POLISH ACADEMY OF SCIENCES INSTITUTE OF BIOCHEMISTRY AND BIOPHYSICS

RESEARCH REPORT ISBN-83-906782-4-1 INSTITUTE OF BIOCHEMISTRY AND BIOPHYSICS Polish Academy of Sciences http://www.ibb.waw.pl

Director: Włodzimierz Zagórski-Ostoja

Head of the Scientific Council: Andrzej Paszewski

Deputy Scientific Director: Piotr Cegłowski

ADDRESS, E-MAIL, FAX AND TELEPHONE:

5a Pawińskiego Street, 02-106 Warsaw, Poland

E-Mail: [email protected] International telephone and Fax network: (48) 39-12-16-23 Domestic telephone: (48-22) 658-44-99, (48-22) 659-70-72 Domestic Fax: (48-22) 658-46-36 Switchboard: (48-22) 658-44-99, (48-22) 659-70-72 Director/ Secretariate: (48-22) 658-47-24; Fax: (48-22) 658-46-36 Scientific Council: (48-22) 658-44-99 ext. 24-12 or 21-40 Scientific Secretariate: (48-22) 658-47-53 Polish-French Center of Plant Biotechnology:(48-22) 658-46-60 School of Molecular Biology: (48-22) 658-47-02 Warsaw University Affiliates: Department of Genetics: (48-22) 658-47-54 Institute of Experimental Plant Biology: (48-22) 658-39-49 fax: (48-22) 658-48-04 Administration: (48-22) 658-47-00 Guest rooms: (48-22) 658-47-94 The Institute of Biochemistry and Biophysics of the Polish Academy of Sciences was founded in Warsaw in 1957. Initially a biochemical research center, the Institute presently specializes in advanced molecular biology of plants, fungi and bacteria and in state-of-the-art computer modeling and bioinformatics.

Specialized areas of interest include:

gene sequencing and mapping yeast and bacterial genomics regulation of gene expression at DNA and RNA levels protein biosynthesis and post-translational modification mutagenesis and DNA repair metabolism of nucleic acids protein kinases regulation of enzyme activity protein structure-function relationships computer modelling of peptides and proteins NMR studies of proteins and peptides antiviral and anticancer nucleotides polyprenoid structure and function nucleus-mitochondria interaction molecular plant virology 'plant transformation molecular basis of plant and microbial biotechnology Several research and educational organizations associated with the Institute are located in modern quarters of IBB with over 12,000 square meters of laboratory space

They include:

IBB Polish-French Center for Plant Biotechnology Research sponsored by the Governments of both countries

Interdisciplinary Center for Advanced Computer Modeling, Warsaw University (equipped with Cray computers)

Institute of Experimental Plant Biology and Department of Genetics Warsaw University which complement IBB expertise

IBB Post-graduate School of Molecular Biology with almost 100 students working towards the Ph.D. degree

IBB NMR Laboratory (equipped with the 500MHz spectrometer)

IBB Polish National Node of European Molecular Biology Network (EMBNet) provides access to major gene banks and related programs and information for over 600 scientists in Poland

Regional Node of International Center for Cooperation in Bioinformatics (ICCB) under the auspices of UNESCO CONTENTS

Institute profile 5 Scientific Council 11 Trustee Council 15 School of Molecular Biology 18 Polish-French Center of Plant Biotechnology 19 International Collaboration 20 Library 21 Research Units of the Institute Department of Biophysics 25 Department of Genetics 32 Department of Lipid Biochemistry 39 Department of Microbial Biochemistry 43 Department of Molecular Biology 50 Department of Plant Biochemistry 56 Department of Protein Biosynthesis 61 Laboratory of Mutagenesis and DNA Repair 67 Laboratory of Purine Metabolism 70 Laboratory of Antimetabolites 71 Laboratory of DNA Sequencing and Oligonucleotide Synthesis 76 NMR Laboratory 78 Bioinformatics Unit 80 Laboratory of Molecular Biology 81 (affiliated with the University of Gdańsk) Research Units of the Warsaw University located at the Institute Department of Genetics (associated with the Institute) 87 Institute of Experimental Plant Biology 89 Research grants 99 Meetings, Symposia, Courses & Seminars 103 List of Publications Publications 112 Abstracts. Miscellaneous 133 Patents 164 Dissertations 165 Institute Staff 176 INSTITUTE PROFILE

HISTORICAL BACKGROUND

The Institute of Biochemistry and Biophysics (IBB; (www.ibb.waw.pl1 of the Polish Academy of Sciences (PAS; PAN in Polish) was established in 1957 at the turning point for the development of life sciences in post-war Poland. This was the period of destalinisation, followed by the abrupt fall of the neo-Lamarckian Lyssenko theories and the return to public life of formal genetics, biochemistry and biophysics, which in the previous decade had been wiped off the country's intellectual landscape by party bureaucrats. The efforts of the Institute's founders (professors J. Heller, W. Gajewski, T. Korzybski, D. Shugar), who had been educated in pre-war universities and understood the realm of science, focused on the nation-wide reconstruction of these domains, which for many years thereafter concentrates in the Institute laboratories. Still, during the succeeding decades, the general state policy gave priority to heavy industry and the development of basic sciences was on the whole neglected. Under the communist system, the Institute never received appropriate funding and was located in run-down premises rented from an industrial science unit. Against these odds, however, the Institute teams kept pace with the developments of world science, contributing in several fields of molecular biology, biochemistry, biophysics and genetics. Until 1989 the Institute published around 2000 publications and granted around 200 Ph.D. degrees. It has to be noted that in this period some 100 Institute Ph.D. holders emigrated, creating in the late 1980's a generation gap. In the initial period of Institute activity, Professor J. Heller (founder and first Director of the Institute), developed research in insect comparative biochemistry. Since the late 1950's, D. Shugar's team has been making significant contributions in the area of chemistry of nucleotides and nucleosides, and the mechanisms of base-pairing that underlie mutagenesis. This experience is now of prime importance for constructing inhibitors exhibiting antiviral activity. In the 1970's, underP. Szafranski's leadership, the mechanisms of protein translation were actively analysed. This led to the involvement of the Institute in the studies of RNA splicing. The Institute team led by W. Filipowicz and Magdalena Konarska was the first to show the existence of RNA-ligase and contributed to our understanding of lariat formation. Former members of this team are presently working abroad, directing research groups in Friedrich Miescher Institute (W. Filipowicz), Rockefeller University (Magdalena Konarska), University of North Carolina (R. Kole), which have added profoundly to our understanding of splicing mechanisms. Indeed, these eminent scientists, who today serve as Trustees of the Institute (Trustee Council) represent the IBB-derived, internationally recognised scientific school. Lipid chemistry was actively developed in the domain of dolichols, novel at the time (the group of T. Chojnacki - since 1987 Foreign Assistant Professor, Karolińska Institute). Yeast formal genetics was a strong Institute topic with the group of Aleksandra Putrament, who in the late 1960's introduced methods for selective mutagenisation of mitochondria and supplied the world collection with over 700 genetically characterised mutants. This collection has been in use in numerous foreign laboratories studying mitochondria! genome expression. At the same time bacterial genetics was developed under the leadership of T. Kłopotowski and Danuta Hulanicka in the field of aminoacid biosynthesis. After 1989, with political changes in Poland, the intellectual potential of the Institute and the importance of life sciences were recognised, and the Institute received state support that enabled rapid construction of a new, well equipped site, occupied since 1994-5. A formal network of former Institute employees working abroad was immediately created, and the inviting Institute granting system and excellent working conditions attracted back (middle-aged) scientists in their prime years with experience obtained abroad. This is the new generation of the Institute's group leaders, who create new dynamics in the Institute research. With up-graded conditions and an attractive research and education programme, the Institute became a truly living center, with over 90 Ph.D. students selected from among several times as many applicants, and around 300 undergraduate students per year learning specialised techniques in the Institute's dedicated laboratories directed by associated University teams.

THEMATIC FOCUS

In general, scientific interests of the Institute have evolved over the years from classical biochemistry, biophysics and physiological chemistry toward up-to-date molecular biology. Research interests focus on: replication, mutagenesis and repair of DNA; regulation of gene expression at various levels; biosynthesis and post- translational modifications of proteins; gene sequencing and functional gene analysis; structure and function of enzymes; conformation of proteins and peptides; modeling of structures and prediction of functions of proteins; mechanisms of electron transfer in polypeptides. Although basic research is considered the primary function, attention of several research groups is simultaneously oriented to potential clinical and agricultural applications. The main research interests of the Institute's biophysics groups concern structural, conformational, kinetic and thermodynamic determinants of intra- and intermolecular interactions in polypeptide and polynucleotide systems, underlying folding in native forms and biological functions of proteins and nucleic acids. Both experimental (optical and NMR spectroscopy, steady-state and fast kinetic methods, electrophoretic and chromatographic techniques) and theoretical computer modelling (molecular mechanics and dynamics and quantum mechanical calculations, threading .modelling methods) approaches are employed. Lipid biochemistry is actively pursued, with a focus on biosynthesis and function of polyisoprenoids mainly in protein glycosilation. Microbial genetics is followed in the areas of sulfate metabolism in Enterobacteriaceae, sugar metabolism in Lactococci and mechanisms of stable plasmid maintenance. Formal and molecular genetics of lower fungi, traditionally a strong point in Institute's activity, is studied by several teams. They concentrate on genes functioning in nucleo-mitochondrial interactions, peroxisome formation and activity, and aminoacid synthesis. Other objects of analysis are nuclear genes controlling protein transport to mitochondria, as well as interrelations among regulatory circuits governing enzymes involved in sulphur and folate metabolism. Yeast genomics is actively pursued. Our previous participation in the European yeast genome sequencing effort is followed today by functional analysis of 26 new ORFs, identified by Institute teams. This research is conducted within the EURO- FAN program. Plant molecular biology has become during the last 5 years one of the Institute's focal programmes. Plant pathogen genome expression is studied in vivo and in vitro, appropriate virus-resistant plants are constructed, patented and, since recently, undergoing field trials under the control of the Ministry of Agriculture, first such tests in Poland. Plant transformation (Arabidopsis, potato, tobacco, carrot, lettuce, corn) became a standard procedure. Signal transduction is studied with appropriate transformants, the protein kinases involved are identified and isolated in work carried out within EU consortia. A NATO-funded programme on oral plant vaccines has recently been initiated. Chromatin function in plant development is analysed with transformants carrying foreign histone genes. Nucleoside and nucleotide advanced chemistry is actively pursued, synthesis of prospective anti-HlV compounds and potent specific kinases inhibitors has recently been achieved. Fidelity of DNA repair and mechanisms of mutagenesis induced by chemicals and environmental stresses in bacteria, yeast and plants are the subject of work of our internationally recognised teams of molecular biologists. Gradually, however, these teams have been shifting their interest to human genes that control mutagenesis. The aim of these studies is the understanding of the mechanisms of oncogenesis and ageing. Gene engineering programs implemented in all the activities enumerated above, receive support from expert groups in overexpression of proteins in E.coli, S.cerevisiae, Pichiapastoris and Baculovirus systems. The Institute's activities include bioinformatics. Current releases of major molecular biology databases, updated daily, are available on the Institute network, along with tools for software analysis. Our internal network permits access to software and data maintained on a multiprocessor Silicon Graphics system from more than 100 computers and terminals located throughout the Institute. The Institute network is accessible nation-wide through the Internet, and over 800 molecular biology database users are registered at the Institute mainframe. The quality of our bioinformatics set-up has led to the recognition of IBB as the national node of the European Molecular Biology Network, which means that IBB is recognised as a prime bioinformatics facility in Poland. Bioinformatics is also being pursued within a Polish-Israeli program, supported by UNESCO, and co-directed by IBB and the Weizmann Institute of Science in Rehovot. As a result of this co-operation, since April 1997, the Institute has been acting as a Regional Node of UNESCO ICCBnet - a network for promoting bioinformatics to biotechnologically developing countries (http://dapsas 1 .weizmann. ac.il/bcd/iccb.htmD. IBB has undertaken responsibilities of the regional node in Central and Eastern Europe. ICCBnet has been approved as the official UNESCO network by the resolution of the General Conference in November 1997.

EDUCATIONAL ACTIVITIES

The Institute's core activity remains basic research, but teams are working towards enabling the transfer of the accumulated knowledge to the higher education system. This has led to a formal agreement between IBB and Warsaw University and the creation of a division of the Institute at the University of Gdansk. These affiliations with university centers have contributed not only to an expansion of the Institute's research activities, but also to a programme of advanced education in molecular biology through the IBB School of Molecular Biology, with more that 90 young researchers enrolled for the Ph.D. degree.. The Institute is formally and closely associated with the Faculty of Biology of Warsaw University, leading to the location at the Institute site of the University's Department of Genetics and Institute of Experimental Plant Biology. Recently, a division of the Institute has been created at the University of Gdańsk. These affiliations with university centers, as well as collaboration with other Polish research centers (Institute of Bioorganic Chemistry PAS in Poznań, Institute of Plant Breeding and Acclimatisation in Radzików, Departments of Biochemistry and Biotechnology of the Universities of Wrocław, Lublin and Łódź) have contributed to an expansion of the research activities of the Institute, mainly in fungal and bacterial genetics and molecular biology, and in plant biotechnology. This is evidenced by several collaborative research projects and joint publications. Specialised courses for undergraduate students are organised in collaboration with the Departments from the Warsaw University Faculty of Biology. The courses include training in basic methods of molecular biology and biotechnology, e.g. gene cloning and sequencing, analysis of gene expression etc., with special emphasis on bioinformatics. These educational activities received partial support from the PHARE SCI-TECH Project. The Institute actively works towards improving the public perception of science. Three years ago our staff members (D. Shugar, Magdalena Fikus) initiated a nation-wide event - the Warsaw Science Festival. In 1998, over 20,000 and in 1999 - over 25,000 participants attended different activities of the Festival, which featured 274 panel discussions, presentations, open-laboratory days and special demonstrations.

ORGANISATION

IBB (according to the Polish law) is a legal body, governed by its own statute approved in 1998 by the President of the Polish Academy of Sciences. The staff of the Institute currently numbers 260 persons, 186 of whom are involved in research, and the remainder in services and administration. The Departments and independent Laboratories, along with their research and other activities, are listed in full detail below. The Institute is administered by the Director, supported by Board (consisting of Heads of departments, laboratories and research teams) and the Scientific Council, the member of which include scientists from other centers in Poland (see below for the list of members). There is also a Trustees Council, consisting of former Institute staff members who now reside permanently abroad at various academic or other research centers, but who desire to maintain close scientific contacts with the Institute (see below for the list of members). It should be noted here that IBB possesses the necessary authorisation to award higher degrees (Ph.D., Dr. Sc.) in the fields of biochemistry, biophysics and genetics. The Institute has at its disposal several funds for the expansion and promotion of research activities: (a) Stipends for candidates formally registered in study programs leading to a higher degree. (b) Repatriation fund, in the form of financial assistance to former members of the Institute, presently working in academic or other research centers outside Poland, who have expressed the desire to return to IBB. (c) The European Fellowship Fund: This Fund offers fellowships to scientists from other research centers in Central and Eastern Europe, enabling them to spend periods of 3 to 6 months at our Institute or affiliated Polish scientific centers, working either on an IBB research project, or on one already being conducted in collaboration with the candidate's parent institution. This is facilitated by the availability, in our new buildings, of several comfortable guest rooms, also used by invited lecturers. Collaboration with foreign laboratories, carried out since the Institute's establishment, has been particularly accentuated for the past seven years, especially with centers in France. In 1991 IBB became formally associated with the Center de Genetique Moleculaire (COM) of the Center National de la Recherche Scientifique (CNRS), and the Institut Jacques Monod of the Universite de Paris VII. The Polish- French Center of Plant Biotechnology (see below for further details) was established in 1994 by an accord between the CNRS, the Polish Academy of Sciences, and the Polish State Committee for Scientific Research (KBN). This has already enabled many members of our staff to spend working visits in French molecular biology laboratories, which has led to several collaborative research projects, while many French scientists have visited our Institute, mainly to deliver lectures and seminars to our Ph.D. students in the School of Molecular Biology. Within the framework of the Polish-French collaboration, a special program of co-directed Ph.D. theses was initiated. Several Institute graduates are working toward their Ph.D. degrees under the joint direction of Polish and French professors, carrying out experiments both in IBB and in French laboratories. Further details of international collaborative projects are outlined below. During the last several years, the Institute activities enabled the creation of a modern research unit, equipped according to world standards and cumulating human potential in up-to-date techniques of molecular and structural biology. Today, the Institute's aim is to utilise this potential by implementing new programs, solving specific problems indicated by end-users active in bio-medicine, food production and environment protection. We believe that in biology, there is no sharp divide between basic and applied research. Applications have to stem from a sound theoretical background. On the other hand, identifiable end product(s) of market value, resulting from a research programme is a built-in test of the robustness of employed methodology. The Institute is in the process of gradual transformation into such a center, where synergy between theory and application would work towards achieving the highest scientific standards.

10 SCIENTIFIC COUNCIL 1999 - 2002

Members of the Polish Academy of Sciences:

Czesław CIERNIEWSKI Corresponding Member, Institute of Physiology and Biochemistry, Medical Academy ul. Lindleya 6, 91-131 Łódź

Tadeusz CHOJNACKI Corresponding Member, Institute of Biochemistry and Biophysics, PAS ul. Pawińskiego 5a, 02-106 Warszawa

Leszek KACZMAREK Corresponding Member, Institute of Experimental Biology, PAS ul. Pasteura 3, 02-095 Warszawa

Włodzimierz OSTROWSKI Member, Jagiellonian University, Collegium Medicum, Institute of Medical Biochemistry ul. Kopernika 7, 31 -034 Kraków

David SHUGAR Foreign Member, Institute of Biochemistry and Biophysics, PAS ul. Pawińskiego 5a, 02-106 Warszawa

Przemysław SZAFRAŃSKI Member, Institute of Biochemistry and Biophysics, PAS ul. Pawińskiego 5a, 02-106 Warszawa

Piotr WĘGLEŃSKI Corresponding Member, Warsaw University, Department of Genetics/Institute of Biochemistry and Biophysics PAS, ul. Pawińskiego 5a, 02-106 Warszawa

11 Kazimierz L. WIERZCHOWSKI Member, Institute of Biochemistry and Biophysics, PAS ul. Pawińskiego 5a, 02-106 Warszawa

Lech WOJTCZAK Member, Institute of Experimental Biology, PAS ul. Pasteura 3,02-095 Warszawa

Maciej ŻYLICZ Corresponding Member, Division of Molecular Biology, University of Gdańsk ul. Kładki 24, 80-822 Gdańsk

Outside Members of the Council:

Ryszard W. ADAMIAK Institute of Bioorganic Chemistry, PAS ul. Noskowskiego 12/16, 60-637 Poznań

Krystyna DOMAŃSKA-JANIK Center of Experimental and Clinical Medicine, PAS ul. Pawińskiego 5, 02-106 Warszawa

Jerzy DUSZYŃSKI Institute of Experimental Biology, PAS ul. Pasteura 3, 02-095 Warszawa

Marek GNIAZDOWSKI Medical Academy ul. Lindleya6, 90-131 Łódź

Marian KOCHMAN Wrocław Polytechnical University ul. Wybrzeże Wyspiańskiego 27, 51-114 Wrocław

Liliana KONARSKA Medical Academy, Warsaw, Institute of Biopharmacy ul. Banacha 1, 02-097 Warszawa

12 Bogdan LESYNG Warsaw University, ICM ul. Pawińskiego 5a, 02-106 Warszawa

Stefan MALEPSZY Agricultural Academy, Warsaw, Department of Genetics and Plant Breeding ul. Nowoursynowska 166, 02-789 Warszawa

Jacek OTLEWSKI University of Wrocław, Faculty of Natural Sciences, Institute of Biochemistry ul. Tamka l, 50-137 Wrocław Andrzej PŁUC1ENNICZAK Institute of Antibiotics and Biotechnology ul. Starościńska 5, 02-216 Warszawa

Krzysztof STAROŃ Faculty of Biology, Department of Biochemistry, Warsaw University ul. Żwirki i Wigury 93, 02-089 Warszawa

Zygmunt WASYLEWSKI Institute of Molecular Biology, Jagiellonian University Al. A. Mickiewicza 3, 31-120 Kraków

Institute Staff:

Ewa BARTNIK (Warsaw University/IBB PAS) Andrzej BIERZYNSKI Jerzy BUCHOW1CZ Piotr CEGŁOWSKI Zygmunt CIEŚLA Andrzej EJCHART Magdalena FIKUS Jacej HENNIG Monika HRYNIEWICZ Danuta M. HULANICKA-DZIECHCIŃSKA Witold JACHYMCZYK Celina JANION Grażyna JAGURA-BURDZY Andrzej JERZMANOWSKI (Warsaw University/IBB PAS) Ewa KULA-ŚWIEŻEWSKA Tadeusz KULIKOWSKI Jarosław KUŚMIEREK Grażyna MUSZYŃSKA Grażyna PALAMARCZYK

13 Andrzej PASZEWSKI Irena P1ETRZYKOWSKA Magdalena RAKOWSKA-BOGUTA Joanna RYTKA Piotr P. STĘPIEŃ (Warsaw University/IBB PAS) Jan W. SZARKOWSKI Ewa ŚLEDZIEWSKA-GÓJSKA Bernard WIELGAT Włodzimierz ZAGÓRSKI-OSTOJA Zofia ZARĘBSKA Piotr ZIELE1MKIEWICZ Jerzy ŻUK

Representatives of Junior Scientists:

Jerzy BRZYWCZY Małgorzata ŁOBOCKA Andrzej PALUCHA Jerzy SMAGOWICZ Anna WÓJCIK

14 Members of the Trustee Council of IBB:PAS (a group of former staff members of the Institute now residing abroad)

Witold Filipowicz Jerzy Barankiewicz Fredrich Miescher Institut Cypros Pharmaceutical Corporation 4002 Basel, Switzerland 2714 Loker Avenue West tel: (41 61)6976993 Carlsbad, CA 92008, USA tel: lab. (41 61) 6974 128 tel: (619) 929 95 00 tel: home (4161) 69 73 976 fax:(619)9298038 E-mail: [email protected] E-mail: [email protected] Marcin Filutowicz Katarzyna Bębenek University of Wisconsin Laboratory of Molecular Genetics, Department of Bacteriology National Institute of Environmental 1550 Linden Drive Health Sciences, PO BOX 12233, Madison, WI 53706, USA Research Triangle Park, NC 27709, USA tel: (608) 262 6947 tel: (919) 54126 44 fax: (608) 262 9865 fax: (919) 541 76-13 E-mail: [email protected] E-mail: [email protected] Leszek Kleczkowski Jadwiga Chroboczek Department of Plant Physiology Institut de Biologie Structural Umea University, Umea, Sweden 41, Avenue des Martyrs tel: (46) 90 19 34 39 38027 Grenoble Cedex 1, France fax: (46) 90 16 66 76 tel: (033 4) 76 88 95 80 E-mail: [email protected] fax: (033 4) 76 88 54 94 E-mail: [email protected]

15 Jerzy Paszkowski Włodzimierz Mandecki Fredrich Miescher Institut Corporate Molecular Biology P.O. Box 2543 Department 93 D, Building 9A CH 4002 Basel, Switzerland Abbott Park, IL 60064, USA tel: (41 61)6979144 tel: (708) 937 22 36 fax: (41 61)6973976 fax: (708) 938 60 46 E-mail: [email protected] Ryszard Kole Lineberger Comprehensive Cancer Norman Pieniążek Center, School of Medicine Division of Parasitic Diseases The University of North Carolina at Biology and Diagnostic Branch Chapel Hill, Campus Box 7295 4770 Buford Highway NE, Chapel Hill, NC 27599-7295, USA Mailstop F 13 tel: (919)966 11 43 Atlanta, GA 30341-3724, USA fax:(919)96630 15 tel: home (770) 822 40 12 E-mail: [email protected] tel: (404) 488 40 73 fax: (404) 488 41 08 Magda Konarska E-mails: The Rockefeller University [email protected] 1230 York Avenue [email protected] New York 10021-6399, USA tel: (212) 327 84 32 Anna Radomińska fax: (212) 327 83 70 University of Arkansas for Medical E-mail: Sciences konarsk@rockvax .rockefeller.edu Department of Biochemistry and Molecular Biology Piotr Lassota 301 WestMarkham, Slot 516 American Cyanamid Company Little Rock, AR 72205-7199 Lederle Laboratories Division tel: 01 (501)68654 14 Oncology and Immunology Research faxl:(501)68681 69 Section, fax2: (501)603 11 46 401 N. Middletown Road E-mail: Pearl River, NY 10965, USA [email protected] tel: (914) 732 21 57 fax:(914)7325695 E-mail: [email protected]

16 Andrzej Stasiak Andrzej Śledziewski Universite de Laussanne Biopharmaceuticals-NovoNordisk Laboratoire d'Analyse Ultrastructurale 1201 Eastlake Avenue East Batiment de Biologie, niveau l Seattle, WA 98102, USA CH-1015 Lausanne-Doringt, Switzerland tel: (206) 442 67 10 tel: (41 21) 692 24 71 fax: (206) 442 66 08 fax: (41 21)6922540 E-mail: [email protected]

Włodzimierz Szer Ludwika Zimniak The Scripps Research Institute University of Arkansas for Medical 10550, N. Torrey Pines Rd. Sciences La Jolla, CA 92037 4301 W. Markham St. slot 567 SBR6, r. C205 Little Rock, AR 72205, USA USA tel: (501) 603 1003 E-mail: [email protected] E-mail: [email protected]

17 SCHOOL OF MOLECULAR BIOLOGY - PH.D. STUDIES Head: professor Grażyna Palamarczyk

The Warsaw School of Molecular Biology was founded in September 1994, to facilitate the attainment of the general requirements for the Ph.D. degree. The Board of the School consists of professors: Grażyna Palamarczyk (chairman), Joanna Rytka, Andrzej Jerzmanowski In 1998-99, 14 students involved in the program have completed the studies and obtained the Ph.D. degree. At the moment attendance includes almost hundred (97) candidates for the Ph.D. degree, among them 17 were addmitted to the first year and 21 began the last year of their studies. The teaching program offers lectures and seminars on various topics. In the last two years lectures on genomics (30 hrs on bacterial, yeast, plant and human genomes) and seminars on non-Mendelian inheritance and regulation of the cell cycle were organized. The lectures were given by the local as well as external specialists and seminars were supervised by the more advanced and experienced Ph.D. students. The students also attended the international symposium, organized together with the Warsaw University, on molecular mechanisms of translation and transcription. All students have numerous opportunities to present their research achievements. In the last two years 43 papers co-authored by Ph.D. students were published and over one hundred short communications were presented at local and international research meetings. From its own budget the School offers mini grants (12 per year) to support best research projects presented to the Board by the Ph.D. students.

18 POLISH - FRENCH CENTER OF PLANT BIOTECHNOLOGY Director: professor Stanisław Lewak

The Polish-French Center of Plant Biotechnology was founded in 1993 by an international agreement between CNRS (French National Center of Science Research), KBN (Polish State Committee for Scientific Research) and PAS (Polish Academy of Sciences) as a continuation of the twin association between IBB and COM CNRS (CNRS Molecular Genetics Center). The Center is financially supported by the governments of both countries. The funds are destinated for the promotion of joint research programmes on plant molecular biology and genetics. The principal Polish participants are the Institute of Biochemistry and Biophysics of Warsaw, the Institute of Bioorganic Chemistry of Poznań and Warsaw University. Polish research teams cooperate with several CNRS and INRA laboratories and French universities. The supported activities concern the following areas: 1. Higher plants: plant - microorganism interactions, regulation of gene expression, nucleic acid metabolism, metabolic regulation. 2. Yeasts and filamentous fungi: sequencing and functional analysis of the yeast genome, studies on nuclear - mitochondrial relationships, regulation of metabolic pathways. 3. Plant viruses - studies on transgenic virus resistant plants. The Center's financial contribution to the Polish - French cooperation consists in supporting short-term (1-3 months) visits (15 persons in 1998 and 7 in 1999) and joint research programs (16 grants in 1998, 14 in 1999). Moreover, the organisation of two conferences was partially financed by the Center in 1998-1999: 1. "Plant transport systems and pathogens" Warsaw, November 16, 1998; 2. "Molecular mechanisms of gene expression - 5 years of Polish - French common research (1993-1998)" Warsaw, June 10-11, 1999.

19 INTERNATIONAL COLLABORATION BASED ON FORMAL AGREEMENTS BETWEEN IBB PAS AND FOREIGN INSTITUTION

1. Max Planck Institut fur molekulare Genetik, Berlin (Germany)

2. Laboratory of Terpenoid Chemistry, Institute of Chemistry of Moldova Academy of Science (Moldova)

3. Institute of Plant Sciences of Leiden University (Netherland)

4. Polish-French Center of Plant Biotechnology, located at IBB PAS, common agreement between PAS, KBN and French CNRS

20 LIBRARY FACILITIES Head: Teresa Żyłka, M. Sc.

The Institute Józef Heller Library, established in 1954, has grown to become one of the best in Poland in the field of molecular biology, biochemistry, and related areas. At present it has almost 7,300 books and 15,428 volumes of periodicals. The journal section includes 335 titles, 174 of which are received on a regular subscription, and 21 titles by exchange with domestic and foreign institutions. This collection comprises the most important international publications in biology, molecular biology, biochemistry, chemistry, biophysics, genetics, biotechnology, virology, structural biology. The Library also has a collection of 485 theses ( M.Sc., Ph.D., Dr.Sci.) delivered at the Institute. The Library prepares and distributes annually a list of publications of the Institute. Computer search services include Life Sciences Collection on CD-ROM, Current Contens and selected periodicals in computerized form, as well as access to The Internet. E-mail address: [email protected]

21

RESEARCH UNITS OF THE INSTITUTE

DEPARTMENT OF BIOPHYSICS Head: professor Kazimierz L. Wierzchowski

The Department's main research interests concern structural, conformational, kinetic and thermodynamic determinants of intra- and intermolecular interactions in polypeptide and polynucleotide systems, underlying folding in native forms and biological functions of proteins and nucleic acids. Both experimental (optical and NMR spectroscopy, steady-state and fast kinetic methods, electrophoretic and chromatographic techniques, biochemical assays) and theoretical computer modeling (molecular mechanics and dynamics, quantum mechanical calculations, threading modeling methods) approaches are employed. Research projects include: (i) mechanisms involved in Ca2+ binding by specific protein carriers, (ii) specific interactions responsible for stabilization of cc-helical conformation of polypeptides, (iii) early steps and conformational properties of intermediates involved in the folding of a trypsin inhibitor (BPTI), (iv) long range electron transfer between radical redox centers in model peptides and proteins, (v) modeling of protein crystallization and structure of proteins, (vi) molecular interactions in the formation of transcriptional complexes in procaryotic systems, (vii) application of recombinant DNA techniques in some of these projects, and (viii) electric field effects on living cells underlying cell electroporation and electrofusion.

1. CONFORMATIONAL STUDIES OF METAL BINDING PROTEINS AND THEIR FRAGMENTS

A. Bierzyński, A. Ejchart, G. Goch, J. Wójcik, H. Kozlowska, E. Soszyńska, I. Joukov

Structure and metal-binding properties of S100A1 protein. The protein is built of two identical subunits with two calcium-binding sites each. Using fluorescence methods binding constants of calcium have been determined for the native protein as well as for its Glu32-»Gln and Glu73-»Gln mutants in which the N-terminal and C-terminal binding sites, respectively, are inactivated. The most important finding is that the affinity to calcium of the N-terminal site of S100A1 protein is strongly pH-dependent within the physiological pH range of 6.5 - 7.5. This effect is probably related to the deprotonation of one of the histidine residues situated in a close vicinity of the binding ioop and strongly conserved in all proteins from the S100 family. Spectral assignment of'H, I5N, and 13C NMR signals in SIOOA1 is in progress; ca. 70% of peaks have been already assigned on the basis of multinuclear ('H/15N/13C) two- and three-dimensional techniques. Investigation of a-helix nucleation by a calcium-binding loop. It has been shown previously by us that a calcium-binding peptide segment with the sequence Ac-DKDGDGYISAAE-, when saturated with lanthanide tons, nucleated with

25 extreme efficiency the a-helical conformation of short peptide segments attached to its C-terminus. Comparative studies of a series of synthetic peptides with the sequences

Ac-DKDGDGYISAAE(A),,-NH2, where 0 > n < 5, were undertaken. The NMR results strongly suggest that very short helices, containing only one or two hydrogen bonds, as well as C-terminal residues of longer helices, assume the 3/10-helical conformation. LaJ+-binding constants increase considerably with the peptide length. From a thermodynamic cycle the Gibbs free energy of helix propagation can be estimated at about 0.6 kcal/mol of residues. Structure and folding of Cucurbita maxima trypsin inhibitor (CMTI-I). The NMR structure of the Met8-»Leu mutant of the inhibitor has been determined and compared with that of the native protein. Although both structures are very similar, the mutant is less stable as evidenced by NH hydrogen exchange measurements. Oxidative folding of the protein is also strongly perturbed by the mutation. These effects were shown to arise from the reorientation of a hydrophobic cluster situated on the protein surface and formed by side chains of residues Leul7, Ile6, and Met or Leu8, respectively. Publications: 3266,3369,3417,1306/A, 1310/A, 106N, 114N

2. LONG-RANGE ELECTRON TRANSFER BETWEEN RADICAL REDOX CENTERS IN MODEL PEPTIDES AND PROTEINS

K. L. Wierzchcrwski, K. Bobrowski*, J. Poznański, K. Majcher

In continuation of our earlier pulse radiolysis studies on the kinetics and pathways of long-range electron transfer (LRET) accompanying intramolecular radical transformation Trp[N*] -> Tyr[O*] in model peptides: H-Trp-(Pro),,-Trp-OH, (n = 1-5) and in hen egg-white lysozyme (cf. for review 3253, 3254), the effect of protonation equilibrium TrpfN*] + H+ o Trp[NH'+] on the parameters of this transformation in the higher members of proline bridged peptides 3 (n =3), 4 (n = 4) and 5 (n = 5) was investigated at pH 2-8 at 298 K and at pH 4 over the temperature range 283-328 K. LRET was found to occur down to pH 2, contrary to expectation based on electrochemical redox potentials for Trp and Tyr in the form of free amino acids. The

first-order rate constants of LRET, kuhs, varied sigmoidally with pH, allowing the

evaluation of p/fai's for deprotonation of Trp[N'] (3.7, 4.1 and 4.3 for 3, 4 and 5, + respectively), and of the intrinsic rate constant, k2, of LRET involving solely Trp[NH' ]

radicals. Variation of pKai was attributed to electrostatic interactions between the indolyl radical and the COOH group of the terminal Tyr, expected to decrease in the order shown, and taken as an indication of variation, in the same order, of the

electrochemical driving force AG° of LRET. In 4 and 5, k2 proved to be about 60-fold larger than the corresponding k\ values characteristic for Trp[N*], which indicates that the kinetics of LRET involving Trp[NH*+] exhibits similar through-bond distance

dependence as that found earlier for the reaction with Trp[N']. For 3 the ratio of k2l k> was found to be about 3-fold lower than for its two longer-bridged analogues, 4 and 5.

26 Moreover, contrary to the the latter two peptides, Arrhenius activation energy for LRET in 3 varied nonlinearly with temeperature indicating a decrease in. pK^ of Trp[NH"+] by one unit in the temperature range studied. Molecular dynamics modeling of conformational preferences of 3 showed that a p -> a transition at the central vj/(Pro3) dihedral angle, which could bring NH (indole) and COOH groups into a close contact compatible with the formation of a hydrogen bond, may occur in 3 on the time scale of the observed electron-transfer reaction. In longer-bridged analogues two such concerted transitions would be necessary to perturb LRET measurably, an event of extremely low probability. The analysis of thermodynamic parameters indicated that in reactions involving both forms of indolyl radical the free energy barrier for activation of LRET is mostly entropie in nature. All these findings indicate that electrostatic interactions and protonation equilibria may play an important role in the regulation of LRET involving indolyl radicals in electron-transfer proteins. Publications: 3445

3. MODELING OF PROTEIN STRUCTURE AND FUNCTION

P. Zielenkiewicz, D. Plochocka, A. Kierzek, S. Gavryushov

Modeling protein crystal growth. A new model of protein crystal growth has been developed. In these studies the reversibility of molecular attachment to the surface of the growing crystal has been taken into account. Lattice simulations of the tetragonal lysozyme crystal growth covered the size scale ranging from the initial small intermediates composed of a few molecules up to microcrystals of the size of 10s molecules. These large complexes exhibit the habit of a macroscopic crystal which constitutes an experimental validation of the model. The simulated growth of the crystal faces agrees with the atomic force microscopy observations. The model provides information concerning the initial events in protein crystal formation. Model of human glucose-6 phosphate dehydrogenase (G6PD). G6PD is the first enzyme in the pentose phosphate pathway and its main physiological role in red blood cells is to produce NADPH necessary for the protection of the cells against oxidative stress. G6PD deficiency is the most common human enzymopathy, affecting over 200 million people worldwide. A model of the human G6PD structure has been built on the basis of high sequence similarity to G6PD from Leuconostoc mesenteroides of known crystal structure. Two mutations of the enzyme: R227W and T336A were analysed. The R227W mutation does not cause changes in the protein structure - the main chain is unaltered - as it is situated on the surface of the enzyme. The influence of the R227W mutation on G6PD structure explains why it is a class 2 variant. The other, T336A, mutation is a class 1 variant. It is located inside an a + (3 domain, far away from the dimer interface. The only structural changes caused by the T336A mutations are in its proximity. The main distortion appears on the protein surface between residues 326 and 335. The changed loop is moved outside of the

27 protein by more than 5A in the mutated enzyme. Probably this causes enzyme destabilisation. Electrostatic interactions of biopolymers with electrolyte solutions. Modified Poisson-Boltzmann equations were numerically solved for model proteins and DNA to calculate the mean electrostatic potential and ionic distributions. It was shown that the classical differ, in the case of multivalent electrolytes significantly, form those obtained using the Poisson-Boltzmann equation. Publications: 3385,3393,3397, 3359,1144/A, 121 I/A

4. MOLECULAR MECHANISM OF TRANSCRIPTION INITIATION IN PROCARYOTIC SYSTEMS

K. L. Wierzchowski, I. Kolasa and T. Łoziński

is routinely used as a chemical probe for footprinting of transcriptional open complexes as it preferentially oxidizes thymine (also cytosine though to a lesser extent) residues to corresponding glycols in the single-stranded DNA region of these complexes. Comparative analysis of KMnO4 footprints of open complexes formed at a number of promoters in the absence and in the presence of Mg2+ ions led to the proposition that specific binding of Mg2+ to the open complex is accompanied by an expansion of the melted DNA region at each of its ends and by exposure of the base residues adjacent to the transcription start point. In neither of the published papers, however, the experimental methods used and the data obtained were presented in sufficient detail to assure the readers that conditions of a single oxidation event per DNA molecule were controlled (single-hit footprinting, SHF). These conditions should be fulfilled for a reliable interpretation of footprints in terms of relative oxidizabilities of the bases in DNA-protein complexes. Therefore, we performed KMnO4 footprinting of the open promoter complex formed on a synthetic consensus-like E.coli promoter Pa, containing only AT base pairs in the bubble region, in the absence and presence of 10 mM MgCl2, as a function of the oxidant dose at 37° C. The location of thymine residues oxidized to corresponding glycols was determined 5'-j2P-labeIed DNA primer extension with the use of the Klenow fragment of DNA polymerase I for both template and nontemplate DNA strands and were PAGE separated on a sequencing gel. End-labeled DNA products were quantified with a Molecular Dynamics Phosphorimager and Image Quant software. The integrated intensities of groups of bands, each proportional to the amount of DNA contained therein and corresponding to a DNA fragment terminated at an oxidized thymine residue in a given strand within the melted DNA region, were corrected for local background radioactivity and normalized to the integrated intensity of the whole lane. The fractions of oxidized DNA were found to vary with the applied dose of the oxidant according to Poisson statistics. This allowed us to distinguish KMnO4 dose ranges corresponding to the single-hit (SH) and multiple-hit (MH) conditions of the oxidation reaction. In the lowest range of the oxidant dose applied, thymine residues in both DNA strands were oxidized under SH conditions. Integrated areas of these groups of bands

28 were deconvolutued into Gaussian components to yield fractions^ of DNA fragments terminated at each of the 6 T residues oxidized in either of the two DIM A strands. The dependence of these fractions, as well as of the fraction l-S/[ of unoxidized DNA, on the oxidant dose was subjected to global kinetic analysis in terms of individual pseudo first-order reactivity rate constants k; for particular Tj residues. In the absence of the added Mg2+ ions, all 13 oxidizable T residues present within the 14 bp long melted

DNA region exhibited measurable reactivity towards KMnO4: T+3, T+2, T-2, T-3, T-4 and T-6+T-7 of the nontemplate strand, and T+l,T-l, T-5, T-8, T-9 and T-ll of the template strand. The most reactive proved to be T+l 1 and T+9 of the template strand, and T-2, T-3 and T-4 of the complementary strand. Addition of Mg2+ caused an increase in the reactivity of all T residues in the template strand: of T+l and T-l by a factor of 3, and of those located more distantly from the transcription start point by a factor of 1.4-1.8. Within the same range of doses applied, the reactivity of T+3 and T+2 of the nontemplate increased also about 2.7-fold, while that of remaining residues by a factor of 1.6-1.8. Thus, contrary to earlier reports, no extension of the melted DNA region upon binding of Mg2+ ions to the open complex was observed. The data obtained fully support, however, earlier qualitative observations that the presence of Mg2+ ions increases primarily the reactivity of T residues located close to the transcription starting point. It can be thus concluded that they are presumably bound in the proximity of the catalytic center of RNA polymerase. The nontemplate strand of the double stranded pDS3 plasmid DNA was also footprinted with KMnO4 and reactivities of a number of its fragments evaluated in terms of kinetics of the reaction. It was found that the reactivity of T residues in dsDNA under SH conditions is by more than two orders of magnitude lower than in single stranded DNA within the melted region of the open complex, profoundly depends on the base sequence context, and is about 2.5-fold higher in the presence of lOmMMgCk

4.1. PREPARATION AND STRUCTURE DETERMINATION OF THE 4.2 a70 DOMAIN INVOLVED IN THE RECOGNITION OF THE -35 PROMOTER REGION

K. L. Wierzchowski, K. Bolewska, J. Poznański K. Majcher and T. Rak

The structure of E.coli RNA polymerase (RNAP) and of its component subunits a, P, (3' and a70 has been a subject of extensive studies for a number of years. We have previously overexpressed and purified two protein fragments (with and 70 without an N-terminal His6 tag) containing the 4.2 domain of cr , implicated in specific recognition of the -35 promoter region. UV-CD and 'H TOCSY NMR investigations carried out subsequently indicated that this domain is almost 100% helical. To elucidate the structure of this domain in more detail, attempts were undertaken to crystallize both protein fragments. Until now, however, they proved I5 15 13 unsuccessful. In parallel, N- and N, C unifonnely labelled His6 -tagged fragments were overexpressed and purified with a high yield and were subjected to further NMR

29 investigations. The analysis of data thus far collected (2D 15N HSQC, 3D spectra: HN- CA-13C TOCSY, HNCACB and HN(CO)CACB, HNCO and HNCACO, and a series of J-modulated [15N-'H]-COSY spectra) indicates that the protein is made of one short (close to the N-terminus) and two longer alpha-helices, separated by short unstructured chains. Both the His6 tagged N-terminal fragment and the C-terminal fragment are rather unstructured. The two longer helices are characterized by the distribution of leucine and arginine residues suggesting that they may be involved in DNA binding. Further NMR and molecular modeling studies are under way.

