel mobility shift assays have re-vealed that sigma54-RNAP, but not sigma54 subunitalone, binds to the regulatory region of the rpoH gene.It is consistent with the data that sigma54 unlike othersigma factors can bind to its promoter in the absence ofthe core RNAP but only if the ‘T-tract’ element withinpromoter sequence is present. The promoter in ques-tion does not contain the ‘T-tract’. Electron microscopyexperiments confirmed this result and showed thatsigma54-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. NtrCinteraction with this site is under investigation. IHFprotein also has been purified for these and in vitrotranscription studies.The project was supported by State Committee for Scientific

Research (KBN, Poland) grant nr 3 P04A 001 23).


The gene expression profiles in patients with abdominal aortic aneurysm (AAA)

Aleksandra Korcz1, Krzysztof Waliszewski2, Daniel Lipinski1, Marcin Gabriel2, Stanislaw Zapalski2,Ryszard Slomski3

1 — Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszyñska 32, 60-479 Poznañ, 2 — Clinics of General andVascular Surgery, University of Medical Sciences, Poznañ, 3 — Department of Biochemistry and Biotechnology, AgriculturalUniversity, ul. Wo³ynska 35, 60-637 Poznañ, Poland

Abdominal aortic aneurysm (AAA), a localized abnor-mal dilatation of aorta, is a life-threatening conditionaffecting 4–9% of population with a risk increasingwith 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 singlegene was shown to be responsible for it. Based on theresults of the studies done so far, it is believed thatpathogenesis 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 aortamay help to understand complex biological processesresponsible for pathogenesis of AAA we applied DNAarray 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 fromAtlasTm Human Stress Array. We have identified sev-eral up-regulated and down-regulated gene expressionchanges in abdominal aortic aneurysm tissues whencompared to control unchanged aortas.


The EcoVIII endonuclease gene expression is modulated by the low-usage argininecodons

Iwona Mruk, Tadeusz Kaczorowski

Katedra Mikrobiologii, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

Each organism represents its own preference of us-age of the 61 available amino-acids (aa) codons. This is

reflected by the particular cell tRNA pool of those. Theaa arginine can be encoded by six codons. In E. coli, the


most frequent arginine codons are CGC and CGU. Theother four belong to the low-usage codons with AGAand AGG codons which are the rarest among all codonsin 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 beeninvestigated. Both genes possess high percent oflow-usage arginine codons: 18 out of 20 for R.EcoVIIIand 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 incase of R.EcoVIII whereas the same tandem is observedat 167–168 aa position in case of M.EcoVIII. It has beenshown that presence of such consecutive sequence ofrare codons close to the gene start site can lead totranslational pausing, frameshifting and aamisincoporating. This effect is not observed when thesame rare codons occure beyond the first 25 triplets.The insufficient tRNA population for rare codons mayexplain our unsuccessful attempts in R.EcoVIII over-production. This was overcome when the overproduc-ing strain BL21(DE3) was transformed with theplasmid pRARE (Novagen) carrying the dnaY gene cod-

ing for the proper tRNA that recognises AGA/Gcodons.

The EcoVIII R-M system is a possible result of horizon-tal gene transfer. This finding may support not only thestrong 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 adaptationindex (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 forR.EcoVIII and M.EcoVIII respectively, indicate that bothgenes belong to the E. coli genes that were most probablyacquired by horizontal gene transfer. It is extremely im-portant for bacterial cell to balance these two activitiesof R-M systems: restriction and modification. In anycase, the overrestriction leads to cell suicide.

We show that the abundance of the low-usage codonsand their position within the EcoVIII endonucleasegene may seriously affect its expression. We hypothes-ise that the activity of the endonuclease gene can bemodulated depending on the host genetic context.Thus, we propose another mode of R-M system regula-tion which allows to control the potential lethal actionof the restriction enzyme.


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 cauliflowergene of subunits of complex I (nad3, nad6 and nad9)and genes of complex V subunits (atp6, atp9) as well astheir expression. Although the nad9, nad6 and nad3genes as well as atp6 and atp9 were analyzed in a fewplant species, no data about the structure and expres-sion of these genes are known for cauliflower.

The copy number analysis confirmed the presence ofone copy for each analyzed gene in mtDNA. Our se-quence analysis revealed the open reading frames fornad9, nad6 and nad3 genes of 573, 618 and 357 bp, re-spectively. The 5’ and 3’ flanking region of the nad9varied more in the sequence than the gene. A similarvariability was observed for the 5’ flanking region ofnad6 and 3’ flanking region of nad3.

To examine the expression of analyzed genes, North-ern blots of mtRNA revealed transcripts of about 1.4,

1.2 and 1.1 kb for nad9, 1.6 and 0.8 kb for nad6, 1.6 and1.43 kb for nad3, 1 kb for atp6, and 0.3 kb for atp9 genewere detected. All the transcripts were long enough tocover the entire coding region of all the genes analyzed.The presence of longer transcripts may indicate thatcotranscription with other genes is possible.

The 5’ termini of the nad9 and nad6 mRNAs wereidentified. They were mapped at a distance of about 380and 160 bp from the start codon of nad9 gene and atdistance of about 90 and 190 bp from the start codonfor nad6 gene.

In order to detect proteins which bind to 5’ flankingsequence of nad9 gene, the EMSA analysis was carriedout. In the two analyzed fractions of cauliflower mito-chondrial proteins, some polypeptides showing bindingactivity were detected.



Loss of heterozygosity at the p16 locus in human endometrial carcinomas

Agnieszka Stenzel-Bembenek1, Andrzej Semczuk2, Barbara Marzec3, Anna Szczygielska1, MartaStryjecka-Zimmer1

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 Medycznaw Lublinie, ul. Radziwi³³owska 11, 20-080 Lublin

p16INK4A — tumor suppressor gene, encoding pro-tein associated with regulation of the cell-cycle is oftenaltered in human carcinomas. Some of these alter-ations are caused by point mutations and deletions,some by modifications of DNA sequence as well as byloss of heterozygosity (LOH).

Currently, we examined 50 endometrial carcinomasfrom 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 clinicopathologicalfeatures. All the samples were classified according tothe 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-productswere separated by electrophoresis through polyacry-lamide gels cross-linked with piperazindiacrylamideand visualized by silver staining. The immunoreactionwas performed with a mouse monoclonal anti humanIgG 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 12of 50 (24%) carcinomas with a higher incidence in ad-vanced endometrial carcinomas than in early-stageuterine tumors. p16INK4A-LOH was significantly cor-related with reduced nuclear p16INK4A expressionimmunohistochemically.

287 Poster

Relationship of polymorphism in the apolipoprotein E gene with coronary arterydisease or ischemic stroke in Silesian patients

Anna Balcerzyk1, Pawe³ Niemiec1, Beata Sarecka1, Zbigniew Ciemniewski2, El¿bieta Marsza³3, Iwona ¯ak1

1 — Department of Biochemistry and Medical Genetics, Medical University of Silesia, ul. Medyków 18, 40-752 Katowice, 2 — TheFirst Department and Clinic of Cardiology, Medical University of Silesia, ul. Zio³owa 45/47, 40-635 Katowice, 3 — Departmentand Clinic of Pediatric Neurology, Medical University of Silesia, ul. Medyków 16, 40-752 Katowice

Background: Apolipoprotein E (apoE) is involved inthe 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 affectplasma cholesterol concentrations, what may be one ofthe reasons of its association with coronary artery dis-ease (CAD). The E4 allele increases the risk of mortal-ity after myocardial infarction and susceptibility tocerebrovascular disease.

