Wilhelm bernhard workshop on the cell nucleus

Many morphological changes (i.e. cell shrinkage, cytoplasm and chromatin condensation, cell surfaces exposition of PS and normally hidden sugar residues) characterized lymphocytes apoptosis. In this study we investigated the relationship between nuclear and cell surface modifications in lymphocytes isolated from human, rat and mouse and induced to apoptosis with cycloheximide (CHX) 10^-2M for 24hrs. Cells were monitored for their progressive accumulation of cell surface and morphological modifications. Three types of apoptosing lymphocytes, with different percentages, were found at all times of incubation with CHX irrespective of the species. Type 1 (early apoptosis): the majority of the cells showed no signs of apoptotic morphological modifications, although very few cells show mild margination of chromatin or the stereotypical apoptotic morphology. Increased PS and sugar residue expression on the cell surface were detected, independently of the nuclear modifications. Type 2 (mature apoptosis): the majority of cells showed extensive chromatin condensation and nuclear fragmentation and cellullar blebbing. Cell surface was highly modified, with extensive exposure of PS and sugar residues. Type 3 (late/necrotic apoptosis): almost all apoptotic cells underwent secondary necrosis. A decreased exposure of PS and abundant expression of sugar residues (i.e.fucose), absent from surface of early apoptotic lymphocytes were observed. Nuclei were for the most fragmented and the fragments were seen as micronuclei inside the dead cells, whose cytoplasm was largely vacuolized. When surface modifications are considered, differences were observed between human and rat lymphocytes and between rat and mouse lymphocytes, human and mouse lymphocytes being similar.

FURTHER NOTES ON NUCLEI AND NUCLEOLI IN THE PROGRAMMED CELL DEATH AND PRECEDING TERMINAL MATURATION

Smetana K., Čermák J., and Hrkal Z.

Institute of Hematology and Blood Transfusion, Prague, Czech Republic
Erythroid and granulocytic precursors represent convenient models to study both natural and induced apoptotic process with and without preceding terminal maturation because their maturation stages are well known and defined. Terminal maturation of erythroblasts preceding the programmed cell death is accompanied by the chromatin condensation, transformation of nucleoli to micronucleoli and formation of HERDS (nuclear heterogeneous ectopic ribonucleoprotein derived structures: see, Biggiogera et al., 1999). Pathological erythroblasts in refractory anemia of myelodysplastic syndrome entering to premature apoptosis exhibited structural abnormalities of micronucleoli as well as their premature loss and HERDS. In cultured leukemic granulocytic precursors (HL-60 cells), the apoptotic process without previous terminal maturation induced by photodynamic treatment (see Smetana et al., 2000) was characterized by chromatin condensation, reduction of AgNORs, fibrillar centers and surrounding dense fibrillar components without reduction of the nucleolar size. Thus presented observations indicated that in pathological erythroblasts the apoptotic process was preceded by terminal maturation in which the transformation of nucleoli to micronucleoli with structural abnormalities, loss of nucleoli together with characteristic chromatin condensation and HERDS reflected the irreversible pre-apoptotic state. Such state, however, was absent after induction of the apoptotic process in HL-60 cells by photodynamic treatment.

The present study was supported by the Grant NC/7603/3 of the Inner Grant Agency of the Ministry of Health of the Czech Republic

References:

Biggiogera et al., Histochem. Cell Biol. 112, 473, 1999

Smetana et al., J. Photochem. Photobiol. B: Biol. 59, 80, 2000


AGGRESOME-LIKE INCLUSIONS WITHIN THE NUCLEOPLASM:

PROTEASOMAL DEGRADATION AND SUB-NUCLEAR PATHOLOGY

Chen M., Rockel T., Hemmerich P., and von Mikecz A.

Insitut für umweltmedizinische Forschung (IUF), Heinrich-Heine-University Düsseldorf, Germany
Protein aggregates and inclusions containing a variety of cellular proteins such as ubiquitin, proteasomes, heat shock proteins, and transcriptional coactivator CBP are hallmarks of neurodegenerative diseases. Studies on the composition and dynamics of such aggregates have led to an intriguing new hypothesis for disease, namely, that the sequestration of certain essential cellular proteins by protein aggregates results in a loss-of-function phenotype and ultimately in tissue dysgenesis. We reported previously that (1) proteasomal degradation of nuclear proteins is correlated with their subnuclear localization (J. Struct. Biol. 140: 189, 2002), and (2) recruitment of the nucleolar autoantigen fibrillarin to proteasome-dependent degradation occurs upon xenobiotic-induction in cell culture and animal models (Mol. Biol. Cell 13:3576, 2002). Quantification of fluorescence intensities and measurement of proteasomal activity in nuclear fractions corroborate that nuclear proteins that colocalize with proteasomes are degraded by proteasome-dependent proteolysis within the nucleoplasm. Moreover, upon induction by certain xenobiotics protein aggregates are formed in the nucleoplasm that contain proteasomes, topoisomerase I, fibrillarin, spliceosomal components, PML, and CBP. We term these aggregates aggresome-like inclusions (ALIs). The formation of ALIs correlates with decreased replication, and transcription activity and results in arrest of cell growth. Since ALIs are detectable in primary cells from patients with systemic autoimmune diseases we suggest that ALI formation represents a novel subnuclear pathology.