5, FUNCTIONAL ANALYSIS OF RRD1 (YIL153w) AND RRD2 (YPL152w), TWO PUTATIVE ACTIVATORS OF PHOSPHOTYROSYL PHOSPHATASE ACTIVITY OF PP2A IN SACCHAROMYCES CEREVISIAE

B. Rempola

In the context of the cooperative project for functional analysis of novel genes uncovered during the systematic sequencing of the Saccharomyces cerevisiae genome, we deleted two paralogous ORFs: YIL153w and YPL152w. Based on the resulting phenotypes, the corresponding genes were named RRDI and RRD2, respectively. Rrd proteins show significant similarity to the human phosphotyrosyl phosphatase activator (PTPA). Both single mutants, rrdlA and ndIA, were viable. Deletion of RRD I caused pleiotropic phenotypes under a wide range of conditions, including sensitivity to Ca , vanadate, ketoconazole, cycloheximide, calcofluor white and resistance to caffeine and rapamycin. The only phenotypes found for rrd2A - resistance to caffeine and rapamycin, were weaker than the corresponding phenotypes of rrdlA. The double mutant, rrdlA rrd2A was inviable on rich glucose medium, but could grow in the presence of an osmotic stabilizer. The double mutant rrdlA rrd2A was partially rescued by inactivation of HOG1 or PBS2, suggesting an interaction between the RRD genes and the Hoglp signal transduction pathway. Introduction ofsl(2A into the rrdlA rrd2A background improved the growth of rrdlA rrd2A on sorbitol-containing medium, indicating that the Rrd proteins also interact with the Slt2p/Mpklp signaling pathway. Suppression of the lethal phenotype of the rrdlA rrd2A mutant by overexpression of PPH22 (coding for one of the two catalytic subunits of protein phosphatase 2A) suggested that the products of the RRD genes function positively with catalytic subunits of PP2 A. The synthetic lethality was also suppressed by the Viable' allele (SSDI-vl) of the SSD1 gene. Collaboration: J. Rytka - Department of Genetics; P.P. Słonimski - Centre de Genetique Moleculaire CNRS, Gif sur Yvette, France

30 6. STRUCTURE OF THE FOLDING INTERMEDIATES AND THEIR IMPACT ON THE FOLDING PROCESS

M. Dadlez, A. Wyslouch-Cieszyńska, K. Zdanowski, J. Dyczkowski, A. Jabionowska

We are interested in the elucidation of the factors directing the formation of protein structure during folding and aggregation. Our interest in this area has been strengthened in recent years, as it has been realized that incorrect protein folding may lead to serious diseases both in animals and in humans. In our studies of the folding process of the bovine pancreatic trypsin inhibitor (BPTI) we have shown recently that a group of amino-acid side chains interacts in the unfolded state in a native-like fashion and accelerates the acquisition of the native chain topology in this protein. These residues in the folded protein form a structure called a -hairpin, and our studies suggest that such a hairpin structure is formed also in the first stage of folding in a fraction of the molecules. Currently we have extended our studies to other model molecules: myoglobin - to study the formation of structure in D- helical proteins, and AC peptides responsible for the Alzheimer disease - to study factors responsible for aggregation of these peptides. In cooperation with other groups from the Institute and also from the University of Wrocław (prof. Otlewski's group) and Warsaw University (prof. Darzynkiewicz's group) we are also carrying out several other projects from the field of protein engineering and protein-protein recognition. We have completed a series of more than 50 mutants of BPTI which have been characterized in terms of their affinities to different proteinases. The crystal structures of some of these mutants in complex with proteinases have been obtained. This enabled the structural interpretation of the obtained complex stability data. New inhibitor variants for trypsin and chymotrypsin have also been recently obtained from the selection from a library of ca. 105 different protein variants. These variants are currently under study. In a separate project the method of protein chemical ligation, or in vitro protein splicing, which allows one to obtain protein from its fragments is being used to obtain a specifically phosphorylated variant of the translation initiation factor eIF4E for structural studies. A short protein fragment, containing phosphorylated serine, was obtained by chemical synthesis whereas the remaining part of the protein by overexpression in bacteria. In the ligation process the expected product is formed as judged by SDS-PAGE electrophoresis. Work is in progress to isolate and purify this product. Publications: 3269,3304,3370,3380,3382,3383,3394,3413,119/N

31 DEPARTMENT OF GENETICS Head: professor A. Paszewski

There are five independent research groups in the Department conducting the following lines of investigations: 1. Regulation of sulfur amino acid metabolism in fungi, focusing on cloning and characterisation of structural and regulatory genes in Aspergillus nidulans. 2. Identification and characterization of S. cerevisiae genes involved in the regulation of tRNA biosynthesis and control of translation fidelity in cytosolic and mitochondria! systems. 3. Mechanism of DNA repair and mutagenesis by using mutations in DNA polymerases, mismatch repair and radiation sensitivity genes to study their infulence on the generation of mutations in nondividing, resting cells (adaptive mutations) in S. cerevisiae. 4. Pleiotropic effects of the S. cerevisiae IRR1 gene, particularity on colony formation and sister chromatid cohesion. 5. Regulation of heme synthesis in S. cerevisiae: the analysis of structure-function relationship of ferrochelatase (the last enzyme of the pathway). 6. Role of RSP5 the ubiquitin-protein ligase of S. cerevisiae in protein import to mitochondria and endocytosis of plasma membrane proteins 7. Molecular analysis of the glucose-6-phosphate dehydrogenase gene in Polish 6-GDP deficient subjects. 8. Genetic and phylogenic studies of the Polish Bison bonasus population with the use of nuclear and mitochondrial markers.

1. GENETIC REGULATION OF SULFUR AMINO ACID METABOLISM IN FUNGI

A. Paszewski, J. Brzywczy, M. Grynberg, M. Kacprzyk, I. Lewandowska, R. Natorff, M. Piotrowska, M. Sieńko

The filamentous fungus Aspergillus nidulans presents a rich repertoire of metabolic options regarding sulfur metabolism sharing features of bacterial and animal systems. Our studies concentrate on the characterization and regulation of sulfur metabolism related structural and regulatory genes. Two novel genes - cysA and metE - were cloned and characterized. They both code for proteins structuraly belonging to the homoserine O-acetyltransferase family, however, the CYSA protein functions physiologically as serine O-acetyltransferase. The evidence suggests that this enzyme originated by differentiation of a homoserine O-acetyltransferase of bacterial type with aquirement of a novel function. A positive acting sulfur regulatory gene - metR - was identified, cloned and characterized. It encodes a sulfur-specific transcriptional factor which plays a similar role to that of the Neurospora crassa CYS3 protein, however, homology between these two proteins is limited to the DNA binding domain. Mutations

32 in the metR gene are epistatic to mutations in the negative acting scon genes and lead to methionine auxotrophy. Cloning and sequencing of metH revealed that it codes for methionine synthase. Since mutations in this gene have a pleiotropic effect on the regulation of other folate-dependent enzymes synthesis it remains to be determined whether the METH protein has, besides enzymatic, also regulatory functions or whether changes in folates pool due to these mutations are responsible for the observed regulatory effects. It was found that Khiyreromyces lactis homocysteine synthase gene is expressed and regulated in S. cerevisiae under its own promoter in spite of the fact that it differs markedly from its S. cerevisiae counterpart. This indicates a substantial conservation of the sulfur regulatory systems between both yeasts. We have also obtained an evidence that cysteine, but not S-adenosylmethionine, is the effector in the regulation of sulfur metabolism in S. cerevisiae. Publications: 3283, 3378, 3412, 3420, 3427,1168/A, 1173/A, 1175/A, I196/A, 1268/A, 137/N

2. NUCLEAR GENES INVOLVED IN MITOCHONDRIAL BIOGENESIS IN YEAST

M. Boguta, A. Chacińska, A. Konopińska, B. Szcześniak

We have shown that Nam9p is a nuclear encoded protein of the small subunit of the mitochondria! ribosome. Nam9p was localized in mitochondria! ribosomal fraction using specific antibodies. Conditions dissociating the two ribosomal subunits resulted in the co-immunoprecipitation of Mrp!3, a marker protein of the small ribosomal subunit, whereas Mrp20, a marker of large subunit, remained in the supernatant. Yeast expressing the point mutant nam9-l have a respiration defect. Using specific antibodies we have shown that the steady state levels of several subunits of the respiratory chain and ATP synthase were decreased in the mutant, whereas cytochrome c oxidase subunit CoxII, was undetectable. De novo synthesis of Coxll in the nam9-l strain was unaffected. Pulse-chase experiments showed, however, that CoxII and some other mitochondrially encoded proteins are unstable when synthetized on ribosomes with the nam9-l mutation. As reported previously, the respiratory defect of nam9-l could be suppressed by overexpression and also by deletion of the HSPI04 gene encoding a cytosolic chaperone. Now we found that CoxII stability was restored in the nam9-l strain by transient overexpression as well as by deletion of the cytosolic chaperone Hspl04. In addition, low concentrations of guanidine hydrochloride enable the nam9-l strain to respire. The effects of Hspl04 and guanidine hydrochloride are reminiscent of the conditions that cure yeast from prions and let us conclude that nam9-l phenotype is prion-dependent. First we excluded the possibility that the mutated Nam9-lp is a prion-like protein itself. We showed that mutated and wild type Nam9 synthetized in vitro have the same aggregation properties, protease resistance and are imported to mitochondria

33 with the same efficiency. In vivo, Nam9-lp, like Nam9, is localized to the small mitoribosomal subunit. In contrast we were able to show that nam9-l phenotype is directly dependent on the prion [psi+] which is a form of the cytosolic translation termination factor Sup35p. We found that, beside HSP104, the nam9-l phenotype is suppressed by the C- terminal domain of Sup35p which is known to inactivate the [psi+] prion. We also checked that nam9-l phenotype appeared when cytoplasm of a [psi+] strain, but not the isogenic [psi-] was introduced. These findings provide evidence for the existence of a mutual control system for cytosolic and mitochondria) translation in yeast. Publications: 3360,1164/A, 1165/A, 1272/A, 88/N, 142/N

3. MECHANISMS OF DNA REPAIR, RECOMBINATION AND MUTAGENESIS IN SACCHAROMYCES CEREVISIAE

) W. Jachymczyk, J. Żuk, H. Baranowska-Wyszomirska, A. Hałas, A. Ciesielski, - ż. Policińska i O i Cvi l o l.The involvement of DNA polymerases a and 8 in DNA repair-synthesis L. during mitotic gene conversion was confirmed. This involvement appear to be independent of the kind of mutagen used as the inducer intragenic conversion, since experiments with MMS, UV-!ight or mono- and bifunctional psoralens showed similar dependence on the same polymerases. Only a marginal dependence of gene conversion on the activity of DNA polymerase e was observed, which suggests that this enzyme plays a minor or auxiliary role in the intragenic conversion. Results are in press in Acta Biochimica Polonica. 2. Studies on the influece of replicative and nonreplicative DNA polymerases on stationary phase (adaptive) mutations in Saccharomyces cerevisiae showed that the DNA polymerases 5 and s, both responsible for DNA elongation, may replace each other during DNA repair in starving cells, but at the cost of an increased frequency of stationary phase mutations. Based on DNA sequence analysis in the mutants obtained the majority of reversions observed under these conditions were 1-bp deletions in short mononucleotide tracts, mostly A. It was also demonstrated that besides polymerases 5 and s, DNA polymerase |3 is similarly involved in adaptive mutations in yeast, most probably during the recombinational DNA repair in starving cells. Other yeast DNA polymerases are not involved in this process. Studies on the influence of the 3'-»5' exonucleolytic proof-reading activity of DNA polymerases 8 and e on the formation of stationary-phase mutations in yeast are in progress. 3. Similar studies on the influence of genes responsible for mismatch repair on the formation of adaptive mutations in starving cells were continued. The increase in the frequency of selection-induced reversion to Lys prototrophy in mismatch repair mutants during prolonged incubation on selective medium was significantly higher than that observed in wild-type cells. In Amsh mutants this effect was especially pronounced, even in comparison with other mismatch repair mutants like A/W//J/ or ApmsL It was

34 also found that the Msh3 and Msh6 proteins can substitute each other in the formation of active repair complexes with Msh2, discovered earlier by others. Publications: 3402,1176/A, 1177/A, 1178/A

4. HEME AND HEMOPROTEINS BIOSYNTHESIS IN THE YEAST SACCHAROMYCES CEREVISIAE

J. Rytka, A. Chelstowska, M. Góra, E. Grzybowska, I. Smaczyńska

The chelation of ferrous iron into protoporphyrin IX to form protoheme is a crucial reaction of the heme biosynthesis pathway. Understanding the structural aspects and the molecular mechanism of ferrochelatase activity is a prerequisite to elucidating the regulation of iron and heme metabolism in the cell. In order to investigate the domains of ferrochelatase that are important for activity and/or association with the membrane we have constructed a series of chimeric yeast/mouse and ysast/B.subtilis ferrochelatase genes. These genes were expressed in a yeast mutant lacking ferrochelatase and the in vivo and in vitro activity and location of the chimeric enzymes were studied. An internal ~ 45-amino acid segment located at the N-terminus of yeast ferrochelatase was identified which when replaced with the corresponding 30-amino acid segment of the B.subtilis enzyme caused the yeast enzyme to be located in the mitochondria! matrix as a soluble protein. The fusion protein was inactive in vivo and had residual activity in vitro. We speculate that this segment, which shows the greatest variability between species, is responsible for the association of the enzyme with the membrane. Exchanging the ~ 40-amino acid C-terminus between the yeast and mouse ferrochelatases caused a total loss of activity and the hybrid proteins were unstable when overproduced in E.coli. Analysis of S.cerevisiae truncated ferrochelatase devoid of its presequence revealed that the leader sequence is not required for mitochondrial targeting and functioning in vivo. To identify genes encoding putative heme- and/or iron-carrier proteins interacting with ferrochelatase the two-hybrid system was employed. The HEM15 gene (encoding ferrochelatase) was fused with a DNA-binding domain (lexA) and used as a bait against yeast gene library (containing the transcription activating domain from GAL4 fused to yeast genes in all 3 reading frames). About 20 million transformants were tested during the library screen. A set of positive clones found in the screen was subjected to further investigation, in order to prove (or exclude) their interaction with ferrochelatase. Surprisingly, most of the proteins found were cytoplasmic (the most reliable ones are: 26S proteasome regulatory subunit and clathrin heavy chain) and only one was mitochondrial (subunit b of ATP synthase). Of the 2 unknown ORFs found in the screen, one seems to have mitochondrial localization. Collaboration: prof. R.Labbe-Bois, Institut Jacques Monod, Universite Paris VII, France. Publications: 3361,3369,1141/A, 1273/A, 1282/A, 1296/A, 95/N, 136/N

35 5. SUPRESSORS OF IRR1 - A GENE INVOLVED IN COLONY FORMATION AND SISTER CHROMATID COHESION

A. Kurlandzka, A. Biaikowska

We have previously cloned and characterized the gene IRRl responsible for colony formation on solid media, zygote formation and spore germination. We demonstrated the pleiotropic function of this gene by expressing it from the CTA1 regulatory promoter. We also found that Irrlp is not directly involved in the cell-solid support interactions. Recently it has also been shown by K. Nasmyth and collaborators that Irrlp is an element of the cohesin complex required for establishing and maintaining cohesion between sister chromatids up to the anaphase transition. In a search for proteins interacting with Irrlp we have isolated 6 spontaneous mutants suppressing the inability to form colonies in Irrlp - depleted cells. The absence of ]RR1 transcripts in the suppressors proved that they are not false-positives resulting from the CTA1 promoter escape from glucose repression. These strains, with the original gene deleted and bearing the CTAl-IRRl regulatory fusion, form colonies on solid medium under non permissive condition. However, the suppression is only partial and mutants are still disturbed in the mating process, which confirms that the original Irrlp is multifunctional. Genetic analysis shows that these suppressors resulted from single-gene mutations. We have cloned 3 of them and began their analysis. We have also found two proteins interacting with Irrlp in the two-hybrid system: LipSp (lipoic acid synthetase) and a kinesine-like protein of as yet unidentified function. Publications: 3333,1269/A, 1281/A, 128/N

6. THE ROLE OF UBIQU1TINATION IN PROTEIN TRANSPORT IN SACCHAROMYCES CEREVISIAE CELLS

T. Żołądek, B. Gajewska, J. Kamińska

RSP5 encodes an ubiquitin-protein ligase. Mutations ofRSP5, mdpl, affect the mitochondrial/cytoplasmic distribution of ModSp-I, growth at elevated temperatures, fluid phase endocytosis, pH and paromomycin sensitivity. To understand the role(s) of RspSp in these cellular processes we studied its interaction with Panlp, involved in actin cytoskeleton organization, determined its intracellular location and studied the function of individual RspSp domains. To test for RspSp-Panlp interaction we used the two-hybrid system. We were able to observe interaction between particular fragments od Rsp5p and Panlp fused to Gal4p as assessed by p-galactosidase assay and growth on -his medium of appropriate transformants. Functional tagged Rsp5p was obtained by appending the N-terminus with the triple hemagglutinin (HA) epitope. HA-Rsp5p localization was studied by indirect

36 immunofluorescence. It is located in the cytoplasm in a punctuate pattern that does not change during the cell cycle. HA-Rsp5p is excluded from the nucleus. Previously characterized mdpl mutations cause alterations of the hect domain. In addition to this C-terminal domain, Rsp5p has three WW domains that mediate protein-protein interactions. Characterization of the mdpl-9 allele showed that it has a substitution of a conserved P in the third WW domain. Mutations inactivating other individual WW domains or combinations of them were obtained by in vitro mutagenesis. The mutant alleles were expressed in low copy from the RSP5 promoter in a mdpl-13 hect mutant strain. The rsp5-wwl and rsp5-ww2 variants complement the growth defect at 37°C, pH 5.2 sensitivity and paromomycin sensitivity whereas variants with mutations in multiple WW domains failed to complement any of the phenotypes. The rsp5-wwl, rsp5-ww2 and rsp5-ww3 mutants complemented also the rsp5::kan deletion at 30°C indicating that individual WW domains have nonessential functions. The results support the model that individual W W domains could be important for subsets of Rsp5p functions. Collaboration: prof. A.K. Hopper, The Pennsylvania State University College of Medicine, Hershey, PA, USA. Publications: 1179/A, 1276/A, 1277/A, 1280/A, 104/N, 133/N, 152/N

7. REGULATION OF tRNA BIOSYNTHESIS IN YEAST

M. Boguta, K. Pluta

Mutations in the Saccharomyces cerevisiae MAPI gene lead to decreased efficiency of tRNA suppression and conditional respiratory growth. Genetic studies reveal that mutations in RPO31, encoding the C160 subunit of RNA polymerase III, suppress these phenotypes. We also identified TEFI, coding for translation elongation factor EF-la, as a high copy suppressor of mafJ-J. However, Maflp is not associated with ribosomes or polysomes, as shown by Western blotting of cellular fractions with specific antibodies. Immunofluorescence microscopy revealed that Maflp is located primarily in the nucleus, mafl-l cells possess elevated amounts of correctly processed and modified tRNAs. Suppression ofmafl by mutations in RPO3I is accompanied by a reduction in tRNA levels. In contrast, overexpression of TEFI does not alter tRNA levels in mafl-l cells. According to our model, increased tRNA levels in mafl-l disturb the formation of ternary complexes by altering the balance between tRNA and EF-la. Mutations in RPO31 decrease the amount of tRNA while overexpression of TEFI increases the amount of the elongation factor. Both would change the proportion in the ternary complex in the same direction. We propose that Maflp limits RNA polymerase III tRNA gene transcription and thus plays a role in controlling cellular tRNA levels. A database search reveals that a variety of organisms have sequences similar to MAPI indicating that this type of polymerase III regulation may not be limited to yeast. Publications: 1149/A, 1271/A

37 8. NOVEL MUTATIONS OF THE GLUCOSE-6-PHOSPHATE DEHYDROGENASE GENE IN POLISH G6PD DEFICIENT SUBJECTS

B. Burzyńska, E. Jablońska- Sk\viecinska, I. Lewandowska, D. Plochocka, D. Grabowska

G6PD is the first enzyme in the pentose phosphate pathway and its main physiologic role in the red blood cells is to produce NADPH necessary for the protection of the cells against oxidative stress. G6PD deficiency is the most common human enzymopathy affecting over 200 million people worldwide. In Poland, G6PD deficiency has been observed very infrequently. DNA sequencing revealed seven different glucose-6-phosphate dehydrogenase mutations in G6PD deficient subjects from ten Polish families. Among them we found two novel mutations: 679 C^-T (G6PD Radiowo, class 2) and 1006 A-»G (G6PD Torun, class 1). Variant G6PD Radiowo was characterised biochemically. Both novel mutations were analysed with respect to the tetriary structure of the enzyme. The main chain of G6PD Torun shows substantial structural differences compared with the wild type G6PD. Collaboration: Department of Laboratory Diagnostics, Medical Center of Postgraduate Education, Warsaw, Poland. Publications: 3452

9. GENOTYPING OF BISON BONASUS BY MOLECULAR MARKER ANALYSIS

B. Burzyńska, D. Grabowska

All extant European bisons, Bison bonasus, descend from 12 founder animals, among which those belonging to the lowland line or the Białowieża line stem from only seven founders (4 males and 3 females). This results in a very limited gene pool and high inbreeding within populations. The aim of our studies was: - to select DNA markers useful in genetic and phylogenetic studies, - to assess genetic heterogeneity within and between different herds, and - to genotype individuals by molecular markers. MtDNA control region and nuclear markers like the K-casein gene, the ITBG 2 gene and microsatellite markers were sequenced and analysed. Nuclear genes and mitochondria! markers were found unsuitable to be for genotyping Bison bonasus populations. On the other hand four microsatellite loci were found very variable. They can be used in genetic and phylogenetic studies. Publications: 3420

38 DEPARTMENT OF LIPID BIOCHEMISTRY Head: professor Tadeusz Chojnacki

The continuing interest of this Department in the biosynthesis and function of polyisoprenoids is represented by two groups studying different aspects of this problem. The group directed by professor Grażyna Palamarczyk is studying the regulation of formation of polyprenols, dolichols and steroids in yeast and dolichol- dependent transglycosylations in filamentous fungi. It was demonstrated that overexpressions of a Saccharomyces cerevisiae mannosylphosphodolichol synthase- encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnorma cell ultrastructure. Several genes encoding proteins in mannose metabolism were isolated from a T. reesei cDNA library. Other achievements concern the role of farnesyl diphosphate synthase in the biosynthesis of dolichols in yeast. A mechanism of chain termination dependent on the interaction of farnesyl diphosphate synthase with endoplasmic reticulum membranes is currently being worked out. The other group headed by associate professor Ewa Świeżewska deals with protein prenylation and biochemistry of plant isoprenoids. Members of this group solved the problem of the site of biosynthesis and intracellular flow of ubiquinone and plastoquinone in spinach cells and continue research on the occurence of polyprenols in plants. Studies on the specificity of mammalian dolichol-dependent glycosyltransferases are carried out using artificial lipid acceptors. Apart from these lines of research a member of the department (Dr. W. Jankowski) cooperates in the field of lipid research with other teams active in forest research and neurology. The role of farnesyl diphosphate synthase in the regulation of the mevalonate pathway is being elucidated by Dr. Sc. Anna Szkopińska.

1. OCCURENCE AND FUNCTION OF TERPENOID LIPIDS IN PLANT TISSUES: STUDIES ON STRUCTURE AND BIOSYNTHESIS

T. Chojnacki, J. Hertel, W. Jankowski, K. Skorupińska-Tudek, E. Świeżewska, M. Wanke, Vo Si Hung

Studies on the prenylation of proteins in plants were continued. Rab prenyl transferase (heterotrimeric) from wheat seedlings was purified 7000-fold. The 60kDa subunit of the plant enzyme was recognized by an anti human a/pRabPTase antibody (Dr. M. Seabra, London). Experiments on in vivo isoprenylation of proteins in transgenic tobacco leaves expressing bacterial cysE gene (Dr. A. Sirko, IBB) and A.thaliana showed similar patterns of labeled proteins (major band of approx. 30kDa). identification of the products is in progress. Studies on the biosynthesis and transport of plastoquinone and ubiquinone in spinach cells revealed the vesicular mechanism of PQ and UQ translocation from ER towards plastids (PQ) and mitochondria (UQ) involving the Golgi membrane system. Transport of UQ was energy — dependent in contrast to

39 PQ. The mechanisms of biosynthesis of polyprenols were investigated. Root culture of Cohtria geoides grown in the presence of selected biosynthetic precursors ([1-'3C] or [6-'JC]glucose) was initiated. Samples of chromatographically purified lipids were prepared for further NMR analyses. In cooperation with Indian research centers (Dr. R.Ranjan, T.P.Varma College, Bihar) and botanical collections in Poland a search for polyprenols in leaves was performed in several groups of plants, e.g. Indian medicinal plants, the succulents, and in various taxonomic groups (on over 400 species). These studies together with our previous findings enabled us to postulate a general rule of the occurence of polyprenols in plants especially with respect to the problem of aging. Lipids of seeds of forty species of gymnosperm and angiosperm trees and shrubs typical for Poland were characterized. The polyprenol component was isolated and quantitatively determined. The content of lipids in long-term stored seeds of Abies sp. from heavily polluted and endangered region of Sudety mountains was estimated. It was found that the drop in lipid content and the rate of their hydrolysis are relevant to the deterioration of the plant's vitality. Phosphonate and sulphate analogues of dolichyl phosphate were tested as acceptors of sugars in the reaction catalyzed by enzymes from rat brain and liver. Only the phosphonate analogues exhibited acceptor activity. Products of brain lipids catabolism were estimated in human brain tumors by 1-H-NMR spectrometry. Publications: 3299, 3330,3353,3358, 3418,3446,3447,3448,1123/A, 1124/A, 1225/A, I136/A, 1138/A, 1139/A, 1140/A, 1141/A, 1242/A, 1243/A, 1244/A, 1254/A, 1294/A, 1327/A, 100/N, 115/N

2. DOLICHOL PHOSPHATE MANNOSE SYNTHASE FROM THE FILAMENTOUS FUNGUS TRICHODERMA REESE1 BELONGS TO THE HUMAN AND SCHIZOSACCHAROMYCES POMBE CLASS OF THE ENZYME*

J. Kmszewska, G. Palamarczyk

Dolichol phosphate mannose synthase (DPMS), which is required in N- glycosylation, 0-mannosylation and glycosylphosphatidyl inositol (GPI) membrane anchoring of proteins, has been postulated to regulate the T. reesei secretory pathway. We have cloned T. reesei, Trdpml, cDNA, that encodes a 243 amino acid protein whose amino acid sequence shows 67% and 65% identity, respectively, with the Schizosaccharomyces pombe and human DPMSs, and which lacks the COOH-terminal hydrophobic domain characteristic of the Saccharomyces cerevisiae class of synthases. Expression levels of Trdpml were directly correlated with the protein secretory capacity of the fungus. *In collaboration with VTT-Biotechnology Center of Finland

40 3. POSSIBLE INVOLVEMENT OF 26S PROTEASOME IN DEGRADATION OF THE YEAST FARNESYL DIPHOSPHATE SYNTHASE (FPPS)*

K. Grabińska, G. Palamarczyk

Yeast Yta7p, which is a member of the ATPase protein family and a regulatory subunit of the yeast cytoplasmic 26S proteasome, was found by the yeast two hybrid system to interact with FPPS encoded by the ERG20 gene. Moreover, mutated FPPS (encoded by the erg20-2 gene), which has been shown to be several fold more stable compared to native FPPS, did not interact with Yta7p. Thus we postulate that the regulation of FPPS activity occurs on the protein level by proteasome-dependent proteolytic degradation. Analysis of FPPS stability in YTA 7-deleted and proteosomal proteases-defficient strains is underway. *In collaboration with the University of Poitiers (France)

4. OVEREXPRESSION OF a-D-1-PHOSPHATE GUANYL-TRANSFERASE ENCODING GENE (MPG1) RESTORES VIABILITY OF S.CEREVISIAE MUTANTS AFFECTED IN EARLY STEPS OF GLYCOCONJUGATE FORMATION

A. Janik, M. Sosnowska, U. Lenart, G. Palamarczyk

The final reaction in GDPMan assembly is catalysed by guanyl (MPG)- transferase encoded by the MPG1 gene. Our results indicate that the availability of GDPMan affects the early steps of glycoprotein formation ascribed to the endoplasmic reticulum, i.e. the assembly of the dolichol-linked oligosaccharide as well as dolichol phosphate mannose (DPM) formation. Overexpression of the MPG1 gene was able to suppress mutations of the genes Dpml, Algl, Alg2 coding for DPMS (Dpmlp) and mannosyltransferases responsible for the addition of (3-1,4 (Alglp) and a-1,6 (Alg2p) linked mannose residues to the dolichol-linked oligosaccharide (DolPP-GlcNAc2 and DolPP-

GlcNAc2Man2). All these proteins are involved in the steps which utilise GDPMan as a substrate. Based on the results obtained so far we postulate that the availability of GDPMan in the cell cytoplasm affects the mannosyltransferases located in the endoplasmic reticulum by changing their substrate affinity. Publications: 3301,3322,1142/A, 1145/A, 1146/A, 1147/A, 3377,3392, 3403, 1283/A, 1284/A, 1285/A, 1286/A, 1287/A, 134/N

41 5. REGULATION OF BIOSYNTHESIS OF MEVALONIC ACID PATHWAY DERIVATIVES IN THE YEAST SACCHAROMYCES CEREVISIAE

A. Szkopińska

The mevalonic acid pathway is a cascade of reactions in which as a result several groups of compounds are synthesized, i.e., sterols, dolichols, ubiquinones as well as farnesyl, geranyl, geranylgeranyl and isopentenyl diphosphates. Besides their structural functions they participate in respiration, as well glycosylation and covalent modifications of proteins and tRNAs. Investigations performed during the last twenty years point to 3-hydroxy-3- methylglutaryl-CoA reductase as the main enzyme regulating the first steps in the mevalonic acid pathway and determining the level of its final products. However, recent results suggest that FPP synthase, the branch point enzyme of the mevalnic acid pathway, and squalene synthase, the first committed enzyme in sterol synthesis, also influence the level of the products synthesized. The consequences of squalene synthase gene disruption on polyprenol synthesis in yeast have been documented. The co-regulation of activity of the three enzymes mentioned above has been described. Research concerning peroxisome participation in dolichol synthesis and localization of mevalonic acid pathway enzymes in this organell are being pursued. The computer-designed 3 dimensional model of yeast FPP synthase is being used to investigate of long dolichol synthesis and to design point mutations leading to the increase of geranyl and geranylgeranyl diphosphate synthesis. Publications: 3321, 1141/A, 1142/A, 1143/A, 1144/A, 1294/A

42 DEPARTMENT OFMICROBIAL BIOCHEMISTRY Head: professor Maria Danuta Hulanicka-Dziechcińska

The Department consists of several research groups, all involved in studies on the mechanisms of microbial gene expression. Bacteria, bacterial plasmids and plant viruses are used as model systems. Genome expression analysis of the potato leafroll virus (PLRV) is conducted by the group of D. Hulanicka. Recent work concerns the use of infectious clones of PLRV for identification of some ORF products and verification of the importance of their sequence motifs. There are two groups studying bacterial metabolism. The group of M. Hrynievvicz works on regulatory mechanisms involved in sulfur metabolism of bacteria. One line of research is focused on the expression of sulfate starvation-induced genes, the other project concerns the systematic mutational analysis of the CysB regulator. The group of J. Bardowski works on sugar metabolism in lactococci, in particular on the role of the CcpA protein in the regulation of !actose/p-glucoside-coupled catabolism and on plasmid-encoded pullulanase. Three groups work on the mechanisms of stable plasmid inheritance in bacteria. The group of P. Cegiowski uses the postsegregational killing system of the plasmid pSM 19035 from Gram-positive bacteria as a model system. Two other groups are involved in studying the active partitioning of plasmids in Gram-negative bacteria. The group supervised by M. Łobocka works on plasmid prophage PI and the group of G. Jagura-Burdzy - on broad-host range plasmids from the IncP family. Current studies of both groups are focused on the identification of host factors involved in partitioning and on characterization of interactions between partition proteins and plasmid DNA in vivo. Recently, the interest of the latter group has been extended to chromosomal partition in the genus Pseudomonas. Another goal of M. Lobocka's group is to determine and analyze the complete nucleotide sequence of two related plasmids, PI and P7.

1. REGULATION OF THE POTATO LEAFROLL VIRUS GENOME EXPRESSION

D. M. Hulanicka, E. Sadowy, M. Juszczuk

The potato leafroll polerovirus (PLRV) genome consists of a single nonpolyadenylated (+)RNA molecule. Sequence analysis revealed the presence of six open reading frames. The functions of some of these ORFs have been identified. The understanding of PLRV life cycle is of economical importance as it causes significant crop loss world-wide. Infectious clones are the basis of the molecular research on RNA viruses. Introduction of a full-length cDNA copy of the viral genome into plant cells

43 and its expression initiates a viral life cycle. Introduction of mutated cDNA clones enables the studies of the functions of particular ORFs. Continuing our studies on PLRV we used the previously constructed plasmids (pBPF and pBOK) carrying a full-length cDNA copy of the PLRV genome. When introduced into plant cells these plasmids were able to initiate a replicative cycle of the virus. We used site directed mutagenesis of the above infectious clones to modify putative functional motifs in the products of ORF1 (proteinase) and ORF2 (replicase), and to prevent translation of ORFO without an effect on expression of other ORFs. The results obtained indicate that: 1 - Mutagenesis of the ORF2 sequence encoding the GDD motif into VHD in the pBOK clone completely abolished viral replication, thus confirming the biological significance of this motif for polerovirus replication. 2 - Mutagenesis of the proteolytic triad (HDS) of serine proteinase either reduced the synthesis of both genomic and subgenomic (sg)RNA (H-»L) or abolished RNA synthesis, only slightly affecting the synthesis of genomic RNA (D—>A). The change S-»A completely inhibited viral RNA synthesis. These results demonstrated the requirement for proteinase activity at different stages of the PLRV life cycle. In vitro transcription-translation (system) applied to the full-length PLRV genome confirmed that the mutation S353->A changes the pattern of proteolytically processed proteins. 3 - Infectious clones, carrying different mutations preventing translation of ORFO without an effect on other ORFs, were not able to replicate in potato leaf discs. This finding indicates that the 28 K protein (the ORFO product) is essential for viral replication. The predicted structure of the 28 K protein suggests that this protein may facilitate the replication complex formation and target it into cell membranes. Publications: 3291,3295,3343, 3408,3443,122/N, 135/N, 1167/A, 1318/A, 1319/A

2. REGULATION OF SULFATE METABOLISM IN ENTEROBACTERJACEAE

M. Hryniewicz, R. Iwanicka-Nowicka, A. Łochowska, T. Bykowski

Enteric bacteria are able to assimilate sulfur from various sulfur compounds both inorganic and organic. Regulation of genes involved in the utilization of alternative sulfur sources differs from the regulation of cys regulon genes engaged in sulfate assimilation. In one of our projects we continued studies on the regulation ofssi (sulfate starvation-inducible) genes engaged in the utilization of organic sulfur sources in E. coli. In particular, we have shown that the expression of this class of genes requires concurrent action of two transcriptional regulators, CysB and Cbl. However, the relations of these two regulatory proteins in the activation of particular ssi promoters are not the same. Both CysB and Cbl are required for the tau operon expression in vivo and both proteins bind to the tan promoter region. In the case of the ssit operon, both CysB and Cbl bind the promoter region but only Cbl is directly required for in vivo expression of the ssu operon. We have also shown that an

44 additional regulatory factor for the ssi gene expression is the histone-like protein IHF. We identified experimentally several IHF-binding sites in tau and ssu regulatory regions. DNA-binding studies using highly purified Cbl revealed that sulfide inhibits the interaction of Cbl with tan and ssu promoter regions. We conclude that the strict repression of ssi genes in the presence of sulfate is caused by generation of sulfide in the sulfate reduction pathway and that sulfide, not sulfate, is the negative cofactor of the Cbl activator. Another line of research concerned the structure/function relationship of the CysB regulator. Using random and directed mutagenesis we obtained several cysB mutant alleles. CysB mutant derivatives were overproduced and, when possible, their interactions with cys promoter regions were studied in vitro. Four mutations (El IK, S20F, T221, and I48T) resulted in loss of DNA-binding ability of CysB. Four mutations in the central region of CysB (Ml601, T196I, A244V and A247E) did not affect the DNA-binding ability but resulted in the loss of the conformational change of the protein in response to the inducer, acetylserine. These mutations also abolished the positive control (pc) function of CysB. Two mutations (Y164N and A227D) rendered the protein constitutively active, independent of the presence of the inducer. Several C- terminally truncated derivatives of CysB were also obtained. Their characteristics are consistent with the view that the C-terminal domain of the protein is engaged in the oligomerization function of CysB and that it is also required for DNA binding. Publications: 3425,1180A, 85/N

3. GENETICS OF LACTOSE AND STARCH CATABOLISM IN LACTOCOCCUS

J. Bardowski, T. Aleksandrzak, M. Kowalczyk

In Lactococci the capacity to use lactose as a carbon source is encoded by plasmid-located genes. However, we found that in a plasmid-free strain cellobiose induces a new lactose catabolic pathway. We observed that this induction was abolished through interruption of a gene homologous to the ccpA gene from B. subtilis and to its counterparts from other Gram-positive bacteria. The presence of the ere sequence upstream of ccpA and overlapping the putative promoter region suggests autoregulation of the ccpA gene in L. lactis. Moreover, the location of ere between ccpA and pepQ, located upstream of the former and divergently transcribed, hints that the CcpA protein can also regulate the expression of the latter gene. These observations together with growth tests and enzymatic assays for p-glycosidases performed for the wild type L. lactis strain and its ccpA mutant permitted us to propose a model for the dual role of the CcpA protein in the regulation of lactose/p-glucoside coupled sugar catabolism in L. lactis. This represents a potential tool for genetic modifications of lactose catabolism in lactococci. Pursuing genetic and biochemical studies on amylase produced by the L. lactis IBB500 strain we obtained complete nucleotide sequence of the gene and found that the encoded protein is homologous to the family of pullulanases. Analysis of the DNA

45 sequence revealed several potential promoter sequences upstream of the pullulanase gene, one of them overlapping a putative ere site which is a remnant of the catabolic repression mechanism operating in some Gram-positive bacteria. The lack of a conserved transcription terminator sequence downstream of the pul gene together with the presence of an open reading frame following the pul gene and transcribed in the same direction suggest that the pni gene is located in an operon. We also found that among various sugars tested starch is the most potent inducer of pullulanase production. Publications: +3298, +3310,3319,3320,1133/A, 1226/A, 1227/A, 1228/A, 1240/A, 1241/A, 136/N, 137/N

4. FUNCTIONAL ANALYSIS OF PLASMID pSM19035 FROM GRAM- POSITIVE BACTERIA

P. Ceglowski, I. Kern, I. Sitkiewicz, U. Zielenkiewicz

The low-copy-number and broad-host-range plasmid pSM 19035 and its deletion derivative pDBlOl are very stable in Gram-positive bacteria of low G+C content. Previously we have shown that high segregational stability of pDBlOl depends mainly on the activity of plasmid genes s and £. The products of these genes, Epsilon and Zeta, constitute a plasmid addiction system which acts post-segregationally by selectively killing plasmid-free cells. Zeta is a toxin; its lethal activity is neutralized by the antidote protein, Epsilon. It has been demonstrated for several plasmid addiction systems that genes encoding toxins and antidotes form operons that are autoregulated at the level of transcription either by the antidotes alone or by the toxin-antidote complexes. We have found that, unlike in those systems, the genes e and £, form one transcription unit together with the regulatory gene o>, and that the co-e-E; operon is regulated only by co. The product of this gene, Omega, represses transcription by binding to heptameric repeat sequences located upstream of the plasmid genes co, 8 and copS, the latter being a negative regulator of plasmid copy number. Further characterization of the pSM19035 addiction system revealed that: (i) within the co-s-J; operon there is a weak, constitutive promoter which directs the basal transcription of genes e and £; (ii) genes e and ^ are sufficient to fully stabilize segregationally unstable replicons; (iii) in cells overproducing Epsilon, i.e. under conditions of excess of the antidote, genes E and J; fail to act as the plasmid addiction system; (iv) in cells providing an excess of Omega, the copy number of plasmids carrying the copS gene becomes elevated. Interaction between Epsilon and Zeta was studied in the yeast two-hybrid system. Using truncated proteins it was found that the C-terminal part of Epsilon and the N-terminal part of Zeta are most likely involved in the complex formation. Zeta mutant protein in which the nucleotide binding motif (P- loop) was destroyed retains the ability to bind Epsilon. This mutant, however, exhibits a non-toxic phenotype. Experiments on the determination of the cellular target of action of Zeta are in progress. Publications: 1132/A, 1292/A

46 5. MECHANISMS OF ACTIVE SEGREGATION OF THE LOW COPY NUMBER PLASMID PI TO DAUGHTER CELLS AT CELL DIVISION

M. Łobocka, O. Bugajska, A. Dobruk, A. Janowicz, K. Krajewska-Grynkiewicz

Partition of PI plasmid to daughter cells depends on plasmid proteins, ParA and ParB, and a centromere analog, parS. ParA and ParB form a complex at parS. They act in concert with unidentified host proteins to move sibling plasmid molecules from the center of mother cell to the centers of future daughters, Binding of ParB to parS is an initial step of partitioning. It is followed by polymerization of ParB on DNA flanking parS. The polymerization may cause transcriptional silencing of genes flanking parS and destabilization of certain parS-c&rrymg plasinids. Whether it leads to partitioning or partition dysfunction depends on the concentration of ParB, the context of parS, the ratio of ParA to ParB, and unknown host components. We focused on characterization of this step of partitioning. To elucidate the ways the intracellular ratio of ParA to ParB is regulated we looked for internal promoters of par operon and identified, by primer extension and promoter probe techniques, promoters that drive transcription of parB independent of parA. To study the polymerization in vivo a partitionally active fusion of parB to the gfp gene encoding the green fluorescent protein was constructed. The fusion protein was uniformly distributed within the cytoplasm in cells without the mini-Pi. In cells with the mini-Pi, the fusion protein formed fluorescent dots in the middle of the cell or at about 0.25 cell length from each of the poles (in longer cells). We demonstrated by in vivo crosslinking of ParB-Gfp to DNA that the fluorescent dots represented aggregates of ParB on DNA flanking parS. Active partitioning occurred until the entire DNA of parS-carrying plasmids was titrated by ParB. To identify host proteins involved in PI partition we used a biochemical and a genetic approach. Genes encoding His-tagged ParA and ParB were constructed. Both fusion proteins appeared to be active in partitioning. Methods for fast purification of His-tagged ParA and ParB were developed for future isolation of fixed complexes of ParA or ParB with other proteins. Chromosomal mutants were isolated that are defective in both the destabilization of certain pars-carrying plasmids by ParB and active partitioning of P7 plasmid, related to PI. They appeared to have aberrations in distribution of chromosomal DNA. This suggests that the mutations are in genes essential for active partitioning of both low copy number plasmid and chromosomal DNA. Publications: 1151/A, 1152/A, I202/A, 1203/A, 156/N

47 6. COMPLETE NUCLEOTIDE SEQUENCE OF PI, A BACTERIOPHAGE THAT LYSOGENIZES AS A PLASMID

M. Łobocka, M. Rusin and A. Samojedny* *Laboratory of Tumor Biology, Institute of Oncology, Gliwice, Poland

PI bacteriophage lysogenizes E. coli by cyclization of a double-stranded DNA molecule to form a plasmid that is stably maintained at about one copy per bacterial chromosome. We determined the nucleotide sequence of Plcl.100 mod::IS5 strain. The PI genome is 93,601 bp long and encodes at least 110 genes organized in 50 operons. Genes occupy 91% of the genome with rare overlaps. Translation of most of the ORFs is initiated independently from their own ribosomal binding sites and translation start sites. Genes dedicated to plasmid functions are largely limited to those involved in plasmid maintenance. Plasmid replication and partition genes are located separately from multimer resolution and post-segregational killing genes. Not all phage genes are clustered by function, similarly as in T4 phage. A cascade of regulators controls the lysis/lysogeny switch via the represser Cl that binds to 22 operators controlling at least 19 operons. Eleven operons are transcribed from late promoters activated by the product of the C l-regulated gene, Ipa. Additional control of timing of phage development is achieved at a level of translation. One of the Cl regulated operons encodes three tRNAs that recognize codons rare in E. coli genes but overrepresented in certain genes of PI. Phage replication is initiated from an origin separate from the plasmid origin and appears to involve unlinked homologs of bacterial replication genes dnaB and holE. DNA packaging depends on methylation at the pac site by a unique enzyme that contains adeninę and cytosine methyltransferase domains in one polypeptide. A gene essential for processing of certain head proteins is a kinase, possibly involved in the activation of a host protease. Homologs of protein-modifying enzymes among predicted products of PI genes indicate the involvement of protein modification in the process of phage morphogenesis. Recognition sites for the majority of restriction enzymes are present within PI DNA. The antirestriction system of PI appears to be based on DNA methylation. A giant protein essential for antirestriction, DarB, may also be involved in active transport of PI DNA into cells during the process of infection. Homologs of known genes of different provenance scattered throughout the PI DNA suggest that PI was created by a strikingly eclectic process of assembly. Publications: 1204/A, 1205A

48 7. ACTIVE PARTITIONING OF INCP PLASMIDS AND PSEUDOMONAS AERUGINOSA CHROMOSOMES

G. Jagura-Burdzy, M. Adamczyk, A. Balcerzyk, K. Lasocki

Low copy number plasmids rely on active distribution to ensure the presence of at least one copy of plasmid DNA in each daughter cell. The active partitioning system consists of a pair of proteins: ParA and ParB and a centromere-like DNA sequence. Active pardoning of IncP plasmids depends on IncC (a putative ATPase - ParA homolog) and KorB (ParB). KorB acts as a global represser recognizing the OB palindrome present 12 times in the plasmid genome. IncC potentiates KorB repression at all tested promoters and enhances the KorB DNA binding activity in vitro. Using the yeast two hybrid system we demonstrated direct interaction between IncC and KorB and mapped the IncC interaction domain to central 45aa of the KorB protein. Another putative component of the IncP partitioning apparatus is KfrA, a protein homologous to eukaryotic motor proteins. kfrA genes from two IncP subgroups (a and P) show low conservation at the DNA or aa level, but the encoded proteins have very similar predicted structures (almost 100% cc-helical). The IncPp kfrA ORF was cloned and shown to autoregulate kfrAp. The ability of KfrA (342aa) to polymerise was demonstrated in vivo (by the two hybrid system) and in vitro (by glutaraldehyde cross! inking of His-tagged protein). The deletion of central 70aa impairs neither the regulatory properties nor the ability to dimerize. In vitro constructed kfrA deletion mutants are being introduced into the plasmid genome for testing. The plasmid encoded ParA and ParB proteins have homologs in many bacterial genomes sequenced to date. They have been shown to play a role in chromosome partitioning in B.subtilis, C.crescentus and P.putida. We plan to characterise the ParA and ParB proteins of the pathogenic bacterium P.aeruginosa, to identify their DNA target and to attempt to interfere with the process of chromosome separation to stop the pathogens from spreading. We cloned par A and parB, His- tagged and purified both proteins as well as their truncated derivatives. Using the yeast two hybrid system we demonstrated a direct interaction between ParA and ParB as well as the ability of ParB to dimerize. In vitro cross-linking defined 60aa from the C- termini of ParB as responsible for multimerization. We interchanged the C-termini of KorB (IncPa: plasmid) and ParB from P.aeruginosa. Replacing the C-terminal dimerization domain of KorB (essential for repression at a distance) by the C-terminal 60aa of ParB did not restore full KorB represser activity although it facilitated dimer formation in vitro. We are also involved in the analysis of a plasmid pool from P.aeruginosa clinical isolates as well as DNA sequencing of naturally occuring plasmids under the Plasmid Genomics project. Collaboration: prof. C.M.Thomas, The University of Birmingham, UK Publications: 3332,3387,3390

49 DEPARTMENT OF MOLECULAR BIOLOGY Head: professor Jarosław Kuśmierek PL0201950

The Department's continuing interest is centered on the mechanisms of mutagenesis and DMA repair. Part of the studies concerns the effects of the interdependence of mutator and antimutator genes on the response of E. coli cells to various mutagens. The phenomenon of mutation frequency decline (MFD) was studied and recently distinct response of this system to UV light and to alkylating agents was shown. Studies on the isfA gene, which is involved in the regulation of the SOS response, including its precise localization on the bacterial chromosome, were continued. Also, studies on the mechanism of DNA replication-independent mutagenesis in lambda phage were continued. Halogen light mutagenesis and the role of the hem gene in the near UV and UVA irradiation mutagenesis were investigated. New experimental approaches were adapted to study adaptive mutations. Another part of the studies was focused on the relation between the structure of DNA bases adducts and their coding and mutagenic properties, as well as their properties as substrates for DNA repair enzymes. These studies include instrumental methods, such as NMR, MS, HPLC and GC/MS, to investigate structure and the kinetics of formation and excision of adducts, and organic chemistry methods to synthesize adducts. The coding and mutagenic properties of several oxidized and alkylated bases (including imidazole-ring opened purines) were determined by employing in vitro enzymatic and phage/bacteria systems. The sites of reaction of chloroacetaldehyde, a metabolite of carcinogenic vinyl chloride, with a fragment of human p53 gene, were studied using the method of polymerase fingerprint analysis. Ethenoadducts, the products of the reaction of chloroacetaldehyde with adeninę and cytosine, and also products of solvolytic degradation of ethenoadenine, were studied as substrates of various DNA glycosylases. Several purine and pyrimidine derivatives were synthesized and tested as inhibitors of DNA glycosylases. Studies on the mechanisms of photoreaction of psoralens with lipid components of cell membrane were continued. Lecithin-psoralen adducts were characterized and their role in photochemotherapy is being investigated.