Aim: The aim of this study was to assess a possible as-sociation between the epsilon polymorphism of apoEgene and CAD or ischemic stroke.

Materials and Methods: Sixty seven adult patientswith angiographically documented CAD, 15 ischemicstroke children and 105 control subjects were studied.

The apoE genotypes were determined using PCR-RFLPanalysis 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-icantly higher than in controls (0.27 vs. 0.07, chi2=12.6,p=0.0004). Carriers of the E4 allele were more frequent inthe stroke group (0.33) than in the CAD patients (0.19)and controls (0.13). The E4E4 genotype was not found inthe CAD patients and controls, while 20% of ischemicstroke children were E4E4 homozygotes.

Conclusions: Our data suggest a strong associationbetween carrier-state of the E4 allele in the apoE geneand ischemic stroke in children, but further analysis oflarger group is needed.


288 Poster

Direct sequencing of plasmids isolated from hospital environment using cassettescarying antibiotic resistance genes inserted randomly

Agnieszka Dekowska, Ewa Chêæ, Tadeusz Kaczorowski


There are two basic strategies of genomic DNA se-quencing. First is based on random shotgun methodsemploying DNA fragmentation, subcloning of smallfragments, 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 newoligonucleotide primer for each run, or by using antibi-otic resistance gene cassettes containing primer bind-ing sites. Random insertion of such elements enablesDNA sequencing by producing overlapping sequencesthat are used to obtain the total nucleotide structure ofthe analyzed DNA. The overall goal of each of thesestrategies is to make DNA sequencing faster, more ac-curate, and less expensive. The approach based on theuse of the chloramphenicol resistance marker has beensuccessfully applied in our laboratory for direct se-quencing of bacterial plasmids isolated from entero-pathogenic strains of Escherichia coli (EPEC). Thesebacteria are responsible for infantile diarrhea, espe-cially in less developed countries. Epidemiological stud-ies revealed that EPEC strains are especially abundantin plasmids. Some of them are associated with pathoge-nicity (e.g. EAF plasmid), while function of others isstill unclear. In our studies we are interested in howplasmids enable bacteria to adapt in the hospital envi-

ronment where antibiotic pressure and other factorspromote fast evolution of strains with novel properties.

Plasmids analyzed in our laboratory were processedby partial digestion with restriction endonucleaseSau3AI (a frequent cutter) in the presence of ethidiumbromide which severly inhibits the enzyme’s activitypromoting creation of linear forms of plasmid DNA. Inthe next step, after excision from delivering plasmidwith BamHI enzyme, a chloramphenicol cassette[pKRP10; Reece and Phillips, Gene 165 (1995)141–142] was inserted into natural plasmids partiallydigested with Sau3AI, as described earlier. Both en-zymes after digestion produce the same 5’ protrudingends. Recombinants were introduced into laboratory E.coli strain (MM294) and selected on plates supple-mented with chloramphenicol. Individual colonies werescreened for the presence of plasmid DNA. Selectedrecombinants were sequenced bidirectionally using asingle set of sequencing primers (cat1 and cat2, respec-tively). Products were analyzed on ABI310 DNA se-quencer. The described procedure enabled us to obtainnucleotide sequences of several medium-size E. coliplasmids (4-10 kb) at low cost and minimum laboratorywork. However, initial analysis of their structure didnot reveal presence of any open reading frames thatcould be associated with virulence.

289 Poster

Cloning and characterization of the genes of the restriction-modification systemfrom Neisseria cuniculi

Beata Furmanek-Blaszk, Tadeusz Kaczorowski


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 oftwo separate enzymes, a restriction endonuclease and amethyltransferase. Genes of R-M systems have some-times been found to be located on transferable ele-ments such as plasmids and bacteriophages, and insome cases, genes encoding proteins involved in DNAmobility, such as transposases, integrases andinvertases, are found in the vicinity of the genes forR-M systems. These genetic structures may facilitatethe 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 proposalthat R-M systems are likely to be mobile genetic ele-ments. However, a few examples in which genes forDNA transfer are not colocalized with a gene for DNAtransfer are known, indicating that other mechanismsare involved in the spread of R-M systems.

Neisseria cuniculi ATCC 14688 is a gram-negativecoccus 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


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 fromMoraxella bovis. The search for homologous se-quences using the computer program CLUSTAL Wshowed significant homology to MboII endonuclease.A comparison of the DNA sequences revealed simi-larities between NcuI and MboII coding regionswhich differ in 189 nucleotides. The overall nt se-quence similarity is 84%. Homology searches showedthat 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 NcuImethyltransferase (M.NcuI) which recognizes andmethylates the same sequences as M.MboII. The nucle-otide sequence for M.NcuI was determined and showed87% identity to M.MboII; the two methyltransferasesM.NcuI and M.MboII are 90% identical at the aminoacid sequence level. We were interested in a compara-tive analysis of the structural and functional features ofM.MboII and M.NcuI.

290 Poster

C�T substitution in the promoter region of CYP17 gene and prostate cancer risk

Monika Gos1, Ma³gorzata Sadowska2, Marek Grzegrzó³ka3, Tomasz Demkow2, Przemys³aw Janik1

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, iscommonly diagnosed among elder Polish men. Manyfactors are involved in prostate cancer etiology, but themost important from them is age. In the year 2000, theincidence rate was about 252/100 000 for men aftertheir seventies and was about 28 times higher as com-pared to values calculated for younger men. Also theandrogen metabolism influences prostate cancer devel-opment, therefore any changes in genes correlated withsex-hormone synthesis can be important for itsetiology. A single-nucleotide change (T�C) in the pro-moter region of CYP17 gene encoding cytochromeP450c17 was described as well as its possible role inprostate 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 changein patients with prostate cancer and age-matched con-

trols using PCR technique followed by digestion withMspAI endonuclease and the electrophoresis inagarose gel.

The prevalence of TT, TC and CC genotypes amongpatients did not differ from values obtained for the con-trols. When we divided examined population accordingto age (<70 and =70) the CC genotype appeared to bemore 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 occurrencein the promoter region of CYP17 gene can be correlatedwith higher risk of prostate cancer development amongmen before their seventies, although further studiesare necessary to confirm this hypothesis.

291 Poster

Polymorphism analysis of vulpine (Vulpes vulpes) androgen receptor gene

Piotr Gronek1, Dariusz Brzezinski2, Robert Kalak3, Karolina Doba2, Piotr Przysiecki4, S³awomir Nowicki5,Katarzyna Nuc2, Ryszard S³omski6

1 — Department of Pigs Breeding, Agricultural University of Poznan, ul. Wo³yñska 33, 60-637 Poznañ, 2 — Department ofBiochemistry and Biotechnology, Agricultural University of Poznan, ul. Wo³yñska 35, 60-637 Poznañ, 3 — Katedra Biochemiii 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, PolskaAkademia Nauk, ul. Strzeszyñska, 60-479 Poznañ

Androgens are important steroid hormones for ex-pression of the male phenotype. The actions of andro-gens are mediated by the androgen receptor.

The androgen receptor gene is located on theX-chromosome at Xq11–12 and codes for a protein witha molecular mass of approximately 110 kDa.


The gene consists of eight coding exons. The aminoacid sequence encoded by exon 1 is partly involved intransactivation of androgen target genes, the region en-coded by exons 2 and 3 enables DNA-binding, and theregion encoded by exons 4 to 8 is involved in ligandbinding. The first exon of the human androgen receptor(AR) contains a translated CAG (poly-glutamine) re-peat. The repeat length is polymorphic in the normalpopulation ranging from 8 to 35 repeats. Short tractsare associated with high intrinsic AR activity and in-

creased severity and earlier age of onset of the andro-gen-regulated tumor prostate cancer, whereas longerCAG tracts are associated with low AR activity andoligospermic infertility. There is possibility of involv-ing AR in aggression behaviour.

