Oral Presentations

O11.1

Mapping of the functional phosphate groups in the catalytic core of DNAzyme 10-23

Barbara Nawrot, Kinga Widera, Marzena Wójcik, Beata Rębowska, Wiesława Goss, Wojciech J. Stec

Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences, Łódź, Poland e-mail: bnawrot@bio.cbmm.lodz.pl

To date, an active structure of the DNAzyme 10–23 in complex with its substrate RNA is not yet known and the details of the mechanism of the RNA cleavage reaction are not fully understood. Much effort has been devoted to the determination of the role of individual nucleosides of the 10-23 catalytic core in the process of catalysis [1]. However, the role of particular phosphates within this core has not been investigated to date. Therefore, we have attempted to study this issue by systematic modification of each phosphate of the core (from P1 between A0 and G1 to P15 between A15 and T16) by introducing sulfur instead of oxygen in non-bridging positions. A metal-specifi-city switch approach permitted to identify non-bridging phosphate oxygens (pro-RP or pro-SP) located at seven po-sitions of the core (P2, P4 and P9-13) suggesting their pos-sible direct coordination with a divalent metal ion(s). By contrast, thio-phosphates at positions P3, 6, 7, 14 and 15 displayed no functional relevance. Interestingly, PS modi-fications at positions P1 or P8 stimulated deoxyribozyme catalytic activity. Among the tested deoxyribozymes, thio-substitution at position P5 had the largest deleteri-ous effect on the catalytic rate in the presence of Mg2+ and this was reversed in the presence of Mn2+ ions. Further experiments with thio-deoxyribozymes of stereodefined P-chirality suggested the direct involvement of both oxy-gens of the P5 phosphate and the pro-RP-oxygen at P9 in the metal ion coordination.

References:1. Zaborowska et al. (2002) J Biol Chem 277: 40617-40622; Schubert et al. (2003) Nucleic Acids Res 31: 5982-5992; Zaborowska (2005) FEBS Lett 579: 554-558.

O11.2

A Novel 2’-deoxy-2’-fluoro-D-arabinonucleic acid (2’F-ANA) modification of DNA creates an efficient gene silencing oligodeoxynucleotide (ODN)

A. Kalota1, L. Karabon1, E. Viazovkina2, M. Elzagheid2, C. R. Świder1, M. J. Damha2, A. M. Gewirtz1

1University of Pennsylvania School of Medicine, Philadelphia, PA, USA; 2McGill University, Montreal QC, Canada e-mail: akalota@mail.med.upenn.edu

Antisense oligodeoxynucleotides (AS ODN) remain at-tractive as gene silencing agents because of their relative ease and low cost of synthesis when compared to siRNA. In addition, because AS ODN do not enter the RISC path-way, they may be less subject to ‘off target’ silencing. AS ODN should be nuclease resistant, form stable DNA/RNA hybrids, and support of RNase H mediated cleavage of heteroduplexes, with negligible non-specific effects on cell function. Fulfilling all these criteria has proven difficult but 2’-deoxy-2’-fluoro-D-arabinonucleic acid (2’F-ANA) modi-fications of DNA may well address these issues in living cells as a result of their ability to simultaneously increase the strength of DNA:RNA hybrids, resistance to nucleases, and elicit efficient RNase H mediated degradation of target mRNA. In the present study, we compared the gene silenc-ing efficacy of 2’F-ANA modified ODN with unmodified AS-ODN in a cellular system. In this study, we targeted the c-myb gene with an AS ODN that was predicted to have improved hybridization accessibility compared to randomly targeted ODN. As predicted, the 2’F-ANA ODN were highly resistant to nuclease degradation. Nucleofec-tion of c-myb targeted 2’F-ANA ODN into K562 cells dem-onstrated their ability to efficiently silence c-myb mRNA and protein expression, even when compared to AS ODN. Of significant interest, and in contrast to AS ODN, gene silencing with 2’F-ANA AS persisted up to 72 h. These data suggest that 2’F-ANA AS are efficient gene silencing mol-ecules, and in contrast to AS ODN, their effects are long lived. We conclude that 2’F-ANA will function as efficient gene silencing agents in living cells and could have signifi-cant therapeutic potential.

O11.3

Arabidopsis thaliana LSM proteins function in mRNA splicing and degradation

Katarzyna Kruszka, Joanna Kufel

Session 11. Nucleic acids

Vol. 53 14541st Meeting of the Polish Biochemical Society

Institute of Genetics and Biotechnology, Warsaw University, Warsaw, Poland

A key component of the eukaryotic RNA machinery is a conserved family of LSM (Sm-like) proteins. Two distinct LSM complexes exist in yeast and humans: the nuclear complex is a core component of the U6snRNP and is in-volved in mRNA splicing, while the cytoplasmic one in-teracts with exonuclease Xrn1 and decapping enzymes Dcp1/Dcp2 and functions in mRNA decay. Additional roles of LSM complexes include degradation and process-ing of various stable RNAs in yeast and trans-splicing in trypanosomes. In contrast, little is known about RNA processes in plants. Recent data implied involvement of AtLSM5 in abscisic acid signalling and drought suggest-ing a link between RNA metabolism and plant response to stress. We have identified and cloned Arabidopsis LSM homologs. Subcellular localization of GFP-AtLsm con-structs in Arabidopsis protoplasts showed that AtLSM2-7 had a double nuclear-cytoplasmic localization, whereas AtLSM1 was cytoplasmic and AtLSM8 – nuclear. This points at the existence of two LSM complexes also in plants. We have selected homozygous knock-out mutants in AtLSM8 and two AtLSM1 proteins and currently we are testing RNA processing/degradation defects in these lines, as well as in a mutant in AtLSM5, sad1. Analysis of sad1 and lsm8 plants by primer extension revealed decreased U6snRNA level. However, semi-quantitative RT-PCR of pre-mRNAs showed that, in contrast to yeast, this had no general effect on splicing but affected only a group of tested transcripts, including those involved in ABA transduction. Similarly, preliminary data for mRNA stability by transcriptional shut-down in sad1 mutant show that only a subset of all mRNAs are subject to 5’-3’ degradation and that these include ribosomal proteins, transcription factors, ARE- and DST-containing mRNAs. Our results indicate the role of AtLSM5 and AtLSM8 in splicing and of AtLSM5 in mRNA decay of specific tran-scripts and suggest that AtLSM proteins may participate in regulation of cellular response processes.

O11.4

The molecular studies – from gene to patient

B. Marzec1, J. Kotarski2, A. Filip1, D. Koczkodaj1, K. Wysocka1, J. Kotarski2, J. Wojcierowski1

1Zakład Genetyki Medycznej Akademii Medycznej, Lublin, Poland; 2I Klinika Ginekologii Operacyjnej, Lublin, Poland

The integration of molecular biologic techniques into studies and diagnosis of cancer offers an opportunity to better understand the causes of cancer, the natural his-tory of different types of cancer, estimate survival rate once the cancer is diagnosed. Most of all it may allow for prevention or appropriate treatment of patient. Regular diagnosis should be commenced from the separation of patients that can be considered as a risk group meaning

with family cancer history. The first line of proceeding can be regarded as direct diagnosis based on blood ex-amination towards germline point mutation (e.g. BRCA in breast or ovarian cancer) or other genetic alterations (e.g. chromosome aberration in hematological neopla-sias) that are widely accepted as a common cancer risk or “forecast” factor. Secondly there should be determined typical cancer corresponding makers (characteristic gene expression on its mRNA or protein level or other recently found out candidate like microRNA expression). Still we don’t know much about many cancer e.g. ovarian caner due to their heterogeneity. It would be eligible to apply molecular tools and diagnostic results to subsequent ther-apy in order to stratify patients and to predict a tumor’s behavior as well as the response to different treatment modalities. Here we review the achievement of standard and the most up-to-date molecular techniques (e.g. SSCP, micro/makrochip, aCGH, SKY, FISH) in studies and di-agnosis of human cancer or as the methods in describing and revealing the nature of different cancer types. Still cancer presents a daunting challenge.

O11.5

The genome – scale cellular responses to ionizing radiation

Joanna Rzeszowska-Wolny1, 2, Joanna Polańska2, Robert Herok1, Andrzej Polański2

1Department of Experimental and Clinical Radiobiology, Centre of Oncology, M.Sklodowska-Curie Memorial Institute; 2 Institute of Automation, Silesian University of Technology, Gliwice, Poland

The relative abundance of different transcripts is a mo-lecular phenotype characteristic of cell type and its physiological state. Global gene expression profiling is now performed by oligonucleotide and cDNA microar-ray methods that produce massive amounts of data and need specialized mathematical tools. We have developed a procedure to study changes in genome-wide expression profiles which are induced in cells exposed to ionizing radiation (IR) and applied it to study expression profiles of two human cell lines, K562 erythroleukemic and Me45 melanoma cells. IR damages cellular macromolecules by direct ionization or indirectly through oxyradical prod-ucts of water radiolysis, with DNA as the most critical target, and damaged cells initiate DNA repair processes and intra- and inter-cellular signaling pathways that de-lay the cell cycle and modulate gene expression. Signals released by irradiated cells may cause heritable genome instability in neighbouring unirradiated cells. Charac-terization of the transcriptional reprogramming induced by radiation is an important step in understanding these processes and differences of response to irradiation ob-served among different cell types. A new approach for analysis of microarray signals which were expressed as histograms and decomposed into Gaussian components

146 2006Abstracts

allowed for clusterization of genes that reacted similarly to IR. In both cell types, two big groups of genes reacted very fast to irradiation by increase or decrease in tran-script level. Genes coding for proteins that take part in regulation of translation, ribosome structure and biogen-esis, proteasome were mainly found in the first group whereas mRNAs coding for proteins that take part in sig-nal transduction and intercellular communication were decomposed immediately after irradiation.