1. INDUCED AND ADAPTIVE MUTATIONS

E. Grzesiuk, C. Janion, A. Wójcik, A. Nowosielska, A. Gozdek

^ We continued the study of the MFD (Mutation Frequency Decline) repair phenomenon in MMS-treated E.coli K12 bacteria. We used the argE3->A.rg+ reversion I CM system in the AB11 SlargES strain to investigate the level and specificity of Arg+ _l revertants under MFD conditions. These studies revealed that the repair process °- observed in MMS-treated and transiently starved AB1157 cells was not directly UvrA- dependent which means that it differs from the MFD repair observed in UV-irradiated bacteria. Instead we found that MFD repair in MMS-treated bacteria depends on two

50 glycosylases, AlkA and TagA, and that the UmuD'C proteins are also involved. Our results lead to the conclusion that 7meGua, which until recently had been construed as a non injurious MMS-induced lesion, in the presence of inactive AlkA protein, is removed from DNA in a process which increases mutagenesis. Adaptive mutations arise in non-dividing or slowly growing stationary phase cells. They are detectable after exposure to non-lethal selection and have been found only in genes whose functions were being selected for. We have adopted the arg-£3-»Arg+ reversion system and replica plating method to study stationary phase mutations in E.coli AB\\57argE3 strains on minimal plates lacking arginine (E-Arg). In the AB1 \51dnaQ49 strain mutated in the e subunit of DNA poi III the amount of Arg+ revertants was the same during 10 days of incubation on E-Arg plates. Surprisingly, starvation-associated mutations appeared in AB1157 dnaQ49AumuDC. We have continued the attempts to explain why the Tn5/Tn/0 transposon (but not Tn9), when inserted into the E. coli AB1157 chromosome, makes the bacteria more sensitive to and less mutable by halogen and UV light irradiation. These effects are most probably caused by the depletion of the UmuD and UmuC proteins. The Tn70/Tn5 transposon insertions have no influence on the NER system and do not increase cell filamentation after induction of the SOS system. The phenomena resulting from the presence of the transposons in the bacterial chromosome are not suppressed by the sulA mutation (the SuIA protein is a cell division inhibitor). Our results suggest that the mechanisms leading to UmuDC-dependent mutagenesis and UmuDC-dependent protection of cell survival are different from each other since transformation of bacteria deleted in umuDC with plasmids carrying umuD(D')C leads to full recovery of halogen light induced mutability but not survival. It was found that in the E. coli hemHl mutant the level of UVA + visible light-induced argE3->Arg+ and thr-/-»Thr+ reversions was much higher in comparison to the hem+ strain. The frequency of mutations was strongly increased in the bacteria transformed with a plasmid overproducing the UmiD'C proteins. Publications: 3282,3306,3308,3314,3327, 3371,3431,3433,1126/A, 1127/A, 1129/A, 1130/A, I131/A, 1233/A, 1234/A, 1235/A, 1236/A, 1237/A, 1262/A, 107/N, 126/N, 141/N, 143/N

2. CHEMICAL BASIS OF MUTAGENESIS AND REPAIR OF CYCLIC BASE ADDUCTS

J. T. Kuśmierek, A. Maciejewska, E. Borys-Brzywczy

Isoguanine (iG) is formed from adeninę in the reaction of DNA with oxygen radicals. We have shown recently (3121) that the presence of iG in n'^otemplates causes the incorporation of dTTP and 5-methyl-2'-deoxyisocytidine-5'-triphosphate (diCMTP) in a ratio 1:10 catalyzed by AMV reverse transcriptase (RT). This ratio corresponds to the ratio of the enol (iGE) and keto (iGK) forms of iG, which suggests the formation of iGE-T and iGK-iCM pairs. We have undertaken studies on the

51 influence on the coding properties of iG of such factors as neighbouring bases in the template, temperature, and the type of polymerase. Four 25-mer deoxytemp\ates

containing iG in four different base neighbourhoods were prepared. The KM and Vmax for the incorporation of dTTP and diCMTP opposite iG were determined with RT at 37°C and Tag polymerase at 37, 50, 65 and 80°C. This allowed us to compare the relative efficiency of iGE-T and iGK-iCM pairs formation. Among the factors studied, only neighbouring bases in the template have a significant, albeit rather small, influence on the coding properties of iG. Several 3-substituted purines were prepared to study the structural requirements of inhibitors of E. coli Tag glycosylase. The diameter of the substituent at

N3 (benzyl is optimal) and the presence of an effective H-bond donor at C6 of purine

ring (as - NH2 in adeninę which is optimal) are essential for inhibition. Subtitutions at

6-NH2> or the replacement of 6-NH2 in adeninę with O or S, or introduction of 2-NH2 and 6-oxo (guanine), diminishes (or abolishes) the activity of 3-benzylpurines. Neither of the derivative tested was inhibitory to the functionally analogous E. coli AlkA and human ANPG glycosylases. A method was elaborated to compare excision by glycosylases of ethenocytosine (sC) and its hydrate (eCH) from DNA. E. coli AlkA excises eCH with marginal efficiency, whereas sC is not excised at all. The kinetics of degradation of ethenodeoxyadenosine and the structure of products were determined using HPLC, NMR and MS. This study was carried out in relation to the study of the properties of these producs as substrates of various DNA glycosylases (in collaboration with Dr. B. Tudek from this Department and Dr. J. Wójcik from the NMR Laboratory). Publications: 3293,3326,3329,1206/A, 101/N, 157/N

3. THE MECHANISM OF PUVA (Psoralen + UVA) PHOTOCHEMOTHERAPY JCN ; g} Z. Zarębska, E. Waszkowska, D. Barszcz

' C^N^ Our investigations on the photoreactions of psoralens with lipids were I _i successfully completed. In the model studies unsaturated fatty acids and lecithins ! °~ having them in the p-position were applied. These compounds upon UVA (320-400 I mn) irradiation in the presence of psoralens undergo cycloaddition reactions of - psoralens to the vinylene bond. Among the TLC isolated products identified principally by NMR and mass spectra analysis were the cyclobutane adducts: oleic acidopsoralen (dloPSO), linoleic acid psoralen (d2<>PSO), and phosphatidylcholineopsoralen (PCdlpaloPSO). Mass spectra evidenced the

formation of double adducts of linoleic acid containing lecithins, PCd2pal(oPSO)2. 8-Methoxypsoralen, the most frequently applied drug, was shown to form similar cycloadducts as psoralen but of a less stable structure in vitro.

52 The multishunt photolysis of lecithins involves photooxidation of double bonds of fatty acids, and release of fatty acid cycloadducts from lecithin. Besides the cyclobutane adducts, the photolyzed mixture revealed two types of adducts with decomposing psoralen. Our findings, together with data of others, enabled us to propose a hypothetical scheme for the lecithin photolysis. We suggest that photolysis of lecithins in the presence of psoralens may play a role in the chain of reactions triggered in the membrane of the cell submitted to PUVA (psoralen + UVA) treatment. The characterization of primary psoralen adducts of fatty acids made possible their application in our further studies on the human lymphocytes cultured in vitro. The apoptosis of lymphocytes induced by physiological doses of PUVA treatment will be compared with the apoptotic influence of the linoleic acidoPSO adduct. These investigations are carried out in cooperation with the Immunology Department of Warsaw University and the Laboratory of Pathophysiology and Immunology of the Institute of Rheumatology in Warsaw. Most of our recent results were presented in the Ph. D. Thesis of Ewa Waszkowska (1999) and in publications. The program was conducted in collaboration with the Department of Pharmaceutical Sciences of the Padua University within the framework of cooperation between the Polish Academy of Sciences and the National Research Center of Italy (1990-99). Publications: 3362,123/N, 1111/A, 1137/A, 1221/A, 1239/A

4. THE MECHANISMS OF UV-INDUCED MUTAGENESIS PL0201953 /. Pielrzykowska, M. Felczak, J. Krwawicz, K. Lasocki

Studies on the isfA gene, presumably involved in the regulation of the switch- off of the SOS response were continued. It has been shown previously that mutation in the isfA gene leads to the inhibition of SOS dependent mutagenesis and other SOS functions in E. coli cells. The effect of the isfA mutation on the specificity of mutations generated in the mutator strains recA730, mutL and recA730imitL was studied by using the LacZ system of Cupples and Miller. The results show that the isfA mutation inhibits transitions and transversions generated with the involvement of the SOS system, both UV-induced and „spontaneous", in the recA730 mutator strain. On the other hand, the isfA mutation has no antimutagenic effect on mutations generated during DNA replication in the mutL strain, indicating that the antimutagenic activity of the isfA mutation is related to the mutagenic pathway but not to the type of mutations, transitions or transversions. Cloning of the isfA gene is under way. However, it is complicated due to the dominant character of the isfA mutation. Therefore, a special cloning strategy was needed. The gene isfwas localized in the 19.5 kbp region between 85.5 - 86.3 min of the E. coli chromosome. Using PCR several fragments of this region from the isfA mutant chromosome were cloned into a low copy number plasmid, recA730 mutator cells were transformed with the plasmid DNA, and the transformants with the isfA phenotype were identified. Several open reading frames, o!6l, yigB, yigA, cyaY, and known genes and operons, dapF, xerC and cyaAXfrom the middle part of the

53 region, did not give the isfA fenotype. Other parts of the region with ORFs and genes are under examination. Studies on the mechanism of UV-induced mutagenesis, which is independent of DNA replication in the lambda phage, were also continued. We have previously shown that UV and MMS-induced mutagenesis of XsusOS may occur under conditions nonpermissive for phage DNA replication in the E. coli 594su~ strain. This new mutagenic pathway differs in several aspects from that occurring under conditions permissive for phage DNA replication. It requires high level of the UmuD' protein and low uninduced chromosomal level of the UmuC protein, and mutations formed under this pathway are not subject to mismatch repair. To learn more about the mechanism of this type of mutagenesis, sequence analysis of UV-induced mutants was undertaken. Sixty percent of UV-induced mutations in the XsusOS phage in the host nonpermissive for phage DNA replication consist of transversions. On the contrary in the permissive host most of the mutations are transitions. The results further support our hypothesis that different mechanisms are involved in UV-induced mutagenesis under conditions permissive and nonpermissive for DNA replication. Publications: 3386,3434,1128/A, 1197/A,3331, 1295/A, 1311/A

5. DNA OXIDATIVE DAMAGE

B. Tudek, J. Cieśla, M. A. Grąziewicz, E. Speina, P. Kowalczyk, T. Obtulowicz

Research of the laboratory is focused on the elucidation of biological effects of damaged DMA bases and pathways of their repair. We continued studies on the formation of Fapy-7-methyladenine, the lesion previously shown to arise in alkaline conditions and inducing SOS-dependent A-»G transitions. Fapy-7MeAde is formed also spontaneously in nucleic acids at pH 7.00, 37°C from 7MeAde (TW2 in poly(A) 24 hrs) and similarly to Fapy-7MeGua, occurrs in two rotameric forms separatable by HPLC. The Oxidatively formed FapyAde also induced SOS-dependent A-»G transitions. Similar biological effects of these structurally related lesions is explained by the existence of different rotameric forms with similar hydrogen bonding faces. 1 ^-Ethenoadenine (sA) is also an unstable lesion and in alkali undergoes conversion ,to 3 products eA-H>B-»C->-D. The products of sA degradation were identified by HPLC, MS and NMR by Prof. J.T. Kuśmierek (this Department) and Dr. J.Wójcik (the NMR Laboratory). We have shown that bacterial and human 3- methyladenine-DNA glycosylases eliminating the parental eA from DNA are inactive towards products of sA degradation. Compound B, the pyrimidine ring-opened C(2)- N(3)-hydrated sA derivative, was excised from DNA by the Fpg protein and E.coli endonuclease HI (Nth protein). The search for inhibitors of DNA repair glycosylases resulted in finding a base analog, 2-thioxanthine, which inhibited the Fpg protein (I50 = 12 uM). It was also observed that a change of the DNA secondary structure by tryptophane pyrolisate (Trp- P-l) inhibited the activity of DNA glycosylases. The most effective was the inhibition

54 of the AlkA protein (I50

55 DEPARTMENT OF PLANT BIOCHEMISTRY Head: professor Jerzy Buchowicz

The Department is engaged in studies on various aspects of gene expression in higher plants. Considerable attention has been given to the role of chromosomal DMA in cell differentiation and to factors that influence this process in plants transformed with the biolistic method. Significant inhibition of somatic embryogenesis in wheat calli was found to be a remote effect of the biolistic bombardment with tungsten microprojectiles. Independently, knowledge relating to the role of protein kinases in the regulation of gene expression during plant ontogenesis and in response to environmental stimuli has continued its vigorous growth. In particular, it was shown that osmotic stress induces a rapid activation of two protein kinases in tobacco BY-2 cells. One of them, identified as the Salicylic Acid Induced Protein Kinase, is a member of the MAPK family, whereas the other is a member of SnRK2, a subfamily of Sucrose Non-Fennenting-related PK. Systematic investigations on plant-pathogen interactions were of particular importance since transgenic potato plants of increased resistance to to the fungus Phytophthora infestans were obtained. The highly resistant plant lines appear to show an increased content of lipid peroxides. Parallelly, Agrobacterium tumefaciens-mediated transformation was performed to obtain virus-resistant potato lines. A large fragment of the viral replicase gene was inserted into a plasmtd vector and the construct obtained was successfully used to protect greenhouse-grown plants from infection with potato leafroll virus (PLRV). Experiments with cDNA coding for potato p-glucanase allowed us to demonstrate that this enzyme is essential for plant response to the infection with potato virus Y (PVY) and other RNA viruses. Further attempts were made to use transgenic plants for experimental production of antigenic proteins. As a result, tobacco and carrot plants producing Helicobacter pylori urease subunits were obtained. These transgenic plants are currently being tested for their immunogenetic potential. Another transformant has been recently used to investigate the role of serine acetyltransferase and acetylserine lyase in the regulation of sulfur metabolism in tobacco.

1. CHROMOSOMAL AND EXTRACHROMOSOMAL DNA IN PLANT CELL DIFFERENTATION

J. Buchowicz, B. Mazuś, M. Dobrzańska, E. Kraszewska, B. Kroczyńska, M. Bucholc, B. Szurmak. C. Krysiak

Some impairment in cell differentiation was observed earlier in plants transformed with DNA-coated tungsten particles by the biolistic method. This impairment appears to result from the interaction between native DNA and the surface of tungsten metal particles (tungstate and tungsten ions have no effect). When intact plasmid DNA is adsorbed on these particles, a self-nicking activity of supercoiled DNA is induced. As a result, circular plasmids are linearized. Similar DNA double-strand

56 breaks are generatad also in target cells and tissues. The rate of their repair is, apparently, not high enough to allow rapid cell proliferation. Hence, although both bombarded and nonbombarded wheat embryos germinate equally well, the formation of somatic embryos is significantly inhibited by bombardment with uncoated (no DNA) tungsten particles. The unexpected effect of such a nondiffusive agent as tungsten metal particles on DNA integrity, cell divisions and cell differentiation seems to deserve further attention. Publications: 3336,3436,3437,3439,1184/A, 1185/A, 1253/A, 1289/A

2. PLANT PROTEIN KINASES AND SIGNAL TRANSDUCTION

G. Muszyńska, G. Dobrowolska, J. Szczegielniak, J. Trojanek, M. Mikolajczyk, O. S. Awotunde, A. Liwosz, I. Jvrkowski

In the animal kingdom Protein Kinase C (PKC), dependent on calcium and phospholipids, is involved in transducing the signal from the membrane to other components of the signal transduction pathway. The presence of elements required for inositol phospholipid-based signaling in plant cells has prompted us to look for a calcium and phospholipid-dependent protein kinase resembling mammalian PKC. We have identified calcium and phospholipid-dependent protein kinase of apparent molecular mass 54 kD (designated as ZmCPK.54). The enzyme was partially purified from etiolated maize seedlings. The activity of ZmCPK54 is stimulated by phosphatidylserine and phosphatidylinositol, but in contrast to PKC it is not essentially affected by diolein and phorbol esters. The enzyme cross-reacts with polyclonal antibodies against the calmodulin like domain of CDPK (Calcium Dependent Protein Kinase) and does not react with antibodies against the catalytic nor the regulatory domain of PKC. Comparative studies revealed that maize protein kinase is not able to phosphorylate the specific substrates of PKC and its activity is not inhibited by specific PKC inhibitors. The substrate specificity and sensitivity to the inhibitors of the maize enzyme resemble those of CDPK. The biochemical and immunological properties indicate that ZmCPK54 belongs to the CDPK family. We also searched for protein kinases which are involved in osmotic stress signal transduction. We have shown that in tobacco cells the osmotic stress induces a rapid activation of two protein kinases of 48 kD and 42 kD. The 48 kD kinase has been identified as the Salicylic Acid Induced Protein Kinase (SIPK), a member of the MAPK family. Biochemical and immunological properties of the 42 kD protein kinase, named p42OSAK (Osmotic Stress Activated Protein Kinase), indicate that this kinase does not belong to the MAPK family. Therefore, the enzyme was purified to apparent homogenity, and subjected to microsequencing. Sequence analysis indicated that this kinase is a member of the SnRK2 subfamily of SNF1 (Sucrose Non-Fermenting) - related kinases. ft is the first evidence of a role of SIPK and p42OSAK in osmotic stress signal transduction in plants. These results were recently published (Mikolajczyk et al. The Plant Cell 12, 165-178,2000).

57 Collaboration: Prof. Pia Ek from Uppsala University (Sweden), Prof. Alice Harmon from Florida University (USA), Prof. Daniel Klessig from Waksman Institute (USA) Publications: 3268,1191/A, 1298/A, 1299/A, 1313/A, 124/N, 130/N, 151/A

3. TISSUE AND CELL CULTURES OF POTATO: PATHOGENS AND TRANSFORMATION

B. Wielgat, U. Maciejewska, A. Szczerbakowa, |M. BorkowsktĄ L Polkowska-Kowalczyk, D. Boltowicz, L. D. Wasiiewska

Studies on the resistance of potato to the pathogenic fungus Phytophthora infestans have been continued. During the recent two-year period we focused on the somatic hybridization of different potato species possessing varied levels of susceptibility to P. infestans, followed by regeneration, selection and genetic analysis of the interspecific hybrids. Over one hundred of potential hybrids were produced as a result of fusion between protoplasts derived from wild Solanum species (6x S. nigrum and 2x S. bulbocastanum), both resistant to P. infestans, and protoplasts of the cultivated potato S. tuberosum (4x cv. Bzura, 2x H8105 and 2x ŻEL 1136) more or less susceptible to the pathogen. Approximately 60 % of the regenerants recovered after fusion of S. nigrum with ŻEL 1136 exhibited the intermediate morphology and their ploidy level appeared to be about 8x, as determined by flow cytometry. Using RAPD analysis (with 2 primers) the regenerants were found to contain DNA from both parental species. Preliminary results indicate that the somatic hybrids obtained exhibit an increased level of resistance to P. infestans close to that of S. nigrum. Research on the oxidative burst, a very early event in the plant/pathogen interaction which results in the generation of active oxygen species (AOS) has also been conducted. Studies of early responses induced by an elicitor from P. infestans, both in cultured cells and in detached leaves derived from Solanum species, resistant or susceptible to this pathogen, showed significant differences in the kinetics and intensity of AOS production. This was correlated with the level of plant resistance and suggested that in all Solanum species studied so far the membrane bound NADPH oxidase complex (mammalian like) is responsible for the level of AOS production. The increased level of AOS led in turn to higher lipid peroxidation yielding lipid superoxides (playing a role of signal and antimicrobial molecules) implying a contribution of lipoxygenase in this process. Collaboration: Department of Genetics, Plant Breeding and Acclimatisation Institute in Młochów; Flow Cytometry Lab. Drug Institute, Warsaw. Publications: 3351,3372,1121/A, 1188/A, 1213/A, 1245/A, 1255/A, 1256/A, 105/N

58 4. MECHANISMS LEADING TO THE ACTIVATION OF PLANT DEFENSE GENES

J. Hennig, D. Konopka, M. Krzymowska, A.Drobek, M. Koter, A. Talarczyk

A growing body of evidence suggests that salicylic acid (SA), reactive oxygen species (ROS), nitric oxide (NO), and ethylene are important signal molecules involved in the synthesis of plant Pathogenesis Related proteins (PR-s). These proteins are crucial for the complex defense response in order to reduce infection. The activity of glucan endo-l,3-p-glucosidase ((3-1,3-glucanase; EC 3.2.1.39), a member of the PR-s family, is believed to be involved in the plant defense against pathogens. We have isolated and characterized a full-length cDNA coding for P-1,3-glucanase from potato (Solatium tuberosum L. cv. Desiree) different from those reported earlier by others. To obtain the cDNA, total RNA isolated from potato leaves infected with potato virus Y (PVY) was used. Our cloning strategy was to obtain a composite full-length cDNA by reconstituting two partial cDNAs obtained by 5'- and 3'-RACE (Rapid Amplification of cDNAs). The coding region contains 1044-nucleotides, resulting in a protein of 347 a.a., designated GLUB (PVY- induced glucanase B; ace. no. AJ009932) The GLUB protein shows 91.9 % identity with tomato glucanase (GLUA) and 74.3 % identity with acidic tobacco glucanase (PR-2d). The expression of p-l,3-glucanase mRNA (gluB) in response to PVY infection and salicylic acid treatment was similar to the pattern of expression of mRNAs for other PR proteins, indicating that it is a part of general and coordinated mechanism of the potato plant response to viral infection. In recent years several strategies have been described to utilize viral genes in constructing virus resistant plants. We generated a deletion of 126 nt (42 amino acid residues) at the 5' of Potato Leafroll Virus (PLRV) replicase gene (ORF2). This construct was cloned into a binary vector (pROK2) and the Agrobacterium tumefaciens - mediated transformation was performed to introduce it into potato (5. tuberosum cvs. Bzura and Ibis). The modified PLRV replicase could not be detected in spite of the presence of ORF2 specific transcripts in the transgenic plants. Over 40 greenhouse- grown transgenic lines have been infected with PLRV (Polish isolate - strain L7) using aphids as a vector. Transgenic plants containing the PLRV A-replicase gene showed varying degrees of protection against PLRV. One line showed no visible symptoms of infection despite high virus concentrations. It was determinate that the Ns gene is responsible for the hypersensitive reaction and the resistant phenotype of potato to Potato Virus S (PVS) infection. Using the RAPD method and bulk segregant analysis we identified four RAPD markers linked to the Ns gene. The sequence of one RPAD marker was identified in order to develop SCAR (Sequence Characterized Amplified Region). SCAR primers were designed and used towards improvement of diploid potato plants through marker-assisted selection of PVS-resistant lines. Publications: 3288,3297, 3346,3351,3352,3437,3429,1110/A, 1116/A, 1120/A, 1208/A, 1209/A, 1210/A, 97/N, 109/N, 129/N

59 5. EXPRESSION OF BACTERIAL GENES IN PLANTS

R. Brodzik, A. Blaszczyk, F. Liszewska, A. Sirko

Production of antigenic proteins in edible tissues of transgenic plants presents a very attractive possibility for obtaining large amounts of inexpensive vaccines. In order to use plants as bioreactors for vaccine production we constructed transgenic tobacco (a model plant for transformation) and carrot (edible crop) producing Hdicobacter pylori urease subunits, UreA and UreB. The transgenic plant material is currently being tested for its immunogenic potential in laboratory animals. In the course of analysis of these plants it appeared that bacterial urease is enzyinatically active in the transgenic tobacco plants. Plants with the highest amount of the protein contained several-fold increased levels of ammonia. We are planning to investigate the significance of this observation for nitrogen metabolism of transgenic plants. Finally, plant expression cassettes for the production of H. pylori HspA protein (another example of a strong antigen) were constructed and we are in the process of transformation of tobacco plants with this cassette. Combined expression of ureB and hspA in transgenic plants is also being considered. Our aim is to obtain plant material that would be able to induce an immunological response sufficient to protect against H. pylori infection or to inactivate existing bacteria. Parallelly, tobacco plants producing bacterial SAT (serine acetyltransferase) and OAS-TL (O-acetylserine (thiol) lyase) in either cytosol or chloroplasts were constructed in order to investigate sulfur metabolism in higher plants. The heterogenic proteins are enzymatically active and plants with several-fold increased activity of SAT and OAS-TL (transformed with Escherichia coli cysE and cysK genes, respectively) were obtained. Molecular characterization of plants overproducing bacterial SAT revealed higher levels of cysteine and glutathione in leaf tissues in comparison with controls. The most striking physiological consequence of the modification of sulfur metabolite levels in these plants was their increased resistance to oxidative stress generated by exogenous hydrogen peroxide. Plants overproducing OAS-TL and plants overproducing both SAT and OAS-TL will be characterized and compared with those overproducing SAT. We expect that comparison of the above groups of transgenics will help to reveal the role of OAS in the regulation of cysteine and other sulfur compounds in higher plants. Publications: 3291,3354,3355, 3448,1154/A, 1155/A, 116/N, 127/N

60 DEPARTMENT OF PROTEIN BIOSYNTHESIS Head: professor Włodzimierz Zagórski-Ostoja

The Department traditionally was implicated in studying in vitro protein synthesis. The accumulated expertise is put today into use for studying overexpression of selected proteins and testing in vivo phenotypes created by de-regulation of the synthesis of specific polypeptides. Pichia pastoris, Saccharomyces cerevisiae and Baculovirus expression systems are in hand, phenotypes are tested in model systems including insect cells and A.thaliana.Solanaceae of industrial interest (potato, tobacco, tomato) are successfully transformed and selection of productive cultivars with appropriate traits is actively followed. Expression of viroids (Potato Spindle Tuber Viroid) and viruses (Potato Virus Y, Potato Leafroll Virus, Potato Virus M) is tested in host plants. Sequence analysis allows the definition of phenotype diversity in virus pseudo-strains. Viral cDNA libraries are used in constructing transgenic potato and tobacco cultivars resistant to local variants of viral pathogens. Our interest in gene expression at RNA level induced studies on local RNA structures and their functions. The Department incorporates four specialised laboratories and recently gave rise to an independent Laboratory of DNA Sequencing and Oligonucleotide Synthesis. The Department actively follows a policy of collaboration with external units, creating mixed groups with the Warsaw University Department of Biology and running long-term joint project on transgenic plants with Młochów Division of the Institute of Plant Breeding and Acclimatisation PAS.

1. STRUCTURE OF POTATO SPINDLE TUBER VIROID (PSTVd) AND POTATO VIRUS Y (PVY) IN RELATION TO THEIR PATHOGENECITY IN PLANTS

W. Zagórski, P. Szafrański, A. Chachulska, A. Góra-Sochacka, A. Palucha, J. Pawlowicz, M. Milnei; W. Podstolski, E. Nowak, I. Rosa

Several lines of transgenic tobacco and potato plants (cultivar Irga) carrying a truncated or full-length polymerase gene of potato virus Y in either (+) or (-) orientation were constructed. Thirty-four potato clones were tested for resistance to PVY. The disease symptoms started to develop in the fourth week post inoculation. They were limited to a systemic mosaic. Six clones were symptom-free with very low ELTSA values, similar to those of the non-infected control plants and of inoculated resistant standard plants. In all of the resistant transgenic potato plants the presence and the copy number of the transgene were confirmed by detailed molecular analysis based on Southern hybridisation. The expression of the transgene was checked by Northern hybridisation.

61 Two of the resistant clones carry the sense RNA-expressing transgene, the other four are antisense RNA expressing clones. This shows the potential of antisense RNA expression in engineering potato resistance against novel versions of viral genomes. Potato Virus Y virion is a rod-shaped, flexuous particle, consisting of RNA, 2000 copies of identical coat protein units and 1 copy of the VPg protein. It has frequently been assumed that coat proteins of rod shaped viruses may share structural features with TMV coat protein i.e. that they have a four helix bundle architecture. The topology fingerprint method (MATCHMAKER module of the SYBYL 6.3 package ) shows that the sequence of the PVY coat protein fits into the jelly-roll fold of the Rhinovirus coat protein with a statistically significant score. CD spectroscopy of the virus shows small amount of helical structure, confirming the hypothesis that the structure of the protein belongs to all-beta rather than all-alpha class of structures. This is an evidence against using the structure of TMV coat protein as the model for designing site-directed mutagenesis experiments of potyviral coat proteins. The work also rises the question about the possible evolutionary relationship of two groups of RNA viruses: potyviridae and picornaviridae. In order to confer resistance against PSTVd a truncated (355bp) cDNA copy of severe molecular variant S23 was introduced into the plant genome. The 355bp cDNA was cloned into the binary vector pKYLX71-35S2 under control of the double CaMV 35S promoter. Constructs with inserts in (+) and (-) orientations were obtained and were used in Agrobacteriwn mediated transformation of Solarium tuberosum cv. Irga. A total of 113 plant lines were regenerated and grown in vitro. PCR analysis of the plant DNA confirmed the presence of the introduced PSTVd cDNA. Fifty selected transgenic plants were tested to evaluate their degree of resistance against PSTVd infection at the Plant Breeding and Acclimatisation Institute, unit Młochów. None of them was resistant to PSTVd. Further analysis of the transformants led to an unexpected observation. Cell sap extracted from several transgenic potato plants (expressing (+) RNA of the truncated transgene) was able to cause PSTVd infection. The analysis of viroid's progeny from these transgenic plant lines showed the presence of the full- length repaired infectious pathogen. Molecular analysis of the transgenic plants is in progress. Work on the genetic stability of PSTVd sequence variants was continued. Sequence analysis of viroid progeny from individual infected plants confirmed high genetic stability of the S23 molecular variant. Publications: 3295,3408,3442,3455,1106/A, 1118/A, 1119/A, 1122/A, 1156/A, 1157/A, 1159/A, 1160/A, 1161/A, 1162/A, 1231/A, 1314/A, 1316/A, 1317/A, 91/N, 92/N, 94/N

62 2. ROLE OF HORMONES IN GENE EXPRESSION OF GALLERIA MELLONELLA

K. Grzelak, B. Kludkiewicz, K. Gocman

Studies on the expression of the EcR and USP proteins in Pichia pastoris were continued (in co-operation with the Biochemistry Department of the Technical University of Wrocław). The proteins are elements of the functional ecdysteroid receptor. Conditions have been worked out of the efficient synthesis and secretion of the EcR and USP proteins from Pichia cells transformed with pPICZaC-EcR and pPICZaC-USP plasmids. The recombinant proteins secreted into the medium were detected by Western analysis using the anti-His, anti-myc and anti-USP antibodies. The proteins were visible on Coomassie stained SDS-PAGE gels. Ca. 15mg/l EcR and ca. 20mg/l USP proteins were obtained. The molecular weight of the recombinant EcR and USP proteins secreted from Pichia cells are ca. 108kDa and 74 kDa, respectively. This is, to our knowledge, the first time, that the EcR and USP proteins have been obtained in a eukaryotic expression system. Studies on the influence of cold stress on the synthesis of Galleria mellonella proteins of mol.wt. ca. 70kDa to 82 kDa were continued (in co-operation with the Department of Invertebrate Physiology, Warsaw University). Using Northern analysis and hybridisation with specific cDNAs probes the presence of Lhp 76 and Lhp 82 transcripts in larvae, which were exposure to low temperature (larval diapause), was proven. The results of sequencing of the N-termini of proteins with mol.wt. ca. 76 kDa and ca. 82 kDa (which were purified from the hemolymph of diapausing larvae) indicated that these proteins are LHP-76 and LHP-82. Thus, the proteins which are synthesised in diapausing Galleria larvae belong to the LHP protein class (without any doubt). Co-operating scientists: Prof. M. Kochman, dr. hab. A. Ożyhar - Technical University of Wrocław; Prof. B. Cymborowski, J. Godlewski - Warsaw University. Publications: 1189/A, 1252/A, 1260/A

63 3. BACULOV1RUSES AND THEIR RECOMBINANTS

J. Michalik, E. Szołajska, K. Rybicka

Baculovirus from Stilpnotia salicis was propagated in the cell line IPL-LD- 65Y from Lymantria dispar. This enabled us to characterize the genome of SsMNPV by making a physical map as well as obtaining the sequence of the polyhedrin gene and neighbouring areas in the genome. The results suggest that SsMNPV is a variant of the Orgyia pseudotsugata isolate (OpMNPV), a baculovirus isolated in North America. The system Bac-to-Bac was used to express several heterologous proteins with satisfactory yield. These are: human prolactin, Vpg, convertase. Characterisation of the above protein products is under way. Publications: 3186,3273,3404,1186/A, 1187/A, 1232/A, 1231/A, 1261/A, 1251/A, 1257/A, 1258/A, 146/N

4. NUCLEOLYTIC ENZYMES FROM HIGHER PLANTS AND HUMAN CARCINOMA CELLS

A. Przykorska, J.W. Szarkowski, E. Kuligowska, D. Klarkowska, K. Olszak

Two single-strand-specific nucleases isolated from higher plants were used as structural probes of the secondary and tertiary structure of RNA. In many Candida species, the leucine CUG codon is decoded by a tRNA with two unusual proprietes: it is a ser-tRNA and, uniquely, has guanosine at position 33. Using a combination of enzymatic (Rnl nuclease, VI RNase ) and chemical (Pb2+, imidazole) probing of the native Candida albicans ser-tRNAcAG> we demonstrated that the overall tertiary structure of this tRNA resembles that of a ser-tRNA rather than a leu-tRNA, except within the anticodon stem. Using non-modified in vitro transcripts of the C. albicans ser-tRNAcAo carrying G, C, U or A at position 33, we demonstrated that it is specifically a G residue at this position that induces the atypical anticodon stem structure. Using Rnl nuclease which cleaves single-stranded RNA regions we demonstrated that the variable loop of the C. albicans ser-tRNACAG is similar to that of the S. cerevisiae ser tRNAiGA, yet is completely resistant to Rnl hydrolysis, whereas the variable loops of a number of leu-tRNAs examined are subject to Rnl hydrolysis. We conclude that the anticodon arm distortion, induced by a guanosine base at position 33 in the anticodon loop of this novel tRNA, results in reduced decoding ability which has facilitated the evolution of this tRNA without extinction of the species encoding it. Unmodified tRNAs are powerful systems to study the effects of posttranscriptional modifications and site-directed mutations on both the structure and function of these ribonucleic acids. To define the general limitations of synthetic constructs as models for native tRNAs, it is necessary to elucidate the conformational states of unmodified tRNAs as a function of solution conditions. We have studied the conformational properties of unmodified yeast tRNAPhe as a function of ionic strenght, [Mg++], and temperature using a combination of spectroscopic measurements along

64 with chemical and enzymatic probes. The enzyme of choice for this studies was Rnl nuclease from rye germ nuclei, which is active at broad pH and temperature range and does not need the addition of divalent cations to the reaction mixture. Our findings suggest that although the tertiary structures of various tRNAs are broadly comparable to one another, the intrinsic stability of the tertiary fold, the conformational properties of intermediate states, and the stability of intermediate states can differ significantly among tRNA sequences. Thus, the use of unmodified tRNAs as models for native molecules can have significant limitations. A new sequence-specific RNase was isolated from human colon carcinoma T84 cells. The new RNase selectively cleaves the phosphodiester bonds at AU or GU steps at the 3'side of A or G and the 5' side of U. 5'AU3' or 5'GU3' is the minimal sequence required for T84 RNase activity, but the rate of cleavage depends on the sequence and/or structure context. Publications: 3318,3344,3442,1201/A, 1305/A, 1304/A, 1249/A

5. IDENTIFICATION OF A PUTATIVE CHROMATIN REMODELING COMPLEX IN ARABWOPSIS

A. Jerzmanowski, J. Brzeski, K. Olczak, T. Sarnowski (mixed group: IBB PAN, Department of Biology, Warsaw University)

Multiprotein complexes involved in active disruption of chromatin structure, homologous to the yeast SWI/SNF complex, have been described for human and Drosophila cells. In all SWI/SNF-class complexes characterised so far, one of the key components is the SNF5-type protein. We have isolated a plant (Arabidopsis thaliand) cDNA encoding a 27 kDa protein which we named BSH, with high structural and functional homology to yeast SNFSp. Analysis of the whole cell and nuclear protein extracts with antibodies against recombinant BSH indicates that the protein is localised in nuclei. BSH co-elutes from a gel filtration column together with 12 other polypeptides, in a complex with an apparent molecular mass of about 2 MDa. Publications: 3389

65 6. HISTONE HI-MEDIATED TRANSCRIPTIONAL REPRESSION AND THE FUNCTION OF LINKER HISTONE VARIANTS IN PLANTS

A. Jerzmanowski, R. Tomaszewski, M. Prymakowska-Bosak, M. Przewloką, T. Calikowski (mixed group: IBB PAN, Department of Biology, Warsaw University)

Histone HI plays an active role in establishing the transcriptionally repressed chromatin state of the oocyte-type 5S RNA genes in the early stage of Xenopus development. We have shown earlier that the binding of HI results in protection of DNA sites within the AT-rich oocyte-type repeat and in spaced nucleosomes that are partly stabilised in specific positions over template DNA. Performing in vitro transcription on reconstituted minichromosome templates we have now established that the above effects of HI are absolutely dependent on the presence of the!20 bp oocyte 5S rRNA gene in its native position within the flanks. We have also analyzed the molecular and morphological phenotypic consequences of a marked change in the proportions of linker histone variants achieved by using the antisense approach with transgenic tobacco. We established that in plants the native stoichiometry of linker histones is critical for the correct course of male meiosis and the subsequent development of functional pollen grains. Publications: 3350,3423,3430

66 LABORATORY OF MUTAGENESIS AND DNA REPAIR Head: professor Zygmunt Cieśla PL0201954

Two main lines of research were continued: the first one concerned the mechanisms controlling the fidelity of DNA replication in Escherichia coli; the second concerned cellular responses of Saccharomyces cerevisiae to DNA damaging agents. We have been investigating the question whether during chromosomal DNA replication in Escherichia coli the two DNA strands may be replicated with differential accuracy. To address this question we set up a new system that allows the examination of mutagenesis either of the leading strand or the lagging strand. Our results suggest that the lagging strand replication of the E. coli chromosome may be more accurate than leading strand replication. More recently, we studied mutagenesis of the two strands in recA730 strains which exhibit constitutive expression of the SOS system. Our results clearly indicate that in recA730 strains there is a significant difference in the fidelity of replication between the two replicating strands. Based on our data we propose a model describing a possible mechanism of SOS mutagenesis. To get more insight into cellular responses to DNA damage we have isolated several novel genes of S. cerevisiae, the transcription of which is induced by DNA lesions. Main effort was concentrated on the characterization of the DIN7 gene. We found that Din7p specifically affects the metabolism of mitochondria! DNA (mtDNA). The elevated level of Din7p results in an increased frequency of mitochondrial petite mutants, as well as in a higher frequency of mitochondrial point mutations. Din7p affects also the stability of microsatellite sequences present in the mitochondrial genome. As expected, Din7p was found to be located in mitochondria. In another project, we found that the DIN8 gene isolated in our laboratory is identical with the UMP1 gene encoding a chaperone-like protein involved in 20S proteasome maturation. Interestingly, induction of UMP1 expression in response to DNA damage is subject to regulation independent of cell cycle checkpoint genes. Since the din8/umpl mutants show an increased sensitivity to UV light, we speculate that the proteasoine may have a role in the repair of UV damage.

1. REGULATION OF THE FIDELITY OF DNA REPLICATION IN E. COLI

I. J. Fijafkowska, P. Jończyk, M. Maliszewska-Tkaczyk, D. Gaweł, M. Białoskórska

Despite progress in understanding the components of SOS mutagenesis, the mechanism of error-prone replication is still unknown. In our laboratory we have been studying mutagenesis on the E. coli chromosome in a recA 730 strain. recA 730 strains exhibit constitutive expression of the SOS system and a spontaneous mutator effect without the need for activation of the system by DNA damage. We measured possible differences in the fidelity of synthesis of the leading and lagging strands of DNA during SOS-modified chromosomal replication in vivo. The possibility of differential replication fidelity for the two strands arises from the distinct modes of replication of

67 the two strands. Two strands may present different opportunities for forming the SOS mutasome. Several pairs of strains were constructed differing in the orientation of a mutational target, the lac operon, with respect to the origin of chromosomal DNA replication. Within each pair, a defined lac sequence is replicated as the leading strand in one strain and as the lagging strand in the other. We used several lacZ alleles constructed by Cupples and Miller with different missense mutations at the same coding position that can be reverted only by a single, specific base-pair substitution. Our results clearly indicate that in recA730 strains there is a significant difference in the fidelity of replication between the two replicating strands. Based on these data we propose a model describing a possible mechanism of SOS mutagenesis. First, the requirement of the SOS response for the RecA nucleofilament is much more readily satisfied by the lagging strand of replication than by the leading strand. By its very nature, the lagging strand is characterized by the presence of single-stranded DNA, both behind and ahead of the growing Okazaki fragment. Second, in contrast, in the leading strand, the polymerase is connected directly via the T subunit to the helicase-primase complex that motors the opening of the replication fork and no single-strand DNA is exposed. The single stranded DNA in the lagging strand, in particular the stretch ahead of the growing Okazaki fragment, would provide a ready entry point for the generation of the RecA/UmuD'C nucleofilament. On this basis we propose that the strong enhancement of mutagenesis in cells consititutively induced for the SOS response results from a disproportionately large increase in fixed replication errors in the lagging strand of DNA replication. Publications: 3285,3309,1200/A, 1300/A, 1301/A, 1320/A, 1321/A, 1322/A, 117/N, 118/N, 132/N, 155/N.