MOLECULAR CHARACTERIZATION AND DEVELOPMENTAL EXPRESSION OF THE XENOPUS EMERIN

Gareiß M.¹, Zentgraf H.², Wilken N.¹, Müller C.R.³ and Dabauvalle M.-C.<sup>1

1</sup>Department of Cell and Developmental Biology, and ³Department of Human Genetics, Biocenter, University of Wuerzburg, and ²German Cancer Research Center, Heidelberg, Germany
Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked disease due to mutations in emerin. Emerin is an ubiquitous transmembrane protein of the inner nuclear membrane. While the protein is expressed in the majority of human tissues analyzed, the pathology predominates in cardiac and skeletal muscle of EDMD patients. For a better understanding of the functions of emerin in the organisation of the cell nucleus, we decided to characterize and to investigate its role in vivo and in vitro in Xenopus laevis as a model system.

By cDNA cloning and sequenzing we have characterized X-emerin, a Xenopus homologue of the mammalian emerin. Analysis of the amino acid sequence resulted in a polypeptide with 180 residues and a calculated molecular mass of 21,6 kDa (24 kDa on SDS-PAGE). A motif screen of the protein showed an LEM domain (N1-C44) and a transmembrane motif in the C-terminus (N147-C169). Sequence comparison showed a homology of 52 % to the human and 53 % to the murine emerin. We have generated monoclonal antibodies specific for Xenopus emerin. The analysis of emerin expression during development revealed that X-emerin was absent from oocytes and from early developmental stages and was first detectable at tadpole stage 43. Interestingly, the expression of the lamin A started approximately at the same developmental stage (stage 40). Whole mount in situ immunolocalisation and hybridizations were performed to study the expression pattern of emerin. Since emerin as well as lamin A is not expressed in oocytes and eggs, it is possible to study the effect of exogenously expressed emerin, in the presence or absence of lamin A, on the nuclear envelope assembly, on the nuclear structure and on the chromatin organization in oocytes, in early embryogenesis and in a cell free extract prepared from Xenopus eggs.

THE WILHELM BERNHARD

JUNIOR LECTURE

NUCLEAR MYOSIN I AND ACTIN ARE REQUIRED FOR RNA POLYMERASE I TRANSCRIPTION

¹Philimonenko V.V., ²Iben S., ¹Dingová H., ¹Kyselá K., ²Grummt I., ³de Lanerolle P., and ¹Hozák P.

¹Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic; ²German Cancer Research Center, Heidelberg, Germany; ³University of Illinois at Chicago, USA
Recently, nuclear myosin I (NMI) was shown to be involved in RNA polymerase II transcription. We report for the first time that actin and NMI are required for rRNA synthesis. Electron microscopy reveals that both actin and NMI are localized within the nucleoli, and their distribution is highly specific. NMI colocalizes with nascent rRNA in the dense fibrillar component and its presence here is transcription-dependent. When nuclear extracts are fractionated, significant amounts of actin and NMI are detected in fractions containing partially purified Polymerase I (Pol I) and its transcription factors. Experiments of co-immuno-precipitation demonstrate association of actin with murine initiation-competent Pol I holoenzyme complexes. The rate of Pol I transcription in vitro is dramatically reduced by antibodies directed against NMI, while addition of purified NMI stimulates transcription from mouse rDNA promoter in a dose-dependent manner. Anti-actin antibodies specifically inhibit formation of full-length transcripts, but not ACU trimers in abortive initiation assay, indicating that actin plays a role in promoter clearance or transcript elongation. Microinjections of anti-actin and anti-NMI antibodies significantly reduce the level of nucleolar transcription in cultured HeLa cells, demonstrating the requirement for these proteins for transcription in vivo. We conclude that the acto-myosin molecular motor can be generally required for the movement of DNA relative to transcription complexes.








CELL CYCLE

50 YEARS NOBEL PRIZE FRITS ZERNIKE IN PHASE CONTRAST MICROSCOPY (1953 – 2003)

Gundllach H.

Carls Zeiss, Central Research and Technology, Oberkochen, Germany
The exact design of the microscope was established from the theory for image formation developed by Ernst Abbe in 1873. With the introduction of apochromatic lenses and oil immersion, a theoretical limit of resolution of 200 nm (0,2µ) became available at this early stage. The back focal plane of the objective is one of the most important planes in the microscope, in relation to illumination and especially contrast. In 1935, the Dutch physicist Frits Zernike published his work on phase contrast microscopy, which is based on recognising the difference between the diffraction pattern formed by an absorbing and non absorbing object.Because of the importance in the application in biology and medicine, Zernike wae awarded with the Nobel price in physics in 1953. The first prototype of a phase contrast microscope was constructed in 1936 by Zeiss in Jena. Following this, in 1941, Dr. Kurt Michel, head of the department of microscopy in Jena, produced the first movie in phase contrast on 35 mm of the Meiosis in spermatogenesis of the grass hopper (Psophus stridulus L.). This film is now available on a DVD and will be shown during the congress.