O11.6

The influence of proteolytic enzymes on remodeling the extracellular matrix during wound healing

Magdalena Jurzak, Katarzyna Gojniczek, Agnieszka Garncarczyk

Department of Cosmetology Science, Medical University of Silesia, Sosnowiec, Poland

e–mail: magda@slam.katowice.pl

The wound healing is dynamic phenomenon connected with the influence of many factors that may lead to res-toration of integrality and function of damaged tissues. Prolonged wound healing is connected with elongated inflammation state and high activity of fibroblasts and macrophages. The production of new extracellular ma-trix is increased as well. The amount and the kind of syn-thesized during wound repair matrix is a result of two antagonistic processes: matrix synthesis and their enzy-matic degradation. These processes are termed matrix remodeling. Disturbing of remodeling phase of wound healing can conduct to abnormal scars formation. The most important enzymes participated in this process are extracellular matrix metalloproteinases (MMPs). The ma-trix metalloproteinases (MMPs) comprise a family of at least 25 proteolytic enzymes that play an essential role in tissue remodeling. The function and regulation of matrix

metalloproteinases in normal and abnormal wound heal-ing is discussed. Aim of the study was analysis of profile of gene expression coding different classes of MMPs and comparison transcriptive activities of these genes during normal and impaired wound healing. In this study, we present the results of applying real-time RT-PCR for as-sessing the MMPs mRNA level in the skin segments of patients with normal and impaired wound healing. We hope that results received in this research help as to deter-mine relationship between MMPs gene expression and its influence on excised wound healing pattern and rate.

O11.7

Dinucleotide 5’ mRNA cap analogues with modified triphosphate bridge: useful tools to study decapping and translation

E. Darzynkiewicz1, E. Grudzien,

J. Kowalska1, M. Kalek1, J. Jemielity1, J. Zuberek1, M. Lewdorowicz1, E. Bojarska1, Z. Darzynkiewicz1, J. Stepinski1, R. Stolarski1, R. E. Rhoads2, R. E. Davis3

1Department of Biophysics, Warsaw University, Warsaw, Poland; 2Department of Biochemistry and Molecular Biology, LSU, Shreveport, LA,USA; 3Univ. Colorado School of Medicine, Aurora, CO, USA e-mail: edek@biogeo.uw.edu.pl

Eukaryotic mRNAs are modified at their 5’ ends by ad-dition of a 7-methylguanosine attached by a 5’-5’ triphos-phate bridge to the next nucleoside forming a cap structure: m7G(5’)ppp(5’)N. Caps fulfill a variety of functions in the synthesis, translation and degradation of mRNA. Synthet-ic in vitro capped RNA transcripts have been widely used in a variety applications. However, using bacteriophage RNA polymerases, more than 30% of the caps are incorpo-rated in the reverse orientation, that is, with m7G moiety of m7GpppG linked by a 3’-5’ phosphodiester bond to the first nucleoside residue of the RNA chain. This problem was solved by blocking the ribose 3’ or 2’ hydoxyl group with methyl substituent in 7-methylguanosine moiety. We synthesized series of anti reverse cap analogs (ARCA) modified in the C2’ and C3’ positions of m7Guo and in the number of phosphate residues: m2

7,2’Gp3G, m27,3’Gp3G,

m27,2’Gp4G, m2

7,3’Gp4G and m27,3’Gp5G. Furthermore, we

designed and synthesized two cap dinucleotides (type of ARCA), in which a methylene group was substituted be-tween the α- and β-phosphate moieties, m2

7,3’GppCH2pG and m2

7,3’GpCH2ppG, that are predicted to be resistant to cleavage by decapping enzymes: Dcp1/Dcp2 and DcpS, re-spectively. The above compounds were analysed for their conformation in solution, binding affinity to eIF4E, inhibi-tion of in vitro and in vivo translation, degree of reverse cap-ping during in vitro transcription, capping efficiency and the ability to stimulate cap-dependent translation (in vitro and in vivo) when incorporated into mRNA. The results show that modifications at C2’, like those at C3’, prevent reverse incorporation, tetra- and pentaphosphate cap dinu-cleotides bind eIF4E and inhibit translation more strongly than their triphosphate counterparts, and tetraphosphate ARCAs promote cap-dependent translation most effec-tively (2.5-fold better than the conventional cap, m7Gp3G) what makes them valuable tools in biotechnology. mRNAs capped with m2

7,3’GppCH2pG appeared to be resistant to cleavage by Dcp1/Dcp2 and were more stable in vivo, indi-cating that 5’ to 3’ pathway makes a major contribution to overall degradation in mammalian cells. The synthesis and some biochemical properties of the new serie of phospho-rothioate ARCAs will be also presented.

O11.8

Isoenergetic RNA microarrays, a new method for study of the structure and interactions of RNA

Ryszard Kierzek

Vol. 53 14741st Meeting of the Polish Biochemical Society

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland

There are a growing number of RNAs for which biologi-cal function and structure are not known. The knowledge of RNA structure is crucial to the understanding of its biological function. It is also helpful for applying RNA as a potential agent or target for therapeutic treatment. The current methods used to solve the native structure of large RNAs (enzymatic and chemical mapping) are complicat-ed and time consuming. Application of RNA microarrays (which should soon become commercially available) to study RNA structure should make this process fast, cheap and accurate. In native RNA, single stranded regions are short (4–6 nucleotides on average) and are often involved in weak non-Watson-Crick and tertiary interactions. Most regions of RNA interact with complementary strands, forming duplexes and are unable to bind to the single stranded probes on RNA microarrays. Present methods are based on RNA microarrys built with short (5–6 mers) of highly modified RNA probes that are covalently linked to agarose coated microscope slides. The 2-O-methylated and LNA-modified oligomer probes have been designed in such a way, that the interaction of each probe with its complementary single stranded fragment of target RNA has the same thermodynamic stability (is isoenergetic). The thermodynamic stabilities of modified probes bind-ing to complementary fragments of target RNA were predicted based on the measurement of the thermody-namic stabilities of many model LNA-2’OMeRNA/RNA duplexes. Moreover, the influence of numerous single nucleotide mismatches on thermodynamic stabilities of model LNA-2’OMeRNA/RNA duplexes were evaluated as well. A library of 850 isoenergetic pentamers (from 1024 possible pentamers) was chemically synthesized, deprotected and purified. The probes contained an ami-nohexyl linker at the 5’-side for covalent linking with ac-tivated agarose coated slides. The RNA microarays were prepared by manual spotting, as well as by using a Spot-Array 24 printer. The target RNAs were radioactively la-beled on their 5’- or 3’-side and used for hybridization on the microarray. The hybridization was performed under various conditions in order to optimize the interaction of the RNA microarray and target RNA. The model target RNA was the 5S rRNA from E. coli. Experiments were also performed on the 323 nucleotide R2 5’RNA from Bombyx mori, whose structure was unknown. The hybridization results were used as constrains in the RNAstructure pro-gram to predict RNA secondary structure. In the case of 5S rRNA, microarray experiments allowed for 93% cor-rect prediction of base pairing. Microarray experiments on the R2 5’RNA lead to the proposal of a secondary structure which is consistent with the one determined from chemical mapping experiments. Methodological studies on RNA microarrays seem to be promising and encouraging for further studies. However, the method still requires some improvement.

O11.9

Regulation of RNA activity by low molecular ligands

Jan Wrzesinski1, Jerzy Ciesiołka1, Małgorzata Jeżowska-Bojczuk2, Magdalena Brzezowska2, Wojciech Szczepanik2, Stanisław Jóźwiakowski1

1Institute of Bioorganic Chemistry, PAS, Poznań, Poland; 2Faculty of Chemistry, University of Wrocław, Wrocław, Poland e-mail: wrzesinj@ibch.poznan.pl

Regulation of gene expression involves complicated net-work interactions of RNA molecules with proteins, other RNAs as well as small molecules such as metabolites and metal ions. Participation of riboswitches in such regula-tion is well documented in prokaryotes. Riboswitches are highly structural domains located in 5’ UTR regions of certain mRNAs which are able to bind low molecular ligands with high affinity and specificity. Binding of these ligands changes RNA structure regulating gene expres-sion on the level of transcription or translation. However, it is conceivable that also metal ions, the simplest ligands present in the cell which are required for proper folding of RNA and RNA catalysis, may modulate RNA activ-ity. Searching for such metal, we focused our efforts on two exceptional transition metal ions: cobalt and cooper. The in vitro selection methodology was applied to search for Co(II)-binding RNA aptamers and two such aptam-ers were found. The important role of the N7 position of some purine residues of the selected aptamers in direct binding of Co(II) ions and in organization of the aptam-ers structure was clearly shown using the NAIM method. Moreover, we tried to determine the structural changes that occurred upon Co(II) binding and we found that in the presence of Co(II) the aptamers were folded into com-pact more stable structures. It turned out that other tran-sition metal ions might replace Co(II) bound to aptamers; however, alkaline metal ions did not show such proper-ties. In another series of experiments, we showed that some antibiotics i.e. sisomycin, vancomycin and actino-mycin D in complex with Cu(II) inhibit catalytic activity of delta ribozyme much stronger than the same antibiot-ics in their free state. In the case of actinomycin D–Cu(II) complex, determined Ki value was reduced 35-fold upon metal ion complexation. On the basis of the presented in-formation we propose that coordination of small ligands such as metal ions may substantially change functional properties of RNA.

O11.10

Barley stripe mosaic virus (BSMV) – based functional characterization of genes associated with powdery mildew resistance in barley

Maria Barciszewska-Pacak1, Ingo Hein2,

148 2006Abstracts

Katarina Hrubikova3, Ken Shirasu4, Christophe Lacomme3, Artur Jarmołowski1

1Department of Gene Expression, Adam Mickiewicz University, Poznań, Poland; 2Programme of Genome Dynamics, 3Programme of Cell-to-Cell Communication, Scottish Crop Research Institute, Invergowrie, Dundee, UK; 4The Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK e-mail: marbp@amu.edu.pl

Virus Induced Gene Silencing (VIGS) is currently one of the essential methods successfully applied for functional genomics in plants. The most successful VIGS applica-tion in monocots was achieved using a vector based on an RNA virus, barley stripe mosaic virus (BSMV). For testing BSMV-derived VIGS vectors, barley pds silencing was performed. The results showed that sense, antisense and direct inverted-repeats silencing-inducing sequenc-es triggered significant gene silencing. We have applied BSMV VIGS system for the functional characterization of barley genes associated with the Mla13-mediated re-sistance towards fungal pathogen Blumeria graminis f. sp. hordei. In barley Clansman cultivar the VIGS approach targeted Rar1, Sgt1 and Hsp90 genes, known to play a role in the Mla-mediated resistance towards powdery mildew. Their silencing was detected at the RNA and protein level in leaves of main stem and axillary shoots. A resistance breaking phenotype on the BSMV-silenced plants was also observed when treated with an incompatible powdery mildew strain carrying Avr13 gene. The BSMV VIGS sys-tem occurred to be a useful tool for characterizing genes involved in pathogen resistance in barley. Our findings represent also the first evidence in monocots that Hsp90 is a required component for Mla13-mediated race specific resistance. Due to its role in the mechanism, we intend to investigate the interactions of barley Hsp90, SGT1 and RAR1 proteins in vivo and in vitro. For the latter, so far we have cloned HvRar1 and overexpressed HvRAR1-GST fu-sion protein.