2. CELLULAR RESPONSES OF SACCHAROMYCES CEREV1SIAETO DNA DAMAGE

Z. Cieśla, M. U. Fikus, P. Koprowski, P. Mieczkowski,

Our approach to study cellular responses of S. cerevisiae cells to DNA damage is based on the isolation of novel genes, the expression of which is induced by lesions in DNA. We have focused on studying the function of one of these genes, DIN7, the product of which belongs to a large family of proteins involved in DNA repair and mutagenesis. A turning point in our work on DIN7 was the observation that enhanced synthesis of Din7p results in an increased frequency in the formation of mitochondria! petite mutants as well as in a higher frequency of mitochondrial mutations conferring resistance to eryrthromycin. These results suggested that Dtn7p may function in mitochondria. This expectation was fulfilled by the experiments with the use of Din7p fused to the green fluorescent protein (GFP) and cell fractionation experiments which clearly indicated that the protein is specifically located in mitochondria. In order to get insight into the mechanism of the Din7p-mediated effect on the metabolism of mitochondrial DNA (mtDNA) we examined whether the level of Din7p

68 affects mtDNA recombination. Recombination is known to be required for the stability of the mitochondria! genome. However, we could not find any significant difference in the frequency of recombination between mitochondria! unlinked markers in strains either defective in the Din7p function or overproducing the protein, as compared with the wild-type strain. We also considered the possibility that the effect of Din7p on the stability of mtDNA may be due to its interference with maintaining microsatellite sequences present in the yaest mitochondrial genome. To examine this hypothesis, we used the system of T. Petes designed to determine the stability of various microsatellite sequences within the ARGSm gene inserted into mtDNA. Interestingly, we found that poly GT sequences are less stable in din? mutants compared with the DIN7 strain. In light of the finding that the DNA mismatch repair is involved in maintaining the stability of nuclear microsatellites, this observation raises the possibility that Din7p may affect the functioning of mitochondrial DNA mismatch repair. Publications: 3285, 1273/A, 1274/A, 1296/A, 1297/A

3. CELLULAR SYSTEMS ENGAGED IN THE REPAIR OF ALKYLATION

E. *Sledziewska-Gojska, ••*,„ A. Gamszewska,, . ,A. pi no n -1955 We have established that the DNA damage inducible gene of S. cerevisiae DIN8, previously isolated in our laboratory, is identical with UMPI encoding a small chaperone-like protein involved in 20S proteasome maturation. The transcription of UMPI is induced both in response to DNA damage as well as in response to the treatment with the DNA replication inhibitor hydroxyurea (HU). Analysis of the expression of UMPI revealed that the induction in response to DNA damage is independent of the regulatory cell cycle checkpoint genes: MEC1, RAD53 or DUN1. The only known (until now) DNA damage inducible gene, the expression of which is independent of all the above regulatory genes, is UBI4. However, unlike UBI4, which is induced under general stress conditions, our results show that the expression of UMPI is not induced by heat shock or H2O2 treatment. The products of the majority of DNA damage inducible genes of S. cerevisiae are involved in the processes engaged in the maintenance of genetic stability of the cell. We investigated the effect of the disruption of UMPI on the sensitivity of S. cerevisiae cells to agents causing DNA damage. It was found that the lack of Umplp leads to an increased sensitivity of S. cerevisiae cells to UV light. We did not observe any meaningful effect of the UMPI mutation on the cellular sensitivity to MMS or HU treatment. These results suggest a role of proteasome in the repair of UV damage in DNA. Publications: 3285, 87N, 131 /N, 148/N, 1273/A, 1274/A, 1297/A

69 LABORATORY OF PURINE METABOLISM Head: professor Maria M. Jeżewska

PROPERTIES OF SELECTED ENZYMES OF PURINE METABOLISM

M. M. Jeżewska and G. Jewdoszuk

Previously we found in H. pomatia hepatopancreas a S'-methylthioadenosine (MTA) deaminating activity not reported to occur in the animal kingdom before. Fractionation of hepatopancreatic proteins on Cellulose DE-52, DEAE-Sephacel and Sephadex G-200 under various conditions was performed, but the separation of this activity from that deaminating Ado and 2'dAdo was not achieved. Instead, several peaks of the activity deaminating always all three adeninę nucleosides were found. Two main peaks corresponded to the proteins of m. wt. 145 000 and 132 000, the latter representing the previously described H. pomatia adenosine deaminase. We verified that similar proteins deaminating MTA, Ado, 2'-dAdo (and several their analogues) occur also in the H. aspersa hepatopancreas. During fractionation of hepatopancreatic proteins on Cellulose DE-52 a specific MTA phosphorylase (resembling that occurring in vertebrates) was found and separated from Ado/2'-dAdo phosphorylase described by us previously. These data suggest that at least in the Helicidae snails MTA could be catabolised in two ways: by a non-specific adeninę nucleoside deaminase, and by the specific MTA phosphorylase. Deficiency of hypoxanhine-guanine phosphoribosyltransferase (HGPRT), the biochemical defect underlying the Lesch-Nyhan Syndrome (LNS), a rare X-linked familial disorder was being diagnosed in our laboratory between 1972-1997. The data obtained about the level of HGPRT in Polish patients were summarised and presented in the 7th Symposium of the European Society for the Study of Purine and Pyrimidine Metabolism in Man, held in Gdańsk, 15-19 September 1999. Among 47 patients (age range 4 months - 14 years) with various symptoms of neurological disorders and uric acid overproduction, HGPRT activity was 0 - 2 % of the normal activity in 12 cases, and only 6 boys presented full LNS with self-mutilation developed between 2-4 years after birth. The incidence of LNS in Poland approached the minimum frequency estimated for Canada as 1 in 380 000. In Polish patients with LNS 7 different mutations in the HGPRT gene were identified, and 5 of them are novel. In the world LNS population, over 50 % of mutations determined resulted in single amino acid substitutions or a change to a stop codon, found at 50 of the 218 positions of the amino acid sequence of HGPRT monomer. We compared the distribution of those mutations in the HGPRT sequence with the already described HGPRT structure and found that the mutations are clustered, but not necessarily located in the sequence areas strictly conserved or involved directly in catalysis. In turn, comparing this mutation spectrum of the hprf germ-line with the published data on the somatic HGPRT" mutations arising in vivo in human T-lymphocytes, we found several differences in the mutation frequency and distribution. The basis of these differences between the germ-line and somatic mutations is not known. Publications: 3307,3313,1331/A

70 LABORATORY OF ANTIMETABOLITES Head: professor Tadeusz Kulikowski

The research activity of the Laboratory of Antimetabolites in the past two years continued to focus on two basic, major areas including: (1) the synthesis and properties (physico-chemical and biological) of new pyrimidine and purine nucleoside and nucleotide analogues with potential antitumor .and antiviral activities; and (2) the interaction of these analogues with various enzymatic systems, especially key enzymes in antitumour and antiviral chemotherapy such as thymidylate synthase and reverse transcriptase. Of special interest were studies on the design of new selective inhibitors of human RNA viruses such as human immunodeficiency virus (HIV) and, recently, hepatitis C virus (HCV). Some of them will be currently subjected to in vivo evaluation as potential anti-HIV/AIDS drugs. Of interest were also investigations of new fluorinated analogues of pyrimidine deoxyribonucleosides and acyclonucleosides with anti-leukemic activity, decreased cytotoxicity against host cells, and active against fluorouracil- and fluorodeoxyuridine- resistant cell lines. These investigation were supplemented by crystallographic and NMR studies of the conformation of biologically important analogues, as well as their complexes with key enzymes of antitumor therapy.

1. dUMP ANALOGUES AS INHIBITORS OF MURINE LEUKEMIC CELLS AND THEIR THYMIDYLATE SYNTHASE

K. Felczak, A. Miazga, M. Bretner, D. Shugar and T. Kulikowski,

Thymidylate synthase (TS, E.G. 2.1.1.45) catalyzes the reductive methylation of dUMP in the de novo biosynthesis of dTMP and is a useful target in cancer chemotherapy with the use of 5-substituted dUMP analogues. We synthesized and investigated the structure and biological properties of a range of 5-substituted N4-hydroxy-2'-deoxycytidines and their 5'-monophosphates, untypical dUMP analogues with CH3, CH2OH, F, Cl, Br, and I substituents, exhibiting different electronic, steric and hydrophobic properties. Inhibition was studied of two TS forms differing in sensitivities towards FdUMP inhibition, isolated from parental and FdUrd-resistant L1210 cell lines. N4-hydroxy-dCMP and all its 5-substituted analogues were competitive vs dUMP 8 4 slow-binding inhibitors with K; values ranging from I0~ M for N -hydroxy-dCMP and its 5-fluoro congener, 10"6 M for the 5-iodo- and 5-hydroxymethyl derivatives, and 10~5 M for the 5-chIoro and 5-bromo analogues, to 10"4 - 10"3 M for the 5-methyl-substituted compound. None of the 5-haIogeno analogues became dehalogenated in the TS reaction. Attempts to correlate the latter results with the 5-substituents physical properties pointed to the decisive role of hydrophobicity, as the compounds with substituents of low hydrophobic constants, such as -F, -H and -CH2OH

71 were stronger TS inhibitors than those with highly hydrophobia -Br, -Cl, and -CH3 substituents. Certain effect of electronic properties should also be considered. The nucleoside forms of the analogues were potent L5178Y leukemia cell 9 8 growth inhibitors with IC50 ranging from 10" M for 5-fluoro-dUrd and 10~ M for its N4-hydroxy-dCMP congener to 10'5 M for 5-chloro-dUMP, 5-bromo-dCMP and N4- hydroxy-5-methyl-dCMP. A second class of dUMP analogues, potential TS inhibitors, involved acyclic derivatives of 5-fluoro-dUMP and 5-fluoro-dUrd. We synthesized l-[(2-hydroxyethoxy)-methyl]-5-fluorouracii (HEMFU) and l-[(l,3-dihydroxy-2-propoxy)methyI]-5-fluorouracil (DHPFU) as well as their monophosphates and investigated their interaction with leukemie TS and growth inhibitory properties vs leukemia cell culture. DHPFUMP proved to be the strongest inhibitor non-competitive vs dUMP with K-, 2.8 //M. HEMFU, despite the weak interaction of its nucleotide analogue with the enzyme, proved to be a strong LSI78Y 5 cell growth inhibitor with IC50 in the range of 10" M. A novel class of further potential TS and tumor growth inhibitors is represented by selenopyrimidine nucleosides and nucleotides, compounds with decreased pKa values for the dissociation of N(3) proton. We synthesized a range of 2- and 4-selenouraciI nucleoside isosters of thionucleosides, which we recently investigated. Initial results of biological studies proved the nucleotide forms of 2- and 4-selenonucleosides to inhibit leukemie TS in the range of 10~5 - 10"6 M. These results confirm the hypothesis stating that the increase of the pKa value of N(3)H dissociation in dUMP analogues leads to the increase of the potency of TS inhibitors. Collaboration: W. Rode et a!., Nencki Institute of Experimental Biology, PAS, Warszawa Publications: 3286,3303, 3407,110/N, 1302/A, 1328/A, 1329/A

2. INHIBITORS OF HUMAN IMMUNODEFICIENCY VIRUS AND ITS REVERSE TRANSCRIPTASE

K. Felczak, A. Miazga and T. Kulikowski

The inhibitors of HIV reverse transcriptase (RT), the key enzyme in anti-HIV chemotherapy, fall into two categories: (I) RT competitive inhibitors and/or DNA chain terminators i.e. dideoxynucleosides without 3'-OH group; they must be phosphorylated by cellular enzymes to their 5'-triphosphates to exert the inhibitory activity; (II) non-competitive RT inhibitors, which do not need phosphorylation and bind allosterically to its hydrophobic pocket, close to the catalytic site, and distort the conformation of RT to the point where it cannot function as the enzyme. The most important representatives of this class of compounds are the recently discovered 6-arylthio- and 6-arylselenenyl acyclonucleosides. In contrast to 6-arylthio acyclonucleosides, which inhibit only HIV-1, some 6-phenylselenenyl analogues are also active against HIV-2. 5-Alkyl-6-selenenyl pyrimidine nucleosides have been

72 intensively studied by the Schinazi group, and l-(ethoxymethyl)-6-phenylselenenyl)-5-ethyl uracil (D seems to be a promising candidate for AIDS chemotherapy. To improve the hydrophobia properties of (1), important in the treatment of HIV infections in the central nervous system (CNS), we decided to synthesize and investigate hitherto unknown 5-propyluracil derivatives. l-[(2-Hydroxyethoxy)methyI]- 6-(phenyl-selenenyl)-5-propyluracil (HP 10), l-(ethoxymethyl)-6-(phenylselenenyl)-5-propyluracil (EP11) and l-(benzyloxymethyl)-6-(phenylselenenyl)-5-propyluracil (BN12) were prepared with the use of the regioselective lithiation procedure. Preliminary investigation of these compounds in CEM cells points to a substantial anti-HIV-1 activity (EC50 ~1 //m) and a good therapeutic index (SI>100) of EP11. Further investigations are currently under way. Collaboration: A. Piasek et al., AIDS Diagnosis and Therapy Center, Warszawa. Publications: 3422,3406,1182/A, 1332/A

3. STRUCTURAL STUDIES OF BIOLOGICALLY IMPORTANT NUCLEOSIDE AND NUCLEOTIDE ANALOGUES AND THEIR INTERACTION WITH RAT HEPATOMA THYMIDYLATE SYNTHASE

K. Felczak, M. Bretner, D. Shugar and T. Kulikowski

In order to understand the influence on thymidylate synthase (TS) interactions with dUMP analogues of the pyrimidine ring 2- and/or 4-thio, and 5-fluoro substitutions, X-ray diffractions by crystals of 5-fluoro-dUrd and its 2- and 4-thio, and 2,4-dithio analogues were measured, the four structures solved and refined. The effect of 4-thio substitution of FdUMP, altering the specificity of inactivation of thymidylate synthases from various sources, is probably due to weaker proton acceptor power of the 4-thio substituent and the increased acidity (enhanced proton-donor power) of the N(3)- H moiety, resulting in an impaired fitness into the network of hydrogen bonds in the enzyme active center cleft. 2,4-Dithio substitution results in (i) impaired pyrimidine ring recognition by the enzyme active center, due to the 4-thio substituent (ii) increased pyrimidine ring aromaticiry in dUMP, leading to the resistance of C(6) to nucleophilic attack by the enzyme active center cysteine, and (iii) altered planarity of the pyrimidine ring and deflections, with respect to the ring plane, of substituents at C(2), C(4) and C(5). 5-Fluoro substitution apparently activates the pyrimidine ring towards the interaction with thymidylate synthase by producing local strain, which results in an increased reactivity as predicted by the Walsh-Bent rule. In order to learn more about the mechanism of TS inhibition by N4-hydroxy-5- fluoro dCMP (N4-OH-FdCMP), a strong competitive and mechanism-based inhibitor, we followed, with the use of NMR spectroscopy, the interaction of this analogue with 4 rat TS in the presence or absence of CH2FH4. Preliminary study of N -OH-FdCMP in water showed conformational similarity to known pyrimidine deoxynucleoside 5'- monophosphates, with the exception of a larger stabilization of the S (C2'-endo)

73 puckering form of the sugar moiety. The same analogue in a borax buffer appeared to exist as an equilibrium of two slowly exchanging conformational states, pointing to the pyrimidine N4-OH cis-trans interconversion. Increasing boron ion concentration stabilized the N(3)-C(4)-N4-O trans rotamer population (up to 50 %), while in water the only observed rotamer was cis. A relatively large broadening of both 19F and 'H resonance lines pointed to an interaction of the boron ion with C(5)-fluorine and/or 5'- phosphate. Rat TS did not form a covalent binary complex with N4-OH-FdCMP (up to 3 mM). Addition of an excess of CH2FH4 resulted in a covalent complex, clearly visible as a new wide 19F resonance line shifted upfield by 14 ppm. Concentration of the enzyme-bound inhibitor equalled the total enzyme concentration, indicating that one N4-OH-FdCMP molecule is bound per one enzyme molecule. Thus, N4-OH-FdCMP appears to form a covalent complex with rat TS only in the presence of CH2FH4. The latter is consistent with our previous results demonstrating the formation of covalent complexes of rat TS with FdUMP or 4-thio-FdUMP, visible in I9F NMR spectra, only in the presence of CH2FH4. Pyrimidine C(6) substituted nucleosides are of interest as potential antimetabolites, e.g. 5-fiuoro-6-fluoromethyl-2'-deoxy-uridine, and model compounds to determine whether the parent non substituted nucleoside (or nucleotide) is involved in a given enzymie reaction in the syn or anti conformation. This led previously to determination of the structures of a variety of 6-substituted uracil nucleoside analogues. At present we determined the crystal structure of the analog from cytidine series, namely its 6-propyl derivative. The conformation of the above mentioned compound in solid state is syn and the ribose ring is close to planar and almost coplanar with the cytosine ring. The overall structure consists essentially of two planes perpendicular to each other, the plane of the sugar moiety and that of the 6-propyl cytosine. An interesting feature in the crystal structure is the formation of two hydrogen bonds. Both O(2) and H(2), bonded to N(2), in the same molecule are involved in the formation of two hydrogen bonds with a symmetry-related molecule. A similar hydrogen-bonding network is found in the DNA structure. We can conclude that 6-propyl-cytidine could be a carcinogenic compound, and could be a useful tool in molecular recognition because of similarities in the structure and the hydrogen-bond network. Collaboration: J. Poznański, Department of Biophysics, Institute of Biochemistry and Biophysics PAS; A. Jarmuła, W. Rode, Nencki Institute of Experimental Biology PAS; Department of Chemistry, Warsaw University; Department of Chemistry, University of Arizona, Tucson, USA; K.B. Birnbaum, National Research Council of Canada, Ottawa. Publications: 3296, 3367,3421,1181/A, 1326/A, 1332/A

74 4. ATP-BINDING DOMAIN OF RNA HELICASE AS CANDIDATE TARGET FOR HEPATITIS C ANTIVIRAL THERAPY

M. A. Siwecka and T. Kulikowski

Helicases are capable of enzymatically unwinding duplex RNA structures by disrupting the hydrogen bonds that keep the two strands together. This is accomplished by a reaction that is coupled with the hydrolysis of y-phosphate of ATP. All helicases contain conserved amino acid sequences, so-called Walker motifs A and B, which bind the terminal phosphate groups of NTP and the Mg2+ of the Mg-NTP complex, respectively. In previous works it was observed that lp-D-l-ribofuranosyl-1,2,4- triazole-3-carboxamide (ribavirin), a compound that does not contain y- nor p- phosphate groups is a competitive (in regard to ATP) inhibitor of the NTPase activity of HCV and West Nile virus NTPase/helicase. However, the inhibition was efficient at relatively low ATP concentration (<1/10 of Kra measured for ATP). To enhance the inhibitory potential of ribavirin, ribavirin-S'-triphoshate (ribavirin-TP) was synthesized and investigated. Ribavirin-TP was synthesized with the use of the modified Yoshikawa-Ludwig-Mishra-Brown procedure involving direct phosphorylation of unprotected nucleosides. Kinetic analysis revealed enhanced inhibitory potential of ribavirin-TP (IC50= 40 fiM) as compared to ribavirin (IC50>500 fM). Analysis of the inhibition type by means of graphical methods shows a competitive type of inhibition with respect to ATP. In view of the relatively low specificity towards NTP of the viral NTPase/helicases, it could not be ruled out that the investigated enzyme hydrolysed ribavirin-TP to less potent products. Investigations including non-hydrolysable analogs of ribavirin-TP or ribavirin-S'-diphosphate (ribavirin-DP) are currently under way. Collaboration: P. Borowski et al., Abteilung fur Virologie, Bernhard-Nocht-Institut fur Tropenmedizin, Hamburg, Germany. Publications: 1330/A

75 LABORATORY OF DNA SEQUENCING AND OLIGONUCLEOTIDl SYNTHESIS Head: professor Joanna Rytka

An impressive development of gene sequencing has taken place in recent years, involving a wide range of organisms from bacteria to mammals. A unique position is occupied by the yeast Saccharomyces cerevisiae, which for decades has had a prominent position as a model, single-celled eukaryotic organism and is the first eukaryote with a completely sequenced genome. Our laboratory has been involved in the European Community projects on yeast genome sequencing (chromosome II and III) and on the functional analysis of yeast genes (Eurofun). More recently, the interest of the laboratory has focused on identifying the functions of new genes discovered during the sequencing of the yeast genome. The information provided by completely or partially sequenced genomes indicates that most of the core biological functions are carried out by orthologous proteins. In this context the information on the function of unknown yeast genes extends to similar genes in evolutionarily distant species including mammals. Furthermore, the laboratory is involved in a pilot project of sequencing the Paramechtm tetraurelia genome and extrachromosomal genomes of Euglenoid species.

SEQUENCING AND ANALYSIS OF THE YEAST GENOME

J. Rytka, B. Babińska, R. Gromadka, R. Kucharczyk, A. Migdalski, M. Ostrowska, M. Zagiiiski

Within the frame of the EUROFAN network for functional analysis of the yeast genome we constructed deletion mutants of six open reading frames from chromosome II: YBR128c, YBR 131w, YBR 133c, YBR 137w, YBR 138c, and YBR142w. In order to search for possible mutant phenotypes associated with gene inactivations, tetrads derived from heterozygous diploids ORF/orfn::KanMX4 were subjected to phenotypic tests under a wide range of conditions. Tetrad analysis revealed that only one ORF, YBR142w, encoding a putative DEAD-box RNA helicase, is an essential gene. The most interesting appeared the ybrlllcA mutant, which displayed increased sensitivity to caffeine, calcium and zinc. To emphasise this phenotype we named the gene CCZI. The cczJA cells are characterized by altered vacuolar morphology resembling that of yptlA mutant. Multicopy suppressors correcting the deletant phenotypes were isolated. Increased gene dosage of the PMC I, PMR1, and YKLIoOw genes suppressed only calcium sensitivity, whereas the YPT7 gene (encoding a ras-like GTPase) suppressed both zinc and calcium sensitivity and restored normal vacuole morphology. Our results indicate that Cczlp may be involved in cellular

76 functions linked to such processes as the regulation of ion homeostasis, vacuole functioning or vesicular transport. The functional analysis of the newly discovered Saccharomyces cerevisiae gene KRR1 (YCL059c) was continued. It encodes a protein essential for cell viability. Experiments with a Krrlp-HA fusion showed that the protein is localized in the nucleolus. The KRR1 gene is highly expressed in dividing cells and its expression ceases almost completely when cells enter the stationary phase. A conditional allele of the gene was constructed. Experiments with the conditional mutant showed that Krrlp depletion leads to drastic reduction of 40S ribosomal subunits due to defective 18S rRNA synthesis. We propose that Krrlp is required for proper processing of pre-rKNA and the assembly of preribosomal 40S subunits. Collaboration: Prof. P.P.Słonimski, Centre Genetique Moleculaire, CNRS, Gif-sur- Yvette, and Dr, R. Rosine Haguenauer-Tsapis, Institut Jacques Monod-CNRS, Paris FRANCE Publications: 3289,3328,3392,3410,1248/A, 1270/A, 1275/A, 1278/A, 1284/A, 1285/A, 92/N, 137/N

77 LABORATORY OF BIOLOGICAL NMR Head: professor Andrzej J. Bierzyński

The objective of the Laboratory is to provide access to a high-resolution NMR facility, as well as scientific and technical assistance for scientists from Polish laboratories involved in structural studies of biologically important molecules. The laboratory is equipped with a 500-MHz Varian UnityPlus instrument with three channels, ultrashim system, digital processing, two - 5 mm and 8 mm - triple 'H/I3C/15N probes, and a 5 mm broadband probe with gradients. Full hardware and software computer equipment for the analysis of multidimensional spectra, structure simulations, and molecular dynamics calculations is also available. The following projects are being pursued:

Investigation object Principal investigator Affiliation Calcium binding peptides* A. Bierzyński IBB PAS CMTII and its mutant* A. Bierzyński IBB PAS S100A1 protein* A. Ejchart IBB PAS a70 subunit of RNA polymerase* K. L. Wierzchowski IBB PAS Protamine fragments* W.Bal Wroclaw University Cerebrospinal fluid B. Toczyłowska IBB PAS DNA bases etheno adducts J. Kuśmierek IBB PAS Modificated pirimidine nucleotides T. Kulikowski IBB PAS Products of ETAD decomposition B. Tudek IBB PAS Interactions of antimutagenic E. Grzesiuk IBB PAS agents with aflatoxins Psoralen photoadducts Z. Zarębska IBB PAS BPTI mutants M. Dadlez IBB PAS Extracts of plant tissues K. Kamieńska-Trela Inst.Org. Chem.PAS I3C-'H spin couplings in model K. Kamieńska-Trela Inst.Org. Chem.PAS compounds Interactions of calixarenes with M. Pietraszkiewicz Inst. Phys. Chem. PAS nucleotides Cofactor F430 - p-cyclodextrine J. Taraszewska Inst. Phys. Chem. PAS complex 5'mRNA cap analogues: R. Stolarski Warsaw University stability and interactions with proteins Cyclic enkephalin analogues J. Izdebski Warsaw University Daunorubicin and its derivatives I. Oszczapowicz Inst. of Antibiotics

78 Nuclear relaxation of acetylene A. Gryff-Keller Warsaw Technical Univ. derivatives Extracts of cancer tissues Z. Czerniecki Warsaw Medical Acad. Core region ofHafnia alvei A. Gamian Inst. Immunology and lipopolysaccharides Experimental Therapy Serine proteinase inhibitors J. Otlewski Wrocław University dAVP vasopressine analogues K. Rolka Gdańsk University

*Scientific projects carried out in close cooperation with the Department of Biophysics. For more information see the appropriate chapter of this report.

Publications: 3280,3300,3315,3325,3378,3379,3388, 3414,3415,3416,1107/A, 1108/A, 109/A, 1112/A, 1114/A, 1115/A, 1117/A, 1131/A, 1134/A, 1163/A. 1215/A, 1216/A 1217/A, 1218/A, 1219/A, 1239/A, 1222/A, 1223/A, 1230/A, 1323/A, 1324/A, 149/N, 154/N

79 BIOINFORMATICS UNIT Head: associate professor Piotr Zielenkiewicz

P. Zielenkiewicz, A. Kierzek, D. Plochocka, S. Kaczanowski

The Bioinformatics Unit of the Institute runs the Polish National Node of the European Molecular Network (EMBnet), which is a European non-profit organization for data access and distribution of information and software in the fields of molecular biology, genetics and biotechnology. It is composed of a network of nationally mandated and special nodes, each being a recognized biocomputing centre. The Polish National Node located at IBB was authorized by the State Committee for Scientific Research (KBN) and established in autumn 1994. From the fall 1997 the bioinformatics node at IBB serves also as a Regional Node of UNESCO's ICCBnet (International Center for Cooperation in Bioinforniatics network). The latest releases of nucleotide (EMBL - updated weekly and GenBank) and protein (SW1SSPROT, PIR and TREMBL) databases are available locally, together with a restriction enzyme database (REBASE) and a collection of protein sites (PROSITE). The Genetics Computer Group (GCG) package of over 100 computer programs for sequence manipulation, database searching, sequence analysis and structure prediction is the most widely used software. Software tools for molecular evolution studies are available (PHYLIP, CLUSTALW), as well as programs for analyzing genomic information (ACeDB). Searching remote databases is made possible through links to the relevant sites on the World Wide Web (e.g. Software Retrieval System SRS), and for clients of the BLAST and "entrez" services at the National Center for Biotechnology Information (USA). Molecular modeling studies are possible with the use of commercial packages of BIOSYM/MSI and TRIPOS Ass. The node currently serves a community of around 600 users from academic institutions in Poland. User support and training is provided, including bioinformatics courses, both national and international, organized at IBB. The responsibilities of the Unit involve also bioinformatics infrastructure planning and coordination and maintenance of IBB's World Wide Web page at http://www.ibb.waw.pl which can be readily consulted with regard to current activities of the Institute and the Unit. The research activities of the Unit concentrate on the development of new algorithms for sequence comparison and biopolymer structure analysis. Publications: 3335

80 LABORATORY OF MOLECULAR BIOLOGY (affiliated with the University of Gdańsk)

1. MOLECULAR MECHANISMS OF ASSEMBLY, INHERITANCE AND DISASSEMBLY OF BACTERIOPHAGE A REPLICATION COMPLEX

A. Węgrzyn, M. Gabig, A. Czyż, G. Węgrzyn*

In Escherichia coli cells the A replication complex, containing the O protein protected from the ClpP/ClpX protease by other elements of this complex, is inherited by one of the two daughter A plasmid copies after each replication round and can function for many cell generations. We found that the replication complex is inherited randomly by one of the two daughter A. plasmid copies, rather than preferentially inherited by either the copy carrying the parental r strand or that containing the / strand. The heritable replication complex requires transcriptional activation of the origin region to initiate a new replication round. We found that the process of transcriptional activation of or/A is stimulated by the host-encoded DnaA protein due to the activation of the pR promoter. We also demonstrated that this activation requires a contact between DnaA and the P subunit of RNA polymerase. This was the first reported demonstration that the (3 subunit may be a transcriptional activator contact site. Although the A replication complex is a stable structure, a temperature shift from 30 to 43 °C results in its disassembly. On the basis of our studies we have proposed a model for this disassembly. According to our hypothesis, the replication complex dissociates from A DNA during the negative resupercoiling phase, which follows the transient DMA relaxation caused by heat shock. Then, the complex becomes susceptible to the action of the GroEL and GroES proteins, causing its disassembly, and the liberated O protein is degraded by the ClpP/ClpX protease. Interestingly, we found that the A replication complex is resistant to heat shock in the absence of the A-encoded Cro protein. It appears that tight binding of the replication complex to A DNA in the absence of Cro prevents its dissociation after heat shock. We propose that this tight binding is due to the interaction of the replication complex with other DNA-bound proteins. Our genetic analysis suggests that DnaA is one of these proteins. Publications: 3290,3292,3338, 3340,3341,3342,3356,3398,3399, 3400,3401, 1150/A, 1194/A, 1195/A, 1246/A, 1247/A, 1250/A, 1290/A, 1291/A

* Department of Molecular Biology, University of Gdańsk

81 2. ISOLATION OF HUMAN RECOMBINANT HEAT SHOCK PROTEINS AND THEIR INTERACTIONS WITH THE p53 SUPPRESSOR GENE PRODUCT

Frank King and Maciej Żylicz*

The p53 gene is mutated in more than half of human tumors indicating that it plays a role as a tumor suppressor. We cloned the human p53 tumor suppressor gene into appropriate vectors, and the p53 gene products were purified from Escherichia coli bacteria cells and the baculovirus system. Using the same approach, we also purified several p53 "Hot Spot" mutants including: R175H, R248W, and R273H. We also purified human Hsp40 and Hsc70 which were overexpressed in E. coli. For control experiments, natural Hsc70 and Hsp90 were purified from bovine brain. We have shown that purified p53 forms large, salt resistant, aggregates. In initial experiments, we had success in disaggregating p53 by one of the bacterial chaperone machines, DnaK, DnaJ and GrpE. Members of the Hsp 100 family, bacterial ClpB or Yeast Hspl04, catalyze this reaction to a moderate extant but are not required for dissagregation. Following experiments showed that two human cytosolic chaperones, Hsp40 and Hsc70 (but not Hsp90), could also disaggregate p53 in an ATP-dependent reaction. Human Hsp40 and Hsc70 can also dissociate mutant p53 proteins, R273H, R248W and R175H in a similar manner. Modified ELISA assay was used to monitor any direct binding interactions between p53 and cytosolic chaperones. We showed that p53 displays high affinity for Hsp40, but not for Hsc70 and Hsp90. These p53-Hsp40 interactions occur in the absence of ATP. Hsp40 binding to p53 is not conformationally specific, as Hsp40 has similar binding affinity to both wild-type and mutant p53. These experiments show for the first time that not only mutant but also wild type p53 interacts functionally with heat shock proteins.

* Department of Molecular and Cellular Biology, University of Gdańsk. Present address: International Institute of Molecular and Cell Biology, Warsaw

3. MECHANISMS OF FtsH-DEPENDENT DEGRADATION OF THE a32 TRANSCRIPTIONAL REGULATOR OF ESCHERICHIA COLI AND THE ROLE OF CHAPERONE PROTEINS IN THIS PROCESS

Adam Btaszczak, Krzysztof Liberek* and Maciej Żylicz*

The Escherichia coli cr32 transcriptional regulator has been shown to be degraded by the FtsH (HflB) protease, a member of the AAA protein family. Using purified proteins we reconstituted in vitro the a32 -dependent RNA synthesis reaction in the presence of the FtsH-protease. We show that the wild type cr32 is protected from degradation by FtsH when complexed to the RNA polymerase core. Addition to this in vitro transcription system of DnaK, DnaJ and GrpE stimulates the proteolysis of cr32. We propose that the DnaK chaperone machine autoregulates its own synthesis by

82 sequestering o32 from RNA polymerase, thus making it accessible to degradation by the FtsH protease. Publications: 3435,3451,1190/A, 99/N

* Department of Molecular and Cellular Biology, University of Gdańsk

83

RESEARCH UNITS OF THE WARSAW UNIVERSITY LOCATED AT THE- INSTITUTE

DEPARTMENT-OF GENETICS (associated with the Institute) Head: professor Piotr Wegleński

1. GENETIC REGULATION OF THE ARGININE CATABOLISM IN ASPERGILLUS NIDULANS

P. Wegleński, P. Borsuk, A. Dzikowska, J. Empel, A. Grzelak

The expression of the otaA gene coding for ornithine transaminase has been characterised. One of the regulatory genes of the arginine catabolic pathway, suDpro, has been cloned and sequenced. This gene seems to code for a glycoprotease. The gene of Saccharomyces cerevisiae homologous to suDpro was disrupted and the phenotype of the diruptant is now under study. The nature of two mutations of suDpro (suD19 and suD25) was established. Functional analysis of promoters of otaA and agaA (arginase structural gene) has been advanced. The function of several DNA sequences putatively important for either general or specific regulation of these genes was established. Among others we are trying to find out the role of the arginine aptamer-resembling structures which are present in the 5'UTR of the agaA and otaA messenger RNAs. In cooperation with Dr. Anna Przykorska we found that this structures bind arginine in vitro. Preliminary results of our experiments also show that insertion of the aptamer-containing DNA fragment from Aspergillus in front of the p-galactosidase gene results in a dependence of this enzyme synthesis in bacterial cells on the presence of arginine. These results seem to support our hypothesis on the direct involvement of arginine in the regulation of expression of genes coding for arginine catabolising enzymes. Publications: 3374,3411

2. REGULATION OF mRNA TURNOVER AND PROCESSING IN YEAST MITOCHONDRIA

P. Stępień, E. Bartnik, A. Dmochowska, P. Golik, A. Dziembowski, M. Mińczuk

Regulation of gene function in yeast mitochondria is achieved mainly by processing and controlled decay of messenger RNAs. Our research concentrates on yeast mitochondria! degradosome: a multiprotein complex of nuclear origin, which interacts with mitochondria! RNAs. We have previously cloned and sequenced the yeast nuclear gene DSS1, which codes for the second subunit of the degradosome and shows RNase protein motifs. Our data indicate that in the absence of DSS1 both the processing and turnover of mRNA are altered. We isolated the PET127 gene as a suppressor of the DSS1 deletion. Our data suggest that in the yeast mitochondria there is a functional coupling between the 3' and 5'ends of mRNA. Publications : 3334,153/N, 3335

87 3. ANALYSIS OF HUMAN MITOCHONDRIAL DNA AND OF GENES INVOLVED IN MITOCHONDRIAL FUNCTION

E. Bartnik, P. Stępień, K. Mroczek, A. Dmochowska, P. Golik

Our research is aimed at analyzing pathogenic mitochondria! DNA (mtDNA) mutations in persons affected with mitochondrial diseases. As there were no data on normal mtDNA polymorphism in the Polish population we have analyzed DNA collected from 200 persons for polymorphisms for two restriction enzymes - BamHI and Hpal. Six different types of mitochondrial DNAs were found. We are also interested in analyzing human genes which are involved in the regulation of mitochondria. Our strategy was to look for human homologs of 5. cerevisiae genes known to affect mitochondrial function. We have cloned the human cDNA for one such gene, SUV3, analyzed its sequence and expression in various tissues and also located it by the FISH technique to chromosome 10. Publications: 3364,3376, 3381,144/N, 145/N INSTITUTE OF EXPERIMENTAL PLANT BIOLOGY Director: professor Andrzej Jerzmanowski

DEPARTMENT OF PLANT BIOENERGETICS Head: professor Anna Rychter

REGULATION OF PLANT ENERGY ECONOMY BY ENVIRONMENTAL CONDITIONS

A. Rychter, L Ciereszko, A. Gniazdowska, I. Juszczuk, E. Malusa, B. Szal

Phosphate deficient growth medium was used to study the regulation of oxidative metabolism (mitochondrial respiratory chain), sugar metabolism and the mechanisms which control nitrate uptake in bean plants. Phosphate limitation increased the capacity for cyanide resistant respiration and the level of alternative oxidase protein (immunoblot analysis). Mitochondrial respiration and the activities of cytochrome and alternative pathways were examined in Pi sufficient and Pi deficient roots. Lower respiration rates and the participation of the non phosphorylating alternative pathway decreased ATP concentration in the roots and leaves. The mechanisms which may control the nitrate uptake rate under low ATP concentration were investigated. In isolated plasma membrane fractions from phosphate deficient roots a decrease in total phospholipid content was observed. No changes in ATPase and proton pumping activities were noted between phosphate sufficient and phosphate deficient roots. The lower nitrate uptake rate may be explained by the increased NO 7 concentration in the root tissue resulting from decreased nitrate reductase activity and lower transport of nitrate from roots to shoots. Publications: Mikulska M, Bomsel J-L and Rychter A. 1998. The influence of phosphate deficiency on photosynthesis, respiration and adeninę nucleotide pool in bean leaves. Photosynthetica, 35, 79-88 Ciereszko I., Zambrzycka A. and Rychter A. 1998. Sucrose hydrolysis in bean roots (Phaseolus vulgaris L.) under phosphate deficiency. Plant Sci., 133, 139-144 Gniazdowska A., Mikulska M. and Rychter A. 1998. Nitrate uptake, growth and respiration during phosphate deficiency in bean roots. Biol. Plantarum, 41, 217-226 Wanke M, Ciereszko I., Podbielkowska M. and Rychter A. 1998: Response to phosphate deficiency in bean (Phaseolus vulgaris L.) roots. Respiratory metabolism, sugar localization and changes in ultrastructure of bean root cells. Ann. of Bót.-London, 82, 809- 819. • - »• ;

89 Juszczuk I. M, Malusa E. and Rychter A. M. 1998. Alternative oxidase from phosphate deficient bean roots. In: Plant Mitochondria from Gene to Function I. M. Moller, P. Gardestrom, K. Glimeiius and E. Glaser, ed. Blackhuys Publishers, Leiden, The Netherlands, pp. 53-57 Gniazdowska A., Krawczak A., Mikulska M. and Rychter A. M. 1999. Low phosphate nutrition alters bean plant ability to assimilate and translocate nitrate. J. Plant Nutr., 22, 551-563. Ciereszko I., Farrar J. F. and Rychter A. M. 1999. Compartmentation and fluxes of sugars in roots of bean (Phaseolus vulgaris L.) plants under phosphate deficiency. Biol. Plantarum 42, 223-23 I Gniazdowska A. Szal B. and Rychter A. M. 1999. The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots. Acta Physiol. Plant., 21, 263-269 Ciereszko I., Milosek I. and Rychter A. M. 1999. Assimilate distribution in bean plants (Phaseolus vulgaris L.) during phosphate limitation. Acta Soc. Bot. Pol. 68: (4) 269- 273

DEPARTMENT OF PLANT RESISTANCE Head: professor Alina Kacperska

At the Department there are two research groups working in two different research fields: Laboratory of Plant Resistance and Laboratory of Plant Molecular Biology (the later cooperating directly with IBB)

PLANT RESISTANCE TO ABIOTIC STRESSES: PHYSIOLOGICAL AND BIOCHEMICAL MECHANISMS INVOLVED IN PLANT ACCLIMATION TO LOW TEMPERATURE AND DEHYDRATION

A. Kacperska, S. Egierszdorff, M. Kitbacka-Zebalska, D. Solecka, B. Dobrska

Studies performed in 1998/99 concerned: - the role of phenylpropanoids in acclimation of biennial plants, such as winter oilseed rape (Brassica napiis L. var. oleifera L), to cold (>0°C), freezing temperatures, and radiation stress. Changes in the activity of phenyl ammonium lyase (PAL) and in the amount and composition of soluble and cell wall-localized phenylpropanoids were studied. The project was supported by K.BN and ended in 1999 by the preparation of Ph.D. thesis (by D. Solecka). - low temperature-induced modifications in mechanical properties of leaf cell waits. Experiments were performed in the cooperation with dr Jacek Żebrowski of the Institute of

90 Plant Breeding and Acclimation (IHAR). - freezing effects on membrane electric potential in leaves (in the cooperation with Dr. Maria Filek of Plant Physiology Department, PAS, Kraków). - low temperature-induced modifications in the structure of actin cytoskeleton in suspension cells (from winter oilseed rape tissues). - chilling and freezing sensitivity of young seedlings and leaves of maize (Zea mays L) of different genotypes, as indicated by changes in membrane permeability, water status and in modifications of IP3 level. Studies on maize capability to acclimate in cold were performed as well. The project was supported by the INCO-COPERNICUS grant. Publications: Kozbiat P.Z, Jerzinanowski A., Shirsat A.H., Kacperska, A. (1998) Transient freezing regulates expression of extensin-type genes in winter oilseed rape. Physiol. Plant. 103:264-270 133-146. Kacperska A. (1999) Plant responses to low temperature: signalling pathways involved in plant acclimation. In; Cold-Adapted Organisms. Ecology, Enzymology and Molecular Biology. (R. Margesin and F. Schinner, eds), Springer Verlag, pp: 79-101. Kubacka-Zębalska M., Kacperska, A. 1999. Low temperature-induced modifications of cell wall content and polysaccharide composition in leaves of winter oilseed rape (Brassica napus L., var. oleifera L.). Plant Sci. 148: 59-67. Stefanowska M., Kuraś M., Kubacka-Zębalska M., Kacperska, A. (1999) Low temperature affects pattern of leaf growth and structure of cell walls in winter oilseed rape (Brassica napus L., var. oleifera L.) plants. Ann. Bot. 84: 313-319. Sołecka D., Boudet A., Kacperska A. (1999) Phenylpropanoid and anthocyanin changes in low-temperature treated winter oilseed rape leaves. Plant Physiol. Biochem. 37(6): 491-496

PLANT MOLECULAR BIOLOGY LABORATORY Head: professor Andrzej Jerzmanowski

STRUCTURE AND FUNCTION OF PLANT CHROMATIN PROTEINS

A. Jerzmanowski, M. Prymakowska-Bosak, M. Przewloką, P. Koźbial

To examine the function of linker histone variants in plants we constructed transgenic tobacco in which major somatic histone variants H1A and H1B were decreased in chromatin by about 75%. The decrease of major variants was accompanied by a compensatory increase of the four minor variants: H1C-F characterised by considerably smaller and less charged molecules. This offered a unique chance of examining the consequences at the organismal and molecular level of a major remodelling of the

91 chromatin set of linker histones. The plants with drastically changed proportions of HI variants retained normal spacing of nucleosomes but their chromatin was less compacted than that of control plants. They grew normally but showed characteristic aberrations in flower development and almost complete male sterility. These mutations were correlated with changes in temporal but not spatial pattern of expression of developmental genes that could be linked to the abnormal flower phenotypes. Preceding these changes in flower morphology were strong aberrations in male gametogenesis. The earliest symptoms could be accounted for by the disturbances in correct pairing and/or segregation of homologous chromosomes in meiosis. No aberrations were observed during mitosis. We conclude that in plants the physiological stoichiometry and distribution of linker histone variants is crucial for correct course of male meiosis and the subsequent development of functional pollen grains. Publications: Ślusarczyk, J., Prymakowska-Bosak, M, Przewloką, M., Jerzmanowski, A. and Kuras, M. (1999) Ultrastructural Organization of Leaves of Transgenic Tobacco Overexpressing Histone HI from Arabidopsis thaliana. Ann. Bot. 84, 329-335. Prymakowska-Bosak, M., Przewloką, M.R., Ślusarczyk, J., Kuraś, M., Lichota, J., Kiliańczyk, B. and Jerzmanowski, A. (1999) Linker histones play a role in male meiosis and the development of pollen grains in tobacco. Plant Cell 11, 2317-2329.