We research group of 135 foxes. The aim is searchingof polymorphism distribution in exon 1 and poly-morhisms in following exons. All exons were amplifiedand 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 humanmelonoma cells

Robert Herok1, Krzysztof Fujarewicz2, Maria Wide³3, Joanna Rzeszowska-Wolny1

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 Doœwiadczalneji Klinicznej, Centrum Onkologii Oddzia³ Gliwice, Wybrze¿e Armii Krajowej 15, 44-101 Gliwice

Melanomas are very malignant cancers capable offorming distant metastases. Usually, albeit not always,they are ionizing radiation-resistant. During periodsbetween therapy sessions as well as during metastaticspread, selective expansion of neoplastic cell clones oc-curs. Due to great genetic instability of these neo-plasms both genotype and phenotype of arising clonesmay differ (for example in their radiosensitivity). Thegoal of our experiments was to check gene expressionpattern changes during growth of a new cell populationand whether clones obtained differ indeed in theirradiosensitivity. Answer to these questions is essentialfor understanding mechanisms of metastasis. The pres-ent study is the first of the planned series.

Starting with Me45 melanoma line, three subcloneswere obtained. Ionizing radiation sensitivity of thestarting line and that of the subclones was compared.Cell survival tests indicated no statistical differences inradiosensitivity. Total RNA was then isolated from thestarting cell line as well as from the subclones and ex-pression levels of some 22 thousand genes werechecked using high-density DNA microarrays fromAffymetrix.

Among genes with significantly altered expressionlevel were those coding for G antigens and other cellsurface 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/PO4/2001and 4PO5015190.

293 Poster

Ionizing radiation-induced changes of gene expression patterns in human melanomacells

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 riskof malignancy as well as an important antitumor ther-apy tool. Damages induced by radiation trigger in cellsvarious signaling pathways, repair mechanisms as wellas cause changes in gene expression patterns. Knowl-edge of processes induced in cells by radiation shouldhelp 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 cellsexposed to ionizing radiation as well as to monitor thetime course of these changes. Material used in ourstudy was human melanoma Me45 cell line (obtained atthe Gliwice Center of Oncology). Cells were irradiatedwith 4Gy dose and total RNA was then isolated fromthe cells (a) immediately; (b) after 12 h and (c) after 24


hours following radiation exposure. As a control,non-irradiated cells were used. Expression levels ofsome 22 thousand genes were then assessed using highdensity oligonucleotide microarrays, hybridizationequipment and procedures from Affymetrix.

Groups of genes were selected for which gene expres-sion pattern differed in a specific manner following var-ious exposure times. Among genes for which expres-

sion was substantially increased or decreased in irradi-ated cells were those coding for:

— transcription factors— cell cycle control proteins— proteins participating in metabolic processes— signal transduction proteins— repair proteins

The study was financed by the State Committee for ScientificResearch (KBN, Poalnd) grants no. PBZ-040/PO4/2001and 4PO5015190.

294 Poster

Analysis of transcription in the region encoding protein phosphatase PrpEin Bacillus subtilis

Krzysztof Hinc1, El¿bieta Ratajczak2, Adam Iwanicki1, Micha³ Obuchowski1

1 — Katedra Biologii Molekularnej, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk, 2 — Miêdzyuczelniany Wydzia³Biotechnologii, Uniwersytet Gdañski i Akademia Medyczna, ul. K³adki 24, 80-822 Gdañsk

In spite of huge progress in research on proteinphosphorylation, still the new representatives of pro-teins kinases and phosphatases are being described.One of these enzymes is protein phosphatase PrpEfrom Bacillus subtilis. The existence of characteristicmotifs in the amino-acid sequence of this protein sug-gests that it belongs to the PPP protein phosphatasesfamily. Although, PrpE possesses different propertiesfrom 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-pends on Ni2+ ions, which is not common property.Also it is not inhibited by typical inhibitors of PPPphosphatases: 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 proteinphosphatase PrpE.

The PrpE gene is a single open reading frame localised inthe 106 of B. subtilis chromosome. Preliminary computeranalysis 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 thermostablebeta-galactosidase and integrated into the amyE locus ofthe B. subtilis chromosome. Obtained results confirmedpresence of the promoter in the PrpE region. The promoterbecomes activated during the osmotic stress. Results of ex-periments with strains deleted for prpE and sigB genesprovide indication that this phosphatase is involved in re-sponse to changing environment osmolarity. Elucidationof the conditions in which expression of the prpE gene be-comes activated may help in establishing the cellular role ofthis 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 in1997. During realisation of this task a putative tran-scription unit spanning from the yloI to the yloS genewas 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/Thrkinase. The gene prpC overlaps 3 bp with the next gene,prkC. Product of these genes may regulate one or morecellular processes, since autophosphorylated form ofthe PrkC kinase is a good substrate for PrpC


phosphatase. Genes encoding these enzymes areco-transcribed and form with adjacent gene yloQ a con-served genes layout phosphatase-kinase-gene. Productof the yloQ gene is a protein of unknown function thatdoes not have any known homologues. It appears to bethe essential gene in some specific conditions (growthin 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 theYloQ protein. This promoter was previously shown tobe induced by the ethanol stress. Presence of the se-quence similar to the sigma H consensus near the startsite of transcription suggests that this is sigmaH-dependent promoter. We show that transcription ofthe yloQ gene depends neither on this sigma factor norsigma B, which is involved in transcription of generalstress response genes.

296 Poster

Molecular diagnostics of medullary thyroid cancer in Polish patients

Marta Kaczmarek1, Katarzyna Ziemnicka2, Justyna Hoppe-Golebiewska3, Karolina Wielgus4, Jerzy Sowinski2,Ryszard Slomski4

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 andBiotechnology, 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 themultiple endocrine neoplasia type 2 syndromes (MEN2). Hereditary form of MTC may occur as familiarmedullary thyroid cancer (FMTC) or more commonly isassociated with phaeochromocytoma and hyper-parathyroidism in multiple endocrine neoplasia type2A (MEN 2A) and also with mucosal neuromas,ganglioneuromatosis of the gastrointestinal tract inmultiple endocrine neoplasia type 2B (MEN 2B). It isan autosomal dominant cancer syndrome, caused bymutation in RET protooncogene, mapped to thecentromeric region of chromosome 10q11.2. Proto-oncogene RET encodes a member of the receptor tyro-sine kinase family of transmembrane receptors and isexpressed in various tissues as thyroid, adrenal, devel-oping kidney, nerve tissue and in some humanneoectodermal tumors as well. It is possible that RETplay also a role in the differentiation and proliferationof neural cells.

In case when the medullary thyroid cancer is recog-nized, genetic tests are performed, independently fromdata obtained on basis of family interview and physicalrecognition, indicating hereditary form of cancer. It isestimated that individuals with negative interview indirection of hereditary form show 10% probability of ge-netic predisposition.