O11.11

tRNAs in translation: individualism or conformism?

Mikołaj Olejniczak1, Sarah Ledoux2, Olke C. Uhlenbeck2

1Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland; 2Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, USA e-mail: mikolaj@ibch.poznan.pl

Although tRNAs differ from one another with their esteri-fied amino acids, modifications and nucleotide sequence, they are all equally good substrates in translation. Indeed, it has been recently reported that different E. coli aminoa-cyl-tRNAs have remarkably uniform binding affinities to

the ribosomal A and P sites. While this result is consistent with their role in translation, an important challenge is to understand how a diverse set of tRNAs can interact with ribosomes in a way that yields similar affinities. We propose that each tRNA body has evolved to offset dif-ferent contributions of esterified amino acid and antico-don-codon interaction that are unique for each tRNA. In-deed, the mutagenesis of E. coli tRNA2

Ala revealed that its highly conserved A32-U38 nucleotide pair is a negative binding determinant, and it was proposed that it serves to compensate for very tight anticodon-codon interaction of this tRNA. Interestingly, mutations in the same posi-tions of the anticodon stem-loop are important not only for ribosomal affinity, but also for the kinetics of tRNA progression through the translational mechanism, and for the accurate codon recognition. It appears that tRNAs are not interchangeable adaptors but have individually evolved to be uniformly translated.

O11.12

An attempted enzymatic reactions of base modificated pyrimidine ribosides

Marcin Wróblewski1, Iwona Kaczyńska2, Renata Kołodziejska1, Renata Studzińska1, Elżbieta Sochacka2, Marcin Dramiński1

1Department of General Chemistry, Collegium Medium, Bydgoszcz, UMK Toruń, Poland; 2Department of Organic Chemistry, Technical University, Łódź, Poland e-mail: marcinw8572@wp.pl

Modified nucleosides are of interest because of the hope to obtaining therapeutically useful agents.In our experiments, we used three modified pyrimidine ribosides 1-3:1 – 2-pyrimidinone ribonucleoside2 – 4-pyrimidinone ribonucleoside3 – 6-pyrimidinone ribonucleosideThe compound 1 is commonly known as zebularine [1]. Zebularine – bacteriostatic agent – is a potent cytidine deaminase inhibitor. How the selected enzymes recog-nise modified base in nucleoside is a topic of our study. The following methods for the transformation of ribo-sides 1–3 were used: 1) an enzymatic phosphorylation by nonspecific nucleoside phosphotransferase from a wheat shoot (EC 2.7.1.77) [2, 3]; 2) an enzymatic acetylation by lipase B from Candida antarctica (transfers acetyl group from vinyl acetate) [4]. We obtained 5’-monophosphate and mono-, di- and triacetylated pyrimidine ribosides. Compounds 1-3 was synthesized by Department of Or-ganic Chemistry, Technical University in Łódź and details of synthesis will be published soon.References:1. Marquez VE, Kelley JA, Agbaria R, Ben-Kasus T, Cheng JC, Yoo CB, Jones PA (2005) Ann NY Acad Sci 1058: 246-254.2. Rutkowski M, Dramiński M (1991) Acta Biochim Polon 38: 449-457.

Vol. 53 14941st Meeting of the Polish Biochemical Society

3. Brunngraber EF, Chargaff E (1967) J Biol Chem 242: 4834-4840.4. Kołodziejska R, Dramiński M (2004) Pol J Appl Chem 3-4: 75-81.

Posters

P11.13

Conformational changes of small subunit rRNA during elongation of polypeptide in plant ribosomes

Kamilla Bąkowska-Żywicka, Tomasz Twardowski

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland e-mail: bakowska@ibch.poznan.pl

The interaction and conformational relationships between rRNAs and ribosomal proteins are responsible for the ri-bosome activity. Most important for understanding the translational mechanism is how the ribosome interacts with the substrates and other important factors involved in polypeptide elongation. One of the methods allowing to investigate this mechanism is the antisense strategy. The basic idea of this strategy is to introduce short an-tisense DNA oligomers complementary to an important part of rRNA into a ribosome. These short oligomers in-teract through the Watson-Crick hydrogen bonds with single-stranded rRNAs and block the activity of target rRNAs. We tested 7 different deoxyoligonucleotides com-plementary to the selected, highly conserved sequences of 18S rRNAs important in protein biosynthesis. We car-ried out a reaction of binding Phe-tRNA to A site on the ribosomes converted either to pre- or to post-transloca-tional states (with or without pre-hybridized oligonu-cleotides). We found a correlation between the level of oligomer’s hybridization and the inhibition of AA-tRNA binding. We observed well defined structural changes of ribosome’s conformation during different steps of the elongation of protein biosynthesis. The summary of our results is as follows:• the interactions between 18S rRNA and the elongation factor EF-2 are necessary for AA-tRNA binding,• helix h18 of 18S rRNA is important during all steps of protein biosynthesis, but after forming a peptide bond is no longer exposed to so many interactions,• the E site is important for the formation of required ri-bosome conformation,• the proper functioning of the decoding center is impor-tant during the AA-tRNA binding.

P11.14

Construction of a MboII R-M system for the study of the genetic stability of bacterial hosts

Robert Boratyński, Zbigniew Hanas, Beata Furmanek-Blaszk, Marian Sęktas

Department of Microbiology University of Gdansk, Gdańsk, Poland e-mail: sektas@biotech.univ.gda.pl

We studied the contribution of the restriction-modifica-tion (R-M) system to genetic recombination and horizon-tal gene transfer, to better understand genetic variation among and between bacterial species. One of the most interesting (R-M) system from the functional point of view is MboII R-M from Moraxella bovis. In contrast to the common class II R-M, this system consists of three genes code for R.MboII restriction endonuclease, and two DNA methyltransferases, M.MboIIA – recognizing 5’GAAGA3’ sequence, and M.MboIIB specific to 5’TCTTC3’, a strand complement of the canonical site. We created experimen-tal conditions containing only one functional methyl-transferase (M.MboIIB), to analyze the genetic stability of various E. coli hosts under such conditions (insufficient methylation [hemimethylation] of the canonical sequence in the presence of a surplus activity of R.MboII). This was done in such a way that one of the complimentary strands of the canonical sequence could never be meth-ylated, giving rise to completely unprotected sites at the early post-replication stage. By application of the PCR technique we have isolated, cloned and overexpressed in E. coli host mboIIMB gene. We constructed plasmids carrying the mboIIMB gene under the control of various promoters. We set the molecular conditions to enable us to keep control of gene expression of the “uncompleted” MboII R-M. Then, we studied the restriction-dependent plasmid co-transformation efficiency, the stability of the cat(CmR)-gene used as a molecular marker containing cassettes abundant with MboII restriction sites (restric-tion hot-spots), as well as, the cellular SOS response to N4-cytosine methylation and unbalanced restriction activity.

P11.15

Silencing of CBP20 and CBP80 genes expression in Solanum tuberosum

Joanna Cichocka, Jarosław Nowicki, Paulina Piontek, Artur Jarmołowski, Zofia Szweykowska-Kulińska

Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland e-mail: cichocka@amu.edu.pl

CBC is the nuclear cap-binding protein complex which consists of two subunits: CBP20 and CBP80. Together these two proteins recognize the cap structure at the 5’-end of all RNA polymerase II transcripts. The CBP20/CBP80 complex participates in the splicing of pre-mRNA, mRNA 3’-end formation, export of RNA from the nucleus to the cytoplasm, mRNA translation. A small subunit of the complex, CBP20, shows very high homology in differ-ent eucariotic organisms. All CBP20s contain a very con-

150 2006Abstracts

servative RNA binding domain (RBD) with two motifs: RNP2 and RNP1 in N-terminal part. At the C-terminal end of plant CBP20s two short sequences composed of basic amino acid residues form two active nuclear locali-sation signals (NLS). The Arabidopsis CBP80 shows less similarity to CBP80s from other eucariotic organisms. The characteristic feature of all CBP80s is the MIF4G domain at their N-terminal part. A. thaliana RNAi mutants with low level of CBP20 or CBP80 have been obtained in our laboratory. Both mutants display the same phenotype: abscisic acid hypersensitivity, enhanced drought resist-ance and slightly serrate leave margins. Identical phe-notypes have been observed in the case of AtCBP20 and AtCBP80 T-DNA insertion mutants. Our main goal is to obtain transgenic Solanum tuberosum plants with silenced genes encoding CBP20 or CBP80. In our laboratory we work with two varieties of the potato Solanum tuberosum: Sante and PS646. We characterized the cDNA sequence of CBP20 from both potato varieties. We characterized the gene encoding CBP20 in Sante variety. It has, as in the Arabidopsis thaliana and Oryza sativa, seven introns. Intron no. 4 is U12 intron, which is spliced in a differ-ent way than the other, classical introns. We have already obtained sequences of 1st, 3rd, 4th, 6th and 7th intron of CBP20 of Sante variety. We are going to use RNAi tech-nology to silence CBP20 gene expression. Gene silencing will be achieved using two vectors: pHANNIBAL and the binary vector pART27. We cloned two ~650 bp frag-ments of CBP20 cDNA to pHANNIBAL, one in “sense” and another one in “antisense” orientation. Subsequently, this cassette was subcloned into the pART27 binary vec-tor. This construct will be introduced into potato plants in order to gain transformants with low expression of CBP20. Transgenic potato plants will be analysed for their drought resistance and sensitivity to ABA.