DEPARTMENT OF PLANT GROWTH AND DEVELOPMENT Head: professor Andrzej Podstolski

1. REGULATION OF SECONDARY METABOLITE BIOSYNTHESIS AND ACCUMULATION IN PLANTS

A. Podslolski, R. Dębowska, J. Malinowski'.

The biosynthetic pathway of benzoate derivatives in Vanilla planifolia cultured in vitro was studied. It was established- that the benzoate pathway branches out of the phenylopropanoid pathway at the level of jD-coumaric acid. The key enzyme regulating input into the benzoate pathway - /)-bydroxybenzaldehyde synthase converting p- coumaric acid into /^hy'droxybenzaldehyde - was purified, sequenced (in colaboration with Dr R.A.Dixon from the-Noble Foundation, Ardmore, OK, USA) and its properties are currently being investigated. Parallely, the properties of polyphenol oxidase (PPO) from vanilla were studied. Preliminary results suggest an involvement of PPO in ort/io-hydroxylation of vanillin precursors in cultured vanilla tissues. Publications:

92 Havkin-Frenkel D., Podstolski A., Mikołajczyk M., Molęcki P., Witkowska E. (1999) Vanillin Biosynthesis - An Overview. Plant Cell and Tissue Culture for Food Ingredients. In: Tong-Jen Fu, Gurmet Singh and Wayne R.Curtis, Eds. Kluwer Academic Plenum Publishers, New York, Boston, Dordrecht, London, Moscow, pp. 35-43. Dębowska R. and Podstolski A. (1998) Properties of polyphenol oxidases from Vanillaplanifolia (Andr.) tissues cultured in liquid medium. Acta Physiol. Plant. 20:17.

2. METABOLIC CONTROL OF SEED DORMANCY AND GERMINATION

S. Lewak, R. Bogatek, B. Maciejewska, E. Ghichowska

\l Studies on apple seed dormancy were continued. Cyanide-mediated hydrolysis of stored oligosaccharides and degradation of its products in relation to embryonic dormancy were investigated. It is postulated that endogenous HCN in apple seeds controls the removal of embryonic dormancy in parallel to the described earlier control executed by light and gibberellin. The results obtained suggest that hydrolysis of oligosaccharides and their catabolism are a target for cyanide. II Studies on osmoconditioning (priming) as a method of seed germination improvement were performed. The results obtained suggest that reinvigoration of aged sunflower seeds by priming was associated with a fall in lipid peroxidation processes and an increase in the cell antioxidative potential (enhancement of SOD - superoxide dismutase, CAT - catalase and GR - glutathione radectase activity). It is proposed that the cell detoxifying system plays a key role in the maintenance of seed vigour. Publications: Bogatek R., Come D., Corbineau F., Picard M.A., Maciejewska B., Lewak S. (1999) Sugar metabolism as related to the cyanide-mediated elimination of dormancy in apple embryos. Plant Physiol. Biochem., 37:577-585. Bailly Ch., Bogatek R., Corbineau F., Come D. (1998) Seed germination and seedling growth as related to scavenging or accumulation of active oxygen species in sunflower. Winter Meeting of the Society for Free Radical Research on Oxygen, Free Radicals and Oxidative Stress in Plants, 17 - 19 of December, Granada, Spain. Bogatek R., Bailly Ch., Corbineau F., Come D. (1999) Involvement of oxidative processes during sunflower seed priming and subsequent seedling growth. International Workshop on Seeds, 24 - 29 of January, Merida, Mexico.

93 DEPARTMENT OF PLANT MORPHOGENESIS Head: professor Mieczysław Kuraś

STRUCTURAL AND ULTRASTRUCTURAL INVESTIGATIONS OF ZYGOTIC AND SOMATIC MORPHOGENESIS

M. Kuraś, R. Hasz, A. Majewska, M. Stefanowska, T. Tykarska, A. Tylicki

The department investigates the following subjects: - developmental regularities of zygotic and somatic embryogenesis and postembryogenesis of Brassica napus L. and Gentiana sp., organogenesis in in vitro culture of Solarium lycopersicoides (structural, ultrastructural, and immunocytochemical investigations; analysis and localization of secondary metabolites - mainly phenolic compounds). - influence of anticancer substances (extracts of Taxus, Rhodiola, Catharanthus and another plants) on mitotic activation and ultrastructure of meristematic cells. Publications: Kuraś M., Stefanowska-Wronka M., Lynch J. M., Zobel A. M., 1999. Cytochemical localization of phenolic compounds in columella cells of the root cap in seeds of Brassica napvs I. Changes in the localization of phenolic compounds during germination. Ann. Bot. 84: 135-143. Pietrosiuk A., Furmanowa M., Zobel A., Kuraś M.,MichalakA., 1999. Cytological changes in meristematic cells ofAllmm cepa L. root tips treated with extracts from callus of Catharanthus roseus (L.). Acta Soc. Bot. Pol 68: 109-118. Ślusarczyk J., Prymakowska-Bosak M. Przewloką M., Jerzmanowski A., Kuraś M., 1999. Ultrastructural organization of leaves of transgenic tobacco overexpressing histone HI from Arabidopsis thaliana. Ann. Bot. 84: 329-335.

94 ENVIRONMENTAL PLANT POLLUTION LABORATORY Head: Dr. Sc. Małgorzata Wierzbicka Dr Danuta Maria Antosiewicz, mgr Renata Załęcka, Ewa Fluks

The main aim of the research is to find the defence mechanism(s) of cells and whole plants againts heavy metals (lead). The uptake, movement and localization of lead in plant cells and tissues is studied mainly with physiological and cytological techniques. The resistance and sensitivity of plants to heavy metals is ivestigated.

1. THE EFFECT OF LEAD ON THE CELL CYCLE

M. Wierzbicka

Allium cepa L. adventitious roots were treated with lead. The cell cycle was studied using pulse labeling with 3H-thymidine. Mitotic activity kinetics, occurrence of disturbed mitoses (c-mitoses), and level of DNA synthesis were examined. It was found that lead prolonged the cell cycle and that cells in two phases of the cycle, G2 and S, differed in their sensitivity to lead. Cells in G2 were more sensitive—lead lengthened their cycle by 216% and disturbed the course of cell division by causing c-mitoses. Cells in S-phase were less sensitive. Their cell cycle was longer by 55%. They went through their G2 phase without major disturbances, mitosis in these cells was normal. During treatment of Allium cepa with lead, its destruptive effects on cells were exerted only during the first few hours (around 6 h) of incubation. That is when the inhibition of mitotic activity, numerous disturbances of cell division, a decline in the number of cells synthesizing DNA, and a lower level of DNA synthesis were observed. As the incubation continued, the above processes were found to return to normal. In the discussion, data are presented supporting the hypothesis that during the initial period of exposure of Allium cepa to lead, this metal enters both the root apoplast and symplast, exerting a destructive effect on cells, while later, lead penetrates only into the root apoplast, and in this way remains harmless to cells. Publications: Michalak E. and Wierzbicka M. 1998. Differences in lead tolerance between Allium cepa plants developing from seeds and bulbs. Plant Soil, 199:251-260. Wierzbicka M., Panufnik D., 1998. The adaptation ofSilene vulgaris to growth on a calamine waste heap (S. Poland). Environ. Pollut., 101: 415-426. Wierzbicka M., 1998, Lead in the apoplast of Allium cepa L. root tips - ultrastructural studies. Plant Sci., 133:105-119. Michalak E., Wierzbicka M., 1998, Accumulation of heavy metals in the storage organ of Allium cepa (onion), In: Ecophysiological Aspects of Plant Reactions to Abiotic Stress. Grzesiak S., Skoczowski A., Miszalski Z., Eds: PAN, Kraków pp 261-266.

95 Wierzbicka M., 1999, Comparison of lead tolerance in Allium cepa with other plant species, Environ. Pollut. 104: 41-52. Wierzbicka M., Obidzińska J., 1998, The effect of lead on seed imbibition and germination in different plant species, Plant Sci. 137: 155-171. Wierzbicka M., 1999, The effect of lead on the cell cycle in the root meristem of Allium cepa L., Protoplasma, 207: 186-194.

2. THE ROLE OF CALCIUM IN HEAVY-METAL ACCUMULATION AND TOLERANCE

D. Antosiewicz

Plants that hyperaccumulate heavy metals could be used in the process of phytoremediation to clean up contaminated soil. It is well known, however, that a high level of heavy-metal tolerance of a plant is not necessarily accompanied by its ability to hyperaccumulate metals. With the phytoremediation in mind, as the practical approach to the problem, the main aim of the research is the role of Ca in both the accumulation of and tolerance to heavy metals. For the experiments four crop species (tomato, flax, mustard and sunflower) and four populations (Pb-to!erant and non-tolerant) of herbs Silene inflata and Biscutella laevigata were used. It was shown that heavy-metal tolerance and high Pb accumulation were accompanied by a specific Ca metabolism (high level of Ca-deficiency tolerance, ability to take up calcium in the presence of Pb); the level of tolerance increases with the rise of Ca concentration in the medium. Tomato, a model plant with the highest Pb- tolerance, was mutagenized with the use of EMS and selected towards Pb-hypertolerance. Seeds were collected from the second generation and further study will be conducted. On the other hand, the research on a clone from wheat roots with the use of yeast revealed that Ca, Cd and Pb ions are transported through plasmalemma into the cell as a result of the activity of the cDNA (named LCT1). The existence of a common (and/or mutually related) route of transport of the above mentioned cations could be one of the reasons of the Ca-Pb relationship found. To get an insight into that relation tobacco transformation with LCT in the collaboration with IBB was being started. Publications: Clemens, S., Antosiewicz, D.M., Ward, J.M., Schachtman, D.P. and Schroeder, J.I. (1998) The plant cDNA LCT1 mediates the uptake of calcium and cadmium in yeast. Proc.Nat.Acad.Sci. USA 95:12043-12048. Antosiewicz D.M. and Wierzbicka M. (1999) Localization of lead in Allium cepa L cells by electron microscopy. J. Microsc. 195:139-146.

96 RESEARCH GRANTS

1. Research projects financed by the State Committee for Scientific Research (KBN)

1.1. Włodzimierz Zagórski "Structural and functional analysis of the genome of the yeast Saccharomyces cerevisiae", 1995-1998;

1.2. Magdalena Boguta "Nuclear control of mitochondrial function in yeast", 1995-1998;

1.3. Celina Janion "Modification of bases - induction of stable DNA replication", 1995-1998;

1.4. Iwona Fijałkowska "Fidelity of replication of the leading and the lagging DNA strands in E.coli", 1995-1998;

1.5. Zygmunt Cieśla "Isolation of a novel Saccharomyces cerevisiae gene, the expression of which is induced by DNA damage", 1995-1998;

1.6. Piotr Cegłowski "Genes functioning in the stability region segB from the plasmid pSM19035", 1995-1998;

1.7. Witold Jachymczyk "The role of DNA polymerases in repair of various kinds of damage in DNA of Saccharomyces cerevisiae", 1995-1998;

1.8. Jarosław Kuśmierek "Mechanism of action and potential inhibitors of 3-methyladenine-DNA glycosylase of bacterial and human origin", 1996-1998;

1.9. Anna Przykorska "RNA structure probing with plant nucleases. Cloning of genes coding for the Rn and ChS nucleases", 1996-1998;

1.10. Anna Kurlandzka "The search for the function of the essential S.cerevisiae gene U17918", 1996- 1998;

97 1.11. Jacek Ba rdowski "Cellobiose - induced (3-galactosidase activity in Lactococcus lactis", 1996- 1998;

1.12. Włodzimierz Zagórski "Constructions of virus-resistant potato transformants", 1996-1998;

1.13. Anna Chaclmlska "Construction of transgenic plants resistant to potato virus Y", 1996-1998;

1.14. Maria Bucholc "Isolation, recombination and application of linear plasmids in plant transformation", 1996-1998;

1.15. Michał Dadlez "Rational design and production of specific serine proteinase inhibitors based on molecular modeling, kinetic data analysis and site directed mutagenesis", 1996-1998;

1.16. Tadeusz Chojnacki "Rubber-like polyprenoid lipids of plant origin", 1996-1998;

1.17. Andrzej Ożyhar (Wroclaw Polytechnic School) Krystyna Grzelak "Expression of protein elements of functional ecdysteroid receptor in bacterial and yeast cells", 1996-1999;

1.18. Tadeusz Kulikowski "Synthesis, investigation of the structure and conformation of novel antitumor, antiviral, and antiparasitic nucleosides and nucleotides and invesigation of their interactions with thymidylate synthase", 1996-1999;

1.19. Grażyna Palamarczyk "Improvement of protein production-secretion in Trichoderma reesei", 1997- 1999;

1.20. Joanna Michalik "Nuclear polyhedrosis virus from Stilpnotia salicis (Lymantriidar) - a more effective biopesticide. Genome characteristics and virulence", 1997-2000;

1.21. Ewa Bartnik "The molecular basis of mitochondria! diseases", 1997-1999;

1.22. Agnieszka Sirko "Oral vaccines from plants: construction of transgenic plants producing urease of Helicobacter pylori", 1997-1999;

98 l .23. Irena Pietrzykowska "SOS dependent mutagenesis: possible mechanism of down regulation of the SOS response and the role of UmuD'C proteins in mutagenesis independent of DNA replication", 1997-1999;

1.24. Ewa Kula-Świeżewska "Prenylation of plant proteins", 1997-1999;

1.25. Urszula Maciejewska "Somatic hybridization - pathogen resistance in potato", 1997-2000;

1.26. Anna Szkopinska "Genetic and biochemical characterization of polyprenol synthesis in eukaryotes", 1997-2000;

1.27. Jacek Hennig "Study of virus resistance in transgenic potato plants expressing the replicase gene of potato leafroll virus", 1997-2000;

1.28. Ryszard Otlewski (Wroclaw University) Michał Dadlez "Selection from protein libraries on the M13 phage applied for the production of new proteinase inhibitors and optimization of their structural specifity and folding", 1997-2000;

1.29. Małgorzata Łobocka "Studies on the partition of low copy number plasmids to daughter cells and cell division", 1997-1999;

1.30. Joanna Rytka "The hem mutants of the yeast S.cerevisiae as a tool in studies of the interacellular transport and regulatory role of heme", 1996-1998;

1.31. Andrzej Jerzmanowski "Studies on the function of histone HI in eukaryotic cell, the identification of protein factors responsible for transcriptional accessibility of plant chromatin and the analysis of the mechanism of SWI/SNF dependent transcription", 1997-2000;

1.32. Grażyna Dobrowolska "The role of protein kinases in transduction of salinity stress in plants", 1997- 1999;

1.33. Andrzej Paszewski "Molecular analysis of structural and regulatory genes in fungal sulphur metabolism", 1998-2001;

99 1.34. Beata Burzyńska "Analysis of European bison population with the use of molecular biology methods, particularly with respect to a male disease of unknown ethiology", 1998-2001;

1.35. Anna Chełstowska "Functional analysis of the newly discovered genes of Saccharomyces cerevisiae", 1998-2001;

1.36. Danuta Hulanicka "Characterisation of transcription activation by the CysB regulator in Escherichia coli", 1998-2000;

1.37. Jacek Hennig

"Role of H2C>2 in the response of tobacco plants to TMV infection", 1998- 2000;

1.38. Magdalena Boguta "Prions in the yeast Saccharomyces cerevisiae", 1998-2001;

1.39. Danuta Hulanicka "Determination of the function of the products of ORFO and ORF1 of potato leafroll virus; construction of infectious clones under the 35S promoter", 1998-2000;

1.40. Krzysztof Pluta " MAPI, a novel yeast gene: functional analysis", 1998-1999;

1.41. Grażyna Jagura-Burdzy "Characterization of plasmids from clinical collection of Pseudomonas aeruginosa", 1999-2002;

1.42. Andrzej Ejchart "Structure determination of the S100A1 protein and conformational studies of calcium binding peptides by means of NMR", 1999-2002;

1.43. Monika Hryniewicz "Molecular mechanisms of gene expression control in sulphur metabolism of Escherichia coll"', 1999-2002;

1.44. Iwona Fijałkowska "Fidelity of the leading and lagging DNA strands replication in Escherichia coli cel Is", 1999-2001;

100 1.45. Teresa Żołądek "Role of ubiquitination in protein transport in the yeast Saccharomyces . cerevisiae", 1999-2002;

1.46. Zygmunt Cieśla "DIN7 and DIN8/UMP1, novel genes involved in the response of Saccharomyces cerevisiae cells to DNA damage", 1999-2001;

1.47. Andrzej Pałucha "Cloning of infectious clones of potato virus Y and construction of chimeric viral cDNA carrying reporter genes", 1999-2001;

1.48. Jacek Hennig "Investigation of the defense mechanisms that are activated in plants during infection", 1999-2001;

2. International co-financed Research Projects

2.1. POLONIUM projects: Polish State Committee for Scientific Research - French Government

2.1.1. Grażyna Palamarczyk "Genetic and biochemical characterization of dolichol chain synthesis in eukaryotic cells", 1996-1999;

2.1.2. Jacek Bardowski "Production and genetic regulation of amylase in Lactococcus lactis", 1998;

2.1.3. Jacek Bardowski "Genetic studies and modification of starch catabolism in Lactococcus lactis", 1999.

3. Research projects financed by International Organizations

3.1. EU COPERNICUS project ERB IC15-CT96-090Q Grażyna Muszyńska "Metabolic regulation in plants. Specificity of signal transduction by the phospholipid-dependent protein kinase from seedlings and prediction of potential substrates of this enzyme among plant proteins", 1997-1999;

101 3.2. EU COPERNICUS project ERB IC15-CT96-0901 Alina Kacperska (Warsaw University) Grażyna Muszyńska "Manipulation of the lipid composition and physical state of plant membranes for improving tolerance to temperature stress", 1997-1999;

3.3. EU-B1OTECH Concerted Action B104-CT-0099 Piotr Ceglowski "Mobile elements' contribution to bacterial adaptability and diversity", 1998- 2000.

3.4. EU Supported International Agency for Researchon Cancer Project ENV4- CT97-0505 Barbara Tudek "Exocyclic adducts as new risk markers for DNA damage to man", 1998;

3.5. EU PHARE SCI-TECH II PL.9611-03.02 Piotr Zielenkiewicz "Commercial molecular methods for potato virus diagnosis", 1998-1999;

3.6. UNESCO project SC/RP 206.902.7 Piotr Zielenkiewicz "The development of Web based bioinformatics services", 1997-1999;

3.7. Stefan Batory (Soros Foundation branch) project 04/06/MT Piotr Zielenkiewicz, Michał Mińczuk (UW) "Molecular biology education on the Internet", 1997-1999;

3.8. The Wellcome Trust project 056022/Z/98/Z Grażyna Jagura-Burdzy "Genome partitioning in Pseudomonas aeruginosa", 1999-2002;

3.9. United States - Polish Maria Skłodowska Curie Joint Fund U project PAN/HHS-96-248 Iwona Fijałkowska "Mutagenesis in Leading and Lagging Strands of DNA Replication", 1996-1999;

3.10. Swiss National Foundation project Magdalena Boguta "Effect of the cytosolic chaperone Msp 104 on the mitochondrial protein Nam9", 1997-1998;

102 MEETINGS, SYMPOSIA, COURSES (organized by the Institute of Biochemistry and Biophysics)

1998

"Plant Transport Systems and Pathogens" Prof. S. Lewak, October 16, 1999

"Inhibitors of Protein Kinases" Prof. G. Muszyńska, Prof. D. Shugar, Dr. G. Dobrowolska, September 15-20, 1999

1999

"Molecular Mechanisms of Gene Expression: Five Years of Polish-French Common Research (1993-1998)" Prof. S. Lewak, June 10-11, 1999

103 INSTITUTE SEMINARS Presented by the Staff of IBB PAS and (*) Invited Guests.

1998

I. In vivo protein interactions within the E. coli DNA polymerase III core. Piotr Jończyk; 06.01.98

*2. Studies of the genes coding for the eukaryotic translation initiation factor 4E (etF4E) mArabidopsis. Christophe Robaglia /Laboratoire du Metabolisme Carbone, CEA-Cadarache, France/; 13.01.98

3. The expression of Potato Leafroll Virus genome. Marek Juszczuk; 20.01.98

4. Control of translation fidelity in yeast. Magdalena Boguta; 27.01.98

5. Plants as bioreactors. Agnieszka Sirko; 03.02.98

6. Electrostatics of macromolecules: a more advanced approach. Sergei Gavriouchov; 10.02.98

7. RTG genes are involved in communication between organelles in the cell of Saccharomyces cerevisiae. Anna Chelstowska; 24.02.98

*8. Transfecting peptide originating from the viral protein. Jadwiga Chroboczek /Institut de Biologie Structurale, Grenoble Cedex, France/; 5.03.98

9. Chromatin and Gene Expression. Radosław Tomaszewski; 10.03.98

10. Regulation of agaA expression in Aspergilhts nidulans. Piotr Borsuk; 17.03.98

I1. Functional analysis of open reading frames (ORFs) of S.cerevisiae. Bożenna Rempoła; 24.03.98

*12. MutatortRNA Małgorzata Słupska/University of California, Los Angeles, USA/; 31.03.98

104 13. Mechanism of UV-induced mutagenesis independent of DNA replication. Irena Pietrzykowska; 07.04.98

14. Proteins controling expression of the cysteine regulon. Anna Węgleńska; 14.04.98

15. Cloning of Trichoderma reesei genes encoding enzymes of early steps of glycosylation. Joanna Kruszewska; 12.05.98

16. Characterization of a novel DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7. Piotr Mieczkowski; 19.05.98

*17. DNA methylation, gene expression and genome stability in Ascobolus. Jean-Luc Rossignol /Institut Jacques Monod, Paris Cedex, France/; 22.05.98

18. Expression ofEcR and USP genes in the Pichiapastoris yeast cells. Krystyna Grzelak; 26.05.98

* 19. Aspergillus nidulans as a model organism to study cell biology and differentiation. Reinhard Fisher /Max-Planck Institut fur terrestrich Mikrobiologie, Germany/; 28.05.98

*20. Molecular analysis of the role of bacteria for the plant-mycorrhizal symbiosis. Natalia Requena /Max-Planck Institut fur terrestrich Mikrobiologie, Germany/; 28.05.98

*21. Molecular events in the initiation of replication of a broad host range plasmid. Donald Helinski /University of California, San Diego, USA/; 29.05.98

22. The genome of PI bacteriophage - analysis of the complete nucleotide sequence. Małgorzata Łobocka; 02.06.98

*23. Protein mobility and electron transfer within a natural protein complex as studied by site directed mutagenesis and with monoclonal antibody. Florence Lederer/Laboratoire d'Enzymologie du CNRS, Gif-sur-Yvette, France/; 03.06.98

*24. Silencing of genes flanking the PI plasmid centromere. Michael Yarmolinsky/National Cancer Institute, Bethesda, USA/;10.06.98

*25. The casein breakdown pathway in Lactococcus lactis as a reason to develop an induced lysis system for this lactic acid bacterium. Jan Kok/Dept. of Genetics, University of Groningen, Haren, Holandia/;15.06.98

105 26. Amyloidosis and prion structure. Andrzej Bierzyński; 16.06.98

27. Potato virus Y (PVY): genome polymorphism and engineering of virus resistant plants. Anna Chachulska; 30.06.98

*28. Elicitor recognition and signal transduction in plant defense. Dierk Scheel /Institute for Plant Biochemistry, Halle (Saale), Germany/; 18.06.98

*29. Bacterial signal transduction: crystal structure of enzyme IIA and models of its interactions with other components of the lactose phosphotransferase system. Piotr Sliz /Protein Crystallography Lab, University of Toronto, USA/; 07.09.98

*30. part 1: A new strange medium for extraction and bioreaction: the supercritical fluid, part 2: lipase-catalysed lipid transformation. Alain Marty /CNRS-INSA, Toulouse, France/; 02.09.98

*31. An in vivo physiological analysis of metabolic regulations in Lactococcus lactis. Pascal Loubiere /CNRS-INSA, Toulouse, France/; 02.09.98

*32. Protein folding in the mitochondria! matrix. Sabinę Rospert /Biozentrum, University of Basel, Switzerland/; 03.09.98

*33. Transcription initiation of stress genes in archea: peculiarities and puzzles. Alberto Macario /Wadsworth Center, Albany, NY,USA/; 10.09.98

*34. GATA factor regulation of nitrogen catabolite gene transcription - control by competition. Terrance G. Cooper/University of Tennessee, Memphis, Tennessee/; 10.09.98

35. Protein phosphorylation and signal transduction. Edwin Krebs /University of Washington, USA/; 15.09.98

*36. Global regulators of the genomic survival in bacteria. Christopher M. Thomas /The University of Birmingham, U.K7; 25.09.98

*37. Progress in anti-HIV chemotherapy. Tadeusz Kulikowski; 20.10.98

*38. Calcium-dependent protein kinases and their roles in signal transduction. Alice C. Harmon /University of Florida, USA/; 22.10.98

39. Chromatin structure transactions and regulation of transcription. Jan Brzeski; 03.11.98

106 40. Protein prenyltransferases of plants. Ewa Świeżewska; 17.11.98

*41. Carnitine in brain: a challenge for a biochemist. Katarzyna Nałęcz /Nencki Institute of Experimental Biology, PAS/; 01.12.98

42. Construction and unexpected properties of a mitochondnal system for expressing nuclear genes in the yeast S. cerevisiae. Paweł Golik; 08.12.98

43. Solution structure of rice iipid transfer protein. Jarosław Poznański; 15.12.98

*44. Ribosomal RNA maturation in yeast. Joanna Kufel /University of Edinburgh, Edinburgh, UK/; 22.12.98

1999

45. Sulphur metabolism regulatory genes in Aspergillus nidulam and possible interactions between their products. Renata Natorff; 05.01.99

*46. The mevalonate-independent route to isopentenyl diphosphate in bacteria and plants: elucidation and distribution. Michel Rohmer /Universite Louis Pasteur, Institut Le Bel, Strasbourg Cedex, France/; 12.01.99

47. Localization and characteristics of chloroacetaldehyde-induced DNA damage in human p53 gene. Jarosław Cieśla; 02.02.99

*48. Protein phosphatase 2A, regulation of activity and the role in signal transduction of eukaryotic cell. Stanisław Żołnierowicz /Intercollegiate Faculty of Biotechnology UG-AMG, Gdańsk/; 09.02.99

49. Safety with ultraviolet sun and lamps. Zofia Zarębska; 16.02.99

*50. Progress in studying the chloride channels in epithelia: the example of CFTR. Aleksander Edelman /INSERM, Faculte de Medicine Necker, Paris Cedex, France/; 23.02.99

107 *51. Role of PPAR 2-nd nuclear receptor in bone-marrow ageing. Beata Łęcka - Czernik /Univ. Arkansas Medical Sciences, Dept. Geriat, USA/; 24.02.99

*52. Cellular signalling in responce to ionizing radiation. Irena Szumiel /Institute of Nuclear Chemistry and Technology, Warszawa/; 26.02.99

53. Signal transducers in plant pathogenesis. Andrzej Talarczyk; 02.03.99

54. Early steps in the folding of proteins; a BPTI study. Konrad Zdanowski; 09.03.99

*55. Mechanisms and factors affecting the genetic instability of human tri-nucleotide repeat sequences (TRS). Paweł Parniewski /Center of Microbiology and Virology, PAS, Łódź/; 30.03.99

56. Danio rerio, a model of the early development of vertebrates. Jacek Topczewski; 30.03.99

57. Regulatory gene omega of plastnid pSM19035. Izabela Sitkiewicz; 13.04.99

58. Adaptive mutations. Aneta Nowosielska; 20.04.99

*59. How do serine proteases work ? Harvey Rubin /School of Medicine, University of Pennsylvania, USA/; 26.04.99

*60. Single-stranded DNA viruses of plants, a multipartite puzzle. Bruno Gronenborn /Institut des Sciences Vegetales CNRS, Gif-sur-Yvette, Francja/; 28.04.99

61. Glycosylation defects corrected by the changes in GDPMannose level. Grażyna Palamarczyk; 04.05.99

*62. Plant proteomics: the tentoxin binding site in ATPase, a model case. Meir Edelman /The Weizmann Institute of Sciences, Rehovot, Israel/; 05.05.99

63. Expression of heterologous genes in Pichiapastoris cells. Krystyna Grzelak; 11.05.99

*64. Molecular aspects of staphylococcal pathogenesis. Gregory A. Bohach /University of Idaho, Moscow, USA/; 18.05.99

108 65. Examples of protein molecular applications: farnesyl synthase, glucose 6-phosphate dehydrogenase and VPg protein. Danuta Płochocka ; 25.05.99

*66. Statistics in experiment planning and conclusion drawing: possibilities and dangers. Daniel Rabczenko /Department of Statistics, National Institute of Hygiene, Warszawa/; 01.06.99

67. The effect of the product of the S. cerevisiae DIN7 gene on the stability of mitochondrial DMA. Marta Fikus; 08.06.99

*68. Birth and death of mitochondrial .proteins. Gottfried Schatz /Biozentrum der Universitat Basel, Switzerland/; 11.06.99

69. Use of proton exchange rates in the investigation of the protein and peptide structures. Jacek Wójcik; 22.06.99

*70. The HIV protein Net disturbs signal transduction and protein sorting. Jacek Skowroński /Cold Spring Harbor, USA/; 22.06.99

*71. Nucleus/cytosol exchange: genetic studies in S. cerevisiae. Anita Hopper /The Pennsylvania State University, Hershey, USA/; 29.06.99

*72. Salicylic acid- and nitric oxide-mediated signal transduction in plant disease resistance. Daniel Klessig /Waksman Institute, RUTGERS, USA/; 02.08.99

*73. Mutagenicity of genotoxic and non-genotoxic carcinogens in Big Blue transgenic mice. Barbara Shane /Louisiana State University, USA/; 09.08.99

*74. On the function of pericentromeric silencing in a plasmid. M. Yarmolinsky /National Institutes of Health, Bethesda, USA/; 28.09.99

*75. Cellular determinants of the inverse cross sensivity of mouse lymphoma L5178Y cell lines to ionizing radiation and hydrogen peroxide. M. Kraszewski /Institute of Nuclear Chemistry and Technology, Warszawa/; 12.10.99

*76. Lessons about plasmid evolution based on studies with IncP-1 and IncP-9 plasmids. Christopher M. Thomas /University of Birmingham, U.K/; 21.10.99

109 *77. Cellular responses to mitochondnal dysfunctions: from genetics to genomics. Ronald A. Butow /The University of Texas, Southwestern Medical Center, USA/; 22.10.99

78. Search for genes responsible for neuron plasticity in bees. R. Kucharski; 26.10.99

*79. How does a bacteriophage cross the bacterial cell wall? The lytic transglycosylase of bacteriophage PI. Hansjoerg Lehnherr/Uniwersytet w Odensee , Denmark/; 29.10.99

80. Transgenic plants as a source of vaccine against Helicobacter pylori. R. Brodzik; 16.11.99

*81. Mechanisms of action of glutathione transferase that prevents the oxidative stress - information from the crystal structure. P. Zimniak /University of Arkansas, USA/; 24.11.99

82. Genes affecting mitocondrial translation identified as the suppressors of defective mitochondria! ribosome in yeast. A. Konopinska;30.11.99

83. Biological effects induced by halogen light irradiation in Escherichia coli K12 strains. A. Wojcik;02.11.99

84. Unusual structure of M. (; ) BssHlI - procaryotic DNA-methyltransferase. R. Iwanicka-Nowicka; 09.11.99

*85. Regulation of glycolytic genes in L lactis. Emanuel Jamet/CRJ-INRA, Jouy-en Josas, France/; 07.12.99

86. Genetic regulation in Lactoccocus lactis — research and applications. J. Bardowski;7.12.99

87. Sister chromatid cohesion and colony formation in Saccharomyces cerevisiae. Anna Kurlandzka; 14.12.99

88. Synthesis and biological activity of a new inhibitor of HBV. Maria Bretner; 21.12.99

89. Replication fidelity of the RB69 phage DNA polymerase. Anna Bębenek; 22.12.99

110 LIST OF PUBLICATIONS

This list comprises publications of Institute Staff in 1998-1999. Publication numbers are referred to in reports, above. Abstracts of oral and poster presentations at scientific meetings are marked A (e.g. 1105/A) and patents are marked P (e.g. P4). The list includes also titles of Dissertations for the M. Sc., Ph. D. and Doctor Habilitatus (Dr. Sc.) degrees. M. Sc. degrees are issued by collaborating Institutions of Higher Education, Ph. D. and Dr. Sc. - by the Institute. Dissertation numbers are marked N.

Ill PUBLICATIONS

3269. KROKOSZYŃSKA,!., DADLEZ,M., OTLEWSKI,J. Structure of single-disulfide variants of bovine pancreatic trypsin inhibitor (BPTI) as probed by their binding to bovine (i-trypsin. J. Mol. Biol. (1998) 275. 503-513

3273. MICHALIKJL, SZOŁAJSKA.E., LOMBARSKA-ŚLIWIŃSKA,D., ROSIŃKI.G., KONOPIŃSKA,D. Hypertrehalosaemic insect peptide periplanetic CC-2 and its analogues: Synthesis and biological evaluation. Eur. J. Entomol. (1998) 95, 1-7

3278. NATORFF,R., PIOTROWSKA.M., PASZEWSKI.A. The Aspergillus nidulcms sulphur regulatory gene sconB encodes a protein with WD40 repeats and an F-box. Mol. Gen. Genet. (1998) 257, 255-263

3279. POKORSKI,M, MATYSIAK,Z. Fatty acid acylation of dopamine in the carotid body. Med. Hypotheses (1998) 50, 131-133

3280. EJCHART.A., SIEDLECKA,M., STICHT,H., BIERZYNSKI,A. NMR studies of secondary structure in calcium binding hexadecapeptide. Bull. Pol. Ac.: Sci. Chem. (1998)46, 1-8

3282. GRZESIUK,E., JAN1ON,C. Mutation frequency decline in MMS-treated Escherichia coli K12 mutS strains. Mutagenesis (1998) 13, 127-132

3283. SIEŃKO,M., TOPCZEWSKI.J., PASZEWSKI.A. Structure and regulation of cysD, the homocysteine synthase gene of Aspergillus nidulans. Curr. Genet. (1998) 33, 136-144

3284. KRAWIEC.K., KIERDASZUK.B., KALINICHENKO,E.N., MIKHAILOPULOJ.A., SHUGAR,D. Unusual nucleoside triphosphate donors for nucleoside kinases: 3'- Deoxyadenosine-2'-triphosphate and 2'-deoxyadenosine-3'-triphosphate. Acta Biochim. Polon. (1998)45, 87-94

112 3285. JOŃCZYK,?., NOWICKAA, FUAŁKOWSKA.I.J., SCHAAPER,R.M., ,CIEŚLA,Z. In vivo protein interactions within the Escherichia coli DNA polymerase III core. J. Bacteriol. (1998)180, 1563-1566

3286. FELCZAK.K., GOŁOS,B., DZIK,J.M., RODE,W., BRETNER,M., SHUGAR,D., KULIKOWSKI.T. Acyclic analogues of 5-fluoro-dUMP and 5-fluoro-2'-deoxyuridine: Synthesis and inhibition of thymidylate synthase and tumour cell growth. Acta Biochim. Polon. (1998) 45, 75-82

3287. BRZESKU., GRYCUK,T., LIPKOWSKI,A.W., RUDNICKI,W., LESYNG,B., JERZMANOWSKI,A. Binding of SPXK-and APXK-peptide motifs to AT-rich DNA. Experimental and theoretical studies. Acta Biochim. Polon. (1998) 45, 221-231

3288. KNOESTER.M:, van LOON,L.C., van den HEUVEL.J., HENNIG,J., BÓL, J.F., LINTHORST.H.J.M. Ethylene-insensitive tobacco lacks nonhost resistance against soil-borne fungi Proc. Natl. Acad. Sci. USA (1998) 95, 1933-1937

3289. KUCHARCZYK,R., ZAGULSKI.M., RYTKA,J., HERBERT,Ch.J. The yeast gene YJRO25c encodes a 3-hydroxyanthranilic acid dioxygenase and is involved in nicotinic acid biosynthesis. FEBS Lett. (1998)424, 127-130

3290. WĘGRZYNA, HERMAN-ANTOSIEWICZ,A., TAYLOR,K., WEGRZYN,G. Molecular mechanism of heat shock-provoked disassembly of the coliphage A, replication complex. J. Bacteriol. (1998) 180. 2475-2483

3291. SIRKO.A., WĘGLEŃSKA,A., HULANICKA,D. Integration host factor positively regulates cysJIH transcription. Mol. Gen. Genet. (1998) 258. 174-177

3292. SZALEWSKA-PAŁASZ,A., WĘGRZYNA, BŁASZCZAKA, TAYLOR,K., WEGRZYN,G. DnaA-stimulated transcriptional activation of on A: Escherichia coli RNA polymerase (3 subunit as a transcriptional activator contact site. Proc. Natl. Acad. Sci. USA (1998) 95, 4241-4246

113 3293. TUDEK.B., van ZEELAND,A.A., KUSMIEREK,J.T., LAVAL,J. Activity of Escherichia coli DNA-glycosylases on DNA damaged by methylating and ethylating agents and influence of 3-substituted adeninę derivatives. Mutat. Res. (1998)407, 169-176

3294. HAKENBECK.R., KÓNIG.A., KERN,!., van der LINDEN,M., KECK,W., BILLOT-KLE1N,D., LEGRAND,R., SCHOOT,B., GUTMANN.L.

Acquisition of five high-Mr penicillin-binding protein variants during transfer of high-level (3-lactam resistance from Streptococcus mitis to Streptococcus pneiimoniae. J. Bacteriol. (1998)180, 1831-1840

3295. PAŁUCHA,A., ZAGÓRSKI.W., CHRZANOWSKA,M., HULANICKA,D. An antisense coat protein gene confers immunity to potato leafroll virus in a genetically engineered potato. Eur. J. Plant Pathol. (1998) 104.287-293

3296. JARMUŁAA, ANULEWICZ,R, LEŚ,A., CYRANSKI.M.K., ADAMOWICZ,L., BRETNER,M., FELCZAK,K., KULIKOWSKI,!., KRYGOWSKI,T.M., RODE.W. Crystal structures of 5-fluoro-dUrd and its 2 and/or 4-thio analogues: models of substituted dUMP pyrimidine ring interacting with thymidylate synthase. Biochim. Biophys. Acta (1998) 1382. 277-286

3297. BRODZIK,R., HENNIG,J. Adeninę methylation at GATC sequences regulates activity of tobacco PR-1 and PR-2 promoters in electroporated protoplasts. Plant Physio!. Biochem. (1998) 36,401-406

3298. RAYA,R., BARDOWSKl,]., ANDERSEN,P.S., EHRLICH, S.D., CHOPIN,A. Multiple transcriptional control of the Lactococcus lactis trp operon. J. Bacteriol. (1998) 180. 3174-3180

3299. BENTINGER,M., GRUNLERJ., PETERSON,E., ŚWIEŻEWSKA.E., DALLNER,G. Phosphorylation of farnesol in rat liver microsomes: properties of farnesol kinase and farnesyl phosphate kinase. Arch. Biochem. Biophys. (1998) 353, 191-198

3300. KAMIENSKA-TRELA,K., WÓJCIK,J. Applications of spin-spin couplings. Nucl. Magn. Reson. (1998)27,143-198

114 3301. KRUSZEWSKA,J.S., SALOHEIMO,M., ENTTILA,M.,PALAMARCZYK,G. Isolation of a Trichoderma reesei cDNA encoding GTP:a-D-mannose-l- phosphate guanyltransferase involved in early steps of protein glycosylation. Curr. Genet. (1998) 33,445-450.

3302. NIEDŹWIECKA-KORNAŚ, A., KIERDASZUK.B., STOLARSKI ,R., SHUGAR.D. Tautomerism, acid-base properties and conformation of methylated analogues of the promutagenic W'-hydroxycytosine. Biophys. Chem. (1998) 7L. 87-98

3303. FELCZAK.K., BRETNER,M., DZIK,J.M., GOŁOS.B., ZIELINSKI,Z., RODE,W., KULIKOWSKI.T. N^-hydroxy-S-halogeno-I'-deoxycytidines and their 5'-mono-phosphates as inhibitors of thymidylate synthase and in vitro antileukemic agents. In: Purine and Pyrimidine Metabolism in Man IX edited by Griesmacher et al. Plenum Press, Press,New York, 1998. Adv. Exper. Med. Biol. (1998) 431. 617-621

3304. POLANOWSKA.J., KROKOSZYŃSKA,!., CZAPINSKA,H., WATOREK,W., DADLEZ,M., OTLEWSKIJ. Specificity of human cathepsin G. Biochim. Biophys. Acta(1998) 1386, 189-198

3305. CHECHŁACZ,M., MICHALIK.J., CYMBOROWSKI,B. Suboptimal temperature-dependent changes in the brain development and activity in Galleria mellonella larvae. Archiv. Insect Biochem. Physiol. (1998) 38, 66-73

3306. GRZESIUKJE. The role of mutation frequency decline and SOS repair systems in methyl methanesulfonate mutagenesis. Acta Biochim. Polon. (1998) 45, 523-533

3307. TREMBACZ,H., JEZEWSKA,M.M. Adeninę nucleoside phosphorylases in trematode Fasciola hepatica, the mammalian parasite. In:Purine and Pyriinidine Metabolism in Man IX. Edited by Andrea Griesmacher,Peter Chiba,and Mathias M.Muller, Plenum Press,New York, 1998 Adv. Exp. Med. Biol. (1998) 431. 137, 711-717

3308. PIERZYNOWSKA.J., GRZESIUK,E. Antimutagenic effects of ellagic acid, rutin and psoralen against aflatoxin Bl. J. Anim. Feed Sci. (1998) 7, 277-283

115 3309. FIJAŁKOWSKA,U., JOŃCZYK,?., MALISZEWSKA-TKACZYK,M., BIAŁOSKÓRSKA,M., SCHAAPER,R.M. Unequal fidelity of leading strand and lagging strand DNA replication on Escherichia colt chromosome. Proc. Natl. Acad. Sci. USA (1998) 95, 10020-10025

3310. FRENK1EL,H., BARDOWSKI,J., EHRLICH,S.D, CHOPIN,A. Transcription of the trp operon in Lactococcus lactis is controlled by antitermination in the leader region. Microbiology (1998) 144. 2103-2111

3311. KIERDASZUK.B., KRAWIEC,K., KAZIMIERCZUK.Z., JACOBSSON,U., JOHANSSON,N.G.., MUNCH-PETERSEN,B., ERIKSSON,S., SHUGAR,D. Substrate/inhibitor specificities of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2). In:Purine and Pyrimidine Metabolism in Man IX. Eds A.Griesmacher et al.Plenum Press, New York ,1998. Adv. Exper. Med. Biol. (1998)431, 120, 623-627

3312. KULIKOWSKA,E., BZOWSKA.A., HOLY,A., MAGNOWSKA,L., SHUGAR,D. Antiviral acyclic nucleoside phosphonate analogues inhibitors of purine nucleoside phosphorylase. Ibid.: 747-752

3313. POPOWSKA,E., SUŁEK.A., KUBALSKA,J., PRONICKA,E., JEZEWSKA,M.,TREMBACZ,H., GORYLUK-KOZAK1EWICZ,B., KRAJEWSKA-WALASEK,M. Different mutations in Polish patients with HPRT deficiency - the Lesch- Nyhan and Kelley-Seegmiller syndromes. J. Appl. Genet. (1998) 39, 103-111

3314. FAB1S1EWICZ,A., JAN1ON.C. DNA mutagenesis and repair in UV-irradiated E.coli K-12 under condition of mutation frequency decline. Mutat. Res. (1998) 402, 59-66

3315 EJCHART,A.,/IMNIAK,A., OSZCZAPOWICZ, I., SZATYLOWICZ,H. Comparative I3C relaxation study of R and S isomers of the 1-acetoxy ethyl ester of cefuroxime. Influence of C - H bond lengths on relaxation data consistency. Magn. Res. Chem. (1998) 36,559-564

116 3316. HOWARD,S.T., KRYGOWSKI.T.M., CIESIELSKI.A., WISIOROWSKI.M. Angular group-induced bond alteration. II. The magnitude and the nature of the effect and its application to polynuclear benzenoid systems. Tetrahedron (1998)_54, 3533-3548

3317. KRYGOWSKLT.M, CYRAŃSKI, M., CIESIELSKI.A., SWIRSKA,B., LESZCZYŃSKI,?. Separation of the energetic and geometric contributions to aromaticity.2. Analysis of the aromatic character of benzene rings in their various topological environments in the benzenoid hydrocarbons. Crystal and molecular structure of coronene. J. Chem. Inf. Comput. Sci. (1998) 36, 1135-1141

3318. PRZEWŁOCKI.G., LIPECKA,J., EDELMAN,A., PRZYKORSKA,A. New sequence-specific human ribonuclease: purification and properties. Nucleic Acids Res. (1998) 26,4047-4055

3319. LACTIC ACID BACTERIA. Classification, metabolism, genetics, application, (in Polish) Eds: Z.Libudzisz, P. Walczak, J.Bardowski, University of Łódź, Łódź, 1998, 215 p.