Genetic tests include analysis of mutation in RETprotooncogene in DNA obtained from whole blood lym-

phocytes. Positive results prove directions to testingpatient’s families. In our laboratory at Institute of Hu-man Genetic in Poznañ we perform screening analysisof six exons of RET gene (10, 11, 13, 14, 15, 16) byPCR-SSCP analysis with using fluorescent-labeledprimers as it enabled analysis of many samples in rela-tively short time. Every detected change in SSCP bandpattern 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 andtheir families. The group included families with FMTC,MEN 2A syndrome. The most frequently identifiedchanges occurred in exon 11 (26 changes) and in exon10 (21 changes). Occasionally we detected irregulari-ties in exon 13, 14 and 16.

Virtually all patients with FMTC, MEN 2A and MEN2B develop MTC, what is a clear rationale for perform-ing thyreoidectomy as soon, as RET mutation has beenidentified. Statistically in families of patients with he-reditary form of MTC, 50% of 1st degree relatives arecarriers of mutations, and genetic analysis should beperformed for all 1st and 2nd degree relatives. Conse-quently full medical characteristics describing develop-ment of the disease should be performed immediatelyto consider necessity of surgery. In case when clinicalsymptoms are not present prophylactic thyroidectomyshould be considered, as much better solution than fre-quent monitoring of blood calcitonin level.


297 Poster

Detection of the mutations in the methylenetetrahydrofolate reductase (MTHFR) andcystathionine �-synthase (CBS) genes

Leszek Kadziñski1, Grzegorz Wêgrzyn2, Zyta Banecka-Majkutewicz3, Walenty Nyka3, Wojciech Sawu³a4,Bogdan Banecki4, Joanna Jakóbkiewicz-Banecka5

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 BiotechnologyDepartment, Institute of Oceanology PAS, Gdynia

Methylenetetrahydrofolate reductase (MTHFR) is anenzyme involved in the metabolism of homocysteine.The homocysteine is formed by demethylation of the es-sential amino acid methionine, and it can be furthermetabolized by cystathionine beta-synthase (CBS).Homocysteine may be remethylated to methionine inthe presence of methyl-cobalamin (Me-Cbl) and5-methyl-tetrahydrofolate (Me-THF) as a cosubstrate.The production of Me-THF requires proper function ofMTHFR and the presence of an adequate supply of re-duced folate. Some mutations in the MTHFR and CBSgenes lead to reduction of the activity of MTHFR andCBS 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-

pitalised in the Department of Neurology of the Medi-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 A1298Cmutations in the MTHFR gene as well as for mutationsin the CBS gene by PCR and restriction enzyme analy-sis. Obtained PCR products and restriction fragmentsare easily detected and separated with a 2.5% agarosegel electrophoresis. These results are compared withthe homocysteine level for each patient.

Heterozygotic genotype C677T of MTHFR was de-tected in 24% of patients, A1298T in 52%. However only1% 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 eithergene (p=0.04). Additionally, significantly higher homo-cysteine level in homozygotes than in heterozygoteswas detected (p=0.01).

298 Poster

Reconstituted mononucleosome as a model to study proteins binding to DNA dam-aged by cis-platinum

Magdalena Kalinowska1, Monika Pietrowska2, Piotr Wid³ak1

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 DNAparticipate in all repair pathways. Another group ofdamaged DNA-binding proteins (DDB-proteins) arechromatin proteins (e.g. HMG-1/2), that do not takepart in the repair directly yet modulate its efficiency. Incontrast to interactions between DDB proteins and na-ked DNA interactions between such proteins andchromatin are poorly understood. The aim of presentedwork was to evaluate the feasibility of using in vitro re-constituted mononucleosomes to detect DDB-proteins.

Mononucleosomes were reconstituted in vitro usingcore histones purified from rat hepatocytes and DNA ofthe Lytechinus variegatus 5S rRNA gene. A 195bp re-striction fragment was end-labeled with 32P-� ATP, pu-

rified from acrylamide gel and damaged withcis-platinum (cis-DDP) before chromatin reconstitu-tion. DNA was damaged by 24 hours incubation with 3and 30 mM cis-DDP. Estimated level of the damage wasabout 1 and 20 of platinum-adducted nucleotides perDNA molecule, respectively. The ratio between his-tones and DNA was established experimentally to getoptimal mononucleosome recovery. Either naked DNAor reconstituted mononucleosomes were complexedwith 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.


DNA damaged by cis-DDP was bound with high strin-gency by proteins from nuclear extracts. TheseDDB-DDP proteins were identified as HMG-1/2 byco-migration with complexes between damaged DNAand purified HMG. The presence of platinum-adductednucleotides affected stability of the mononucleosomes— at higher level of the damage mononucleosomes weredisrupted during the storage. When mononucleosomesformed on DNA with lower level of the damage were in-

cubated with nuclear extracts this changed the natureof protein-DNA complexes detected on native gels.Bands specific for mononucleosomes were replacedwith bands specific for complexes between DNA andDDP-DDB proteins. This suggests that binding of HMGproteins 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

Anna Kloska1, Joanna Jakóbkiewicz-Banecka2, Bogdan Banecki3, Grzegorz Wêgrzyn4

1 — Department of Molecular Biology, University of Gdañsk, ul. K³adki 24, Gdañsk, 2 — Genetics and Marine BiotechnologyDepartment, 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 anessential enzyme involved in methionine metabolicpathway. Two common polymorphisms of the humanMTHFR have been identified. One of them is the C677T(Ala222Val) polymorphism that is associated with in-creased thermolability of the enzyme and with 60% lossof its activity. This polymorphism is the cause ofhyperhomocysteinemia — 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 inE. coli MetF is analogous to the C677T mutation in hu-man MTHFR.

In our studies we used the bacterial MetF reductaseas a model to investigate the C530T mutation influenceon the enzyme activity and the effect of the cofactorspresence (folic acid, vitamin B6 and B12) to suppressthe 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 containingthe wild-type metF gene and the metF gene with theC530T mutation (metF177) under the control of differ-ent inducible promoters (plac, pTc, para).

The E. coli delMetF2 strain was transformed by theseplasmids and we are investigating the temperature ef-fect (25oC, 30oC, 37oC, 43oC) and cofactor suppressionon the bacterial growth in strains containing the wildtype metF, the mutant metF177 and in the heterozy-gous strain with both wild-type metF and mutantmetF177.

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 lackingmethionine. The presence of plasmids containing thewild-type metF supports the growth in medium lackingmethionine in every temperature. The strain withplasmid carrying the mutant metF177 gene is unable togrow in 37oC and 43oC. The growth is also poorly sup-ported in lower temperatures (25oC and 30oC).

300 Poster

Radiation-induced bystander effects in human leukemic K562 cells in vitro

Maria Konopacka1, Jacek Rogoliñski1, Robert Herok1, Andrzej Orlef2, Krzysztof Fujarewicz3, JoannaPolañska3, Andrzej Œwierniak3, Joanna Rzeszowska-Wolny1

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 ofAutomation Control, Silesian University of Technology, Gliwice

One of the biological effects of radiation is phenome-non that was termed “bystander effect”. It involves ge-

netic damage occuring in cells that were not directly ir-radiated but responded to signals transmitted from ir-


radiated cells. A number of studies suggest that cell-cellcommunication plays a role in mediating this phenome-non.

In the present study we tested the medium-mediatedbystander 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 leukemicK562 cells were exposed to X-radiation (dose of 4 Gy). Af-ter one-hour incubation at 37oC the medium (ICM) wasremowed, filtered and transferred to non-irradiated flaskscontaining cells from the same line.

Medium transfered from irradiated cells to non-irra-diated ones reduced the number of viable cells after 3days of incubation. The increase of apoptotic andmicronucleated cells was observed after conditionedmedium (ICM) exposure.