P11.16

Analysis of a 35-kb plasmid isolated from enteropathogenic strain of Escherichia coli using randomly inserted cassettes carrying antibiotic resistance genes

Agnieszka Dekowska, Tadeusz Kaczorowski

University of Gdansk, Department of Microbiology, Gdańsk, Poland

Enteropathogenic Escherichia coli (EPEC) are an important cause of diarrhea in the developing countries. These bac-teria contain plasmids carrying genes involved in patho-genesis. Very often these plasmids encode genes that con-fer to bacteria antibiotic resistance. The presence of such genes on mobile genetic elements enables their rapid spread among bacteria. Recent studies have revealed that the large plasmids possess mosaic structure with heavy load of transposable elements. Progress in the efficient DNA analysis depends not only on developement of new technologies for high-speed DNA sequencing, but also on

the improvement of existing methods. The overall goal of all newly arising approaches is to make DNA sequnc-ing faster, more accurate and cheaper. Here, we present the analysis of a 35-kb plasmid pEC986 harbored by en-teropathogenic strain of Escherichia coli. The plasmid en-codes ampicilin and tetracycline resistance. The plasmid contains transposon Tn21 (ApR), a member of a family, which groups mobile genetic elements involved in antibi-otic mulitiresistance and resistance against mercury. Tet-racycline resistance is encoded by Tn1721. The nucleotide sequence of pEC986 was obtained by the use of cassettes carrying antibiotic resistance genes inserted randomly. The method is based on the use a frequent cutter CviJI recognizing specific sequence 5’RG¯CY3’. In the first step of our method, plasmid DNA is digested with CviJI endonuclease, in conditions providing only one, random cut in every DNA molecule. In the second step, an anti-biotic resistance casette, containing primer binding sites is cloned into digested DNA resulting in recombinants possessing randomly inserted cassettes. Finally, recom-binants are sequenced using automated DNA analyzers.

P11.17

Drosophila melanogaster – useful tool for analysis of molecular basis of laminopathies

Magda Dubińska-Magiera, Ryszard Rzepecki

Laboratory of Nuclear Proteins, University of Wrocław, Wroclaw, Poland e-mail: rzepecki@ibmb.uni.wroc.pl

The nuclear lamina plays a role in maintenance of nuclear shape, spacing of nuclear pore complexes, organization of heterochromatin, DNA replication, and regulation of transcription factors. Mutations in genes coding for some components of nuclear lamina are the cause of rare hu-man diseases including muscular dystrophy, lipodys-trophy, cardiomyopathy, neuropathy, Hutchison Gilford progeria and many others. All this disorders are termed laminopathies (envelopathies) and influence whole or-ganism leading to premature aging, alopecia, decreased body weight, organ defects and increase of sensitivity to apoptosis. Mutations responsible for these disorders are mainly connected with following genes: LMNA (coding for lamin A), STA (coding for emerin), LBR and FACE1 (ZMPSTE24 in mice; coding for zinc metalloproteinase involved in maturation of lamin A). Some Drosophila melanogaster proteins which are orthologs of human pro-teins occurring in nuclear lamina including lamin Dm, otefin, lamin C, dLBR and dMAN1 and in which muta-tions cause laminopathies are known. Overall similarity between mentioned proteins from D. melanogaster and from human reaches the level about 50%, but similarity of specific domains that are crucial for proper function-ing of proteins is higher and reaches the level of 70–80%. These advantages and fact that fruit fly has application in genetic research help us to ask the question: how muta-

Vol. 53 15141st Meeting of the Polish Biochemical Society

tion in nuclear proteins lead to laminopathies? We have recently found gene in D. melanogaster genome (CG3748) which product may be a D. melanogaster ortholog of hu-man protein LAP2. Identification and characteristic of unknown so far in Drosophila melanogaster but present in human components of nuclear lamina facilitate under-standing basis of some genetic diseases.

P11.18

Activity of ribozymes at high pressure

Agnieszka Fedoruk-Wyszomirska, Eliza Wyszko, Małgorzata Giel-Pietraszuk, Mirosława Z. Barciszewska, Jan Barciszewski

Laboratory of tRNA Biochemistry, Institute of Bioorganic Chemistry of the Polish Academy of Sciences, Poznan, Poland agaw@ibch.poznan.pl

High hydrostatic pressure (HHP) is a powerful tool for analysis of molecular mechanism of chemical and bio-logical processes [1]. It is a physical factor, which induces conformational changes of nucleic acids and impact on mechanism of molecular reactions [2-5]. With HHP one can influence the conformation of molecules or shift the equilibrium of reaction without addition of a chemical agent. It was shown that high pressure mimics an effect caused by high concentration of monovalent salt [6, 7]. We have applied high hydrostatic pressure technique to ana-lyze the mechanism of RNA catalysis. We showed that at HHP hammerhead ribozymes, targeted against different substrates, specifically hydrolyze RNA in the absence of Mg2+. We also showed that deoxyribozyme is active in the absence of magnesium ions under HHP as well. Accord-ing to our data, catalytic activity of nucleic acids is based on proper structure formation. Magnesium ions are not involved directly in hydrolysis process. These data give new information on the mechanism of RNA catalysis as well as mechanism of high pressure action.

References:1. Balny C, Masson P, Heremans K (2002) High pressure effect on biological macromolecules: from structural changes to alteration of cellular processes. Biochim Biophys Acta 1595: 3-10.2. Krzyżaniak A, Sałański P, Jurczak J, Barciszewski J (1991). B-Z DNA reversible conformation changes effected by high pressure. FEBS Lett 279: 1-4.3. Krzyżaniak A, Fürste JP, Sałański P, Jurczak J, Erdmann VA, Barciszewski J (1993) A-Z conformational changes effected in RNA by high pressure. Acta Biochim Polon 40: 66-68.4. Barciszwski J, Jurczak J, Porowski S, Specht T, Erdmann VA (1999) The role of water structure In conformational changes of nucleic acids in ambient and high-pressure conditions. Eur J Bio-chem 260: 293-307.5. Macgregor RB Jr (1998) Effect of hydrostatic pressure on nu-cleic acids. Biopolimers 48: 253-263.6. Leberman R, Soper AK (1995). Effect of high salt concentra-tions on water structure. Nature 378: 364-366.7. Barciszewski J, Jurczak J, Porowski S, Specht T, Erdmann VA

(1999) The decisive role of the water structure in changes of con-formation of nucleic acids. Acta Biochim Polon 46: 133-144.

P11.19

Isoenergetic RNA microarrays; fluorescent labeling of RNA 3’end

Agata Frątczak, Elżbieta Kierzek, Ryszard Kierzek

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland e-mail: agataf@rose.man.poznan.pl

Identification of secondary structure of native RNA is important for understanding its biological function. The methods which have been used so far to determine RNA structure are laborious and time-consuming. In our lab-oratory, a novel method of using RNA microarrays has been invented with isoenergetic pentamars oligonucle-otides as a probe for determining RNA secondary struc-ture. In order to visualize it after hybridization, target RNA have been radioactively labeled, but the aim of our present study is its fluorescent labeling. The use of fluo-rescently labeled target RNA makes this technique more precise and provides possibility of automating the rea-dout and interpretation of microarray data. Our goal is to prepare efficient fluorescently labeled RNA molecules without changing their native structures. The approach that we used consists in putting multiple fluorescently labeled oligonucleotide on the 3’end of RNA particles. We are exploring two possible ways that seem proper to obtain the desirable effect. One is enzymatic ligation (T4 RNA ligase) of target RNA molecules with fluorescently labeled oligonucleotide, the other is chemical ligation of 3’-oxidited form of RNA with fluorescently labeled oligo-nucleotide via 5’aminoalkyl linker.

P11.20

Mutagenesis of single domains of Maf1, repressor of the yeast RNA polymerase III

A. E. Gajda, M. Boguta

Department of Genetics, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland e-mail: ann_gajda@wp.pl

Maf1 protein was shown to act as a negative regulator of RNA polymerase III (Pol III) in Saccharomyces cerevisiae cells. Comparison of Maf1 sequence with NCBI database using the BLAST server revealed over hundred potential orthologues in human, animals (50 proteins), plants (28 proteins) and fungi (27 proteins). None of them have been investigated before. Also no potential prokaryotic ortho-logues were identified. We have managed to mark off three characteristic domains, A, B and C, common for all of the Maf1 orthologues but the structure of Maf1 protein is still unknown. To gain a better understanding of a func-tion of Maf1 protein in yeast cell we aimed to mutagenize

152 2006Abstracts

MAF1 gene in order to isolate defined mutants localized in each particular domain. The strategy of constructing Maf1 mutants bases on low-fidelity PCR. Sequence cod-ing domain A has been amplified under conditions that allow obtaining 1–2 mutations in this region. Co-trans-formation of the PCR product with a gapped plasmid containing regions of MAF1 flanking domain A enables in vivo recombination to repair the gap with mutagenized DNA encoding domain A. We have screened 10 000 trans-formants for temperature sensitive phenotype character-istic for Δmaf1 strain. We’ve isolated nearly 1000 potential Maf1p mutants that are currently studied using Western-blot, restriction and sequencing analysis. So far we have identified one mutant with single point mutation which causes Phe32Ser amino acid substitution.