3320. BARDOWSKI, J. Molecular biology in studies and improvement of lactic acid bacteria, (in Polish) Ibid.: 25-53

3321. GRABOWSKAA, KARST,F., SZKOPINSKA,A. Effect of squalene synthase gene disruption on synthesis of polyprenols in Saccharomyces cerevisiae. FEES Lett. (1998) 434. 406-408

3322. PALAMARCZYK,G, MARAS,M., CONTRERAS,R., KRUSZEWSKA J. Protein secretion and glycosylation in Trichoderma. In: Trichoderma and Gliocladium vol.1 Edited by Ch. P. Kubicek and G.E.Harman 6, 121-138

3324. FIKUS, M. Gelatin and prions: safety problems, (in Polish) Biotechnologia (1998) 3, (42), 9-13

3325. EJCHART,A., GRYFF-KELLER,A. On the spin-spin coupling constants of trimethylsilylated iodoacetylene. Chem. Eur. J. (1998)4,2072

117 3326. BORYS,E., KUŚMIEREK,J.T. Endogenous and exogenous DNA lesions recognized by TV-alkylpurine-DNA glycosylases. Acta Biochim. Polon. (1998) 45, 579-586

3327. TALJAŃSKl-ZYGMUNT.W., GRZESIUK,E., ZABIELSKI,R., P1ERZYNOWSKI,S.G. Is the use of antimicrobial drugs in agriculture risky for human health? J. Anim. Feed Sci. (1998) 7, 289-295

3328. ZAGULSKI,M., HERBERT,Ch.J., RYTKA,J. Sequencing and functional analysis of the yeast genome. Acta Biochim. Polon. (1998) 45, 627-643

3329. BUKOWSKA-MACIEJEWSKA,M., KUŚMIEREK.J.T. Template-directed base pairing of 2-chloro-2'-deoxyadenosine catalysed by AMV reverse transcriptase. Acta Biochim. Polon. (1998) 45, 587-593

3330. WANKE,M, CHOJNACKI,T., ŚWIEŻEWSKA,E. The diversity of polypreno! pattern in leaves of fruit trees belonging to Ronaceae and Coornaceae. Acta Biochim. Polon. (1998) 45, 811-818

3331. GENES AND THEIR PRODUCTS IN BASIC RESEARCH AND BIOTECHNOLOGY. Abstracts of the 2nd Workshop. Warsaw, November 25-28, 1998 Polish Academy of Sciences, Institute of Biochemistry and Biophysics.

3332. KOSTELIDOU,K., JAGURA-BURDZY.G,. THOMAS,Ch.M. Mutational analysis of the global regulator KorA of broad-host-range plasmid RK2. J. Mol. Biol. (1998) 281, 453-463

3333. KURLANDZKA.A., RYTKA,J., RÓŻALSKA.B., WYSOCKA M. Saccharomyces cerevisiae IRR1 protein is indirectly involved in colony formation. Yeast (1999) 15, 23-33

3334. DZIEMBOWSKIA, MALEWICZ,M., MINCZUK.M., GOLIK.P., DMOCHOWSKA,A., STĘPIEŃ.P.P. The yeast nuclear gene DSS1, which codes for a putative RNase II, is necessary for the function of the mitochondrial degradosome in processing and turnover of RNA. Mol. Gen. Genet. (1998)260, 108-114

118 3335. WĘGIERSKI/T., DMOCHOWSKA,A., JABŁONOWSKA.A., DZIEMBOWSKI,A., BARTNIK,E., STĘPIEŃ,P.P. Yeast nuclear PET127 gene can suppress deletions of the SUV3 or DSS1 genes: An indication of a functional interaction between 3' and 5' ends of mitochondria] mRNAs. Acta Biochim. Polon. (1998)45, 935-940

3336. KRASZEWSKA,E., DOBRZANSKA,M., TUDEK,B. Tobacco BY-2 cells excise both 3-methyladenine and 7-methylguanine from methylated DNA. Mut. Res. (1998) 409.91-95

3337. RESEARCH REPORT 1996 - 1997, Institute of Biochemistry and Biophysics PAS, Warsaw 1998, 154 p.

3338. WĘGRZYNA, WEGRZYN.G. Random inheritance of the replication complex by one of two daughter A. plasmid copies after a replication round in Escherichia coli. Biochem. Biophys. Res. Comm, (1998) 246 634-639

3339. GABIG,M., OBUCHOWSKI,M., CIESIELSKA,A., LATAŁAJB., WĘGRZYNA THOMAS,M.S., WEGRZYN,G. The Escherichia coli RNA polymerase cr subunit and transcriptional activation by bacteriophagie X Cll protein. Acta Biochim. Polon. (1998) 45, 271-280

3340. GABIG,M., OBUCHOWSKIJVL, WĘGRZYNA, SZALEWSKA- PAŁASZA, THOMAS.M.S., WEGRZYN,G. Excess production of phage A, delayed early proteins under conditions supporting high Escherichia coli growth rates. Microbiology (1998) 144, 2217-2224

3341. HERMAN-ANTOSIEWICZA, WĘGRZYNA, TAYLOR,K., WEGRZYN,G.

DnaA-mediated regulation of phage A.-derived replicons in the absence ofpR and Cro function. Virology (1998) 249, 98-107

3342. SZALEWSKA-PAŁASZA, LEMIESZEK.E., PANKIEWICZ,A., WĘGRZYNA, HELIŃSKI.D.R., WĘGRZYN,G. Escherichia coli dnaA gene function and bacteriophage A, replication. FEMS Microbiol. Lett. (1998) 167. 27-32

119 3343. SADOWY,E., PLUTA,K., GRONENBORN,B., HULANICKA.D. Infectious transcripts from cloned cDNA of potato leafroll luteovirus. Acta Biochim. Polon. (1998) 45, 611-619

3344. MAGLOTT,E.J., DEO.S.S., PRZYKORSKA.A., GLICK,G.D. Conformational transitions of an unmodified tRNA: Implications for RNA folding. Biochemistry (1998) 37, 16349 - 16359

3345. FIKUS,M. Methods of in vitro DNA recombination (Genetical engineering), (in Polish) In: "Animal Biotechnology" ed. L. Zwierzchowski, K. Jaszczak, J.A.Modliński, Warsaw 1997 Wydawn. Nauk. PWN p. 18 - 59

3346. KRZYMOWSKA.M. Plants resistance genes and their role during infection, (in Polish) Post. Biochem. (1998)44, 318-325

3347. Z1ELENK1EWICZ,A„ WSZELAKA-RYLIK,M, POZNAŃSKI,]., Z1ELENKIEWICZ,W. Thermochemistry of aqueous solutions of alkylated nucleic acid bases. X. Enthalpies of hydration of cytosine and some methylated, hydroxy and methoxy derivatives of cytosine. J. Sol. Chem. (1998) 27, 235-244

3348. ZIELENKIEWICZ,W., POZNAŃSKI,! Partial molar volumes of hydrophobic compounds - insight into the solvation shell? Part I J. Sol. Chem. (1998) 27, 245-254

3349. ZIELENKIEWICZ,W., POZNAŃSKI,:., ZIELENKIEWICZ,A. Partial molar volumes of alkylated uracils - insight into the solvation shell? Part II J. Sol. Chem. (1998) 27 543

3350. TOMASZEWSKI.R., MOGIELNICKA,E., JERZMANOWSKI.A. Both the 5 S rRNA gene and the AT-rich flanks of Xenopits laevis oocyte-type 5S rDNA repeat are required for histone Hl-dependent repression of transcription of pol Ill-type genes in in vitro reconstituted chromatin. Nucleic Acid Res. (1998) 26, 5596-5601

3351. BORKOWSKA,M., KRZYMOWSKA,M., TALARCZYK,A., AWAN,M.F.M., YAKOVLEVA,L., KLECZKOWSKI.K., WIELGAT,B- Transgenic potato plants expressing soybean 1,3-p-endoglucanase gene exhibit an increased resistance to Phytophtora infestans. Z. Naturforsch. (1998) 53c, 1012-1016

120 3352. TALARCZYKA, HENNIG,J. Characterization of cDNA encoding a glucan endo-l,3-a-glucosidase from potato. The Electronic Plant Gene Register. PGR 98-173 (Accession No. AJ 009932). Plant Physiol. (1998) 118. 711-712

3353. SIZOVA,O.V., MALTSEV,S.D., SHIBAEV,V.N., JANKOWSKI,W., CHOJNACKI,!., ZELINSKY,N.D. Dolichyl sulfate and H-phosphonate: Enzymie reactions with activated sugars. Acta Biochim.Polon. (1998)45, 1021-1030

3354. BYKOWSKI,T., SIRKO,A. Selected phenotypes of/A/mutants of Escherichia coli, Biochimie(1998)80, 1-15

3355. SIRKOA IHF as a regulator of metabolism in Escherichia coli. (in Polish) Post. Biochem. (1998) 44, 345-354

3356. WĘGRZYN.A., SZALEWSKA-PAŁASZ,A., BŁASZCZAK,A., LIBEREK,K., WEGRZYN,G. Differential inhibition of transcription from cr70- and <732-dependent promoters by rifampicin. FEBS Lett. (1998)440, 172-174

3357. JANKOWSKAJ., SOBKOWSKA.A., CIEŚLAK,J., SOBKOWSKI.M., KRASZEWSKI A, STAWIŃSKI.J., SHUGAR,D. Nucleoside //-phosphonates. 18. Synthesis of unprotected nucleoside 5'-H- phosphonates and nucleoside 5'-//-phosphonothioates and their conversion into the 5'-phosphorothioate and 5'-phosphorodithioate monoesters. J. Org. Chem. (1998) 63, 8150-8156

3358. SCHEDIN,S., NILSSON,M., CHOJNACKI.T., DALLNER,G. Alterations in the biosynthesis of cholesterol, dolichol and dolichyl-P in the genetic cholesterol homeostasis disorder, Niemann-Pick type C disease. Biochim. Biophys. Acta (1998) 1394. 177-186

3359. GAVRYUSHOV,S., ZIELENKIEWICZ,P. Electrostatic potential of B-DNA: Effect of interionic correlations. Biophys. J. (1998) 75, 2732-2742

121 3360. BOGUTA.M. Interaction between mitochondrial and nuclear genome. Function of specific nuclear genes in mitochondria] translation of Saccharomyces cerevisiae. (in Polish) In: Molecular Biology of Mitochondria. Wydawn. Naukowe UAM ed. H.Augustyniak, M.Oczkowski, Poznań 1998 11-22

3361. RYTKA, J. The yeast Saccharomyces cerevisiae the model organism to study the heme biosynthesis.(in Polish) Ibid.: 23-32

3362. ZAREBSKA,Z., WASZKOWSKA,E., CAFFIERI,S., DALL'ACQUA,F. Photoreactions of psoralen with lecithins. J. Photochem. Photobiol. B: Biology (1998) 45, 122-130.

3363. WANKE, M,. ŚWIEŻEWSKA, E., DALLNER, G. Subceilular localization and sorting of plastoquinone and ubiquinone in spinach cells In: Adv. Plant Res. Eds.: L Sanchez,, E. Cerda-Olmedo, E. Martinez-Force, Universidad de Sevilla, Sevilla, 1998, 450-453

3364. DMOCHOWSKA,A., STANK1EWICZ,P., GOLIK,P., STĘPIEŃ,P.P., BOCIAN,E.,HANSMAHN,I., BARTNIK,E. Assigment of SUPV3L1 to human chromosome band 10q22.1 by situ hybridization. Cytogenet. Cell. Genet. (1998) 83, 84-85

3366. MUS1ELAK, M., POKORSKI, M., ELWICH, S., NOWAKOWSKA, A., WALSKI, M., MATYSIAK, Z., Phenylmethylsulfonyl fluoride abolishes the hypoxic ventilatory response. Curr. Pneumol. (1998)2,201

3367. BIRNBAUM.K.B., SHUGAR,D., FELCZAK,K. I -(B-D-ribofuranosyl) - 6-propylcytosine. ActaCryst. (1998), 54C, 1959-1961

3368. S1EDLECKA,M., GOCH,G., EJCHART,A., ST1CHT,H., BIERZYNSKI,A. -Helix nucleation by a calcium-binding peptide loop. Proc. Natl. Acad. Sci. (1999)96, 903-908

3369. GÓRA, M., RYTKA, J., LABBE -BOIS, R. Activity and cellular location in Saccharomyces cerevisiae of chimeric mouse/yeast and Bacillus swtó/js/yeast ferrochelatases. Arch. Biochem. Biophys. (1999)361.231-240

122 3370. KICZAK, L., KOŚCIELSKA, K., OTLEWSKI, J., CZERWIŃSKI, M., DADLEZ, M. Phage display selection of PI mutants of BPTI directed against five different serine proteinases. Short Communication. Biol. Chem. (1999) 380. 101-105

3371. WÓJCIK, A, JANION, C. Effect of TnlO/Tn5 transposons on the survival and mutation frequency of halogen light-irradiated AB1157 Escherichia coli K-12. Mutagenesis(1999) 14, 129-134

3372. MACIEJEWSKA, U., SKIERSKI, J.S., SZCZERBAKOWA, A. Nuclear DMA content of Sola/mm species grown in vitro, as determined by flow cytometry. Acta Physiol. Plant. (1999) 21, 37-43

3373. POZNAŃSKI, J., SODANO, P., SE WON SDH, JAE YOUNG LEE, PTAK, M., VOVELLE, F. Solution structure of a lipid transfer protein extracted from rice seeds. Comparison with homologous proteins. Eur. J. Biochem. (1999) 259 692-708

3374. DZIKOWSKA, A., SWIANIEWICZ, M., TALARCZYK, A., WIŚNIEWSKA, M., GORAS, M., SCAZZOCCHIO, C., WĘGLEŃSKI, P. Cloning, characterisation and regulation of the ornithine transaminase (ptaA) gene of Aspergillus rtidulam. Curr. Genet. (1999)35, 118-126

3375. DODZ1UK, H., EJCHART, A., LUK1N, O., VYSOTSKY, O. 'H and I3C NMR and molecular dynamics study of chiral recognition of camphor enantiomers by i -cyclodextrin. J. Org. Chem. (1999) 64, 1503-1507

3376. DMOCHOWSKA, A., KALITA, K., KRAWCZYK, M., GOLIK, P., MROCZEK, K., LAZOWSKA, J., STĘPIEŃ P.P., BARTNIK, E. A human putative Suv3-like RNA helicase is conserved between Rhodobacter and all eukaryotes. Acta Biochim.Polon. (1999) 46, 155-162

3377. KRUSZEWSKA, J. Heterologous expression of genes in filamentous fungi. ActaBiochim. Polon. (1999)46, 181-195

123 3378. EJCHART, A., JACKOWSKI, K., GRYFF-KELLER, A. Magnetic shielding tensors of sp-hybridized carbons in butad iyny 1 trimethy Isi lane. |JC nuclear spin relaxation and ab initio study. Bull. Pol. Acad. Sci. Chem. (1999) 47, 78-81

3379. EJCHART, A. Scalar couplings in structure determination of proteins. Ibid.: 1-19

3380. ZDANOWSK1, K., DADLEZ, M. Stability of the residual structure in unfolded BPTI in different conditions of temperature and solvent composition measured by disulphide kinetics and double mutant cycle analysis. J. Mol. Bio!. (1999) 287. 433-445

3382. WIECZOREK, Z., NIEDŹWIECKA-KORNAŚ, A., CHLEBICKA, L.} JANKOWSKA, M., KIRAGA, K., STĘPIŃSKI, J., DADLEZ, M., DRABENT, R., DARŻYNKIEW1CZ, E., STOLARSKI, R. Fluorescence studies on association of human translation initiation factor eIF4E with mRNA cap-analogues. Z. Naturforsch. (1999) 54c. 278-284

3383. HELLAND, R., OTLEWSKI, J., SUNDHEIM, O., DADLEZ, M., SMALAS, A.O. The crystal structures of the complexes between bovine B-trypsin and ten Pj variants of BPTI. J. Mol. Biol. (1999) 287. 923-942

3384. KACZANOWSKI.S.; KIERZEK.A.M., ZIELENKIEWICZ.P. Shuffling alghorithm for protein design. Protein Peptide Lett. (1999) 6, 99-104

3385. KIERZEK.A.M., POKAROWSKI,P., ZIELENKIEWICZ,P. Lattice simulations of protein crystal formation. Biophys. Chem. (1999) 77, 123-137

3386. FELCZAK.M., BĘBENEK,A, PIETRZYKOWSKA.I. The isfA mutation specifically inhibits the SOS-dependent mutagenic pathway and does not selectively affect any particular base substitution. Mutagenesis (1999)14, 295-300

124 3387. JAGURA-BURDZY,G., MACARTNEY,D.P., ZATYKA.M., CUNLIFFE.L., COOKE,D., HUGGINS,C., WESTBLADE,L., KHANIM,F., THOMAS,Ch.M. Repression at a distance by the global regulator KorB of promiscuous IncP plasmids. Mol. Microbiol. (1999)32, 519-532

3388. KAM1EŃSKA-TRELA,K., WÓJCIK,J. Applications of spin-spin couplings. Nucl. Magn. Reson. (1999)28, 151-207

3389. BRZESKI,!, PODSTOLSKI,W, OLCZAK,K., JERZMANOWSKI.A. Identification and analysis of the Arabidopsis thaliana BSH gene, a member oftheSTVFJ gene family. Nucleic Acids Res. (1999) 27, 2393-2399

3390. JAGURA-BURDZY, G., KOSTELIDOU, K., POLE, J., KHARE, D., JONES, A., WILLIAMS, D.R., THOMAS, Ch.M. IncC of broad-host-range plasmid RK2 modulates KorB transcriptional represser activity in vivo and operator binding in vitro. J. Bacteriol. (1999) 9,2807-2815

3391. GRĄZIEWICZ,M.A., ZASTAWNY,T.H., OLIŃSKI.R., TUDEK,B. SOS-dependent A->G transitions induced by hydroxyl radical generating system hypoxanthine /xanthine oxidase/ Fe3+/EDTA are accompanied by the increase of Fapy-adenine content in M13 mpl8 phage DNA. Mut. Res. (1999) 434.41-52

3392. KRUSZEWSKA.J.S., BUTTERWECK.A.H., KURZĄTKOWSKI.W., M1GDALSKIA, KUBICEK,Ch.P., PALAMARCZYK,G. Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure. Appl. Environ. Microbiol. (1999) 65, 2382-2387

3393. SAYERS,Z., BROU1LLLOTM,P., SVERGUN,D.I., ZIELENKIEWICZ,?., KOCH.M.HJ. Biochemical and structural characterization of recombinant copper- metallothionein from Saccharomyces cerevisiae. Eur. J. Biochem. (1999) 262. 858-865

3394. KROWARSCH,D., DADLEZ.M., BUCZEK,O., KROKOSZYŃSKA,!., SMALAS,A.O., OTLEWSKIJ. [nterscaffolding additivity: binding of PI variants of bovine pancreatic trypsin inhibitor to four serine proteases. J. Mol. Biol. (1999)289, 175-186

125 3395. ZIELENKIEWICZ.W., POZNAŃSKI,). Partial molar volumes-Insights into molecular structure. J. Mol. Liq. (1999) 81,37-45

3396. KRZYMOWSKA.M. Microbial factors triggering plant defense response, (in Polish) Post. Bio. Kom. (1999) 38, 83-93

3397. GAVRYUSHOV,S, ZIELENKIEWICZ,P. Electrostatics of a DNA-like polyelectrolyte: effects of solvent dielectric saturation and polarization of ion hydration shells. J. Phys. Chem. B. (1999) 103, 5860-5868

3398. KEDZIERSKA,S., STANISZEWSKA,M., WEGRZYN,A., TAYLOR,A. The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from Escherichia coli cells. FEBSLett. (1999) 446.331-337

3399. WĘGRZYNA, WRÓBEL,B., WĘGRZYN,G. Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants. Mol. Gen. Genet. (1999) 261. 762-769

3400. GLINKOWSKAM., WĘGRZYNA, WĘGRZYN,G. Replication of bacteriophage ••.'} in the Escherichia coli dnaA I'Jrac hosts. Genetics (1999) 151. 1633-1635

3401. WĘGRZYNA, WĘGRZYN.G. Regulation of replication of"! bacteriophage and !."! plasmid DNAs.(in Polish) Post. Biochem. (1999) 45,5-11

3402. HAŁAS, A., POLICIŃSKA, Z., BARANOWSKA, H., JACHYMCZYK, W.J. The essential DNA polymerases 8 and s are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae. ActaBiochim. Polon. (1999)46289-298

3403. KRUSZEWSKAJ., JANIKA, LENART,U., PALAMARCZYK,G. Glycosylation defects corrected by the changes in GDPmannose level. Acta Biochirn. Polon. (1999) 46, 315-324

3404. MICHALIK,;., SZOLAJSKA,E. Baculovtruses - insect specific viruses. Structure, infectivity and foreign genes expression, (in Polish) Post. Biochem. (1999) 45, 137-142

126 3405. KIERDASZUK,B., KRAWIEC,K., KAZIMIERCZUK,Z., JACOBSON,U., JOHANSSON,N.G., MUNCH-PETERSEN.B-, ERIKSSON,S., SHUGAR,D. Substrate/inhibiotor properties of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2) towards the sugar moiety of nucleosides, including O'-alkyl analogues. Nucleosides & Nucleotides (1999) 18, 1883-1903

3406. SHUGAR,D. Viral and host-cell protein kinases: enticing antiviral targets and relevance of nucleoside, and viral thymidine, kinases. Pharmacol. Ther. (1999) 82,315-335

3407. FELCZAK,K., MIAZGA.A., GOLOS,B., RODE,W., SHUGAR,D., KULIKOWSKI,T. Synthesis, conformation and biological properties of selenonucleosides. Nucleosides & Nucleotides (1999) 18, 635-636

3408. JUSZCZUK,M., ZAGORSKI-OSTOJA,W., HULANICKA,D. Gene expression of positive stranded RNA viruses, (in Polish) Post. Biochem. (1999) 2, 87-94

3409. SZWACKA,M., KRZYMOWSKA,M., MALEPSZY,S. Thaumatin expression in transgenic cucumber plants. In: Plant Biotechnology and In Vitro Biology in the 21st Century. Proceedings of the IXth International Congress of the International Association of Plant Tissue Culture and Biotechnology. Jerusalem, Israel, 14-19 June, 1998 Eds Altman A., Ziv M., Izhar S., Kluwer Academic Publishers, (1999) 609-612 Current Plant Science and Biotechnlogy in Agriculture

3410. KUCHARCZYK,R., GROMADKA,R., MIGDALSKI,A., SLONIMSKI,P.P., RYTKA.J. Disruption of six novel yeast genes located on chromosome II reveals one gene essential for vegetative growth and two required for sporulation and conferring hypersensitivity to various chemicals. Yeast (1999)11,987-1000

3411. BORSUK,P, DZIKOWSKA.A., EMPEL,J., GRZELAK,A, GRZEŚKOWIAK,R., WĘGLEŃSKI,P. Structure of the arginase coding gene and its transcript in Aspergillus nidulans. Acta Biochim. Polon. (1999) 46, 391-403

127 3412. SIEŃKO,M., PASZEWSKI.A. The metG gene of Aspergillus nudilans encoding cystathionine pMyase: cloning and analysis. Curr. Genet. (1999) 35, 638-646

3413. DADLEZ,M., Folding initiation sites and protein folding. Acta Biochim. Polon. (1999) 46,487-508

3414. S1DOR.M., WÓJCIK.J., PAWLAK.,D., IZDEBSKIJ. Conformational analysis of a novel cyclic enkephalin analogue using NMR and EDMC calculations. Ibid.: 641-650

3415. WÓJCIK.J., RUSZCZYNSKA,K., ZHUKOV.I., EJCHART,A. NMR measurements of proton exchange between solvent and peptides and proteins. Ibid.: 651-663

3416. ZHUKOV,!., EJCHART.A. Factors improving the accuracy of determination of 15N relaxation parameters in proteins. Ibid.: 665-671

3417. GOCH.G., KOZŁOWSKA,H., WÓJTOWICZ,A., BIERZYŃSKI.A. A comparative CD and fluorescence study of a series of model calcium- binding peptides. Ibid.: 673-677

3418. TALARCZYK,E., VO SI,H„ KOZŁOWSKI.P., TRZCIŃSKA- DANIELEWICZ.J., ŚWIEŻEWSKA,E. Ras protein of the slime mold Physarum polycephalum is farnesylated in vitro. Ibid.: 771-775

3419. TUDEK.B., GRAZIEWICZ,M., KAZANOVA.O., ZASTAWNY,T.H. O BTUŁO WICZ,T.,LAV AL, J. Mutagenic specificity of imidazole ring-opened 7-methyIpurines in M13mpl8 phage DNA. Ibid.: 785-799

3420. BURZYNSKA,B. OLECH.W, TOPCZEWSKI.J. Phylogeny and genetic variation of the European bison Bison bonasus based on mitochondria! DMA D-loop sequences. \Acta Theriol. (1999) 44, 253-262

128 3421. SZESZKOWSKI,W., CIEŚLA,!, STOLARSKI.R., RODE,W., KULIKOWSKI,T. Investigation of the ternary complex formed between recombinant rat hepatoma thymidylate synthase, FdUMP or S4FdUMP and N5, N 10- methyleneterahydrofolate with the use of !H and I9F NMR. Nucleosides & Nucleotides (1999) 18, 865-866

3422. FELCZAK,K., MIAZGA,A., KULIKOWSKI.T. Synthesis of novel acylo 6-phenylselenenyluracils - potential selective anti- HIV agents. In: Collection Symposium Series (1999)2 245-247 Chemistry of Nucleic Acid Components, XIth Symposium, Spindleruv Mlyn, Czech Republic, September 4-9, 1999 Ed Holy, A., Hocek, M.

3423. ŚLUSARCZYK.J., PRYMAKOWSKA-BOSAK.M., PRZEWŁOKA,M., JERZMANOWSKI,A, KURAS,M. Ultrastructural organization of leaves of transgenic tobacco overexpressing histone HI from Arabidopsis thaliana. Ann. Bot. (1999)84, 329-335

3425. van der PLOEG,J.R., IWANICKA-NOWICKA,R., BYKOWSKI,T, HRYNIEWICZ,M.M., LEISINGER,T. The Escherichia coli ssuEADCB gene cluster is required for the utilization of sulfur from aliphatic sulfonates and is regulated by the transcriptional activator Cbl. J. Biol. Chem. (1999) 274. 29358-29365

3426. TUDEK,B, KOWALCZYK,P, CIEŚLA.J.M. Localization of chloroacetaldehyde-induced DNA damage in human p53 gene by DNA polymerase finger-print analysis. In: Exocyxlic DNA adducts in mutagenesis and carcinogenesis Eds Singer B., Bartsch H. IARC Scientific Publications No 150 Lyon 1999 279-293

3427. ONO,B.I., HAZU,T., YOSHIDA,S., KAWATO,T., SHINODA,S., BRZYWCZY,J., PASZEWSKI.A. Cysteine biosynthesis in Saccharomyces cerevisiae: a new outlook on pathway and regulation. Yeast (1999)15, 1365-1375

3428. BRZYWCZY,J., PASZEWSKI.A. Cloning and characterization of the Kluyveromyces lactis homocysteine synthase gene. Ibid.: 1403-1409

129 3430. PRYMAKOWSKA-BOSAK, M., PRZEWLOKĄ, M., ŚLUSARCZYK, J., KURAS, M., LICHOTA, J., JERZMANOWSKI, A. Linker histones play a role in male meiosis and the development of pollen grains in tobacco. Plant Cell (1999)11, 1-14

3431 = PIERZYNOWSKA,J., GRZESIUK.E., Mutagenicity and carcinogenicity of aflatoxin AFBi. (in Polish) Post. Biochem. (1999) 45, 313-319

3432. TUDEK,B. Mechanisms of oxidised DNA bases repair. 8-hydroxyguanine and thymine- glycol - DNA glycosylases. (in Polish) Kosmos (1999) 48, 4 339-352

3433. JANION,C. Mutagenesis - damage and repair of DNA. (in Polish) Ibid.: 289-292

3434. PIETRZYKOWSKA,!., KRWAWICZ.J. Mechanisms of DNA repair in bacteria and humans, (in Polish) Ibid.: 315-328

3435. BLASZCZAK,A, GEORGOPOULOS,C., LIBEREK,K. On the mechanism of FtsH-dependent degradation of the a32 transcriptional regulator of Escherichia coli and the role of the DnaK chaperone machine. Mol. Microbiol. (1999)31, 157-166

3436. KROCZYŃSKA.B., CIESIELSKI.A., STACHNIAK,K. The nucleotide sequence of a cDNA encoding the AtTIR, a TIR-Iike resistance protein in Arabidopsis thaliana (Accession No API 88334) Plant Gene Register PGR99-172. Plant Physiol. (1999) 121. 1057

3438. KRYSIAK, C., MAZUŚ, B., BUCHOWICZ, J. Relaxation, linearization and fragmentation of supercoiled circular DNA by tungsten microprojectiles. Trans. Res. (1990) 8,303-306

3440. PERREAU, V.M., KEITH, G., HOLMES, W.M., PRZYKORSKA, A., SANTOS, M.A.S, TU1TE, M.F. The Ccmadia albicans CUG-decoding Ser-tRNA has an atypical anticodon stem-loop structure. J. Mol. Biol. (1999) 293. 1039-1053

130 3442. WALCZAK, I., PALUCHA, A., JUSZCZUK, M., MUCHALSKI, T., SZYBIAK-STRÓŻYCKA, U. Potato virus M: the cloning of the genomic copy of isolates with different pathogen ity. Biotechnologia (1999) 3, 169-177

3443. GOLANKIEWICZ, B., PERLOVICH, G., POZNAŃSKI,!, SITKOWSKI, J. STEFANIAK, L., ZEIDLER, J., ZIELENKIEWICZ, W. Complexation of antiretroviral nucleosides 2', 3' - dideoxyinosine, 2', 3' - dideoxyadenosine and 2', 3' - dideoxyguanosine with p-cyclodextrin. A *H NMR study. J. Chem. Soc. Perkin Transact. 2 (1999) U., 2533-2538

3444. BOBROWSKI.K., POZNAŃSKI,!., HOLCMAN,!, WIERZCHOWSKI.K.L. Pulse radiolysis studies of intramolecular electron transfer in model peptides and proteins. 8. Trp[NH«+]—> Tyr [O«] radical transformation in H-Trp-(Pro),,- Tyr-OH, n = 3-5, series of peptides. J. Phys. Chem. B (1999)103, 10316-10324

3445. WANKE,M., DALLNER,G., ŚWIEŻEWSKA,E. Subcellular localization of plastoquinone and ubiquinone synthesis in spinach cells. Biochim. Biophys. Acta (2000) 1463. 188-194

3446. JANKOWSK1,W., JANSON, L. Lipids in long term stored seeds of fir. (in Polish) Prace Instytutu Badawczego Leśnictwa, Seria A (1999) 880, 36-49

3447. JANSON,L., JANKOWSKI.W. Long term storage of lyophylized seeds, (in Polish) Las Polski (1999)3,4-6

3448. BŁASZCZYK,A., BRODZIK,R., SIRKO.A. Increased resistance to oxidative stress in transgenic tobacco plants overexpressing bacterial serineracetyltransferase. Plant J. (1999) 20,237-243

3449. JANIK,A., KRUSZEWSKA,J., LENART,U., SOSNOWSKA,M., PALAMARCZYK.G. Overexpression of a-D-mannose-1-phosphate guanyltransferase encoding gene restores the viability of the Sctccharomyces cerevisiae mutants affected in early steps of glycoconjugate formation. In: Recent Advances in Carbohydrate Bioengineering, ed. H.J.Gilbert et al., Royal Society of Chemistry 35-42

131 3450. EJCHART.A. Nuclear relaxation studies of the protein backbone. In: Molecular relaxation phenomena (in Polish), red. J.P.Hawranek, L.Sobczyk Acta Universitatis Wratislaviensis No 2123 Wrocław 1999 117-134

3451. ŻYL1CZ, M., WAWRZYNÓW.A., MARSZAŁEK,K, LIBEREK,B., BANECKI.B., KONIECZNY,!., BŁASZCZAK,A„ BARSKI,?., JAKÓBKIEWICZ.J., GONCIARZ-SWIĄTEK.M., DUCHNIEWICZ,M., PUZEW1CZ,J., KRZEWSKA.J. Role of chaperones in replication of bacteriophage lambda DNA. In: Molecular chaperones and folding catalysts (ed. Bukau, B.) Harwood Academic Publishers (1999) 295-311

3452. JABŁOŃSKA-SKWIECIŃSKA.E., LEWANDOWSKA,!., PLOCHOCKA,D., TOPCZEWSKU., ZIMOWSKI,J.G., KLOPOCKA,J., BURZYNSKA,B. Several mutations including two novel mutations of the glucose-6-phosphate dehydrogenase gene in Polish G6PD deficient subjects with chronic nonspherocytic hemolytic anemia, acute hemolytic anemia and favism. Human Mutat. (1999) M, 477-484

3456. HALAS,A., CIESIELSKI.A., ZUK,J. Involvement of the essential yeast DNA polymerases in induced gene conversion Acta Biochim. Polon. (1999)4, 862-872

132 ABSTRACTS. MISCELLANEOUS,

1105/A MUSIELAK, M, POKORSKI, M., ELWICH, S., NOWAKOWSKA, A., WALSKI M, MATYSIAK Z. Phenyl-metbyl-suifonyl fluoride abolishes hypoxia response in cat. (in Polish). X Jubilee simposium Polish group ERS in Polish Tatra Mountains 12-14 January, 1998

1106/A ZAGÓRSKI, W., VIEIRA da SILVA, M.R., CLAISSE, M.L. Protein syntheses in whole cells and isolated mitochondria from the yeast Sckwanniomyces occidentalis (acastellii): Heat Shock Effects. UNESCO Global Network MCBN Workshop " New Perspectives in Mitochondria! Research" Padova (Italy), September 18-21, 1997, 221-224

1107/A ZHUKOV, 1., WÓJCIK, J., CIURKOWSKI J., GRZONKA Z., KASPRZYKOWSKI F. Conformational studies of [Me,Ala7]- AVP-vasopresin analogue by means of NMR (in Polish). Proceedings of XXX National Seminar on Magnetic Resonance and its Applications 1-3 December, 1997 Institute of Nuclear Physics, Kraków 1998 p..221-224

1108/A EJCHART, A. How to interpret heteronuclear relaxation data? Case of lactose, (in Polish). Ibid.: 141-144

1109/A EJCHART, A. NMR of proteins: isotope labeling and multidimensional techniques, (in Polish). Ibid.: 153-156

1110/A TALARCZYK, A., KRZYMOWSKA, M., HENNIG, J. Tobacco response to TMV infection is modulated by catalase activity. 5th International Workshop on Patogenesis-Related proteins in Plants Signalling Pathways and Biological Activities. March 29-April 2, 1998 Aussois, France Abstr. L-25 oral presentation [rp]

111 l/A ZARĘBSKA, Z., WASZKOWSKA, E., CAFFIERI, S., DALL'ACQUA F. Photoreactions of psoralen with phospholipids on the mode! lecithins. Silver 25lh Jubilee FEES Meeting July 5-10 1998 Bella Center Copenhagen

133 1112/A EJCHART, A. Consistency of the heteronuclear relaxation data. Case of lactose. EFNC 98 BLED 14th European Experimental N.M.R. Conference Bled, Slovenia, May 10-15, 1998 Abstr. p. 124

1113/A BARDOWSKI, J. Genetic modifications of lactic acid bacteria - perspectives of use in biotechnological processes, (in Polish) Summer School "Lactic Acid Bacteria. Classification, metabolism, genetics, application. Organized by the Institute of Fermentation Technology and Microbiology, Technical University of Łódź, Ciechocinek, 18-22 May 1998. Invited lecture Abstr. p.24

1114/A KAMIENSKA-TRELA, K., KANIA, L., KACZMAREK, L., WÓJCIK, J. Structural studies of indolo [2,3-A]quinolines and their derivatives. Xlh Intern. Conf. "Magnetic Resonance in Chemistry and Biology SUZDAL'98 Russian Academy of Sciences, Russia June 1-7, 1998 Abstr. SG.17p.121

1115/A IWANÓW, A., KOZMIŃSKI, W., SIDOR, M., SITKOWSKI, J., WÓJCIK, J. Structure, 'H and I3C NMR assignments of sulfono di-N-oxides of some C30 nuphar alkaloids. Symp. on "Application of Magnetic Resonance in Chemistry and Related Areas" Institute of Organic Chemistry PAS and Polish Chemical Society, June 24-26, 1998 Warsaw Abstr. P9

1116/A M ARCZEWSKI, W., OSTROWSKA, K., TALARCZYK, A., HENNIG, J., ZIMNOCH-GUZOWSKA, E. RAPD and SCAR markers of Ns gene in selection of potato lines resitant to virus S infection, (in Polish) Ist Domestic Seminar on "Mapping and Selection of Genetic Markers in Plants" Institute of Plant Genetics PAS, Poznań 23 April 1998 Abstr. p.14

1117/A ZHUKOV, I., EJCHART, A. 'H and I5N NMR assignments and secondary structure of the Apo-SlOOAl protein. Symp. on "Application of Magnetic Resonance in Chemistry and Related Areas" Institute of Organic Chemistry PAS and Polish Chemical Society, June 24- 26, 1998 Warsaw, Abstr. P54

134 1118/A FLIS, B., CHACHULSKA, A., CHRZANOWSKA, M. Transgenic potato clones resistant to potato virus Y. The 10th EAPR Virology Section Meeting Baden, Austria 5-10* July, 1998 p.37-38

1119/A CHACHULSKA, A.M., MILNER, M., LIPSKA-DWUŻNIK,A., KIERZEK,A. ROBAGLIA, C., ZAGÓRSK1, W. C-terminal poty viral proteins - analysis of structure and function, phylogenetic implications. 5th Intern. Symp. on Positive Strand RNA Viruses, May 23-28, 1998 Renaissance Vinoy Resort St. Petersburg, Florida Abstr. P2-58

1120/A MARCZEWSKI, W., OSTROWSKA, K., TALARCZYK, A., HENNIG, J., ZIMNOCH-GUZOWSKA, E RAPD and SCAR markers in selection of diploid potato for PVS resistance. Intern. Field Day on Potato Improvement in Poland July 16-17, 1998 IHAR Młochów - Poland

112 I/A MACIEJEWSKA, U, SZCZERBAKOWA, A., BORKOWSKA, M, WIELGAT, B. Somatic fusion between wild and cultivated Solarium species. IX Intern. Congress on Plant Tissue and Cell Culture "Plant Biotechnology and in Vitro Biology in the 21st Century" Jerusalem, Israel, June 14-19, 1998 Abstr. p. 185

1122/A DOROSZEWSKA, T, CHACHULSKA, A. Transformation of tobacco cultivars for potato virus Y resistance Ibid.:p.l39

1123/A VO SI,H., ŚWIEŻEWSKA, E. Prenylation of proteins in plants. Occurrence and some properties of GGTase II. FASEB Summer Conference "Lipid Modification of Proteins" Snowmass, Colorado, August 8-13, 1998 Abstr. P-S7

1124/A WANKE, M., ŚWIEŻEWSKA, E., DALLNER, G. Subcellular localization and sorting of plastoquinone and ubiquinone in spinach cells. 13th Inter. Symp. on Plant Lipids July 5th -10th, 1998 Sevilla, Spain Abstr. B9

135 1125/A GRĄZIEWICZ,M.-A., ZASTAWNY,T.H., OLIŃSKI.R., TUDEK,B. SOS-dependent A—>G transition induced by hydroxyl radicals generating system hypoxantineVxanthine oxidase\ Fe3+\ EDTA is accompanied by increase of Fapyadenine content in Ml3 mp 18 phage DNA. 28llh Annual Meeting Faculty of Natural Sciences University of Salzburg, September 7-10,1998 Abstr. p. 37

1126/A GRZESIUK,E., NOWOSIELSKA.A. The role of proofreading subunit of DNA polymerase III in mutation frequency decline (MFD) repair. Ibid.: p.38

1127/A NOWOSIELSKA,A., GRZESIUK,E. The rate of starvation-associated mutations is enhanced in Escherichia coli dna Q49D umuDC strain. Ibid.: p.49

1128/A KRWAWICZ,J., PIETRZYKOWSKA,! UV-induced mutagenesis independent of DNA replication. Ibid.: P015 p.117

1129/A JANION,C., WOJCIK,A. Response of transposon bearing Escherichia coli ABH57 derivatives to halogen light irradiation. Ibid.: P 037 p. 146

1130/A WÓJCIK,A., JAN1ON.C. The effect of sulA and Ion mutations on Escherichia coli K12 AB1157 phenotype and response to halogen light-irradiation. Ibid.: P 039 p. 148

113I/A GRZESIUK,E., PIERZYNOWSKA,J., WOJCIK,J. Antimutagenic activity of ellagic acid, rutin and psoralens against aflatoxin Bl. Ibid.: P 057 p. 174

1132/A SITKIEWICZ,!., ZIELENKIEWICZ.U., PANKIEWICZ,R., KERN,!., ALONSO,J.C., CEGLOWSKI,P. Characterization of the region involved in a better-than-random segregation of streptococcal plasmid pSM19035. International Symposium on Plasmid Biology 1998, October 10-16, 1998, Merida, Yuc. Mexico. Abstr. p.76 Plasmid (1999) 41: 161-162

136 1133/A BARDOWSKI.J. Application of genetic engineering of lactic acid bacteria and its role in food production. XXIX Scientific Session of The Committee of Technology and Chemistry of Food of PAS on "Technological Processes and Quality of Food", Olsztyn 21-23 September 1998 Abstr. p.51

1134/A PAWLAK,D., PACHULSKA.M., SIDOR, M., WÓJCIK,J, SCHILLER,?.W., IZDEBSKI.J. Novel backbone-cyclic enkephalin analogs. 25lh European Peptide Symposium. Budapest, Hungary, August 30-September 4, 1998 J. Peptide Sci. (1998) 4, (Special Issue) p.75 abstr.P236

1135/A SKOWRONSKA,D., BARTNIK.,E., SZABLEWSKA,!., BALINSKA,M. Synergistic, inhibitory effect of antifolates on murine leukemia cells grown in vitro (in Polish). XXXIVth Meeting of Polish Biological Society Białystok, 15-18 September 1998, abstr.L-28s.207

1136/A SKORUPINSKA.K., SZYMAŃSKA,M., ŚWIEŻEWSKA,E., HERTEL.J., CHOJNACKI T. Long chain polyprenols and their phosphates in plant tissues, (in Polish) Ibid.: C-04 p.23

1137/A ZAREBSKA,Z., WASZKOWSKA.E., CAFFIERI.S., DALL' ACQUA,F. Photoreactions of psoralen with phospholipids on the model lecithins, (in Polish) Ibid.: C-27 p.35

1138/A VO SI.H., ŚWIEŻEWSKA.E. Rab geranylgeranyltransferase in plants. Occurrence and some properties, (in Polish) Ibid.:C-28 p.35

1139/A HERTEL.J., CHOJNACKI.T. The search for new plant sources of long chain polyprenols. (in Polish) Ibid.: C-29 p.36