Alterations of gene expression. In order to solve theproblem whether cells grown in conditioned medium

(so called “bystander cells”) have a genome-wide ge-netic expression profile similar or distinct with respectto directly irradiated cells, we examined the genetic ex-pression profiles of these cells using oligonucleotide ar-rays that contained probes representing 22 283 humangenes. Using hierarchical clustering analysis we sepa-rated from all analyzed genes some groups of geneswith up- (or down-) regulated expression.

The results suggest that for genotoxic damages seen incells 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 015190, No. 4T11F 01824, and PBZ-KBN-040/PO4/2001, in2003 year.

301 Poster

Abnormal nucleoid distribution and cell division in cells overproducing partitionproteins 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 moleculesare actively moved apart from the center of the mothercell into positions corresponding to the centers of fu-ture daughters. In the case of P1 it depends on plasmidproteins, ParA and ParB, and a centromeric site inplasmid DNA. ParA and ParB belong to families of con-served plasmid and chromosome partition proteins.Proteins of ParB family interact with their targetcentromeric sites in DNA. Proteins of ParA family areWalker-type ATPases involved in plasmid trans-location within a cell. They bind to centromeric sites viatheir partner ParB proteins. To find out whether the P1partition proteins can compete with bacterial proteinsfor a common target in a host cell we looked for effectsof their overproduction on the cell cycle. Overproduc-tion of ParA caused cell filamentation. Nucleoid DNAin the majority of cells was decondensed and uniformlydistributed. Surprisingly, cells overproducing ParA

and ParB differed from those overproducing ParA.Their nucleoids were only slightly decondensed, butnucleoid DNA was distributed unevenly, often concen-trated in two or more regions of different size sepa-rated by unequal “empty” regions. Many prolongedcells contained single or multiple cell division septa,which were positioned abnormally, usually at regionsof lower DNA concentration. Sometimes, guillotiningof DNA by cell division septa occurred. Cells overpro-ducing ParB alone did not differ from normal cells, in-dicating that ParB influenced cell divisions andnucleoid 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 ofnucleoid condensation or partitioning machinery.


302 Poster

Application of polymerase chain reaction (PCR) amplification in estimation ofmethylation status of selected DNA fragments

Barbara Krawczyk, Dorota Wyczechowska, Krystyna Fabianowska-MajewskaZak³ad Chemii Medycznej IFiB, Uniwersytet Medyczny, ul. Mazowiecka 6/8, 92 215 £ódŸ

DNA methylation as epigenetic modification is one of thecombined 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, numerousstudies of cancer cells were focused on hypermethylation ofpromoter regions of tumour-suppressor genes or tis-sue-specific genes. Promoters of tumour-suppressor genesare characterized by extended density of CpG sequences(i.e. CpG rich islands) or CCGG sequences. Examination ofdifferences between DNA methylation level in normal andcancer cells can concern both genomic DNA (for example,estimation of C-5 methyltransferase activity or genomicability to accept methyl groups) and any fragment of a se-lected gene.

Current studies are designed to adopt a simpleIwase’s method (MSP, methylation-specific polymerasechain 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 recognizeunmethylated CCGG and CGCG sequences, respec-tively; 2) amplification (PCR) of digested fragment ofER alpha gene; 3) electrophoretic analysis of amplifiedDNA fragments on polyacrylamide gel dyed withethidium bromide.

The results demonstrate that when the fragment ofthe gene contains methylated CpG sequences, DNAsamples are not recognized and digested by the restric-tion enzymes (HpaII and BstU1), the fragment of thegene is amplified and bands on the gel corresponding to347 pb are detected. In contrast, the unmethylated frag-ment of the gene was digested and no fragments wasamplified.

The adopted MSP analysis of the level of methylatedCpG sequences is a simple method which, firstly, elimi-nates laborious bisulfite DNA modification and sec-ondly, may be a useful diagnostic tool to estimatemethylation 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 WerelKatedra 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 themost 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 workhas 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 eachhexamer to enzyme binding.

In order to determine the importance and influenceof the promoter –35 and –10 regions on RNA polymer-ase binding, we constructed various promoter DNAfragments which lack the –35 element or contain –10hexamer modified entirely or in part. Functional char-acteristic of the obtained promoter fragments were de-termined by analysis of the effectiveness of promoterDNA — RNA polymerase complexes formation. For thisanalysis we adopted gel mobillty shift assay on a nativepolyacrylamide gel.

304 Poster

Gene constructs for xenotransplantation

Daniel Lipinski1, Wojciech Juzwa2, Joanna Zeyland2, Andrzej Plawski1, Karolina Wielgus2, Ryszard Slomski2

1 — Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszyñska 32, 60-479 Poznañ, 2 — Department ofBiochemistry and Biotechnology, Agricultural University, ul. Wo³yñska 35, 60-637 Poznañ, Poland

The presence in humans of xenoreactive antibodiesdirected against swine Gal antigen present on the sur-

face of xenograft donor cells, leads to the complementactivation and immediate xenograft rejection — as a


consequence of hyperacute immunological reaction.The Gal antigen (Gala1,3Gal) is synthesized by thea1,3-galactosyltransferase. In aim to prevent hyper-acute rejection it is possible to modify swine genome bytransfection of human genes — controlling enzymaticcascade 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 andCD46 genes and human gene encoding 1,2-fuco-syltransferase were prepared. CD59 blocks formationof the membrane attack complex. CD46 blocks forma-tion of C3 convertases in the classical and alternative

pathways of complement activation. CD46 has Factor Icofactor activity, which cleaves and inactivates C4b andC3b compounds. CD55 accelerates decay of C3 con-vertases in the classical and alternative pathways. Itdissociates C2 and C2a from C4b and dissociates B andBb from C3b. Introduction of additional copies of hu-man gene encoding 1,2-fucosyltransferase into theswine genome leads to reduction of the Gal antigenlevel 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

Magdalena Narajczyk1, Sylwia Barañska1, Magdalena Gabig-Cimiñska2, Grzegorz Wêgrzyn1

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 differentmodes: (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 atemplate to produce phage proteins. Late mode oflambda DNA replication produces lambda genomesnecessary to form complete phage progeny.

According to commonly accepted hypothesis, sigmamode of replication may be preceded by one round oftheta unidirectional replication. Switch from the thetato the sigma mode of replication requires a change

from bidirectional mode of theta replication to the uni-directional replication. There are many hypotheses toexplain the role of different proteins and regulation ofthe change from bidirectional to the unidirectionaltheta replication mode. Most of them are based on in vi-tro experiments.

We show in in vivo experiments the effect of someproteins (ClpXP, lambdaP, lambdaCro) and oopRNAon the switch from bidirectional to the unidirectionaltheta mode of lambda plasmid DNA. Thus we concludethat those factors influence the initiation of sigma rep-lication.

306 Poster

DNAzyme to mRNA of the beta1 integrin subunit as an efficient tool to reduceinvasiveness of human carcinoma cells

Magdalena Nawrot1, Jolanta Niewiarowska1, Izabela Papiewska1, Andrzej Okruszek2, Maciej Ugorski3, Czes³awCierniewski4

1 — Zak³ad Biofizyki Molekularnej i Medycznej, Uniwersytet Medyczny, ul. Mazowiecka 6/8, 92-215 £ódŸ, 2 — Centrum BadañMolekularnych i Makromolekularnych, Polska Akademia Nauk, £ódŸ, 3 — Instytut Immunologii i Terapii Doœwiadczalnejim. 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 cellularevents including adhesion, migration, proliferation,invasiveness, and cellular survival. These cellularevents play important roles in biological processes suchas angiogenesis, tumor growth and metastasis. It is be-coming increasingly clear that particularly members of

the beta1 subfamily of integrins may play a crucial rolein all these processes. Therefore, we developed a novelapproach to downregulate beta1 integrins in severalcell lines and provided evidence that DNA enzymes canbe useful gene-inactivating agents to control expressionof membrane proteins. We designed the DNA enzyme


to beta1 mRNA containing a 15 deoxyribonucleotidecatalytic domain that was flanked by two substrate-re-cognition segments of eight deoxyribonucleotides. Toengineer specific cleavage site in mRNA, the flankingdomains were derived from the most inhibitory oligo-deoxynucleotide antisense to beta1, designated beta11053.