P11.21

Polymorphism of genes coding for proteins involved in DNA repair is colon cancer morbidity factor

Agnieszka Gdowicz-Kłosok1, Marcin Kłosok2, Maciej Wideł3, Maria Wideł1, Ryszard Oliński4, Joanna Rzeszowska-Wolny1

1Department of Experimental and Clinical Radiobiology, M. Skłodowska-Curie Cancer Center-Institute of Oncology Branch in Gliwice, Poland; 2Department of Animal Physiology and Ecotoxicology, Silesia University, Katowice, Poland; 3 Oncologic Surgery Clinic, M. Skłodowska-Curie Cancer Center-Institute of Oncology Branch in Gliwice, Poland; 4Department of Clinical Biochemistry, Collegium Medicum UMK, Bydgoszcz, Poland e-mail: agagdowicz@wp.pl

Colon cancer morbidity in Poland affects 12 000 people annually. This number is influenced by lack of appropri-ate prophylaxis and rapid detection methods. Risk factors involve polymorphism of DNA repair genes. Altered ac-tivity of repair proteins can result in increased frequency of mutations as well as risk of developing neoplasms. We analyzed population frequency distribution of genotypes and alleles of repair proteins in subjects affected with co-lon cancer (134), colon polyps (40) and healthy control group (99). Genes for the following proteins were investi-gated: APE1 (codon 145) involved in BER pathway; NBS1 (codon 185) protein of NHEJ/HRR pathways; polymor-phic variants of XPD protein: XPD312 (codon 312) and XPD751 (codon 751) involved in NER pathway and, fi-nally, a XRCC1 (codon 399) protein involved in repair via BER system. We also investigated in 51 colon cancer pa-tients the correlation between DNA repair dynamics and frequency distribution of XPD312 genotypes. Frequency distribution of APE1 genotypes and alleles revealed a sta-tistically significant domination of Asp145 wild allele in both experimental groups. This form of APE1 gene ap-pears to increase cancer morbidity risk more than three-fold. Similar situation was observed for the distribution

of XPD312 genotype frequency; among persons affected the Asp/Asp wild genotype was significantly dominant. We observed that lymphocytes of XPD312 wild genotype are characterized by slow DNA repair, whereas those of XPD312 polymorphic genotype (Asn/Asn) demonstrate a relatively high level of induced damages as well as rapid and effective DNA repair mechanisms. Our study con-tributes to the functional understanding of polymorphic variants of DNA repair-involved proteins.

P11.22

The polymorphism –148C/T β-chain gene of fibrinogen is a potential risk factor for myocardial infarction in young subjects

Anna Gluba, Tadeusz Pietrucha1Department of Medical Biotechnology, Medical University of Lodz, Lodz, Poland e-mail: tpietruch@csk.umed.lodz.pl

Elevated level of plasma fibrinogen is one of the well-know cardiovascular risk factors. Fibrinogen is composed of three non-identical chains (α, β and γ) which are en-coded by different genes clustered along the arm of 4th chromosome. Synthesis of β chain was shown to be the limiting step in the production of the mature fibrinogen protein. Occurrence of two polymorphisms within the β chain (–148 C/T and –455 G/A) affecting gene expression in response to acute-phase stimuli and influencing plas-ma fibrinogen level respectively, has been related to the risk of Coronary Artery Disease (CAD) and Myocardial Infarction (MI). The aim of our study was to evaluate the influence of these two polymorphisms in fibrinogen gene (β chain), –148 C/T and –455 G/A, on the risk of MI among Polish, young (< 45 years) population. In young people suffering from CAD genetic factors seem to play more important role than the classical clinical ones. One hun-dred seventy five patients with documented myocardial infarction (age below 45 yrs.) not suffering from diabetes mellitus or hypertension were included into our study group. The control group contained 104 healthy volun-taries. In our study polymorphism –455 G/A seems not to play an important role in increasing the risk of prema-ture MI. However, in the case of –148 C/T polymorphism the incidence of heterozygotes with the mutated allele T (C/T) is much higher in the case group in the comparison with controls (53% vs 43%). It suggests, it is a good candi-date for one of the genetic risk markers for MI in young Polish population (e.g. below 45).Supported by grant Medical University of Lodz, No. 502-19-298.

P11.23

Stable maintenance of the plasmid pEC156 is dependent on the presence of the cer locus and a gene encoding the

Vol. 53 15341st Meeting of the Polish Biochemical Society

EcoVIII restriction endonuclease

Tadeusz Kaczorowski, Robert Boratyński, Michał Presz

University of Gdansk, Department of Microbiology, Gdańsk, Poland

Stable maintenance of plasmids is dependent on existence of molecular mechanisms based on genetic elements en-coded either by the particular plasmid itself or by bacterial chromosome. One of such mechanisms which is responsi-ble for efficient resolution of plasmids multimers is based on site-specific recombination requiring plasmid encoded cer locus and bacterial recombinases (XerC/XerD). Anoth-er molecular strategy involved in stable plasmid infere-tance is dependent on the function of type II restriction-modification systems. This observation can be explained by the fact that descendants from cells that have lost the plasmid carrying the R-M system cannot survive due to the reduced cellular pool of DNA methyltransferase mol-ecules. As a model we have choosen a natural ColE1-type plasmid (pEC156) isolated from Escherichia coli E1585-68 which carries type II R-M system EcoVIII. To investigate a role of particular genetic elements affecting pEC156 stability we have constructed several derivatives of this plasmid. Each of them was deffcient either in plasmid resolution system (cer locus) or elements of the EcoVIII R-M system. Our experiments revealed that, a gene en-coding EcoVIII restriction endonuclease indeed stabilizes plasmid pEC156 maintenance. Also, the presence of the cer locus increases the plasmid stability. Moreover, we have shown that derivatives which lacked cer locus and gene encoding EcoVIII endonuclease as well as a gene for EcoVIII methyltransferase were highly unstable in E. coli after growth for 100 generations in the abscence of selec-tion. Similar results were obtained when Klebsiella oxytoca which is phylogenetically related to E. coli was used as a host.

P11.24

The effects of clofibrate, a PPARα agonist, on adiponectin and resistin gene expression in adipose tissue

Joanna Karbowska, Zdzislaw Kochan

Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland e-mail: jonka@amg.gda.pl

Adiponectin and resistin are adipocyte-derived plasma proteins that can affect insulin sensitivity. Both adipok-ines are expressed and secreted predominantly by adi-pose tissue; little is known, however, about the regulation of their expression. The promoter region of the resistin gene contains several putative peroxisome proliferator-activated receptor (PPAR) response elements (PPRE). Recently, a functional PPAR-responsive element in the

promoter region of the adiponectin gene has also been identified. Moreover, it has been shown that PPARs may be required for adiponectin and resistin gene induc-tion. To examine the effects of PPARα activation on adi-ponectin and resistin gene expression, we administered a PPARα agonist, clofibrate (250 mg/kg body wt/d), to male Wistar rats. After 14 days of treatment, adiponectin and resistin gene expression in epididymal white adipose tissue (WAT) of rats assigned to either the control group (n = 8) or the clofibrate-treated group (n = 8) was meas-ured by real-time RT-PCR. Pharmacological activation of PPARα by clofibrate induced the expression of resistin in adipose tissue by 1.8-fold (P < 0.01). In contrast, clofibrate treatment had no effect on adiponectin gene expression in rat WAT. These results suggest that the resistin gene, but not the gene encoding adiponectin, is a downstream tar-get of PPARα and its transcription in adipose tissue may be activated by pharmacological agonists of this nuclear receptor.

P11.25

Eucaryotic ribosome as a dynamic molecular machine

Anna Kietrys1,2, Kamilla Bąkowska-Żywicka1, Tomasz Twardowski1

1Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland; 2Department of Biochemistry and Microbiology, Faculty of Commodity Science, The Poznań University of Economics, Poznań, Poland e-mail: annakietrys@wp.pl

Conformational changes of rRNA’s domains are of critical importance for regulatory process of protein biosynthesis within ribosome. Comparison of three-dimensional maps for translating ribosomes by cryo-EM with that of a non-translating ribosome pinpoint specific structural differ-ences. In our laboratory, we test this dynamic mechanism with the antisense oligodeoxynucleotides (a-DNA). The most evident proof for the activity of antisense oligomer is blocking the activity of the enzymatic system through the hybridization of a-DNA to selected fragments of target nucleic acid domain. Using antisense strategy simultane-ously with mini-tRNA molecules, we tested structure-function correlation of the 26S rRNA fragments which interact with tRNA molecules. The effect of hybridization of selected antisense oligomers has been analyzed after the ribosomal transition from post- to pre-translocational state. We set different tRNA/mini-tRNA molecules into sites:• P-site or P and additionally A-site at pretranslocational state;• P-site or P and E-site at posttranslocational state.We observed higher ratio of a-DNA hybridization to the ribosomes occupied with the mini-tRNA (compar-ing to tRNA). In our opinion, according to smaller size of mini-tRNA, more space in the ribosome is available

154 2006Abstracts

for a-DNA. We concluded that oscillation of the ribos-ome between the main states during the elongation cy-cle causes the exposition of individual functionally im-portant sites of L-rRNA. The structure of the ribosome seems to be more “open” with two sites blocked before the translocation and the sites of functional importance are much more accessible at pretranslocational state. The functionally important sites are less accessible for complementary oligonucleotides in the “closed” con-formation at posttranslocational state.

P11.26

Nutritional regulation of glycerol kinase gene expression in adipose tissue is associated with up-regulation of PPARγ

Zdzislaw Kochan, Joanna Karbowska

Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland e-mail: kochanz@amg.gda.pl

Multiple cycles of alternating caloric restriction and re-feeding promote lipolysis in adipose tissue. We have previously reported a significant increase in hormone-sensitive lipase (HSL) gene expression in rat epididy-mal white adipose tissue (WAT). The hydrolytic actions of adipocyte lipases lead to the production of glycerol, which may be released into the blood or converted to glycerol 3-phosphate by the action of glycerol kinase (GyK). Since little is known about the nutritional regu-lation of glycerol kinase in adipose tissue, we examined the effects of alternating caloric restriction and refeed-ing on GyK gene expression in WAT of 15 male Wistar rats randomised into 3 groups: multiple cycles of alter-nating food deprivation and refeeding (MFR); pair-fed (PF); and ad libitum feeding (control). Compared with the control group, rats subjected to multiple cycles of alternating food deprivation and refeeding showed a considerable (3.37-fold, P < 0.01) increase in GyK gene expression in adipose tissue, as measured by real-time RT-PCR. GyK appears to be regulated by peroxisome-proliferator-activated receptor γ (PPARγ), a major adipocyte-specific transcription factor. In this study, PPARγ gene expression was induced in adipose tissue of MFR rats (1.59-fold, P < 0.01). Moreover, the up-reg-ulation of PPARγ was correlated with the induction of GyK. No such changes in GyK and PPARγ gene expres-sion were observed in adipose tissue of PF rats. These results suggest that multiple cycles of alternating food deprivation and refeeding, but not caloric restriction, induce GyK gene expression in adipose tissue and that PPARγ may participate in the nutritional regulation of glycerol kinase.