1140/A VO SI,H., OLSZOWSKA.O., FURMANÓW A.M., WANKE,M., CHOJNACKI.T., ŚWIEŻEWSKA,E.. Polyprenols in roots of Coluria geoides cultivated in vitro, (in Polish) Ibid.:C-30 p.36

137 114l/A GRABOWSKA,D., SKONECZNY.M., ŚWIEŻEWSKA,E., SZKOPIŃSKA ,A. The effect of induction of peroxisomes by oleic acid on dolichol synthesis in the yeast Saccharomyces cerevisiae. (in Polish) Ibid.:C-31 p.37

1142/A GRABIŃSKA,K., SZKOPINSKA,A., BERGES,Th., KARST,F., PALAMARCZYK,G. Protein/Protein interaction of yeast farnesyl diphosphate subunits. (in Polish) Ibid.: C-32 p.37

1143/A GRABOWSKA,D, KARST,F., SZKOPINSKA,E. Effect of squalene synthase gene (erg9) disruption on synthesis of polyprenols in the yeast S. cerevisiae. (in Polish) Ibid.: C-33 p.38

1144/A GRABOWSKA,D., PŁOCHOCKA,D., ŚWIEŻEWSKA.E., SZKOPIŃSKA,A. Molecular modelling as a tool for explanation of long dolichol formation in the yeast S.cerevisiae. (in Polish) Ibid.: C-34 .p.38

1145/A KRUSZEWSKA,J., JANIK.A-, LENART,U., PALAMARCZYK,G. Regulatory role of GDPMan in biosynthesis of glycoconjugates. (in Polish) Ibid.:G-02 p. 106

1146/A JANIK.A-, PALAMARCZYK.G. Role of mannosylphosphodolichol synthase in the regulation of protein secretion in S. cerevisiae. (in Polish) Ibid.:G-13 p.112

1147/A KRUSZEWSKA,J.S., PALAMARCZYK,G. Isolation of cDNA encoding mannosylphosphodolichol synthase in Trichoderma reesei. (in Polish) Ibid.:G-15 p.113

1148/A PRYMAKOWSKA-BOSAK, M., ŚLUSARCZYK, L, KURAŚ M., PRZEWLOKĄ, M., KILJAŃCZYK, B., LICHOTA, J., JERZMANOWSKI,A. Histone H l has structural function in meiosis and possible role in regulating of developmental genes, (in Polish) lbid.:I-01 p.144

1149/A PLUTA,K., SZCZEŚNIAK,B., BOGUTA, M. Mafl protein in yeast involved in tRNA biosynthesis, (in Polish) Ibid.: 1-09 p.148

138 1150/A GLINKO WSKA.M., WĘGRZYNA, WĘGRZYN,G. Inhibition of phage K growth in Escherichia coli C cells suggests the presence of unusual restriction system, (in Polish) Ibid.: 1-45 p. 166

1151/A JANOWICZA, ŁOBOCKA, M. PI plasmid partition: A method for selection of chromosomal mutants defective in partition.(in Polish) Ibid.: 1-51 p. 169

1152/A DOBRUK, A., ŁOBOCKA, M. PI plasmid partition: Expression of alternative products of PAR operon from an internal promotor, (in Polish) Ibid.: 1-52 p.169

1153/A KOWALCZYK,P., CIEŚLA,J., TUDEK, B. Localization of chloroacetaldehyde induced damage in human p53 gene by DNA polymerase finger print analysis, (in Polish) Ibid.: L-21 p.204

1154/A BRODZIK,R., GAGANIDZE.D., SIRKO.A. Overexpression of Helicobacter pylori urease subunit B in transgenic tobacco plants, (in Polish) Ibid.: M-12 p.226

1155/A BLASZCZYK.A., BRODZIK,R., SIRKO,A. Cloning of bacterial cysE gene, encoding serine acetyltransferase (SAT) and its expression in tobacco plants, (in Polish) Ibid.:M-39 p.240

1156/A LIPSKA-DWUZNIK,A., CHACHULSKA,A.M., ROSA,!., ZAGORSKI.W. Potato virus Y Nib gene - sequence analysis and plant transformation, (in Polish) Ibid.:M-42 p.241

1157/A SZYBIAK-STRÓŻYCKA,U., WALCZAK,!., PAŁUCHA.A., JUSZCZUK,M. Analysis of sequence variability of the potato virus M. (in Polish) Ibid.: N-113 p.306

1158/A WALCZAK,!., PAŁUCHA,A., SZYBIAK-STRÓŻYCKA,U. RT PCR amplification and cloning of cDNA of two isolates of potato wirus M. Ibid.:N-114 p.306

139 1159/A LIPSKA-DWUŻNIK,A., CHACHULSKA,A.M., ZAGÓRSKI.W. Potato virus Y (PVY) polymerase gene - molecular analysis and phylogenetic considerations. 6th International Students' Scientific Conference 7-9 May 1998 Abstr.61 p.37

1160/A MILNER,M, KIERZEK.A., CHACHULSKA,A.M., ZAGORSKI,W. Modelling of the structure of the potato virus Y (PVY) coat protein. Ibid.: Abstr.62 p.37

1161/A LIPSKA-DWUŻNIK, A., CHACHULSKA , A.M., ZAGÓRSKI, W. Molecular analysis of potato virus Y Nib gene and engineering of virus resistance in tobacco and potato. 7th Intern. Congress of Plant Pathology [ICPP98] Abstr. 5.3.5

1162/A CHACHULSKA,A.M., FLIS,B., CHRZANOWSKA,M., MILNER,M., GÓRA-SOCHACKA,A., ROBAGLIA.C., ZAGÓRSKI.W. RNA-mediated potato virus Y resistance in potato and tobacco. Ibid.: 5.3.6

1163/A BIERZYŃSKI.A., ZHUKOV,!., JAROSZEWSKI.I. Conservative mutation Met8 —> Leu effects the folding process and stability of squash trypsin inhibitor CMTI-I. XIIth Symp. San Diego, CA July 25-29, 1998 Abstr. 318-S Protein Sci. (1998)7 suppl. 1

1164/A BOGUTA,M., CHACINSKA,A., ROSPERT,S. Genetic evidence for a functional dependence between mitochondria! protein Nam9 and cytosolic chaperones. Gordon Conference on Mitochondria and Chloroplasts, Les Diablerets (Switzerland) September 22-25, 1998 No 14

1165/A SZCZESNIAK,B., BOGUTA,M. Screening for genes involved in propagation of the yeast prion (PSI). The 13"' Symposium of the Polish Genetics Society, Warsaw, September 22-25, 1998 J. Appl. Genet. (1998) 39A, 174 D51

1166/A BARTNIK, E. Diseases due to changes in mitochondria! DNA. Ibid.: 32 2.(plenary session)

1167/A HULANICKA,M.D. Mechanisms of gene expression from viral genomes. Ibid.: 33 1. (plenary session)

140 1168/A JABŁOŃSKA-SKWIECIŃSKA,E., BURZYŃSKA,B., ZIMOWSKI.J.G., KŁOPOCKAJ., BISKO,M., HOFFMAN-ZACHARSKA,D. Molecular analysis of glucose-6-phosphate dehydrogenase variants in Poland. Ibid.: 69 A 57.

1169/A MROCZEK,K., NOWAK.M., BARTNIK,E. Mutation or polymorphism- that is question. How to distinguish between natural variety and pathogenic changes in human mitochondria! DNA. Ibid.: 74 A 68.

1170/A MARCZEWSKI.W., OSTRO WSKA,K., TALARCZYK,A-, HENNIG.J., ZIMNOCH-GUZOWSKA,E. Application of molecular markers linked to the Ns locus in potato improvement. Ibid.: 88 B16.

117l/A LEWANDOWSKA,!., BALIŃSKA.M., PASZEWSKI,A. Genetic regulation of folate enzyme synthesis in Aspergillus nidulans. Ibid.: 148 D 01

1172/A PIOTROWSKA,M., NATORFF,R., PASZEWSKI.A- SconC, an Aspergillus nidulans sulphur metabolism regulatory gene is homolog of the yeast SKP1 essential gene. Ibid.: 149 D 02

1173/A NATORFF,R., SIEŃKO,M, BRZYWCZY.J. The Aspergillus nidulans METR sulphur positive regulator belongs to bZIP transcriptional factors. Ibid.: 149 D 03

1174/A GRYNBERG.M., TOPCZEWSKI,J., PASZEWSK1,A. Cloning and molecular analysis of Aspergillus nidulans cyslOS gene encoding the homoserine O-acetyltransferase-like protein. Ibid.: 150 D 04

1175/A BRZYWCZY,J., SIENKO,M., KUCHARSKA.A. Structure and regulation of homocysteine synthase encoding genes in Kluyveromyces lactis and Schizosaccharomyces pombe. Ibid.: 150 D 05

1176/A HAŁAS.A., CIESIELSKI.A., ŻUK.J. Involvement of the essential yeast DMA polymerases in induced gene conversion in yeast Saccharomyces cerevisiae. Ibid.: 151 D 06

141 1177/A HAŁAS,A., BARANOWSKA,H., POLICIŃSKA, Z., j ACHYMCZYK,W.J. The role of nuclear DNA polymerases in selection-induced mutations in yeast Saccharomyces cerevisiae. Ibid.: 151 D 07

1178/A JACHYMCZYK,W.J., BARANOWSKA,H., HAŁAS,A., ZUK,J., POLICINSKA,Z., CIESIELSKI,A. Different DNA polymerases are involved in nucleotide and base excision repair systems in yeast Saccharomyces cerevisiae. Ibid.: 152 DOS

1179/A KAMIŃSKA.J., GAJEWSKA,B., ŻOŁĄDEK, T. Rsp5 ubiquitin-protein ligase and Panl involved in actin cytoskeleton organization interact in two-hybrid system. Ibid.: 159 D 22

1180/A LOCHOWSKA,A, HRYNIEWICZ.M., HULANICKA,D.M. Probing the functional regions of the Escherichia coli CysB transcriptional regulator by using its mutant derivatives. Ibid.: 159 D 23

118I/A STOLARSKI.R., CIEŚLA,!, RODE,W., KULIKOWSKI.T. 'H and I9FNuclear Magnetic Resonance investigation of the ternary complex formed between recombinant rat hepatoma thymidylate synthase, 5-fluoro- 2'-deoxyuridine 5'-monophosphate and N5 ,Nlo-methylene tetrahydrofolate. XIII International Round Table "Nucleosides, Nucleotides and their Biological Applications", September 6-10, 1998 Montpellier France Abstr. 98

1182/A PIASEK,A., SHUGAR,D, KULIKOWSKI.T. 2',3' -dideoxy-3' -fluoro-2-thiothymidine (31 -FS2ddThd): in vitro potent and selective anti-HIV agent. Progress in Clinical Virology IV, European Society for Clinical Virology, Aug.30 - Sept.2, 1998, Hamburg (Germany) Abstr. 208

1183/A KULIKOWSKI.T. Analogs of thiatwed nucleosides and nucleotides - potential antiviral and antitumor agents, (in Polish) "Modified Nucleic Acids" Scientific conference dedicated to the memory of Professor Bronisław Filipowicz, Łódź, 13-14 November 1998 Abstr. p.28

142 1184/A KRYS1AK,C., BUCHOWICZ.J. Unfavourable effect of microprojectile bornbradment on the formation of somatic embryos in wheat. IX International Congress on "Plant Tissue and Cell Culture", Plant Biotechnology and I n Vitro Biology in the 21st Century, Jerusalem, Israel, June 14-19, 1998 Abstr. p.88

1185/A KROCZYŃSKA.B. ATE1, an arabidopsis homologue of the bacterial GRPE chaperone protein? 25th Silver Jubilee FEES Meeting, July 5-10, 1998, the Bella Center, Copenhagen, Denmark. Abstr. P39.09 p. 194

1186/A MICHALIK,J., SZOLAJSKA,E., CHROBOCZEK,J. Application of baculovirus for control of pest population. FAO/I AEA Int. Conf. on Area-Wide Control of Insect Pests, Penang, Malaysia 28 May - 2 June 1998. Abstr.D-22 p. 131

1187/A LOMBARSKA-ŚLIWIŃSKA,D., KONOPIŃSKA.D., ROSIŃSKI.G., MICHALIKJ., SZOŁAJSKA,E. Biological evaluation of insect hypertrehalosemic peptide periplanetin CC-2 and its analogues. Xlii"1 Congress of the Polish Pharmacological Society, Katowice,September 13-16, 1998. Pol. J. Pharmacol.(1998) 50 suppl. 166

1188/A MACIEJEWSKA,U., SKIERSKI.J., SZCZERBAKOWA,A. Determination of nuclear DMA content in Solcmum species using flow cytometry method. The 4th Conference of the Polish Society of Cytometry: "Application of flow cytometry in scientific research and diagnostics, 18-218' October 1998, Gdańsk. Abstr. P13 i 189/A GODLEWSKU, KLUDKIEWICZ,B., GRZELAK,K., CYMBOROWSKI.B. Expression oflhp76 and Ihp82 genes in diapausing Galleria mellonella larvae. VI"' Eur. Congr. Entomol. Ćeske Budejovice, August 23-29, 1998. Abstr. p.I29

1190/A BLASZCZAK,A., GEORGOPOULOS,C., LIBEREK.K. On the mechanism of FtsH-dependent degradation of the c32 transcriptional regulator of Escherichia coli and the role of DnaK chaperone machine. Intern. Symp. "Molecular Cell Biology of the Heat Stress Response" Oct. 15"' to Oct. 18th 1998 Biocentre of the J.W.Goethe-Universitat Frankfurt/Main, Germany. Abstr. P 28

143 1191/A LIWOSZ.A-, SZCZEGIELNIAK,J., JURKOWSKI.L, DOBROWOLSKA,G., MUSZYŃSKA,G. Differential inhibition of mammalian and plant calcium and phospholipid dependent protein kinases by their autoregulatory domains. 1st International Conference on Inhibitors of Protein Kinases. Cell. Mol. Biol. Lett. (1998) 3, 316

1192/A SHUGAR,D. Viral & host-cell protein kinases: Enticing antiviral targets. Ibid.: 338

1193/A FELCZAK,K., MIAZGA,A, GOŁOS,B., RODE,W., SHUGAR.D., KULIKOWSKI.T. Synthesis, conformation and biological properties of selenonucleosides. XIII International Round Table "Nucleosides, Nucleotides and Their Biological Applications" Montpellier, France, September 6-10, 1998 Poster 36.

1194/A WĘGRZYN,A., WĘGRZYN,G. Escherichia coli dnaA and seqA gene functions and regulation of a plasmid replication. EMBO Workshop "Coupling of DNA replication to cell growth". Geilo, Norway, June 15-21, 1998

1195/A SZALEWSKA-PAŁASZ,A, WĘGRZYN,A., BŁASZCZAK,A., TAYLOR, K., WĘGRZYN.G. P subunit of Escherichia coli RNA polymerase as a transcriptional activator contact site. RNA Polymerase Meeting, Mountain Lake, October 30-Nov.l. 1998. Abstr. p. 1-4

1196/A BURZYNSKA,B., TOPCZEWSKI.J. Genotyping Bison bonasus by analysis of mitochondria! D-loop and VNTR markers. IV*International Symposium on Ecological Genetics in Mammals. Research Institute of Wildlife Ecology Vienna Vet. University, 6-10 September 1998

1197/A KRWAWICZ,J., PIETRZYKOWSKA.I. Comparison of a mutagenic pathway(s) of UV-induced mutagenesis of

XsusOs phage under conditions permissive and nonpermissive for phage DNA replication. 6-th International Students' Scientific Conference, Gdańsk, Abstr. 33 p.22

144 1198/A LASOCKI.K.W. Cloning cDNA ofarcA, the specific transcriptional activator of enzymes from arginine catabolic pathway in Aspergillus nidulans . 6* International Students' Scientific Conference, Gdańsk, Abstr.34 p.22

1199/A SIWECKA,M.A. Base specificity and amino acid sequence of nuclease II associted with ribosomes of rye germs, (in Polish) "Modified Nucleic Acids" Conference in memory of Prof. B. Filipowicz, Łódź, I3-14 November 1998, Abstr. p. 40

1200/A FUALKOWSKAJ.J., JOŃCZYK,?., MALISZEWSKA-TKACZYK,M., BIALOSKORSKA,M., SCHAAPER,R.M. Unequal fidelity of leading and lagging strand replication on E.coli chromosome. Conference Sponsored by the Genetics Society of America "DNA Repair: Bacteria to Humans". April 16-19, 1998, Airlie Conf. Center Warrenton,Virginia. Poster Abstract # 28

120I/A PRZYKORSKA.A, LONGO.A., KULIGOWSKA.E., CARTER.C.W.Jr. The evidence of conformational changes in tRNACys upon binding with aminonoacyl-tRNA synthetase. EMBO Workshop on "Structure and Function of Aminoacyl-tRNA Synthetases." October 10-15, 1998-Mittelwihr, France. Poster Session II.D p.88

1202/A BUGAJSKA,O., LOBOCKA,M. / n vivo ParB-DNA interaction during partitioning of PI plasmid of Escherichia coll 6th Intern. Students Scietific Conference, Gdansk-Poland 7-9 May 1998 Abstr. 47 p29

1203/A DOBRUK,A., LOBOCKA,M. PI plasmid partition: A potential of Par operon of PI to encode two forms of Para protein. Ibid.: 48 p.30

1204/A ŁOBOCKA.M., ROSE.D., RUSIN.M., SAMOJEDNY,A., YARMOLINSKY,M., BLATTNER.F.C. The genome of PI bacteriophage: Analysis based on nucleotide sequence of two related isolates.

6"' International Workshop on P2, P4 and Related Bacteriophages, October 23-26, 1998, Berlin-Dahlem, Germany. Abstr. p.54

145 1205/A ŁOBOCKA.M., ROSE.D, RUSIN.M., SAMOJEDNY.A., YARMOLIŃSKY,M., BLATTNER.F.C. Complete nucleotide sequence of PI, a bacteriophage that lysogenizes as a plasmid: Analysis based on two related isolates. 121'1 Evergreen International Phage Biology Meeting. 'July 16-20, 1998 Abstr. p.7

1206/A BORYS,E., KUSMIEREK.J.T. Inhibitors of N-alkylpurine-DNA glycosylases. (in Polish) Modified Nucleic Acids Scientific conference dedicated to the memory of Professor Bronisław Filipowicz., Łódź 13-14 November 1998

1207/A JERZMANOWSKl,A., PRYMAKOWSKA-BOSAK,M., PRZEWŁOKA,M., SLUSARCZYKJ., KURAŚ.M. Analysis of transgenic tobacco with drastically reduced level of histone HI. Keystone Symposia on Molecular and Cellular Biology."The Nuclear Matrix: Involvement in Genomic Organization, Function and Cellular Regulation." Epigenetic Regulation of Transcription. Copper Mountain, Colorado, April 4-9, 1998 Abstr. 106

1208/A KRZYMOWSKA,M., HENNIG.J. The salicylic acid responsive element I isolated from tobacco PR-2dg&n& specifically interacts with potato nuclear proteins. 5 "'International Symposium on the Molecular Biology of the Potato. August 2-6, 1998 Bogensee, Germany.

1209/A MARCZEWSK1,W., OSTROWSKA,K., TALARCZYK,A., HENNIG.J., ZIMNOCH-GUZOWSKA,E. Identification of RAPD and SCAR markers linked to the Ns locus in diploid potato. Ibid.:

1210/A HENNIG,J. Signals involved in plant response to pathogen attack. Polish-Korean Bilateral Seminar "Current Trends in Biotechnology in Poland and Korea", Poznań, November 12-13, 1998.

121 I/A KACZANOWSKI,S., KIERZEK,A.M., ZIELENKIEWICZ,P. Shuffling algorithm for protein sequence design. 5th in a biannual series International Conference Bioinformatics, Networking and Computing in Molecular Biology "Genes, Proteins and Computers V" York University 14-16 September 1998.

146 1212/A SKOWROŃSKI.K., DYCZKOWSKI,J., KIERZEK,A.M., ZIELENKIEWICZ.P. Rapid estimation of the significance of the Smith & Watermann aiingments. Ibid.:

1213/A MACIEJEWSKA,U., POLKOWSKA,L. Oxidative processes in cell suspensions of Solatium species induced by elicitor from Phytophthora infestans. Winter Meeting 1998 of the Society for Free Radical Research (European Region) "Oxygen, free radicals and oxidative stress in plants " Granada, Spain December 17-19, 1998. Abstr. p.97.

1214/A HENOUX,V, VIEIRA da SILVA,M.R, ZAGORSKI,W., CLAISSE,M.L. Influence of temperature on cell growth, cytochrome syntheses and in vivo and in organello respiratory activity of the yeast Schwanniomyces occidenlalis (castellii). UNESCO Global Network MCBN Workshop "New Perspectives in Mitochondrial Research" Padova (Italy), September 18-21, 1997 A-10

1215/A EJCHART, A. Scalar couplings in protein structure determination, (in Polish) Procedings of XXXI National Seminar on Magnetic Resonance and its Applications. 1-2 December 1998 Nuclear Physics Institute.

1216/A MOLCHANOV, S., EJCHART, A., GRYFF-KELLER, A., Study of KCN-kryptofix222 complex in DM SO solution by means of relaxation of I3C, '4Nand 15N nuclei (part II). (in Polish) Ibid.: 102-104

1217/A EJCHART, A., RUSZCZYŃSKA, K., WÓJCIK, J., GOCH, G., B1ERZYŃSKI, A. Structural dynamics of calcium binding peptides. (in Polish) Ibid.: 325-328

1218/A SIDOR, M., WÓJCIK, J., PAWLAK, D., PACHULSKA, M., IZDEBSKI, J. Confonnational analysis of new enkefalin analogue by means of NMR. (in Polish) Ibid.: 329-332

1219/A ZHUKOV, I., EJCHART, A. Elements of the NMR structure of the S100A1 protein in solution from relaxation of 15N nuclei Ibid.: 337-340

147 1220/A SHUGAR, D. Viral and host-cell protein kinases: enticing antiviral targets. Modified "Nucleic Acids Scientific conference dedicated to the memory of Professor Bronisław. Filipowicz, Łódź, 13-14 November 1998 Abstr. p.9-10 122 I/A DALL' ACQUA, p., CAFFIERI, s., VEDALDI, D., MIOLO, G., ZARĘBSKA, Z. New therapeutic approach for the treatment of vitiligo: psoralen-membrane fatty acids adducts. XV1'1 EFMC International Symposium on Medical Chemistry, Edinburgh, Scotland, 6-10 September 1998. Abstr. P.351

1222/A JOUKOV, I., EJCHART, A. 15N relaxation study of backbone mobility in CMTI-I (M8L) protein. 8th Chianti Workshop on Magnetic Resonance. Nuclear and Electron Relaxation. San Miniato (Pisa), Italy May 30 - June 5, 1999 Abstr.: p.67

I223/A EJCHART, A., ZIMNI AK, A., OSZCZAPOWICZ, I., WĄSOWSKA, M. 'JC relaxation study of daunorubicin and its new formamidine derivative. Ibid.:p.36

1224/A HUNG, Vo Si, ŚWIEŻEWSKA, E. Rab prenyltransferase in plant cells. 4th European Symposium on Plant Isoprenoids. Barcelona, Spain, April 21-23, 1999

1225/A SKORUPIŃSKA, K., ŚWIEŻEWSKA, E., HERTEL, J., CHOJNACKI, T. Long chain polyprenols and polyprenyl phosphates in plant tissues. Ibid.:

1226/A BARDOWSKI, J. Geneticaly modified lactic acid bacteria - a case of sugar catabolism. 102"d Event of the European Federation of Biotechnology. Food Biotechnology. Zakopane. Poland 9-12 May 1999 p.19

1227/A ALEKSANDRZAK, T., KOWALCZYK, M., BARDOWSKI, J., KOK, J. Regulation of carbon catabolism in Lactococcus lactis. Ibid.: 26 A4

1228/A DOMAŃ, M., CZERNIEĆ, E., TARGOŃSK1, Z, BARDOWSKI, J. Production and genetic regulation of an amylase in Lactococcus lactis. Ibid.: 27 A5

148 ] 229/A SZWACKA, M., KRZYMOWSKA, M., KOWALCZYK, M., OSUCH, A. Transgenic cucumber plants expressing the thaumatin gene. Ibid.: 23 Al

1230/A BAL, W., WÓJCIK, J., MACIEJCZYK, M., KASPRZAK, K.S. Structure determination of the N-terminal 15-peptide of human protamine HP2 by nuclear magnetic resonance, distance geometry and simulated annealing. J. Biomol. Struct. Dynamics (1999) 16 1370-1371

123 I/A MICHALIK, J., SZOŁAJSKA, STROKOVSKAYA, L., OSTOJA- ZAGÓRSK1, W. Expression of viral, genome-linked protein, Vpg, in baculovirus eucariotic system. Abstracts of the 26lh Meeting of the Federation of European Biochemical Societies (FEBS) Nice, June 19-24, 1999 Biochimie suppl. 6(1999)

1232/A STROKOVSKAYA, L., MICHALIK, J., KIKHNO, L, SZOŁAJSKA, E., SOLOMKO, A, OSTOJA-ZAGÓRSKI, W. Intra-and extracellular expression of human prolactin. Ibid.: suppl. 6

1233/A GRZESIUK, E., LAUBITZ, D., ZABIELSKI, R., PIERZYNOWSKI, S.G. The influence of a myoelectrical migrating complex-related elektromagnetic field on the growth of Escherichia coli strains. Twenty-first Annual Meeting. The Hyatt Regency Long Beach, California, USA, June 20-24 1999 Abstr. Book P-57

1234/A TALJAŃSKI, W., GRZESIUK, E., PIERZYNOWSKI, S.G., ZIMAK, J., PIEKARCZYK, A. Preliminary health risk assessment in family environment - adverse effects of cytostatics excreted by patients on chemotherapy. I-st Annual Meeting of Polish Pharmacological and Clinical Society, Poznań 13-14 May 1999 Post. Farmakoterapii (1999) J_ 58-59

1235/A GRZESIUK, E., NOWOSIELSKA, A., Spontaneous argE30->Arg+ reversions in stationary phase cultures of Escherichia coll Abstracts from EEMS-99 29* Annual Meeting of the European Environmental Mutagen Society. 4-9 July, 1999 Pharm. Toxicology (1999) 85 suppl. 1 PII-1 p.19

149 1236/A NOWOSIELSKA, A., GRZESIUK, E., Double defect: mutation in e subunit of DNA polymerase III and deletion of SOS unniDC operon increases the rate of adaptive mutations in Escherichia coli. Ibid.: P II-2

1237/A GRZESIUK, E., GOZDEK, A., TUDEK, B., Contribution of 7-MeGua and 3-MeAde in MMss induced mutagenesis in Escherichia coli. Ibid.:P II-3

1238/A KOTER, M., SYLLER, J., ZIMNOCH-GUZOWSKA, E., SLACK, S., HENN1G., J. Transgenic potato expressing a truncated form of the PLRV replicase (ORF2) gene show replicase mediated protection against potato leafroll virus 9'1' International Congress, Amsterdam, July 26-30 1999 Mol. Plant-Microbe Interact. Book of Abstracts (1999) 12 136

1239/A WASZKOWSKA, E., ZARĘBSKA, Z., POZNAŃSKI, J., ZHUKOV, I. Spectroscopic detection of lecithin photoproducts after 8-methoxypsoralen plus UVA treatment. 8lh Congress European Society for Photobiology, Palacio de Exposiciones y Congresos Granada, Spain 3-8 September 1999 Book of Abstracts P143

1240/A ALEKSANDRZAK, T., KOWALCZYK, M., KOK, J., BARDOWSKI, J. CcpA-mediated genetic regulation of p-glucoside catabolism in Lactococcus lactis. Sixth Symposium on Lactic Acid Bacteria Genetics, Metabolism and Applications, Veldhoven, the Netherlands 1999 September 19/23 Book of Abstracts FEMS

1241/A CZERNIEĆ, E., DOMAŃ, M., TARGOŃSKI, Z., BARDOWSKI, J. Extracellular amylase production in Lactococcus lactis. Ibid.:

1242/A SKORUPIŃSKA, K., VO SI, H. OLSZOWSKA, O., FURMANÓW A, M. WANKE, M., CHOJNACK1, T., ŚW1EŻEWSKA, E. Studies on mechanisms of polyprenol biosynthesis, (in Polish) XXXVth Meeting of Polish Biochemical Society, Olsztyn, 13-16 September 1999, abstr. 111-16 p.73

150 1243/A VO Si, H., ŚWIEŻEWSKA, E. Rab geranylgeranyltransferase in wheat seedings. (in Polish) Ibid.: 111-17

1244/A WANKE, M., ŚWIEŻEWSKA, E. Reconstruction of in vitro transport system of plastoquinone and ubiqninone in spinach leaf cells, (in Polish)

1245/A POLKOWSKA, L., MACIEJEWSKA, U., W1ELGAT, B. Plasma membrane reactions in cultivated and wild potato species from Phytophthora infestans. (in Polish) Ibid.: 111-12

1246/A WĘGRZYN, A., WRÓBEL, B., WĘGRZYN, G. Changes in biological properties of cell membranes in Escherichia coli dnaA and sec/A mutants, (in Polish) Ibid.: 111-19

1247/A GLINKOWSKA, M., WĘGRZYN, A., WĘGRZYN, G. Replication of bacteriophage lambda in Escherichia coli dnaA Arac strains. (in Polish) Ibid.: IV-7

1248/A GROMADKA, R., RYTKA, J. The S.cerevisiae gene KRR1 encodes protein involved in ribosome assembly, (in Polish) Ibid.: IV-8

1 249/A PRZEWŁOCKI, G., LIPECKA, J., EDELMAN, A., PRZYKORSKA, A. Sequence-specific human RNase T84. (in Polish) Ibid.: IV-23

1250/A SŁOMIŃSKA, M., WĘGRZYN, A., WĘGRZYN, G. Role of dnaA and seqA proteins in the regulation of replication of lambda

plasmids and in transcription from thepR promoter of bacteriophage lambda. (in Polish) Ibid.: IV-25

125 l/A SZOŁAJSKA, E., ZIEMNICKA, J., MICHALIK, J. Initial characterization of baculovirus SsMNPV genome, (in Polish) Ibid.: IV-27

151 1252/A KŁUDKIEWICZ, B., GRZELAK, K., NIEDZIELA-MAJKA, A., OŻYHAR.A. Expression of heterologous genes in the yeast Pichia pastoris. (in Polish) Ibid.: VF-8

1253/A KRYSIAK, C., MAZUŚ, B., BUCHOWICZ, J. Side effects of biolistic transformation with the use of DNA-coated tungsten particles, (in Polish) Ibid.: Vl-9

1254/A CHOJNACKA, E., CZERNICKI, Z., GRIEB, P., HORSZTYŃSKI, D., JANKOWSKI, W., JÓŻWIK, A., MATYSIAK, Z., SIDOR, M., TOCZYLOWSKA,B- WAŁECKI, J., WÓJCIK, J. 'H-NMR spectra of water soluble metabolites extraced from tumor tissues. Search for a molecular marker of tumor type and malignancy, (in Polish) Ibid.: V1I-2

1255/A WIELGAT, B., SZCZERBAKOWA, A., Somatic hybridization between wild and cultivated potato species, (in Polish) In: Reports of The 1st National Congress on Biotechnology, Wrocław, September 20-25, 1999, p.59-60

1256/A SZCZERBAKOWA, A., BORKOWSKA, M., MACIEJEWSKA, U., WIELGAT, B. Somatic hybrids Solanum tuberosum L.+ Solarium nigrum. L. Ibid.: p.50 1257/A MICH AUK, J. Baculoviruses and their recombinants as natural bioinsecticides. (in Polish) Ibid.:p.342-344

1258/A SZOŁAJSKA, E, ZIEMNICKA, J., MICHALIK, J. The evaluation ofSlilpnotia salicis (SsMNPV) baculovirus as bioinsecticide. (in Polish) Ibid.: p.281

1259/A MARCZEWSKI, W., FLIS, B., LEBECKA, R., SYLLER, J., HENNIG, J., TALARCZYK, A., GEBHARDT, C., ZIMNOCH-GUZOWSKA, E. Mapping and identification of molecular markers of viral and bacterial resistance genes in potato, (in Polish) Ibid.: p. 119

1260/A KŁUDKIEWICZ, B., GRZELAK, K., NIEDZIELA-MAJKA, A., OŻYHAR, A. Expression of USP protein in the yeast Pichia pastoris. (in Polish) Ibid.: p.88

152 126 I/A STROKOVSKAYA, L., GUNKOVSKAYA, N., DUŻYJ, D., SOŁOMKO, A., SZOŁAJSKA, E., MICHALIK, J. Human prolactin raise by baculovirus recombinant in insect ceil lines, (in Polish) Conference on Baculoviruses in Biotechnology. Poznań, 5.02.1999 UAM 18-21

1262/A J ANION, C. Is adaptive mutagenesis, which occurs more frequently when beneficial than when neutral, a reflection oflag in phenotypic expression? Abstracts from FEMS-99 29lh Annual Meeting of the European Environmental Mutagen Society 4-9 July, 1999 Pharmacol. Toxicol. (1999)85, suppl. 1 p.22 P-II-14

1263/A SPEINA, E., CIEŚLA, J.M., GRĄZIEWICZ, M.A., KUŚMIEREK, J.T., TU DEK, B. The inhibitors ofE.coli formamidopyrimidine - DNA glycosylase (fpg protein). lbid.:p.25 P-IIIa-1

1264/A KOWALCZYK, P., CIEŚLA, J., TUDEK, B. Localisation of cyclic adducts to DNA bases in human p53 gene. Ibid.: p.26 P-IIIa-6

1265/A TUDEK, B., MOSPAN, O., GRĄZIEWICZ, M.A., ZASTAWNY, T., OLIŃSKI, R. SZODKOWSKA, J., RUDSIZKI, P., LIKHACHEV, A. DNA repair enzyme activity and oxidative DNA damage in the tumours and normal lung tissues of lung cancer patients. Ibid.: p.8 W-Ia-3

1266/A KO WALCZYK, P., CIEŚLA, J, TUDEK, B. Localization of chloroacetaldehyde induced DNA damage in human p53 gene by DNA polymerase fingerprint analysis. 7th International Students Scientific Conference, 6-8 May, 1999 Gdańsk, Poland Abstract Book p.20

1267/A SPEINA, E., GRĄZIEWICZ, M.A., CIEŚLA, J.M., KUŚMIEREK, J.T., TUDEK, B. Search for the inhibitors of E. coli formamidopyrimidine-DNA glycosylase (FPG protein). Ibid.: p.25

153 1268/A NATORFF, R., SIEŃKO, M, BRZYWCZY, J., PASZEWSKI, A. The Aspergillus nidukms METR sulphur regular belongs to bZIP transcritional factors. Twentieth Fungal Genetics Conference March 23-28, 1999 California Fungal Genet. Newslett, vol. 46 suppl.

1269/A BIAŁKOWSKA, A., KURLANDZKA, A., Suppressors oflRRl- a gene involved in colony formation in Saccharomyces cerevisiae. XIX International Conference on Yeast Genetics and Molecular Biology, Rimini, Italy, 25-30 May 1999 p. 179

1270/A GROMADKA, R., RYTKA, J. The KRR1 gene encodes protein involved in ribosome assembly. Ibid.: p. 190

1271/A PLUTA, K., SZCZEŚNIAK, B., BOGUTA, M. Functional analysis of the MAPI gene of Saccharomyces cerevisiae involved in tKNA biosynthesis and translation elongatin. Ibid.: p.207

1272/A CHACIŃSKA, A., ROSPERT, S., BOGUTA, M. A mutant of the yeast mitochondrial protein Nam9 affects respiration in dependence on the level of cytosolic Hspl04. Ibid.: p.280

1273/A FIKUS, M.U., MIECZKOWSK1, P.A., KOPROWSKI, P., RYTKA, J., ŚLEDZIEWSKA-GÓJSKA.E., CIEŚLA, Z. The DNA damage-inducible gene of Saccharomyces cerevisiae DIN7 encodes the protein which specifically functions in mitochondria. Ibid.:p.351

1274/A MIECZKOWSKI, P.A., DAJEWSKI, W., GALUSZEWSKA, A., CIEŚLA, Z., ŚLEDZIEWSKA-GÓJSKA, E. The Saccharomyces cerevisiae UMP1 gene encoding the molecular chaperone specifically required for maturation of 20S proteasome, belongs to the family od DMA damage inducible DIN genes. Ibid.: p.358

1275/A REMPOŁA, B., KANIAK, A., MIGDALSKI, A., RYTKA, J., SŁONIMSKI, P.P., di RAGO, J.P. Functional analysis ofRRDl (YIL153w) and RRD2 (YPL152w), two putative activators of phosphotyrosyl phosphatase activity of pp2a in Saccharomyces cerevisiae. Ibid.: p.404

154 1276/A GAJEWSKA, B., KAMIŃSKA, J., JESIONOWSKA, A., HOPPER, A.K., MARTIN, N.C., ŻOŁĄDEK, T. Functional analysis of WW domains of RspSp. Ibid.:p.417

1277/A KAMIŃSKA, J., JESIONOWSKA, A, ŻOŁĄDEK, T. Suppressors ofmdpl mutations located in RSP5 gene encoding ubiquitin protein ligase. Ibid.: p.419

1278/A KUCHARCZYK, R., SŁONIMSKI, P., RYTKA. J. Functional analysis of the newly discovered Saccharomyces cerevisiae gene CCZ/(ORF YBR131\v). Ibid.: p.421

1279/A JARUGA, P., TUDEK, B, SPEINA, E., OLIŃSKI, R. Oxidative DNA base modifications analysed with repair enzymes and GC/MS technique. 4'1' Winter Research Conferences, Valloire - France, 21-26 March, 1999

1280/A GAJEWSKA, B., KAMIŃSKA, J., JESIONOWSKA, A., HOPPER, A.K., MARTIN, N.C., ŻOŁĄDEK, T. Studies on functions of WW domaines of Rsp5p. 7lh Conference on Cell Biology, 9-11 September, 1999 Kraków, Poland Folia Histochem.CytobioL, (1999) 37, suppl. 1 p.9

128 l/A BIAŁKOWSKA, A., KURLANDZKA, A. Suppressors oflRRI - a gene involved in colony formation in Saccharomyces cerevisiae. Ibid.: p.59

1282/A CHEŁSTOWSKA, A., RYTKA, J. Yeast mutants provide a model to study human porphyria cutanea tarda. Yeast Genetics and Human Disease II. Vancouver, British Columbia, June 24-27, 1999 abstr. 140

1283/A KRUSZEWSKA, J., JANIK, A., LENART, U., PALAMARCZYK, G. Overexpression of a-D-mannose-1-phosphate guanyltransferase encoding gene restores the viability of the Saccharomyces cerevisiae mutants affected in early steps of glycoconjugate formation. 3rd Carbohydrate Bioengineering Meeting, Lecture and Poster Abstracts, University of Newcastle Upon Tyne 11"1 -14th April 1999

155 1284/A KRUSZEWSKA, J.S., SALOHEIMO, M., MIGDALSKI, A., PENTTILA, M., PALAMARCZYK, G. Isolation of a Trichoderma reesei cDNA encoding annosylphosphodolichol (MPD) synthase. Ibid.: 2.1

1285/A KRUSZEWSKA, J.S., SALOHEIMO, M., MIGDALSKI, A., PENTTILA, M.,PALAMARCZYK, G. Mannosylpbosphodolichol synthase from T.reesei belongs to the human and S.pombe class of the enzyme. 6 International Trichoderma-Gliodadium Workshop 11-13.6.1999 Swedish-Finnish Cultural Centre, Hanasaari, Espoo, Finland p.40

1286/A GRABLŃSKA, K., BERGES, T, KARST, F, PALAMARCZYK, G. Possible involvement of 26S proteasome in degradation of yeast farnesyl diphosphate synthase. 20th International Specialized Symposium on Yeasts. Yeast Cell Surfaces and Membrane Phenomena. May 23-27, 1999 Smolenice, Slovakia p.61

1287/A JANIK, A., SOSNOWSKA, M., KROTKIEWSKI, H., PALAMARCZYK, G. GDPMannose corrects defect in the synthesis of dolichol-linked saccharides in Saccharomyces cerevisiae. Ibid.: p.29

1288/A DOMAŃ, M., CZERNIEĆ, E., ALEKSANDRZAK, T., KOWALCZYK, M., BARDOWSKI, J. Influence of carbohydrate catabolism in lactic acid bacteria on food biotechnology. XXX Scientific Session of The Committea of Technology and Chemistry of Food of PAS on Technological Processes and Quality of Food. Food Science in the Begining of XXI Century. Kraków, 14-15 September 1999, IV-327

1289/A KROCZYŃSKA, B. Higher plant homologues of the bacterial DnaJ proteins. European Research Conferences. Biology of Molecular Chaperones. The role of molecular chaperones in protein biogenesis, transport and misfolding. Acquafredda di Maratea, Italy, May 22-27, 1999

1290/A UTKOWSKA, S., GABIG, M., WĘGRZYN, A., HERNENDEZ, P., SCHVARTZMAN, J.B., HELIŃSKI, D.R., WĘGRZYN, G. Regulation of the bacteriophage \ switch from an early (theta) to a late (sigma) DNA replication mode. Gordon Conference on Plasmid and Chromosome Dynamics, New London, NH, USA, 1-6 August 1999

156 1291/A GABIG, M., KUŻNIACKA, A., CIEJKA, M., WĘGRZYN, G. Regulation of bacteriophage X r j development in Escherichia coli cells growing in conditions resembling natural habitat of this bacterium. The 1st National Congress on Biotechnology. Wrocław, September 20-25, 1999 Poster

1292/A SITKIEWICZ, I., ZIELENKIEWICZ, U., KERN, L, CEGŁOWSKI, P. Plasmid addiction system and copy number control of pSM19035 are under a common pathway of regulation. First symposium of the EU-concerted action on "Mobile elements' contribution to bacterial adaptability and diversity" (MECBAD), Le Mont Ste. Odile, France 23-27 April 1999 p.53

1293/A HENNIG, J., Tobacco response to TMV infection is modulated by catalase activity. Free radicals, nitric oxide and antioxidants in health and disease, Antalya, Turkey 18-24 September 1999 P17 p.66

1294/A SZKOPIŃSKA, A., SKONECZN Y, M., Ś WIEŻE WSKA, E. Proliferation of peroxisomes by oleic acid induction affects dolichol synthesis in S.cerevisiae. 26th Meeting of the Federation of European Biochemical Societies, Nice, June 19-24, 1999 Biochimie (1999)81* suppl. 6 p.133

1295/A KRWAWICZ, J., PIETRZYKOWSKA, I.