In preliminary experiments we found that suchDNAzyme significantly inhibited expression of thebeta1 integrin subunit in endothelial cells at the level ofmRNA and protein synthesis. They also reduced cellsurface 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 DNAzymeabolished microvascular endothelial cell capillary tubeformation in fibrin and matrigel. In several tests, theDNAzyme and its analogues appeared to be strong in-hibitors of adhesion and migration of malignant pros-tate carcinoma cells (PC3) and various human colonadenocarcinoma cells (HT29, CX1.1, SW620, LS180).Furthermore, they blocked the invasiveness of malig-nant cells through the barrier of matrigel matrix in invitro assay.

307 Poster

Synergistic effect of the p22 (phox) C242T polymorphism and ACE insertion/dele-tion polymorphism in Silesian CAD patients

Pawe³ Niemiec1, Beata Sarecka1, Anna Balcerzyk1, Zbigniew Ciemniewski2, Iwona ¯ak1

1 — Department of Biochemistry and Medical Genetics, Medical University of Silesia, ul. Medyków 18, 40-752 Katowice, 2 — TheFirst 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). Thep22(phox) subunit is a critical component of NAD(P)Hoxidase, which plays an essential role in superoxide pro-duction. However the NAD(P)H oxidase is a constitutiveenzyme, 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 genotypeincreases p22(phox) production and ROS generation.Our previously published data indicate that carrier-stateof 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 genotypedistribution of ACE gene I/D polymorphism andp22(phox) gene C242T polymorphism in CAD patientsand controls.

Materials and Methods: Sixty six patients withangiographically proven CAD, and 98 healthy controlswere studied. ACE and p22(phox) polymorphisms wereevaluated by PCR and PCR-RFLP (RsaI) respectively.

Results: The frequencies of p22(phox) genotypes incases and controls were respectively: CC 40.9% vs.46.9%, CT 45.5% vs. 42.9% and TT 13.6% vs. 8.2%, butthese differences were not significant. The DD geno-type of ACE gene I/D polymorphism was significantlymore frequent in CAD patients (p=0,027). Additionally,we found differences in a number of subjects with bothhomozygous genotypes (DD and TT). The frequenciesof double-homozygous genotypes in cases and controlswere respectively: 9.09 vs. 1.02 (p=0.017, OR=9.70; CI1.10-222.84).

Conclusion: The coexistence of ACE DD genotype andp22(phox) TT genotype is more common in patientswith CAD, than in controls and considerably increasesCAD risk. There is a synergistic effect of the ACE I/Dand p22(phox) C242T gene polymorphisms in Silesianpatients with coronary artery disease.

308 Poster

Characteristics of a second gene 27 product — 27 bis of bacteriophage T4

Józef Nieradko


Gene 27 is a member of the main cluster of six genescoding for bacteriophage T4 baseplate constituents; 51,27,28,29,48,54. Transcription experiments have shown

that these six genes are cotranscribed from a commonpromoter located between the gene 26 and 51. Thesegenes were cloned into pT7-5 vector under control of T7


bacteriophage 10 promoter. The level of synthesis ofparticular products was estimated in the strain E. coliBL21 (DE3)pLysS, which differs from its original coun-terpart BL21(DE3) by the presence of a plasmid codingfor T7 lysozyme and by being nonpermissive for the T4amber mutants. These properties make possible exami-nation of expression and estimation of complemen-tation efficiency in the same culture. The examinationof expression of this hybrid and its deletion derivatives

was the base for the following conclusions: the 27 geneof bacteriophage T4 encode two proteins of 44 and 39kDa (designated 27-44 and 27-39bis, respectively) as aresult of independent translation initiation at two dif-ferent start codons with the same reading frame. Thelatter with molecular weight 39 kDa probably plays sig-nificant role in regulation of expression of gene 51. The44 kDa protein is the structural component ofbacteriophage T4 baseplate.

309 Poster

Microsatellite instability at RAD51 locus in breast cancer

Maria Nowacka1, Magdalena Bryœ1, Hanna Romanowicz-Makowska2, Wanda Krajewska1

1 — Department of Cytobiochemistry, University of Lodz, ul. Banacha 12/16, 90-237 Lodz, 2 — Department of Pathology, Instituteof Polish Mother’s Memorial Hospital, ul. Rzgowska 281/289, 93-338 Lodz

Identification of chromosomal regions with alleliclosses is a method for screening genes implicated in thepathogenesis of human tumors. Loss of heterozygosity(LOH) in the region 15q15.1 (microsatellite region withtandem 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 theRAD51 gene in 30 primary breast cancers. Material foranalysis was obtained from paraffin-embedded tissuesand peripheral blood as control. After extraction ofDNA by the phenol-chloroform/proteinase K methodfragments of the RAD51 gene were amplified usingthree microsatellite markers: D15S118, D15S214,

D15S1006. Fluorescent PCR products were analysed inABI PRISM® 377 DNA Sequencer. Size and quantity ofallele markers were evaluated using GeneScan® soft-ware version 3.1.2. The nuclotide sequences of frag-ments showing peak shifts were determined using theABI PRISM® BigDye™ Terminator Cycle SequencingKit and ABI PRISM® 377 DNA Sequencer.

Allelic loss was detected in 11 of the 22 informativecases for microsatellite marker D15S118, in 7 of the 15informative cases for microsatellite marker D15S214,in 4 of the 15 informative cases for microsatellitemarker D15S1006. LOH was observed in the 15q15.1region for at least one microsatellite marker in 27% ofbreast cancer studied cases.

310 Poster

Polimorphism of XPD gene coding for nucleotide excision repair protein and therisk of head and neck cancers

Joanna Polañska1, Joanna Jaworska2, Monika Pietrowska2, Dorota Butkiewicz3, Joanna Rzeszowska-Wolny2

1 — Institute of Automation Control, Silesian University of Technology, Gliwice, 2 — Zak³ad Radiobiologii Doœwiadczalnej iKlinicznej, Centrum Onkologii w Gliwicach, ul. Armii Krajowej 15, 44-100 Gliwice, 3 — Department of Tumor Biology, Center ofOncology, Maria Sk³odowska-Curie Memorial Institute, ul. Wybrzeze AK, Gliwice

We investigated the frequency of single nucleotidesubstitutions changing Asp to Asn in codon 312 or Lysto Gln in codon 751 of the protein XPD in the group of127 donors, 84 healthy and 43 patients with head andneck cancers. To detect differences in polymorphicvariant 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 theanalyzed subgroups (exact p=0.9554). Similar analysisperformed 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 patientsand healthy donors. A significant excess of hetero-zygosity was observed in cancer patients (observedvalue 0.8140, expected value 0.4884). The estimatedvalue of odds ratio OR was equal to 2.692 (exactp=0.0328) with 95% CI from 1.047 to 7.525. The resultssuggest that heterozygotic genotype at XPD 751 codoncould 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


311 Poster

Genetic instability of (CT/AG) motifs isolated from V. bithynica

Tomasz Sakowicz1, Pawe³ Parniewski2

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 repetitivesequences. Of these repeats microsatellites, stretchesof short, tandemly repeated motifs of 1–6 bp are veryunstable and display very high polymorphism. Wefound such sequences are also present in Vicia bithynicagenome. Based on the screening of DNA library, clonesharbouring long tracts of CT/AG motifs have been se-lected. Sequence analyses precisely revealed the lengthand class of the repeating unit, number of repeats, aswell sequence composition flanking repeating motifs.Representative plasmids were used to investigate their

genetic instability. In the study presented herein, weemployed very well identified and tractable bacterialgenetic 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 somefactors influencing transcriptions through the repeti-tive motifs. These observations are in agreement withsimilar studies performed on human repetitive se-quences.