P11.27

Candidate gene association analysis to search for

new risk factors in sporadic Alzheimer’s disease

Anna Kowalska1, Marek Powidzki2, Mieczysław Wender3

1Institute of Human Genetics, Polish Academy of Sciences, 2Department of Bioinformatics and Statistics, University of Medical Sciences, Poznań, 3Medical Research Center, Polish Academy of Sciences, Poznań, Poland e-mail: annkowal@rose.man.poznan.pl

Background: The complex genetics of sporadic Alzhe-imer’s disease (SAD) remains still unknown. Although it is clear that the APOE ε4 allele is a major risk factor for AD, epidemiological studies estimate that 42–68% of late-onset AD patients do not have an ε4 allele, in-dicating that additional genetic and environmental factors are involved in this form of the disease. Thus, many groups are searching for new AD susceptibility genes with the use of large cohorts of patients from dif-ferent ethnic groups. But, a majority of reports shows rather conflicting data. Objective(s): In this study, a to-tal of 10 genes of interest were chosen from among the previously published reports of significant association in AD. The genes chosen for analysis were: Alpha1-An-tichymotrypsin (ACT), Alpha2-Macroglobulin (A2MG), Apolipoprotein E (APOE), Cathepsin D (CatD), Caveolin 3 (Cav3), Interleukin 1 Antagonist Receptor(IL-RA), Inter-leukin 1α (IL-1A), Interleukin 1β (IL-1 B), Low-density-li-poprotein receptor-related-protein (LRP), and Presenilin 1 (PS1). Methods: Genotyping was performed on poly-morphisms within those genes using PCR-RFLP ap-proach in 113 Polish patients with SAD (n = 53 with EOAD and n = 60 with LOAD) and 125 age-related controls from Poznań region in the Western part of Poland. Results: A replication of association between SAD and CatD, A2MG, and IL-1B genes was observed. A strong overrepresentation of CatD*T allele occurred among patients with AD (P < 0.02), especially in pa-tients with LOAD (P < 0.002). Carriers of Cat*T allele had a 2.87-fold increased risk for developing AD than non-carriers (P < 0.02, OR = 2.87, CI = 95%). Among patients with LOAD the risk was 3.8-fold higher carri-ers (P < 0.002, OR = 3.8, CI = 95%). Moreover, a signifi-cant association between a 5 bp deletion in the A2MG gene at the 5’ splice site of exon 18 and AD was found. The carriers of the A2MG*D allele showed a 1.8-fold increased risk for AD (P < 0.04, OR = 1.8, CI = 95%). A polymorphism in IL-1B gene located in a promoter region at the position -511 (IL-1B -511 *2 allele) has been correlated with an increased risk for LOAD (P < 0.02, OR = 2.42, CI = 95%). A lack of replication of as-sociation findings between ACT, Cav3, IL-RA, LRP and PS1 genes and Alzheimer’s disease was demonstrated in the analyzed Polish sample of patients and controls. Conclusions: The DNA variability within CatD, A2MG, and IL-1B genes may influence a risk for a development of sporadic AD.

Vol. 53 15541st Meeting of the Polish Biochemical Society

P11.28

siRNA-mediated depletion of dnmt1 in Jurkat T cell line: a model of abnormalities in T cell receptor signaling in systemic lupus erythematosus

Anna Kozlowska1, Pawel P. Jagodzinski1

1Department of Biochemistry and Molecular Biology, University of Medical Sciences, Poznań, Poland

e-mail: kozlowa@am.poznan.pl

Systemic lupus erythematosus (SLE) is an autoim-mune disease characterized by exaggerated humoral response. T cells from patients with active SLE exhibit approximately 17% decrease in genomic DNA content of deoxymethylocytosine. DNA methylation of cyto-sine-guanine dinucleotides (CpG) is carried out by DNA methyltransferases (DNMTs) and serves as an epigenetic regulation of gene expression. DNMT1 is responsible for maintenance of methylation pattern, whereas DNMT3A and DNMT3B methylate CpG sites de novo. Hypomethylation of DNA is associated with overexpression of several genes and T cells autoreactiv-ity. DNMTs are involved in T cell lineage development, activation and Th1/Th2 helper T cell polarization. De-crease of DNMT1 activity contributes to development of Th2 effector program that supports humoral response. We examined whether depletion of DNMT1 in Jurkat T cells leads to defects in T cell receptor (TCR) signal-ing similar to abnormalities observed in T cells from SLE patients. Using lentiviral vector expressing siRNA we developed a Jurkat T cell line with stable depletion of DNMT1. Western blot showed an average reduction of 90% in DNMT1 protein level. Quantitative analyses of CD4, CD45, CD70, CD3-zeta, perforin, Fyn, Lck, Itk, PKC-theta, ZAP-70, LAT, SLP-76, ERK-1, AP-1, and Elf-1 transcripts were performed by real-time PCR. The quan-tity of transcripts was normalized with β-actin or MRPL transcripts level. We observed a significant increase of mRNA level of CD4, perforin, Fyn, Lck and PKC-theta in cells depleted of DNMT1. Our studies indicate that DNMT1 depletion is associated with up-regulation of expression of several genes involved in TCR signaling. This finding also suggests that decrease of cellular con-tent of DNMT1 can be responsible for improper function of T cells resulting in enhancement of humoral response in patients with SLE.This work was supported by grant KBN No. 2PO5B-019-27.

P11.29

Regulation of adiponectin receptor gene expression in the liver by clofibrate

Malgorzata Langowska, Joanna Karbowska, Zdzislaw Kochan

Department of Biochemistry, Medical University of Gdansk,

Gdansk, Poland kochanz@amg.gda.pl

Adiponectin is a hormone secreted by adipose tissue that seems to play an important role in the regulation of glu-cose and lipid metabolism. Lower concentrations of this protein in the blood were found in a number of diseases, such as atherosclerosis, obesity and type 2 diabetes. So far, two adiponectin receptors have been cloned, Adi-poR1 and AdipoR2. AdipoR1 is expressed mainly in the skeletal muscle, but also in the kidney, heart, brain and liver; whereas AdipoR2 is expressed mostly in the liver. The regulation of AdipoR1 and AdipoR2 gene expression remains to be elucidated. It has been proposed that adi-ponectin receptor gene expression may be regulated by PPARα, a nuclear hormone receptor predominantly ex-pressed in liver, intestine, kidney and heart. To test this hypothesis we measured AdipoR1 and AdipoR2 gene expression in the liver of control (n = 8) and clofibrate-treated (n = 8) male Wistar rats. PPARα is activated by diverse group of substances called peroxisome prolifera-tors which induce massive proliferation of peroxisomes in rodent hepatocytes. Clofibrate is a drug used in treat-ment of hyperlipidaemia and the efficient activator of PPARα. In our study, gene expression of adiponectin re-ceptors in the liver of control and clofibrate-treated rats was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by gel electrophoresis. Clofi-brate treatment induced hepatic expression of AdipoR1, whilst suppressing AdipoR2 gene expression. In general, therefore, it seems that AdipoR1 and AdipoR2 are differ-ently regulated by PPARα in the liver.

P11.30

Activation and senescence of CD4+ T cells is associated with methylation and demethylation in cytosine guanine (CpG) islands of cyclin-dependent kinase inhibitor 2B (CDKN2B) promoter

Michał W. Łuczak, Ilona Górska, Paweł P. Jagodziński

Department of Biochemistry and Molecular Biology, University of Medical Sciences, Poznań, Poland e-mail: mluc@amp.edu.pl

Methylation of cytosine in CpG dinucleotides in promoter and first exon of genes belongs to epigenetic regulation of genes expression. In mammals this is catalyzed by DNA methyltransferases (DNMTs) that include maintenance DNMT1 and de novo DNMT3A and DNMT3B. Primary transcript of DNMT3B is alternatively spliced into five distinct isoforms and DNMT3B4 splice variant functions as a negative regulator of DNA methylation. Using reverse transcription Real Time Quantitive PCR, Western blotting and bisulfite DNA sequencing we investigated expression of CDKN2B in CD4+ T cells and HUT78 T cells. Peripheral

156 2006Abstracts

blood mononuclear cells were stimulated for 2 hrs with phytohemagglutinin and cultured. The CD4+ T cells were harvested by positive biomagnetic separation at 3, 6, 10, 13, 17, 21 and 24 days of culture duration. In order to de-termine role of DNMT1 in CDKN2B expression we used HUT78 T cells with stable depleted DNMT1 by lentivirus system containing siRNA. We observed that stimulation of CD4+ T cells correlated with increase in intracellular content of DNMT1 protein, loss of CDKN2B expression and hypermethylation of their promoter. However at 17th day of senescence CD4+ T cells culture duration we observed increase in expression of DNMT3B4 splice vari-ant, demethylation of CDKN2B promoter and initiation of CDKN2B expression. We also found that depletion of DNMT1 in HUT78 T cell resulted in increase in CDKN2B gene expression. Our investigation may indicate that the expression of CDKN2B gene in CD4+ T cells is regulated epigenetically via DNMT1 activity and DNMT3B4 variant as negative regulator of DNA methylation.

P11.31

Stimulation of transcription from the λ pR promoter by Escherichia coli SeqA protein requires downstream GATC sequences and occurs after open complex formation

Robert Łyżeń1, Alicja Węgrzyn2, Grzegorz Węgrzyn1, Agnieszka Szalewska-Pałasz1

1Department of Molecular Biology, University of Gdansk, Gdańsk, Poland; 2Laboratory of Molecular Biology (affiliated with University of Gdansk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Gdańsk, Poland

Escherichia coli SeqA protein is a major negative regulator of chromosomal DNA replication acting by sequestration, and thus inactivation, of newly formed oriC regions. How-ever, other activities of this protein have been discovered recently, one of which is regulation of transcription. SeqA was demonstrated to be a specific transcription factor act-ing at bacteriophage λ promoters pI, paQ and pR. Howev-er, a mechanism for stimulation of pR remains largely un-known. Here we demonstrate that two GATC sequences, located about 100 bp downstream of the pR transcription start side, are necessary for this stimulation both in vivo and in vitro. SeqA-mediated activation of pR was as effec-tive on a linear DNA template as on a supercoiled one. In vitro transcription analysis demonstrated that the most important regulatory effect of SeqA in pR transcription occur after the open complex formation, namely during promoter clearence. SeqA did not influence appearance and level of abortive transcript or the pausing during the transcription elongation. As SeqA may affect DNA topol-ogy, downstream GATC sequences were replaced with IHF protein binding site. The pR activation by IHF (In-tegral Host Factor) in this case was topology dependent, while this alteration in vivo resulted in over 50% decrease of pR activity. Interestingly, SeqA is one of a few known

prokaryotic transcription factors which bind downstream of the regulated promoter and still act as transcription ac-tivators.