DNA sequence analysis of ksusOK revertants UV-induced in a host permissive or nonpermissive for DNA replication. Ibid.: p. 126

1296/A KOPROWSKI, P., FIKUS, M.U., MIECZKOWSKI, P., RYTKA, J. ŚLEDZIEWSKA-GÓJSKA, E., CIEŚLA, Z. The product of the DNA damage-inducible gene of S.cerevisiae, DIN7, specifically affects stability of mtDNA. Ibid.: p.277

1297/A GALUSZEWSKA, A., MIECZKOWSKI, P., DAJEWSKI, W., CIEŚLA, Z. ŚLEDZIEWSKA-GÓJSKA, E. Induction of the expression of Saccharomyces cerevisiae UMP1 gene in the response to DNA damage Ibid.: p.276

157 1298/A LOOG, M., TOOMIK, R., SAK, K., MUSZYŃSKA, G., EK, P., JARV, J. Peptide phosphorylation by Ca++-dependent protein kinase from maize seedlings. Ibid.: p.259

1299/A LIWOSZ, A., SZCZEGIELNIAK, J., JURKOWSKI, I., DOBROWOLSKA, G. MUSZYŃSKA, G. Sensitivity of calcium-dependent protein kinase from maize seedlings to phospolipids. Ibid..: p.259

1300/A MALISZEWSKA-TKACZYK, M., JOŃCZYK, P., BIAŁOSKÓRSKA, M. Unequal fidelity of leading strand and lagging strand DNA replication in SOS-induced Escherichia coli cells. Ibid.:p.l27

1301/A GAWEŁ, D., JOŃCZYK, P, FIJAŁKOWSKA, I.J., SCHAAPER, R.M. Fidelity of replication of leading and lagging DNA strands in the Escherichia coli dnaX mutator. Ibid.:p.l26

1302/A KULIKOWSKI, T., DZIK, J.M., FELCZAK, K., MIAZGA, A., RODE, W. Interactions of 5-substitutedN4-Hydroxy-2'-deoxycytidine 5'- Monophosphates with Thymidylate Synthase. Ibid.: p.118

1303/A KULIKOWSKA, E., BZOWSKA, A., BRYS1AK, A., HOLY, A., SHUGAR, D. (S)-PMPA, a weak bisubstrate inhibitor ofE.coli PNP. Ibid.rp.118

1304/A PRZEWŁOCKI, G., LIPECKA, A., EDELMAN, A., PRZYKORSKA, A. Sequence-specific RNase T84. Ibid.: p.244

1305/A OLSZAK, K., KEITH, G., PRZYKORSKA, A. The influence of Mg++ ions on the Rn nuclease I activity. Ibid.: p.I21

1306/A BIERZYŃSKI, A., GOCH, G., OMELYANYUK, V., WÓJCIK, J., RUSZCZYŃSKA, K. Short polyalanine a-helices nucleated by a calcium-binding loop. The Protein Society Third European Symposium 19-22 September 1999, Garmish-Partenkirchen, Germany

158 1307/A MROCZEK, K., NOWAK, M., BARTNIK, E. Polymorphism analysis of mitochondrial DNA in Polish population. 2nd Meeting of Polish Society for Human Genetics, Poznań, 24-26 maja 1999 P80 p.228

1308/A BARTNIK, E. Telomeres and telomerase. (in Polish) Longevity Congress 16-17 September 1999, PKiN, Warszawa p.41

1309/A BARTNIK, E. Mitochondria in ageing and disease, (in Polish) Basic Sciences in Gynecology II 10-12 June, Lublin-Puławy 1999 Abstr. 61

1310/A MACIEJCZYK, M., RUDNICKI, W.R., LESYNG, B. An effective potential for a mezoscopic model of DNA. 11"' Conversation in the Discipline BiomolecuJar Stereodynamics, 11-15 June 1999, Albany, USA J. Bioinol. Struct. Dynam., (1999) 16, p. 1341 Abstr. 146

131 I/A PIETRZYKOWSKA, I., KRWAWICZ, J. On the mechanism of UV-induced mutagenesis independent of DNA replication. 8th Congress European Society for Photobiology, Palacio de Exposiciones y Congresos, Granada, Spain 3-8 September 1999 p.95

1312/A ZHUKOV, I, EJCHART, A. Factors improving accuracy of I5N relaxation parameters in proteins. Symposium on "Application of magnetic resonance in chemistry and related areas" Institute of Organic Chemistry PAS, June 9-11, 1999

1313/A MIKOŁAJCZYK, M., AWOTUNDE, O.S., MUSZYŃSKA, G., DOBROWOLSKA,G. Abscisic acid-independent protein kinases activated by osmotic stress. Seventeenth Annual Symposium, Current Topics in Plant Biochemistry, Physiology and Molecular Biology. April 14-17, 1999

1314/A GÓRA-SOCHACKA, A., ROSA, L, CANDRESSE, T., ZAGÓRSKI, W. Transgenic potato plants carrying truncated cDNA copy of severe strain PSTVd. NATO Advanced Research Workshop, October 17-21, 1999 Szeged, Hungary

159 1315/A ZAGÓRSKI, W., CHACHULSKA, A., LIPSKA-DWUŻNIK, A. Molecular phylogeny as a tool for controlling potato virus Y spread. Ibid.:

1316/A FLIS, B., CHACHULSKA, A., PALUCHA, A. Performance of virus resistant transgenic potatoes. Ibid.:

1317/A M1LNER, ML, HAENNI, A.L., ZAGÓRSKI, W., PALUCHA, A. In vitro and in vivo expression of a PVY cDN A copy containing a truncated viral polyprotein. Septiemes Rencontres de Virologie Vegctale, March 1999, Aussois, France

1318/A SADOWY, E., DAVID, Ch., GRONENBORN, B., HULANICKA, D. In vivo efficient replication of PLRV genome from cloned cDNA. Troisieme Colloque des 3R: Replication, Recombinaison, Reparation. Villejuif, June 15-18, 1999

1319/A SADOWY, E., DAVID, Ch., GRONENBORN, B., HULANICKA, D. Agroinfectious clones of potato leafroll virus. Septiemes Recontres de Virologie Vegetale, Aussois. France 14-18 March 1999

1320/A JOŃCZYK, P., GAWEŁ, D., SCHAAPER, R.M., FIJAŁKOWSKA, I.J. Fidelity of replication of leading and lagging DNA strands in the E.coli chromosome. ASM Conference on DNA Repair and Mutagenesis: Mechanism, Control, and Biological Consequences, November 1-7, 1999 Hilton Head, South Carolina B46 p.86

1321/A FIJAŁKOWSKA, I.J., GAWEŁ, D., JOŃCZYK, P., PHAM, P.T., SCHAAPER, R.M. The asymmetry of mutagenesis during replication of leading and lagging DNA strands in the E.coli dnaXmutator mutants. Ibid.: B48

1322/A MALISZEWSKA-TKACZYK, M., BIAŁOSKÓRSKA, M., JOŃCZYK, P. SCHAAPER, R.M., FIJAŁKOWSKA, I.J. Unequal fidelity of leading and lagging strand DNA replication in SOS induced E.coli cells. Ibid.:B49 p.87

160 1323/A. KAMIENSKA-TRELA, K., KANIA, L., BECHCICKA, M., KACZMAREK, Ł, WÓJCIK, J. Structure of polyheteroaromatic systems using NMR data (in Polish) Seminar on Nuclear Magnetic Resonance and its Applications, 1-2 December 1998.

1324/A PAWLAK, D., PACHULSKA, M., SIDOR, M., WÓJCIK, J., CHANG, N.N., SHILLER, P.,W., IZDEBSKI, J. Cyclic opioid peptides: synthesis, activity and structure, (in Polish) XV Polish Peptide Sympsium, Waplewo 2-4 September 1999

1325/A POZNAŃSKI, J. Computer simulations of rice ns-LTP protein cavity flexibility. The 1st National Congress on Biotechnology. Wroclaw, September 20-25, 1999

1326/A POZNAŃSKI, J., CIEŚLA, J., RODE, W., KULIKOWSKI, T. 'H and 19F nuclear magnetic resonance investigation of 2'-deoxy - N4- hydroxy - 5-fluorocytidine 5'-monophosphate and its ternary complex with rat thymidylate synthase, and N3, N10 methylenetetrahydrofolate. 7th International Symposium on Molecular Aspects of Chemotherapy Gdańsk, Poland 8-11 September 1999

1327/A MALTSEV, S.D., SIZOVA, O.V., DANILOV, L.L., SHIBAEV, V.N., JANKOWSKI, W., CHOJNACKI, T. Analogues of dolichyl phosphate with modified anionie group: dolichyl sulfate and H-phosphonate. 10th European Carbohydrate Symposium Galway, Ireland, July 11-16, 1999, PD023 p.395

1328/A GOŁOS, B., ZIELIŃSK1, Z., RODE, W., FELCZAK, K., MIAZGA, A., WYSZYŃSKA, K., KULIKOWSKI, T. Interactions with mammalian tumour thymidylate synthase of 2- or 4-seleno analogues of dUMP and FdUMP and inhibition of tumor cell growth by the corresponding nucleosides. 7th International Symposium on Molecular Aspects of Chemotherapy Gdańsk, Poland 8-11 September 1999 Abstr. 35 p. 104

1329/A GOŁOS, B., DZIK, J.M., FELCZAK, K., ZIELIŃSKI, Z., CIEŚLA, J., MIAZGA, A., KULIKOWSKI, T., RODE, W. Inhibition of mammalin tumour thymidylate synthase by 5-substituted N4- hydroxy - 2' - deoxycytidine - 5' - monophosphates and of tumour cell growth by the corresponding nucleosides. Ibid.: Abstr. 51 p. 120

161 1330/A BOROWSKI, P., MUELLER, O., SCHMITZ, H., SIWECKA, M.A, KULIKOWSKI, T. ATP-binding domain offlaviviridae RNA helicase as a target for antiviral therapy. Ibid.: Abstr. 56 p. 127

1331/A JEŻEWSKA, M.M. 25 years of enzymatic diagnosis of Lesch-Nyhan syndrome in Poland. 7lh Symposium of the European Society for the Study of Purine & Pyrimidine Metabolism in Man (ESSPPMM), Gdańsk, Poland 15-19 September 1999 Cellular & Mol. Biol. Letters (1999) 4 p.383

1332/A KULIKOWSKI, T. Nucleoside analogues - inhibitors of HIV reverse transcriptase - current and potential anti-AIDS drugs. Multidisciplinary Drug Conference, Muszyna 3-5.03.1999 Abstr. W-22 1333/A TUDEK, B. Localisation of cyclic adducts to DNA bases within human p53 gene, (in Polish) Gliwice Scientific Conference on Genome Structure and Function, 26-27 November 1999 Oncology Center, Gliwice

1334/A GRĄZIEWICZ, M. Fapy - adeninę effect of oxidative adeninę derivative on DNA replication and its mutagenic properties, (in Polish) Ibid.:

1335/A EJCHARTA. Non - classic constraints in protein structure determination: residual dipolar couplings and interference between relaxation mechanisms, (in Polish) Seminar on Nuclear Magnetic Resonance and its Applications, Kraków, 1999

1336/A TOCZYŁOWSKA, B, KIERUL, K. Analysis of twodimensional proton spectra of cerebrospiral fluid, (in Polish) Ibid.: p.31

1337/A ZHUKOV, I. Comparison of two techniques for evaluation of V/w-w« coupling constants in protein by nonlinear fits of ./-modulated [I5N,'H] - HSQC experiments. Ibid.: s.34

162 1338/A KAMIEŃSKA-TRELA, K., DĄBROWSKI, A., NIECZYPORUK, E., BERNATOWICZ, P., WÓJCIK, J.

Substitute influence on the magnitude of "Jcc(n=l-3) and "JCH(n=l-4) couplings in piridines. Experimental and DFT data, (in Polish) Ibid.:p.l3

1339/A PASZEWSKI, A. Regulation of sulfur amino acid biosynthesis in Aspergillus nidulcms: Physiology and genetics. 6"' International Congress on Amino Acids, Germany, Bonn 3-8 August 1999. Amino Acids (1999) 17, p.98-99

1340/A BRZESKI, J., PODSTOLSKI, W., OLCZAK, K., JERZMANOWSKI, A. Chromatin remodeling in plants: Arabidopsis thaliana BS7/gene, a member

1999 Meeting of International Research Scholars from Belarus, Czech Republic, Estonia, Hungary, Latvia, Lithuania, Poland, Russia, Slovakia, and Ukraine. Moscow, Russia, June 22-25 1999

1341 /A HULANICKA, M.D., SADOWY, E., JUSZCZUK, M., GRONENBORN, B. ORF1 of potato leafroll virus encodes a serine proteinase indispensable for viral replication. Xith International Congress of Virology. International Union of Microbiological Societes, 9-13 August 1999, Sydney Convention Centre, Darling Harbour. Sydney, Australia VW15.04

1342/A SHUGAR, D. Purine nucleoside phosphorylases: properties, functions, and chemotherapeutic applications. 7lh Symposium of the European Society for the Study of Purine & Pyramidine Metabolism in Man (ESSPPMM), Gdańsk, Poland 15-19 September 1999 Cellular & Molecular Biology Letters (1999) 4, p.327-328

163 PATENTS

Patent pending P-326571 - BARDOWSKI J. A new strain and a method of its obtaining

Patent pending P-326572 - BARDOWSKI J. A method of obtaining of the amylolytic anzyme

Patent pending P-326573 - BARDOWSKI J. A new plasmid and a method of its obtaining

Patent pending P-331817 - SIRKO A., BŁASZCZYK A., BRODZIK R. Manners of construction of transgenic plants beneficial for breeding

Patent pending P-322518 - CHACHULSKA A, ZAGÓRSKI-OSTOJA W. A new binary vector and a method of its obtaining

Patent pending BPP/3409/CH5 - MICHAL1K J., SZOLAJSKA E, STROKOWSKA L. I, SOŁOMKO A. A, KALININA N. C, KIKHNO I. M, SHVED A. D, MARKELOVA E. Ju. The manner of human prolactin synthesis

164 DISSERTATIONS

Theses for the degree of „doctor habilitatus" (Dr. Sc.)

84/N JAGURA-BURDZY GRAŻYNA Regulation and replication, conjugation and stable maintenance of RK2 plasmid from IncP group of E.coli. Presented November 25, 1997 Research performed in the Department of Microbial Biochemistry IBB PAS

85/N HRYNIEW1CZ MONIKA Molecular mechanism of expression of the cysteine regulon genes in Enterobacteriaceae. Presented November 25, 1997 Research performed in the Department of Microbial Biochemistry IBB PAS

86/N W1ERZCHOWSKI JACEK The search for fluorescent and fluorogenic substrates and inhibitors, and their use for the studies of activity, kinetics and the mechanism action of selected enzymes. Presented January 13, 1998 Research performed in the Department of Physical Chemistry, Faculty of Pharmacy, Medical performed in the Academy of Warsaw

87/N ŚLEDZIEWSKA-GÓJSKA EWA The Escherichia coli cellular systems involved in mutagenesis and repair of DNA lesions induced by alkylating agents. Presented March 3, 1998 Research performed in the Laboratory of Mutagenesis and DNA Repair IBB PAS

88/N RAKOWSKA-BOGUTA MAGDALENA Translation fidelity in Eukariota using the example ofS.cerevisiae. Presented April 28, 1998 Research performed in the Department of Genetics IBB PAS

89/N DOMIŃSKI ZBIGNIEW The role of exons in pre-mRNA splicing. Presented June 23, 1998 Research performed in the University of North Carolina at Chapel Hill, USA

90/N WÓJCIK ANDRZEJ Investigations on post-irradiation adaptive response. Presented October 6, 1998 Research performed in the Institute of Nuclear Chemistry and Technology

165 116/N SIRKO AGNIESZKA The role of integration host (IHF) in the regulation of gene expression in Escherichia coli. Presented April 27, 1999 Research performed in the Department of Plant Biochemistry IBB PAS

117/N FIJAŁKOWSKA IWONA The mechanisms responsible for the fidelity of DNA replication in E.coli strains. Presented January 26, 1999 Research performed in the Laboratory of Mutagenesis and DNA Repair, IBB PAS

118/N JOŃCZYK PIOTR The mechanisms of inducible mutagenesis in E.coli cells. Presented March 16, 1999 Research performed in the Laboratory of Mutagenesis and DNA Repair, IBB PAS

119/N DADLEZ MICHAŁ Early stages of protein folding - the studies of bovine pancreatic trypsin inhibitor (BPTI). Presented November 23, 1999 Research performed in the Department of Biophysics IBB PAS

152/N ŻOŁĄDEK TERESA The Yeast Saccharomyces cerevisiae, a model organism in studies of heme biosynthesis and phototoxic effects of porphyrins. Presented June 15, 1999 Research performed in the Department of Genetics IBB PAS

Doctoral theses (Ph. D.)

91 /N CH ACHULSKA ANNA, MARIA Potato virus Y phylogeny and engineered virus resistance. Presented January 13, 1998 Research performed in the Department of Protein Biosynthesis IBB PAS

92/N ZAGULSKI MAREK Functional analysis of the yeast S. cerevisiae chromosome II open reading frames. Presented April 28, 1998 Research performed in the Department of Protein Biosynthesis IBB PAS

93/N LEWANDOWSKAIRMINA Genetic regulation of folate metabolism mAspergillus nidulans. Presented March 3, 1998 Research performed in the Department of Genetics IBB PAS

166 94/N GÓRA-SOCHACKA ANNA The genetic stability of Potato Spindle Tuber Viroid (PSTVd). Presented June 23, 1998 Research performed in the Department of Protein Biosynthesis, IBB PAS

95/N GÓRA MONIKA Physiological and biochemical consequences of mutations in the gene HEM15 encoding ferrochelatase in the yeast Saccharomyces cerevisiae. Presented January 20, 1998 Research performed in the Department of Genetics, IBB PAS and Laboratoire de Biochimie des Porphyrines, Institut Jacques Monod, CNRS, Paris

96/N TOMASZEWSKI RADOSŁAW The mechanism of HI-mediated selective repression of genes placed in the vicinity of AT-rich DNA sequences. Presented June 2, 1998 Research performed in the Laboratory of Plant Molecular Biology of Warsaw University and Department of Protein Biosynthesis IBB PAS

97/N KRZYMOWSKA MAGDALENĄ Induction mechanisms of the promoter of the PR-2d gene in transgenic potato plants. Presented June 23, 1998 Research performed in the Department of Plant Biochemistry, IBB PAS

98/N GAVRYUSHOV SERGEI Electrostatics of biological macromolecules in electrolyte solutions. Application of the modified Poisson-Boltzmann theory. Presented November 24, 1998 Research performed in the Department of Biophysics, IBB PAS

99/N BŁASZCZAK ADAM Contribution of heat shock proteins in DNA dependent RNA polymerase activity in Escherichia coli. Research performed in the Laboratory of Molecular Biology affiliated with the University of Gdańsk and IBB PAS

120/N BRZESKI JAN Cloning and functional analysis of the Arabidopsis thalicma homolog of the yeast SNF5 gene. Presented March 16, 1999 Research performed in the Department of Protein Biosynthesis IBB PAS

167 121/N KIERZEK ANDRZEJ Theoretical description of the protein crystallization process. Lattice simulations of the formation and growth of the ordered phase. Presented April 26, 1999 Research performed in the Department of Bioinformatics IBB PAS

122/N SADOWY EWA Construction of infectious clones of potato leafroll virus and analysis of their activity. Presented April 27, 1999 Research performed in the Department of Microbial Biochemistry IBB PAS

123/N WASZKOWSKA EWA Products of furocoumarins photoaddition to phosphatidylcholine. Presented September, 1999 Research performed in the Department of Molecular Biology IBB PAS

124/N SZCZEGIELNIAK JADWIGA Calcium and phospholipid dependent protein kinase from maize seedlings. Presented September, 1999 Research performed in the Laboratory of Protein Phosphorylation of Department of Plant Biochemistry IBB PAS

125/N GRĄZIEWICZ MARIA-ANNA Free radical effect on biological properties of DNA and T4 DNA ligase activity. Presented October 19, 1999 Research performed in the Department of Microbial Biochemistry IBB PAS

126/N WÓJCIK ANNA Biological effects induced by halogen light in Escherichia coli K-12. Presented November 23, 1999 Research performed in the Department of Microbial Biochemistry IBB PAS

153/N GOLIK PAWEŁ Nucleo-mitochondrial interactions in the yeast Saccharomyces cerevisiae. Presented April 12, 1999 Research performed in the Department of Genetics U W and Centre de Genetique Moleculaire, CNRS w Gif sur Yvette

168 Theses for the degree of Master of Science (M. Sc.)

100/N WĘGIERSKI TOMASZ Cloning of the PET127 gene as a multi-copy suppressor ofSUYS and DSS1 gene deletion suggests functional interaction of mRNA 5' and 3' ends in mitochondria of S. cerevisiae. Presented January 8, 1998 Research performed in the Department of Genetics, Warsaw University l O l /N BUKO WSKA BOŻENA Synthesis of 3-alkyl-purines - potential inhibitors of N-alkylpurine-DNA glycosylase. Presented March 18, 1998 Research performed in the Department of Molecular Biology IBB PAS and the Department of Chemistry UMCS, Lublin

102/N SKORUPIŃSKA KAROLINA The occurrence of free and phosphorylated forms of polyisoprenoid alcohols in plant tissues. Presented June 30, 1998 Research performed in the Agricultural University of Warsaw and Department of Lipid Biochemistry IBB PAS

103/N MOGIELNICKA ELŻBIETA Effect of histone H l on the transcription of Drosophila Kruppel gene in vitro. Presented July 29, 1998 Research performed in the Laboratory of Plant Molecular Biology of Warsaw University

104/N JESIONOWSKA ALICJA Genetic and molecular analysis ofmdpl/rspS mutants and their suppressors. Presented July 30, 1998 Research performed in the Department of Genetics IBB PAS

105/N POLKOWSKA LIDIA Early response of potato and Solanum nigrum cell suspensions to elicitor from Phytopthora infestans. Presented July 2, 1998 Research performed in the Department of Plant Biochemistry IBB PAS and Agricultural University of Warsaw

106/N WIKTOROWSKA-JEZIERSKA AGNIESZKA An attempt to clone the gene coding for human protamine HP2 in E.coli. Presented July 10, 1998 Research performed in the Department of Biophysics IBB PAS and UMC Dept. of Biology

169 107/N LAUBITZ DANIEL The influence of mutation mphr gene on argE3~* Arg + reversion in Escherichia coli K12 AB 1157 strain induced by halogen light and UV 2541™ Presented July 31, 1998 Research performed in the Department of Molecular Biology IBB PAS and in Dept. of Biology of Warsaw University

108/N KOWALCZYK PAWEŁ Localization of chloroacet-aldehyde induced lesions of human p53 gene by the use of DNA polymerase fingerprinting method. Prersented July 2, 1998 Research performed in the Department of Molecular Biology of IBB PAS and the Dept. of Biotechnology of Agricultural University

109/N MOZOLUK MAŁGORZATA Overexpression and purification of potato leaf roll virus (PLRV) replicase in Escherichia coli. Presented June 26, 1998 Research performed in the Department of Plant Biochemistry IBB PAS and the Department of Biology of Warsaw University

110/N POMIANOWSKI JAN Chemical synthesis of 4-selenouracil nucleosides. Presented April 16, 1998 Research performed in the Department of Biophysics IBB PAS and Department of Chemistry U W

111 /N SKO WRON SKA DOROTA Analysis of the effects ofA.nidulans TBP on S.cerevisiae cells. Presented June, 1998 Research performed in the Department of Genetics U W

112/N KRAWCZYK MICHAŁ Sequence and analysis of the function of the human SUV3P protein. Presented June 30, 1998 Research performed in the Department of Genetics U W

113/N OLSZAK KRZYSZTOF The influence of magnesium ions on the activity of two plant nucleases I. Presented June 30, 1998 Research performed in the Department of Protein Biosynthesis IBB PAN

114/N WÓJTOWICZ ANNA Cloning and expressing of the - GDKDGDGYISAAEM calcium binding peptide. Presented July 10, 1998 Research performed in the Department of Biophysics IBB PAS

170 115/N CHOUDA MARCIN Long-chain polyisoprenoids of seeds. Presented September 23, 1998 Research performed in the Department of Lipid Biochemistry IBB PAS and Department Biochemistry of the Agricultural University of Warsaw

127/N BŁASZCZYK ANNA Overproduction of bacterial serine acetyltransferase in tobacco plants. Presented April 9, 1999 Research performed in the Department of Plant Biochemistry and Department of Biology, Warsaw University

128/N BULKOWSKA URSZULA Construction of Saccharomyces cerevisiae strains expressing DNA methyltransferease from vertebrates Presented June 18, 1999 Resarch done in the Departament of Genetics IBB PAS and Department of Biology, UW

129/N GRZMIL MICHAŁ Morphological and regulatory aspects of the functional analysis of RRD1 and RRD2 genes of Saccharomyces cerevisiae. Presented June 28, 1999 Research performed in the Department of Biophysics IBB PAS

130/N NIŻANKOW1CZ ANNA, M. Purification and characterization of plant protein kinase phosphorylating tyrosine. Presented July 8, 1999 Research performed in the Department of Plant Biochemistry IBB PAS and Agricultural University of Warsaw

131/N DUSIŃSKA MIROSŁAWA Dependence of the expression of MAGI on the POL2, RAD9, RAD24 "checkpoint" gene products of Saccharomyces cerevisiae. Presented July 9, 1999 Research performed in the Laboratory of Mutagenesis and DNA Repair IBB PAS

132/N STOLARCZYK KATARZYNA Studies of the protein-protein interaction of the P subunit (Dna N) of the E.coli DNA polymerase III holoenzyme, in the yeast two-hybrid system. Presented July 15, 1999 Research performed in the Laboratory of Mutagenesis and DNA Repair IBB PAS

171 133/N KOSSOBUDZKI PIOTR Isolation and analysis of multicopy suppressors ofmdp/rspS mutation of the yeast Saccharomyces cervisiae. Presented August 2, 1999 Research performed in the Department of Genetics U W and the Department Genetics IBB PAS

134/N BOROWSKA MAGDALENA Expression of mpgl and dpml, Trichoderma reesei genes coding for mannophosphoguanyltransferase and mannosylphosphodolichol synthase, and the protein secretion. Presented September 9, 1999 Research performed in the Laboratory of Fungal Glycobiology of the Department of Lipid Biochemistry IBB PAS

I35/N WIŚNIEWSKA ANNA The function of the ORFO product in replication of potato leafroll virus. Presented July 15, 1999 Research performed in the Department Microbial Biochemistry IBB PAS

136/N WYSOCKA MONIKA The functional analysis of the newly discovered gene YOR129c from the Saccharomyces cerevisiae genome. Presented June 23, 1999 Research performed in the Department of Genetics IBB PAS

137/N HEJDUK ARKADIUSZ Characterization of the Aspergillus nidulans met A gene. Presented August 13, 1999 Research performed in the Department of Genetics IBB PAS

138/N KOWALCZYK MAGDALENA The ccpA gene and the influence of the ccpA protein on sugar catabolism in Lactococcus lactis. Presented September 29, 1999 Research performed in the Department of Genetics U W and Department of Microbial Biochemistry IBB PAS

139/N KRAKOWIAN DOMINIKA Cloning and expression of urease genes from Helicobacter pylori in lactic acid bacteria. Presented September 29, 1999 Research performed in the Laboratory of Plant Molecular Biology UW and Department Microbial Biochemistry IBB PAS

172 140/N OBTUŁOWICZ TOMASZ Formation, characterization and repair of formamidopyrimidines. Presented September 13, 1999 Research performed in the Department of Genetics UW and Department Microbial Biochemistry IBB PAS

141/N ZIENKIEWICZ MAKSYMILIAN Influence of halogen light on E.coli mutated in hemH. Presented September 27, 1999 Research performed in the Department of Molecular Biology IBB PAS

142/N TAFEL AGNIESZKA The domain structure of mitochondria! ribosomal protein Nam9 in yeast. Presented December 10, 1999 Research performed in the Department of Molecular Biology of the Plants UW and Department of Genetics, IBB PAS

143/N GOZDEK AGNIESZKA The repair of MMS - induced lesions in Escherichia coli AB1157 strains under starvation conditions. Presented September 22, 1999 Research performed in the Department of Genetics UW and Department of Molecular Biology of IBB PAS

144/N NOWAK MAGDALENA Restriction site polymorphism in human mitochondria! DNA in the Polish population. Presented July 10, 1999 Research performed in the Department of Genetics U W

145/N SPODAR KRYSTYNA Attempt to identify hSUVS gene mutations in patients with mitochondria! cytopathies. Presented May 5, 1999 Research performed in the Department of Genetics U W

146/N BARAŃSKAANNA The localization of polyhedrin gene in Stilgnotia salicis SsMNPV. Presented July 5, 1999 Research performed in the Department of Protein Biosynthesis IBB PAS

I47/N ŻAKEWEL1NA Isolation of suppressors of the deletion of the nuclear localization sequence in S.cerevisiae Krrlp. Presented October 12, 1999 Research performed in the Laboratory of Plant Molecular Biology U W and Department of Protein Biosynthesis IBB PAS

173 148/N PTASZYŃSKA ANETA The role of the UMP! (DIN8) gene in the Saccharomyces cerevisiae response to DNA damage. Presented September 29, 1999 Research performed in the Department of Biology and Earth Sciences, The Maria Curie-Skłodowska Memorial University, Lublin and Laboratory of Mutagenesis and DNA Repair IBB PAS

149/N SIDOR MARTA Conformations of cyclic enkephalin analogs in solution by means of nuclear magnetic resonance. Presented June 8, 1999 Research performed in the Department of Physics UW and Laboratory of Biological NMR1BBPAS

150/N MINCZUK MICHAŁ Genetic analysis of the regulatory region of the pyrimidine gene cluster from the extreme thermophile Thermits strain ZO5 using site-directed mutagenesis. Presented June 21, 1999 Research performed in the Department of Genetics, UW and Department of Microbiology,Vrije Universiteit Brussel, Belgium

151/N ONYSZCZUK KATARZYNA Protein kinases involved in osmotic stress signal transduction in plants. Presented June 23, 1999 Research performed in the Department of Biochemistry Warsaw Agriculture University

154/N RUSZCZYŃSKA KATARZYNA NMR study of structure and stability of analogs of the III ref binding loop of calmodulin. Presented June 8, 1999 Research performed in the Laboratory of Biological NMR IBB PAS

155/N KRYSIUK NORBERT The construction of a chromosomal system which allows measurement of deletions frequency during DNA replication on the leading and lagging strands in E.coli chromosome. Presented June 28, 1999 Research performed in the Department of Biology U W and Laboratory of Mutagenesis and DNA Repair IBB PAS

174 156/N BUGAJSKAOLGA Intracellular interactions of the ParB protein with DNA during active partitioning of PI plasmid to daughter cells. Presented June 8, 1999 Research performed in the Institute of Microbiology UW and Department of Microbial Biochemistry IBB PAS

157/N LICHOTA KATARZYNA Study of the coding properties of isoguanosine. Presented December 10, 1999 Research performed in the Department of Molecular Biology UW and Department of Molecular Biology IBB PAS

175 INSTITUTE RESEARCH STAFF

Note: Names of Research Units are abbreviated as follows: Bi - Biophysics, Ge - Genetics, Inf - Bioinformatics, Li - Lipid Biochemistry, Mi - Microbial Biochemistry, Mo - Molecular Biology, Mu - Mutagenesis and DNA Repair, Nmr - Biological NMR, PI - Plant Biochemistry, Pr - Protein Biosynthesis, Pu - Purine Metabolism, , AI - Laboratory of Antimetabolites, UG - University of Gdańsk, UW - Warsaw University.

Baranowska-Wyszomirska Hanna, Ph. D., Ge Bardowski Jacek, Dr. Sc., Mi Barszcz Daniela, Ph. D., Mo Bartnik Ewa, Prof., UW Bębenek Anna, Ph. D., Mo Bierzyński Andrzej, Prof, Nmr Błaszczak Adam, Ph. D., UG Bolewska Krystyna, Ph. D., Bi Borsuk Piotr, Ph. D., UW Bretner Maria, Ph. D., Mo Brzeski Jan, Ph. D., Pr Brzywczy Jerzy, Ph. D., Ge Bucholc Maria Jolanta, Ph. D., Pl Buchowicz Jerzy, Prof., PI Burzyńska Beata, Ph. D., Ge

Cegłowski Piotr, Assoc. Prof., Mi Chachulska Anna, Ph. D., Pr Chetstowska Anna, Ph. D., Ge Chojnacki Tadeusz, Prof., Li Cieśla Jarosław, Ph. D., Mo Cieśla Zygmunt, Prof., Mu Czyż Agata, Ph. D, UG

Dadlez Michał, Assoc. Prof., Bi Dmochowska Aleksandra, Ph. D., UW Dobrowolska Grażyna, Ph. D., Pl Dobrzańska-Wiernikowska Marta, Ph. D., Pl Dzikowska Agnieszka, Ph. D., UW

Ejchart Andrzej, Assoc. Prof., Nmr Empel Joanna Ph. D., UW

176 Fabisiewicz Anna, Ph. D., Mo FeJczak Krzysztof, Ph. D., Mo Fijałkowska Iwona, Dr. Sc., Mu Fikus Magdalena, Prof., Bi Furga Kinga, PI

Gabig Magdalena, Ph. D., UG Goch Grażyna, Ph. D., B i Góra Monika, Ph. D., Ge Góra-Sochacka Anna, Ph. D., Pr Grąziewicz Maria, Ph. D., Mo Gromadka Robert, Ph. D., Pr Grzelak Krystyna, Dr. Sc., Pr Grzesiuk Elżbieta, Dr. Sc., Mo

Haeni Anne Lise, Prof, Pr Hennig Jacek, Assoc. Prof, PI Hryniewicz Monika, Assoc. Prof., Mi Hulanicka-Dziechcińska M. Danuta, Prof., Mi

Iwanicka-Nowicka Roksana, Ph. D., Mu

Jachymczyk Witold, Prof., Ge Jagura-Burdzy Grażyna, Assoc. Prof., Mi Janion Celina, Prof., Mo Jankowski Wiesław, Ph. D., Li Jerzmanowski Andrzej, Prof., UW/Pr Jończyk Piotr, Dr. Sc., Mu

Kelner Anna, M. Sc., Pr Kern Izabela, Ph. D., Mi Kierzek Andrzej, Ph. D., Inf Kiss Anna, M. Sc., Ge Kłopotowski Tadeusz, Prof, Mi Kłudkiewicz Barbara, Ph. D., Pr Kolasa Iwona, M. Sc., Bi Konopka Dorota, Ph. D., Pl Kozłowska Hanna, M. Sc., Bi Krajewska-Grynkiewicz Krystyna, Ph. D., Mi Kraszewska Elżbieta, Ph. D., Pl Kroczyńska Barbara, Ph. D., Pl Kruszewska Joanna, Ph. D., Li Krzymowska Magda, Ph. D., Pl Kula-Świeżewska Ewa, Assoc. Prof, Li Kuligowska Elżbieta, Ph. D., Pr

177 Kulikowski Tadeusz, Prof., Al Kurlandzka Anna, Ph. D., Ge Kuśmierek Jarosław, Prof., Mo

Lewak Stanisław, Prof., UW Lewandowska Irmina, Ph. D., Ge

Łobocka Małgorzata, Ph. D., Mi Łoziński Tomasz, Ph. D., Bi

Maciejewska Urszula, Ph. D., PI Majcher Krystyna, M. Sc., Inf Mazuś Barbara, Dr. Sc., PI Miazga Agnieszka, M. Sc., Mo Michalik Joanna, Dr. Sc., Pr Muszyńska Grażyna, Prof., PI

Natorff Renata, Ph. D., Ge Nowak Leszek, Ph. D., Isotope Facility

Olszak Krzysztof, M. Sc., Pr

Pacocha Monika, M. Sc., Pr Palamarczyk Grażyna, Prof., Li Pałucha Anrdrzej, Ph. D., Pr Paszewski Andrzej, Prof., Ge Pawłowicz Jerzy, M. Sc., Pr Pawłowski Krzysztof, Ph. D., Nmr Pawłowski Piotr, Ph. D., Bi Pietrzykowska Irena, Prof., Mo Piotrowska Małgorzata, Ph. D., Ge Płochocka Danuta, Ph. D., Bi Poznański Jarosław, Ph. D., B i Przykorska Anna, Dr. Sc., Pr

Rabczenko Andrzej, Prof, Bi Rakowska-Boguta Magdalena, Assoc. Prof, Ge Rempoła Bożenna, Ph. D., B i Rybicka Katarzyna, M. Sc., Pr Rytka Joanna, Prof., Ge

Sadowy Ewa, Ph. D., Mi Shugar David, Prof., Al Sieńko Marzena, Ph. D., Ge Sirko Agnieszka, Ph. D., Mi

178 Siwecka Maria Agnieszka, Ph. D., Pr Skoneczna Adrianna, Ph. D., Mu Skoneczny Marek, Ph. D., Ge Smagowicz Wiesław, Ph. D., Bi Soszyńska Elżbieta, M. Sc., Bi Stępień Piotr, Prof., UW Szafrański-Szeliga Przemysław, Prof., Pr Szarkowski Jan W., Prof., Pr Szczegielniak Jadwiga, Ph. D., Pr Szczerbakowa Anna, Ph. D., P! Szczesniak Barbara, M. Sc., Ge Szkopińska Anna, Dr. Sc., Li Szołajska Ewa, Ph. D., Pr Szurmak Blanka, Ph. D., PI

Śledziewska-Gójska Ewa, Assoc. Prof, Mu

Toczyłowska Beata, Ph. D., Nmr Topczewski Jacek, Ph. D., Ge Topczewska Jolanta, Ph. D., Bi Tudek Barbara, Ph. D., Mo

Wasilewska-Dąbrowska Lidia Danuta, Prof, Pl Węgleński Piotr, Prof, UW Węgrzyn Alicja, Ph. D., UG Wielgat Bernard, Assoc. Prof, PI Wierzchowski Kazimierz Lech, Prof, Bi Wójcik Anna, Ph. D., Mo Wójcik Jacek, Ph. D., Nmr Wysłouch-Cieszyńska Aleksandra, Ph. D., Bi Wyszyńska Katarzyna, M. Sc., Al

Zagulski Marek, Ph. D., Pr Zagórski-Ostoja Włodzimierz, Prof, Pr Zarębska Zofia, Assoc. Prof, Mo Zielenkiewicz Piotr, Assoc. Prof, Inf Bi Żołądek Teresa, Assoc. Prof, Ge Żuk Jerzy, Prof, Ge

179 School of Molecular Biology:

Adamczyk Małgorzata, M. Sc., Mu Aleksandrzak Tamara, M. Sc., Mi Balcerzak Aneta, M. Sc., PI Banach Magdalena, M. Sc., Mu Białkowska Agnieszka, M. Sc., Ge Blaszczyk Anna., M. Sc., Pl Bołtowicz Danuta, M. Sc., Pl Borys Ewa, M. Sc., Mo Brodzik Robert, M. Sc, Pl Bugajska Olga., M. Sc., Ge Bykowski Tomasz, M. Sc., Mi Calikowski Tomasz, M. Sc, Pr Chacińska Agnieszka, M. Sc, Ge Ciesielski Arkadiusz, M. Sc, Ge Dobruk Aneta, M. Sc, Mi Drobek Anna, M. Sc, Pl Dyczkowski Jerzy, M. Sc, Bi Felczak Magdalena, M. Sc, Mo Fikus Marta, M. Sc, Mu Flis Krzysztof, M. Sc, Ge Gajewska Beata, M. Sc, Ge Gaweł Damian, M. Sc, Mu Gołębiewski Marcin, M. Sc, Mi Gozdek Agnieszka, M. Sc, Mo Grabińska Kariona, M. Sc, Li Grabowska Dorota, M. Sc, Li Gromadka Robert, M. Sc, Pr Grynberg Marcin, M. Sc, Ge Grzybowska Ewa, M. Sc, Ge Hałas Agnieszka, M. Sc, Ge Jabłonowska Agnieszka, M. Sc, Bi Janik Anna, M. Sc, Li Janowicz Aleksandra, M. Sc, Mi Joukov Igor, M. Sc, Nmr Juszczuk Marek, M. Sc, Mi Jurkowski Ireneusz, M. Sc, Pl Kacprzak Magdalena, M. Sc, Ge Kaczanowski Szymon, M. Sc, Inf Kamińska Joanna, M. Sc, Ge Kęsik Małgorzata, M. Sc, Pr Konopińska Agata, M. Sc, Ge Koprowski Piotr, M. Sc, Mu Koter Marek, M. Sc, Pl

180 Kowalczyk Magdalena, M. Sc., Pl Kowalczyk Paweł, M. Sc., Mo Krwawicz Joanna, M. Sc., Mo Krysiak Cezary, M. Sc., Pl Kucharczyk Róża, M. Sc., Pr Lasocki Krzysztof, M. Sc., Mi Liwosz Aneta, M. Sc., P] Łochowska Anna, M. Sc., Mi Maassen Anna, M. Sc., Pl Maciejczyk Maciej, M. Sc., NMR Maciejewska Agnieszka, M. Sc., Mo Maliszewska-Tkaczyk Magdalena, M. Sc., Mu Mieczkowski Piotr, M. Sc., Mu Migdalski Andrzej, M. Sc., Pr Mikołajczyk Monika, M. Sc., Pl Milner Małgorzata, M. Sc., Pr Nowosielska Anetta, M. Sc., Mo Obtułowicz Tomasz, M. Sc., Mo Olczak Kataczyna, M. Sc., Pr Perlińska-Lenart Urszula, M. Sc., Li Pluta Krzysztof, M. Sc., Mi Podlaska Agnieszka, M. Sc., Mu Podstolski Wojciech, M. Sc., Pr Polkowska-Kowalczyk Lidia, M. Sc., Pl Racki Waldemar, M. Sc., Pr Sarnowski Tomasz, M. Sc., Pr Skorupińska-Tudek Karolina, M. Sc., Li Smaczyńska-de Rooij Iwona, M. Sc., Ge Sidor Marta, M. Sc., NMR Sitkiewicz Izabela, M. Sc., Mi Sosnowska Monika, M. Sc., Li Speina Elżbieta, M. Sc., Mo Talarczyk Andrzej, M. Sc., Pl Trojanek Joanna, M. Sc., Pl Wanke Małgorzata, M. Sc., Li Wieczorek Grzegorz, M. Sc., Inf Wójtowicz Anna, M. Sc., Bi Zdanowski Konrad, M. Sc., Bi Zielenkiewicz Urszula, M. Sc., Mi Zienkiewicz Maksymilian, M. Sc., Mi

181 Holders of the Ministry of Education Scholarships:

Awotunde Olubunmi Sunday, M. Sc., PI King Frank, M. Sc., UG Liszewska Frantz, M. Sc., PI Vo Sy Hung, M. Sc., Li

M. Sc. Students:

Małgorzata Palczewska, Mi Monika Siedlecka, Nmr Magdalena Chełchacz, Pr Jakub Godlewski, Pr Agnieszka Borowska, Ge Celina Kaczor, Mi Piotr Kowalczyk, UW Michał Malewicz, UW Ewa Czernieć, Mi Małgorzata Jaworska, Mo Bernarda Kusztykiewicz, Mo Agnieszka Janisz, Mo Daniel Laubitz, Mo Marcin Chouda, Li Ewelina Żak, Pr Marcin Górski, Pr Agnieszka Kucharska, Ge Alicja Jesionowska, Ge Elżbieta Dziedowiec, Mi Marcin Gołębiewski, Pr Michał Skrzycki, Mi Bożena Bukowska, Mo Anna Wiśniewska, Mi Wojciech Dajewski, Mu Małgorzata Mozoluk, Pl Anna Niżankowicz, Pl Katarzyna Onyszczuk, Pl Sławomir Krawczyk, Inf Magdalena Kowalczyk, Mi Dominika Krakowian, Mi Wojciech Augustyniak, Mo Katarzyna Lichota, Mo

182 Centre of Excellence in Molecular Biotechnology (CEMB)

CEMB is a joint project of IBB PAS and European Commission focused on promotion of scientific exchange with EU and enforcement of IBB role as an international research centre. CEMB consists of ten interconnected work packages, in the frame of their activity 3 international conferences and 10 workshops will be organised, as well as individual scientific exchange with EU and Associated Countries will be supported (60 visitors coming to IBB PAS). It is planned to employ 20 researchers from EU and Associated Countries on professors', post-docs' and Ph.D. students' posts and also 30 visits of IBB scientists to the European laboratories will be founded. CEMB is closely cooperating with School of Molecular Biology at IBB PAS where 20 mini-symposiums will take place. An additional aspect of CEMB activity is an increasing number of applications of research in the field of molecular biotechnology in Poland through close cooperation with Polish research and industrial institutions. All conferences, workshops and individual visits will be announced in order to provide Polish scientific community with maximum advantage of these events. Centre is included in 5th Framework Programme of European Commission - Confirming the Role of Community Research. The founding of the Centre is previewed for 36 months. CEMB has started its activity on the 1 December 2000, as the contract was signed on 9th November 2000. Organisation of the Centre is focused on the creation of a new quality of acting in order to continue its activity as the European funding is over.

CEMB IBB PAN, ul. Pawińskiego 5a, 02-106 Warszawa, Poland tel. + 48 (22) 823 71 89, fax. + 48 (22) 658 46 36, e-mail: [email protected] Centre of Excellence in Molecular Biotechnology (CEMB)

CEMB is a joint project of IBB PAS and European Commission focused on promotion of scientific exchange with ED and enforcement of IBB role as an international research centre. CEMB consists of ten interconnected work packages, in the frame of their activity 3 international conferences and 10 workshops will be organised, as well as individual scientific exchange with EU and Associated Countries will be supported (60 visitors coming to IBB PAS). It is planned to employ 20 researchers from EU and Associated Countries on professors', post-docs' and Ph.D. students' posts and also 30 visits of IBB scientists to the European laboratories will be founded. CEMB is closely cooperating with School of Molecular Biology at IBB PAS where 20 mini-symposiums will take place. An additional aspect of CEMB activity is an increasing number of applications of research in the field of molecular biotechnology in Poland through close cooperation with Polish research and industrial institutions. All conferences, workshops and individual visits will be announced in order to provide Polish scientific community with maximum advantage of these events. Centre is included in 5th Framework Programme of European Commission - Confirming the Role of Community Research. The founding of the Centre is previewed for 36 months. CEMB has started its activity on the 1 December 2000, as the contract was signed on 91h November 2000. Organisation of the Centre is focused on the creation of a new quality of acting in order to continue its activity as the European funding is over.

CEMB IBB PAN, ul. Pawińskiego 5a, 02-106 Warszawa, Poland tel. + 48 (22) 823 71 89, fax. + 48 (22) 658 46 36, e-mail: [email protected] ISBN-83-906782-4-1