312 Poster

Mutations G98T and A561C of the E-selectin gene are not associated with coronaryartery disease in Silesian patients

Beata Sarecka1, Pawe³ Niemiec1, Anna Balcerzyk1, Zbigniew Ciemniewski2, Ewa Rudowska3, Stanis³aw Dyl¹g3,Iwona ¯ak1

1 — Department of Biochemistry and Medical Genetics, Medical University of Silesia, ul. Medyków 18, 40-752 Katowice, 2 — TheFirst Department and Clinic of Cardiology, Medical University of Silesia, ul. Zio³owa 45/47, 40-635 Katowice, 3 — Section ofHematology, Regional Blood Centre, ul. Raciborska 15, 40-074 Katowice

Background: E-selectin mediates in the adhesion ofleukocytes into endothelium. An increased expressionof E-selectin has been observed in activated arterial en-dothelium and appeares to be involved in thepathogenesis of atherosclerosis. Several studies havedemonstrated that the G98T and the A561C mutationsin the E-selectin gene are a strong risk factors for earlysevere atherosclerosis. Some authors suggest also highcorrelation between the G98T and A561C mutations.

Aim: The aim of our study was to investigate relation-ships between the E-selectin G98T or A561C mutationsand coronary artery disease (CAD) in the Silesian pa-tients.

Materials and Methods: Sixty four patients withangiografically documented CAD, and ninety nineblood donors as control group were studied. The G98Tand A561C mutations were analyzed by PCR-RLFP(HphI and PstI respectively).

Results: The allele frequencies of G98T in cases andcontrols 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 inthe patients and 75.8% GG, 21.2% GT and 3% TT in thecontrols. The allele frequencies of A561C in cases andcontrols were respectively: A 85.2% vs. 84.8%, C 14.8%vs. 15.2%. The distribution of A561C genotypes were73.4% AA, 23.4% AC and 3.1% CC in the patients and72.7% AA, 24.2% AC and 3% CC in the control group.Differences in the frequencies of alleles and genotypesbetween 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 inother previously published data. Despite the observa-tion that there was no significant relationship betweenboth mutative genotypes and the disease we found thatthe G98T mutation is highly correlated with the A561Cmutation (p<0.001; correlation coefficient 0.9553) aswell in patients as in control group.

Conclusion: Our data demonstrate that there is no as-sociation between E-selectin G98T or A561C mutationsand coronary artery disease in the Silesian patients.


313 Poster

Limited use of the Cre/loxP recombination system in efficient production ofloxP-containing minicircles in vivo

Marian Sêktas, Maciej Specht


The Cre/loxP recombination system of bacteriophageP1 is one of the most powerful tools in genome engi-neering. We report, however, that activity of Cre/loxPsystem interferes with stability of the multicopyloxP-bearing plasmids. Intermolecular recombinationreduces 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 oftheir copy (overall yield) gradually decreases. We havefound that in the presence of even the slightest amountof Cre activity, loxP-bearing plasmids continuously un-dergo multimerization, which leads to loxP-plasmidfree cells very rapidly. Our results are compatible withthe hypothesis of the multimer catastrophe (Summersand 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 blocsexpression of subset of genes responsible for larvaemetamorphosis, whereas in adult insects activates ex-pression of genes that are necessary for reproductivematuration. A term JH refers to six closely relatedsesquiterpenes containing an ester bond and epoxybond required for the hormone regulatory function. JHis produced in corpora allata and transferred to targetcells by juvenile hormone binding protein (JHBP). Inthe hemolymph more than 99.9% of hormone(s) ap-pears in a complex with JHBP. This protein protectsthe 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 molecularweight (25–30 kDa) monomeric proteins with oneJH-specific binding site.

In our laboratory it has been previously demon-strated that molecule of JHBP from G. mellonellahemolymph is a glycoprotein containing two disulfidebonds and that JH binding induces a profoundconformational change of the protein molecule whichmight 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 anddr G. Bujacz laboratories. Although JHBP(s) have beenisolated and characterized from several insect species,the amino acid sequences are known for four proteinsand a gene sequence has been reported only forManduca 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 genetranscript of G. mellonella jhbp contains a characteris-tic sequence of the initiator (Inr), the downstream pro-moter element (DPE) and four introns, similarly toM. sexta jhbp. However, the introns positioning andtheir length differ between the two genes. The donorand acceptor sequences of all exon-intron boundariesconform 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 55and 56 nt, 296 and 297 nt, 420 and 421 nt, and 573 and574 nt, respectively. Their lengths, calculated fromelectrophoretic mobility, are close to 5 kbp, 3 kbp, 1.2kbp, 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)


315 Poster

Searching for Arabidopsis thaliana mutants resistant to Agrobacterium-mediatedtransformation

Wojciech Strza³ka1, Stanton Gelvin2, Alicja Ziemienowicz1

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 studyinggenomes of various complex organisms. Such a prog-ress gave researchers powerful tools like forward ge-netic screening for studying function of many unknowngenes. Recently, it has been widely engaged for an iden-tification of the plant factors responsible forAgrobacterium-mediated transformation.

Agrobacterium tumefaciens is a plant pathogen charac-terized by an unique feature of interkingdom DNAtransfer. 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 mayresult in a induction of crown gall disease ondicotyledonus plants due to the expression of T-DNAgenes encoding enzymes involved in opines and planthormones synthesis. Although the Agrobacterium fac-tors involved in plant transformation are well charac-terized, our understanding of the T-DNA transport andintegration process into the plant genome still remainsincomplete.

The aim of this study is identification of A. thalianaRAT (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 plantcell.

In this project we use a pool of A. thaliana T-DNAtagged lines (Bressian collection). Sterilized seeds aregerminated in a growth chamber followed by cultiva-tion of seedlings for next 10 days. After this period oftime roots are cut, infected with A. tumefaciens At10,cocultivated on MS basal plate, and then scraped off ona surface of MS basal plate containing timentin. After 6weeks the grown tumors are scored. From a pool of 400tested independent lines 120 candidates were chosenfor second screening and it resulted in 5 potential RATmutants that are likely to show defects in transforma-tion process. These defects might be at different stagesof plant-bacterium interaction including: attachment ofthe bacteria to the plant cell, T-DNA transfer, T-DNAnuclear import and integration. The selected candi-dates will be tested by Southern blot analysis, and theplant DNA sequence interrupted by the T-DNA insertwill 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 sequencesin a promoter region

Barbara Strzelczyk1, Monika S³omiñska1, Grzegorz Wêgrzyn1, Alicja Wêgrzyn2

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 themain negative regulator of DNA replication initiationbut also a specific transcription factor. It binds tohemimethylated GATC sequences and, with somewhatdifferent specificity, to fully methylated GATC regions.Recently, a microarray analysis was reported, in whichtranscriptomes of wild-type and delta seqA strains werecompared. Although in the seqA mutant the levels ofsome transcripts were significantly decreased whilecertain transcripts were evidently more abundant rela-

tive to wild-type bacteria, no correlation between thepresence of GATC motifs in promoter sequences andtranscription activity was found. However, here weshow that when larger DNA fragments, encompassingpositions from –250 to +250 relative to transcriptionstart site, are analyzed, some common features ofGATC distribution near promoters activated by SeqAcan be demonstrated. Nevertheless, it seems thatGATC pattern is not the only determinant ofSeqA-dependence of promoter activity.