P11.32

Degradation of rRNA during oxidative stress in Saccharomyces cerevisiae

Seweryn Mroczek1, Joanna Kufel1

1Institute of Genetics and Biotechnology, University of Warsaw, Warsaw, Poland e-mail: seweryn.mroczek@ibb.waw.pl

Oxidative stress is known to be one of the most important processes leading to the programmed cell death, apopto-sis, and it’s involved in regulation of chronological aging. We identified specific degradation of 25S ribosomal RNA as one of the marks of cell response to oxidative stress in Saccharomyces cerevisiae. 25S rRNA is endonucleolyti-caly cleaved and characteristic degradation products ac-cumulate. Major cleavage sites have been mapped using primer extension and Northern blotting. Surprisingly, this degradation pattern was observed only for some of reactive oxygen species (ROS) generators tested, includ-ing hydrogen peroxide and menadione. We show that this process is correlated with mitochondrial respiration status and components of cellular stress defense systems, since mutations in these elements visibly altered the ex-tent of rRNA degradation. In contrast, expression of an-tiapoptotic and antioxidant proteins from mammalian Bcl-2 family led to increase stability. Moreover, specific rRNA degradation was also observed during aging of wild-type cells and even more profoundly of rho0 yeast. This strongly supports the main role of mitochondria in rRNA degradation as a oxidative response process. Nota-bly, we also report here that the main yeast mitochondrial nuclease Nuc1p, a homologue of human EndoG, is one of the enzymes involved in rRNA degradation during oxi-dative stress. We conclude that described rRNA degrada-tion might be considered as a new molecular outcome of oxidative stress.

P11.33

Regulation of the ibpB gene transcription of Escherichia coli

Beata Nadratowska, Anna Janaszak, Wiktor Majczak, Grażyna Konopa, Agnieszka Szalewska-Pałasz, Alina Taylor

Department of Molecular Biology, University of Gdańsk, Poland e-mail: nadra@biotech.univ.gda.pl

Expression of the heat shock proteins genes in E. coli de-pends on two regulons controlled by σ32 and σ24, RNAP subunits. Recently, it has been reported that the third reg-

Vol. 53 15741st Meeting of the Polish Biochemical Society

ulon of heat shock response, which includes pspA-E oper-on and a gene encoding a small heat shock protein IbpB, is under the control of σ54 promoters. The putative σ54

binding site also was found upstream of the E. coli rpoH coding region. RNAP-σ54 holoenzyme can form a closed promoter complex but is incapable of proceeding to open complex formation in the absence of an activator protein and ATP. IHF mediated DNA bend is frequently essential for efficient induction of the transcription. Our interest is focused on studying the control of the ibpB gene tran-scription. The pσ54 of the ibpB gene is activated only by temperature upshift from 30°C to 42°C. Regulation of this promoter is not known. We analyzed the upstream se-quence of the ibpB gene and found IHF and NtrC binding sites. Binding of NtrC, IHF and RNAP-σ54 holoenzyme to the ibpB DNA fragments was detected by a gel mobility shift assay. DNase I footprinting method was employed for the identification of the NtrC and IHF binding se-quences in the promoter region of the ibpB gene. Prelimi-nary experiments show that NtrC and IHF provide DNA protection against DNase I digestion. Nitrogen starvation inducing NtrC activity was tested in the primer extension assay, but these conditions did not activate ibpB promoter. Primer extension assays revealed additional transcription start sites of the ibpB gene. Identification of the corre-sponding promoters needs further examination.

P11.34

A mutational study of individual functional groups on the G6 base of the 10-23 deoxyribozyme core

Barbara Nawrot, Kinga Widera, Marzena Wojcik, Beata Rebowska, Wieslawa Goss, Barbara Mikolajczyk and Wojciech J. Stec

Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies of Polish Academy of Sciences, Lodz, Poland e-mail: bnawrot@bio.cbmm.lodz.pl

The deoxyribozyme 10-23, first selected and characterized by Santoro and Joyce, has been examined most extensive-ly both in vitro and in vivo. Since an active structure of substrate-deoxyribozyme 10-23 complex is not yet known and the details of the reaction mechanism are not fully understood, mutagenesis of the 10-23 deoxyribozyme core has been employed extensively to explore the role of individual nucleotides [1]. We analyzed the function of the oxygen moiety at the carbon 6 of nucleoside G6 positioned within the catalytic loop by either its removal (substitution of G6 with 2-aminopurine nucleoside) or its replacement with a sulfur atom by using the 6-thiogua-nosine mutant enzyme. Kinetic measurements of these deoxyribozyme variants were performed in the presence of either 3 mM Mg2+ or 3 mM Mn2+ ions. The observed rate constants (kobs) revealed that the stimulation of the catalytic activity in a presence of Mn2+ ions was the same

for unmodified and s6G-substituted enzyme. The ob-served thio effect (the ratio of kobs for unmodified enzyme to kobs of mutated enzyme in the presence of Mg2+ ions) was about 20 and the rescue effect (kobs

Mn/kobsMg for the

same s6G-zyme) was 28, implying that the oxygen atom of the carbonyl moiety may constitute a metal ion ligand and participate in a direct metal ion coordination. In ad-dition, the replacement of the guanine at the position 6 with 2-aminopurine resulted in a complete loss of the catalytic activity, independently on the metal ion used. These findings, along with data obtained by Kurreck et al. (1), confirmed that the exo amino group of G6 is not of functional importance, while the oxygen at the C6 is es-sential for the catalytic activity of the deoxyribozyme 10-23. Moreover, we created ΔN7-zyme by replacement of G6 with 7-deaza-dG nucleoside. This substitution resulted in a 91-fold loss of activity in a presence of Mg2+, suggest-ing that N7 nitrogen may participate in the formation of a functionally important intramolecular hydrogen bond within the deoxyribozyme 10–23 catalytic core. The kobs of this enzyme increased almost three orders of magnitude upon addition of Mn2+ ions.References:1. Zaborowska Z, Furste JP, Erdmann VA, Kurreck J (2002) Se-quence requirements in the catalytic core of the “10-23” DNA enzyme. J Biol Chem 277: 40617-40622.

P11.35

Interaction of antitumor triazoloacridone C-1305 and its analogs with telomeric DNA

Jakub Olewniak, Krzysztof Lemke, Marcin Wojciechowski, Andrzej Skladanowski

Laboratory of Molecular and Cellular Pharmacology, Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, Gdansk, Poland e-mail: jolew@wp.pl

C-1305 is a triazoloacridone derivative with potent activ-ity in lung and colon cancer models. We have recently re-ported that compound C-1305 binds to DNA by intercala-tion and induces structural distortions in DNA regions containing guanine triplets GGG, at concentrations as low as 0.1 µM. This is a unique property of C-1305 since similar results were not observed for any of the structur-ally related triazoloacridone derivatives tested or other DNA binding compounds. Human telomeres consist of tandem repeats of the TTAGGG fragments. Most of telo-meric DNA is double-stranded except the most extreme part where 3’ region of the G strand is single-stranded and may form so-called G-quadruplex structures. In this study, we wanted to clarify whether compound C-1305 and its structural analogs are able to bind telomeric DNA and stabilize G-quadruplexes. The binding affinities of studied compounds to telomeric DNA were tested against different DNA substrates using microdialysis and DNA melting temperature assays and stabilization of G-quad-

158 2006Abstracts

ruplexes by FRET (fluorescence resonanse energy trans-fer). All studied compounds bind to double-stranded oligonucleotides containing telomeric TTAGGG repeats and to a oligonucleotide folded into intramolecular G-quadruplex structure. However, C-1305 showed highest binding specificity toward telomeric DNA. Compound C-1305 increased melting temperature of intramolecular (propeller-like) G-quadruplex structure by 8.4 degrees at optimal concentration (10 µM). In contrast, structurally related triazoloacridones compounds C-1303 and C-2007 showed only marginal effect. We also performed mo-lecular modelling studies and based on our results, we propose a molecular model of complexes between C-1305 and single-stranded oligonucleotide folded into intramo-lecular G-quadruplex.

P11.36

GeneChip array-based expression profile analysis of transcriptomes of Arabidopsis thaliana cbp20 and cbp80 mutants

Paulina Piontek1, Paweł Herzyk2, Artur Jarmolowski1

1Department of Gene Expression, Adam Mickiewicz University, Poznan, Poland; 2The Sir Henry Wellcome Functional Genomic Facility (SHWFGF), University of Glasgow, UK e-mail: ppiontek@amu.edu.pl

The nuclear cap-binding complex (CBC) consists of two subunits, the so called cap-binding proteins: CBP20 and CBP80 and is involved in several aspects of RNA metabolism. The CBC binds to the cap structure of all polymerase II transcripts and promotes efficient splic-ing of pre-mRNA, nuclear export of U-rich small nu-clear RNAs, mRNA 3’ end formation and also plays a role in nonsense-mediated mRNA decay (NMD). In our laboratory we have previously characterized CBC from Arabidopsis thaliana (AtCBC), which is also composed of two subunits, AtCBP20 and AtCBP80. An increas-ing number of genetic mutations that contribute to the plant hormone signalling have been characterized. Two of them, T-DNA insertion mutations in AtCBP20 and AtCBP80 genes have been described recently; both mu-tations affect the response to abscisic acid (ABA). Cbp20 and cbp80 insertion mutants are recessive and show ABA hypersensivity in the early steps of ABA signal-ling. To better understand this phenomenon and the role of cap-binding proteins in it, we performed the whole transcriptome analysis of both mutants and wt plants using microarray technology. The ATH1 array, designed by Affymetrix and the Institute for Genome Research (TIGR), contains probe sets representing 22.748 Arabi-dopsis genes corresponding to approximately 80% of the genes in the genom (29,000). It provides a particularly powerful tool to detect gene expression patterns on the wholegenome scale. To asses the reproducibility of our

experiment and the extent of technical and biological variability, three biological replicate experiments were performed with cRNA from RNAs that were extracted from three independent plant populations for mutants and wt plant. A total of 9 microarray hybridization ex-periments were performed to obtain gene expression profiles of two mutants studied. A comparative analysis of transcriptomes of cbp20 and cbp80 mutants allowed us to select genes whose expression was changed in the case of both mutants. Beyond the great similarity of the transcriptomes we could also select genes preferentially expressed in cbp20 or cbp80 mutant. These genes are sus-pected to play an important role in hypothetical, addi-tional functions of each cap-binding protein alone.

P11.37

Analysis of differentially expressed genes in the liverwort Pellia endiviifolia

Aleksandra Rojek, Izabela Pasternak, Zofia Szweykowska-Kulińska

Department of Gene Expression, University of Adam Mickiewicz, Poznań, Poland e-mail: rojekola@amu.edu.pl

Pellia endiviifolia is a dioecious plant belonging to sub-division Bryophytina, class: liverworts (Hepaticop-sida), subclass: Jungermaniidae. Liverworts are very interesting objects of scientific studies, because they are the oldest land plants living on Earth. In order to check which genes are specifically expressed in male and female gametophyte of the liverwort Pellia endivii-folia we adopted representational difference analysis of cDNA (RDA-cDNA) technique. This method enables us to isolate fragments of cDNA that are differentially expressed in time, different tissues, organs, or under stress etc. It may also be used to isolate the differences between two species or two specimens. RDA-cDNA is a method of detection of cDNA fragments present in one pool (tester) but absent in another pool of cDNA (driver). In our laboratory we isolated total RNA from male and female gametophyte of the liverwort Pellia en-diviifolia. In our experiment, cDNA of the female game-tophyte was the tester and cDNA of male gametophyte – the driver. We performed three rounds of subtractive hybridization and obtained fragments of cDNA rang-ing from ~ 500 bp to ~250 bp that we cloned, sequenced and checked if they are expressed only in the female gametophyte. So far, we have found three fragments of cDNA that contain ORFs which are very similar to LEA, SKP1 and ATP-binding kinase proteins. PCR analysis showed that these fragments are expressed on a lower level in the male gametophyte compared to the female gametophyte. We intend to find cDNA fragments that are specifically expressed in the female gametophyte and then obtain their full length sequences utilising 5’ and 3’RACE method.

Vol. 53 15941st Meeting of the Polish Biochemical Society

P11.38

Chemical mutagenesis of Arabidopsis thaliana T-DNA insertion mutant with disrupted gene encoding cap binding protein 80: searching for suppressors

Marta Sawczak1, Danuta Nabiałkowska2, Zofia Szweykowska-Kulińska1, Artur Jarmołowski1

1Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland; 2Department of Genetics, Faculty of Biology and Environment Preservation, University of Silesia, Katowice, Poland e-mail: msawczak@amu.edu.pl

CBC is a nuclear cap-binding complex which consists of two subunits: CBP20 and CBP80. In animals CBC partici-pates in RNA-processing events mostly by regulation of intron splicing, nuclear export, transcript stability and RNA degradation. Functions of the cap binding complex in plants however remain unknown. Arabidopsis thaliana CBP20 and CBP80 insertion mutants display few devel-opmental changes, like serrated leaf margin or delayed growth and flowering. They also show hypersensitivity to plant hormone abscisic acid (ABA) and display increased drought resistance. ABA plays a primary role in plant re-sponses to many stress conditions. It also participates in many major growth and developmental processes. In our laboratory we are trying to determine the role and molecu-lar mechanism of CBC action in plants. To reach this aim, we performed chemical mutagenesis of cbp80 insertion mutant seeds using ethyl methyl sulfonate (EMS). To ob-tain satisfactory mutation level, but not reduce excessively the fertility of mutant plants, we carfully established the proper dose of EMS. Seeds of the studied mutant do not germinate on medium containing 0.4 µM ABA (concentra-tion that does not inhibit germination of wild type seeds) and its leaves have a characteristic shape. Among the prog-eny of mutagen-treated plants, we looked for plants with mutations that suppress the mutant phenotype. In the next step, we are going to map the mutation sites. We assume, that this approach will help to discover proteins that co-operate with CBC in ABA signal transduction and/or leaf morphogenesis.

P11.39

Targeting BACE1 mRNA with chemically synthesized and plasmid coded siRNAsKatarzyna Sipa, Julia Kazmierczak-Baranska, Wiesława Goss, Barbara Nawrot, Wojciech J. Stec

Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland e-mail: ksipa@bio.cbmm.lodz.pl

β-Secretase, a beta-site APP cleaving enzyme (BACE),

participates in the secretion of beta-amyloid peptides (Aβ), the major components of the toxic amyloid plaques found in the brains of patients with Alzheimer’s disease (AD). According to the amyloid hypothesis, accumulation of Aβ is the primary influence driving AD pathogenesis. Therefore, β-secretase is a primary target for anti-amy-loid therapeutic drug design. Several effective inhibitors of BACE activity (peptidomimetic, non-cleavable sub-strate analogues) have been developed. An alternative approach for future treatment of Alzheimer’s disease is targeting BACE gene with small inhibitory nucleic acids (siNA). Among antisense gene silencing strategies most promising are the ones based on RNA interference tech-nology. RNA interference (RNAi) is a gene expression regulation mechanism, mediated by siRNA molecules (short interfering RNA). In mammalian cells sequence-specific gene silencing can be achieved by delivery of either siRNA or their precursors – short hairpin RNAs (shRNA). Both types of molecules can be generated endogenously (coded in variety of vectors), or synthesized by chemical either enzymatic approaches. Among different vectors encoding shRNA most widely used are plasmids with RNA polymerase III (Pol III) promoters including H1, U6 and tRNAVal. Investigating the most potent anti-amyloid strategy we compared the silencing effects induced by chemically synthesized siRNAs and shRNAs coded in two plasmid systems: commercially available pSilencer-U6 (Ambion) and pPUR-tRNAVal (gift from prof. K. Taira, Japan). Gene silencing effect was determined as a level of fluorescent fusion protein BACE-GFP in HeLa cells trans-fected with plasmid pBACE-GFP (gift from dr W. Song, Canada).

P11.40

The role of E. coli RNA polymerase in the initiation of bacteriophage lambda DNA replication

Anna Szambowska, Monika Glinkowska, Grzegorz Węgrzyn

Department of Molecular Biology ; University of Gdańsk, Gadńsk, Poland e-mail: ANECZKA@biotech.univ.gda.pl

Initiation of λ DNA replication in vivo and in crude in vitro system is strongly dependent on transcription initiated at λ pR promoter. This transcription event is indispensable for, ”transcriptional activation’’ of oriλ and expression of the λO and λP genes. Formation of the lambda replica-tion initiation complex requires both λO and λP initia-tors and many E. coli proteins, including DnaB, primase, SSB, DNA polymerase III holoenzyme as well as DnaK, DnaJ and GrpE chaperones. We performed experiments aiming at explaining the role of transcription and RNA polymerase in initiation of replication at oriλ in more de-tail. Using gel mobility shift assay, we were able to detect stimulation of λO binding to iterones and its self-assem-

160 2006Abstracts

bly into a nucleosome-like structure by RNA polymer-ase. This event was even more pronounced in the pres-ence of NTPs. Glutaraldehyde-crosslinking experiments revealed also a possible direct interaction between λO protein and one of the RNA polymerase subunits. These results encouraged us to propose a hypothesis that direct interactions of these two proteins is required for efficient initiation of DNA replication at oriλ. This hypothesis was subsequently confirmed in in vitro replication experi-ments, in which a plasmid containing phi10 promoter of bacteriophage T7, instead of λ pR promoter, was used as a template. Interestingly, such a plasmid did not replicate in vitro in the presence of T7 RNA polymerase. These re-sults confirm that not only the transcription process but also specific interactions between E. coli RNA polymerase and lambda replication complex are necessary for ,,tran-scriptional activation’’ of oriλ.

P11.41

Promoter A1 of the phage T7 with modified –35 element forms open complex with E. coli RNA polymerase holoenzyme

Katarzyna Turecka, Władysław Werel

Department of Pharmaceutical Microbiology, Medical University of Gdańsk, Gdańsk, Poland e-mail: klewan@wp.pl

Promoter recognition and open complex formation are central events in transcription initiation by RNA polymer-ase. In E. coli promoters two hexameric elements located approximately 10 and 35 bp upstream from the transcrip-tion start site making sequence-specific contacts to σ70 subunit of the RNA polymerase holoenzyme. Although lot of research has been done in this field the functional significance of these sequences still needs to be studied. One of the most critical determinants of promoter recog-nition and following stages in transcription initiation is hexamer –35. In the previous experiments we found that modification of the –35 hexamer sequence did not have big influence on the binary complex formation stage. Bi-nary complex in the presence of heparin was very stable what suggests that it was an open complex. However, the trials of the transcription in vitro have not been successful. In order to determine the nature of binary complex, which is formed by RNA polymerase holoenzyme and promoter A1 of the phage T7 with completely changed –35 hexamer we applied a permanganate assay. KMnO4 is a reagent that has been widely used in nucleic acid sequence analy-sis, for the mapping of unpaired regions in DNA or DNA-protein complexes. Experiments have showed that thym-ines at positions –12, –10, –6 and –4 of the template strand of the modified promoter, as well as wild type promoter A1 of the phage T7, were accessible for the modification by KMnO4. The data indicates that DNA strands within the melting domain are separated, which is a feature of the open complex. It means, that inability of the binary

complex to transcribe reveals in the later stage of the tran-scription than open complex formation.