317 Poster

Biological activity of purified hGH, TNF� and FeldI recombinant proteins expressedin bacterial and eukaryotic systems

Marlena Szalata1, Daniel Lipinski2, Robert Kalak3, Paulina Tobola4, Marek Pienkowski5, Ryszard Slomski1

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 Biochemiii 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 anew field connected with protein structure and func-tion. Now each protein predicted by genome analysiscan be obtained as recombinant protein. Biologicallyactive recombinant proteins can be expressed in bacte-rial or eukaryotic systems. We used for expression ofrecombinant proteins E. coli cells and mammary glandsof transgenic animals. Human tumor necrosis factorTNF and two chains of major cat allergen FeldI werepurified from bacteria. Human growth hormone hGHwas 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 chain1 and 2 by cleavage of sequence recognized bythrombin or enterokinase introduced between tag andprotein sequence. Biological and cytotoxic activity ofrecombinant proteins was estimated. Additionally, forboth FeldI chains immunological analysis was accom-plished by surface plasmon resonance technology. Hu-man growth hormone activity was analyzed towardsgrowth promoting activity using growth hormone de-pendent cells Nb2-11. For all recombinant proteinsamino 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 thepathogenesis of ulcerative colitis, because of inhibitionof butyrate oxidation within the colonocyte. The sul-phate reducing bacteria (SRB) have been shown to bedirectly responsible for the generation of hydrogen sul-phide in the colon. Pitcher et al. (2000) have identifiedon the basis of 16S rRNA sequence the Desulfovibriodesulfuricans as a representative strain in patients withulcerative colitis with clinically active disease. Al-though SRB are not considered as pathogenic bacteriaDesulfovibrio spp. have been isolated from pyogenicliver abscesses, and found to cause septicemia andbacteremia. Langedijk et al. (2001) stated the occur-rence of these bacteries in sites affected by periodontaldisease. The possibility of pathological influences ofDesulfovibrio genus on a human body suggests advis-ability of applying the most appropriate diagnosticmethods with the purpose of identification of this ge-nus strains.

Over the last years techniques involving the analysisof 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 thespacer region in-between within 15 strains (soil and in-testinal) of D. desulfuricans species. Seven enzymes wereevaluated for their discriminatory power in ribotyping16S-23S fragment. The discriminatory power was thegreatest with MboI and HaeIII. Furthermore, these twocases the most complicated banding pattern was re-ceived. The enzymes AluIII, HindIII, HinfIII and EcoR1were 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 areproducible and simple alternative to conventional mo-lecular approaches for typing strains of D. desulfuricans,but the careful choice of appropriate restriction enzymesis indispensable requirement.


319 Poster

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

Alicja Wêgrzyn1, Monika S³omiñska2, Gra¿yna Konopa2, Barbara Kêdzierska2, Grzegorz Wêgrzyn2

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, amain negative regulator of Escherichia coli chromo-some replication initiation, is also a specific transcrip-tion factor. SeqA specifically activates bacteriophagelambda pR promoter while revealing no significant ef-fect on the activity of another lambda promoter, pL.We demonstrate that lysogenization by bacteriophagelambda is impaired in E. coli seqA mutants. Geneticanalysis demonstrated that CII-mediated activation ofthe phage pI and paQ promoters, which are requiredfor efficient lysogenization, is less efficient in the ab-sence of seqA function. This was confirmed in in vitrotranscription assays. Interestingly, SeqA stimulatedCII-dependent transcription from pI and paQ when it

was added to the reaction mixture before CII, whilehaving little effect if added after a pre-incubation of CIIwith the DNA template. This SeqA-mediated stimula-tion was absolutely dependent on DNA methylation, asno effects of this protein were observed when usingunmethylated DNA templates. Also, no effects of SeqAon transcription from pI and paQ were observed in theabsence 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 ofneighboring 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 isorganised as an operon and is responsible for msDNAproduction. Multicopy single-stranded DNA (msDNA)is a structurally unique molecule composed of a sin-gle-stranded DNA (encoded by msd) branched out froma single-stranded RNA (encoded by msr) by 2’-5’ phos-phodiester linkage. msDNAs are common in cells of soilbacteria (Myxococcus, Stigmatella) but occur also in bac-teria from Enterobacteriaceae family. Function ofmsDNA in bacterial cell is unknown.

Single-stranded DNA purified from polyacrylamidegel was sequenced by Maxam-Gilbert method. The se-quence obtained for two strains (W8 and CZD1527) isidentical. It is rather short, i.e. 63 bp, and contains in-

verted repeats of 25 bp. The next aim of research wascloning 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. Totalchromosomal 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 atemplate 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 orientationwere used. For CZD1527 strain, two products havebeen obtained of 350 and 210 bp containing promotor,msd and msr genes.


321 Poster

The optimal eukaryotic signal for translation initiation from non-aug codons, pres-ent upstream of bacteriophage � P cistron, is inactive in Escherichia coli

Borys Wróbel1, Bartosz S³omiñski2, Grzegorz Wêgrzyn2

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�, 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 highlevel. The results presented in this report, suggest con-trary to the previously presented hypothesis, that aAGACUGGAU 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 confirmsthat such a signal is unlikely to be important for P syn-thesis. Instead, a weak Shine-Dalgarno sequence maybe present upstream the P cistron. This weak signalmight be active in the absence of O gene expression.

322 Poster

Demonstration of kidney-specific activity of human uromodulin gene promoter intransgenic mice

Halina ¯bikowska1, Nadia Soukhareva2, Reza Behnam2, Rosemary Chang3, Roman Drews4, Henryk Luboñ2,David Hammond2, Serguei Soukharev2

1 — Katedra Biochemii Ogólnej, Uniwersytet £ódzki, ul. Banacha 12/16, 90-237 £ódŸ, 2 — Plasma Derivative Department, AmericanRed 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 technologyin 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 proteinproduction in the urinary tract is the lack of a reliableand tissue-specific expression vector. The uromodulingene seemed to be a good candidate for this purposesince uromodulin (known as the Tamm-Horsfall pro-tein; THP) is recognized as the most abundant proteinin human urine. The expression of uromodulin is spe-cific to the cells of the ascending limbs of Henle’s loopin the kidney.

In this study, we have isolated and sequenced the6.75-kb fragment of human uromodulin promoter. Inaddition, we have generated transgenic mice for ex-pression of the lacZ marker gene. Analysis of theUro-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 exoncould provide for a highly tissue-specific expression ofthe lacZ gene. All tested transgenic lines showed asteady increase in kidney-specific beta-galactosidase ac-tivity measured spectrofluorometrically. Histologicalevaluation of the beta-galactosidase expression con-firmed high tissue-specificity of the promoter. Signifi-cant activity after staining with X-gal as a substrate forbeta-galactosidase was detected in a medulla of the kid-ney which correlates with the anatomical location ofHenle’s loop.


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