References:

Abbe, E.: Arch. Mikrosk. Anatomie, 9, 413, 1873

Köhler, A., W. Loos:. Die Naturwissenschaften 29, Heft 4, S. 49-61, 1941

Michel, K.: Die Naturwissen-schaften 29, Heft 4, S.61-62, 1941

Michel, K.: Die Reifeteilung (Meiose) bei der Spermatogenese der Schnarrheuschrecke (Psophus stridulus L.). Institut für den Wissenschaftlichen Film, Göttingen, 1958

Zernike, F.: Z. techn. Physik 16, S. 454-457, 1935

DELAYED ENTRY TO QUIESCENCE BY p21^CDKN1A-NULL HUMAN FIBROBLASTS: A POSSIBLE INVOLVEMENT IN THE DISASSEMBLY OF PRE-REPLICATION COMPLEX

Stivala L.A., Perucca P., Cazzalini O. ¹, E. Prosperi ², Madine M., Laskey R.A.L. <sup>3

1</sup>Dipartimento di Medicina sperimentale, Sezione di Patologia generale and ²Istituto di Genetica Molecolare del CNR, Sezione di Istochimica e Citometria, Pavia, Italy; ³MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge, UK.
The cyclin-dependent kinase inhibitor p21^CDKN1A is involved in several cellular processes including cell cycle arrest induced by DNA damage, cell differentiation and senescence. In contrast, the role of this protein in the cell cycle exit to quiescence is more controversial. In order to investigate a possible role of p21 in inducing quiescent cell growth arrest, we have considered the assembly of the pre-replication complex as a possible step targeted by p21. To this end, we have used human embryonic p21-null fibroblasts (obtained by homologous recombination) with extended lifespan induced by expression of the catalytic subunit of human telomerase (htert). Cells were driven to quiescence both by contact inhibition and serum deprivation (0.5%) and residual DNA replicative capacity was assessed by BrdU incorporation. Cellular levels of p21 were determined by immunofluorescence. The results showed that in p21^+/+ parental fibroblasts but not in p21^-/- cells, p21 protein levels as well as the percentage of positive nuclei, were significantly increased after 3 days of serum starvation, and remained high at least up to 7 days. Increased levels of p27^kip1 protein were found in both cell lines, whereas p16 was not significantly modified. Analysis of the residual DNA replicative activity indicated that p21-null fibroblasts retained significant levels of BrdU incorporation, up to at least 10-18 days after serum deprivation, as compared to complete no incorporation observed after 7-9 days in the parental p21^+/+ cells. These findings were confirmed by the capacity to perform DNA replication in vitro, exhibited by nuclei isolated from serum-starved p21^-/- cells, as compared to parental p21^+/+ fibroblasts. Western blot analysis of the pre-replication complex during serum starvation, showed that chromatin-bound MCM2 protein was displaced with faster kinetics in p21^+/+ than in p21^-/- cells. These results suggest that p21 may be involved in the pathway that drive the cells to quiescence.

LIGHT AND ELECTRON MICROSCOPIC STUDY OF REPLICATION SITES IN HELA CELLS DURING S-PHASE

Koberna K., Pliss A., Siegel A.J., Malinsky J., Ligasova A., Dudnik O. Berezney R., and Raska I.

1^st Faculty of Medicine, Charles University and Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Department of Biological Sciences, State University of New York at Buffalo, USA
Replication sites (RSs) in synchronized HeLa cells have been studied with the help of light microscopy (LM) and electron microscopy (EM) detection of labeled nascent DNA. In agreement with previous studies, distinct patterns of replication are observed on the LM level during S-phase. The replication begins in non-numerous small fluorescence foci scattered throughout nucleoplasm, but the number of RSs becomes very high within several minutes. At later stages of S-phase the RSs are less numerous and often larger. Towards the very end of S-phase, the number as well as the size of RSs gradually decreases. The EM analysis of the early replicated chromatin identifies the replication pattern with the small RSs with a maximum size 240 nm, which we refer to as replication units (RUs). RUs of about the same size are clustered into larger domains during later stages of S-phase. These domains correspond to the large RSs observed on the LM level during both the mid and late periods of S-phase. Importantly, the stereological evaluation of the images indicates that the number of RUs is similar in the early andmid S-phase cells (with exception of very early S-phase cells) and declines towards the end of S-phase. Only individual RUs are observed at the very end of S-phase. About 1100 RUs are seen in the early and mid S-phase cells. The analysis of the overall fluorescence replication signal shows, however, an increase in the fluorescence replication signal during the transition from the early to mid S-phase cells. We cannot comment on the number of active replicons in early and mid S-phase cells. However, based on the analysis of the labeled and stretched DNA fibers from the early and mid S-phase cells, the elevation of the replication signal definitely reflects the higher speed of replication forks in the mid S-phase cells.

This work was supported by grants 304/01/0729, 304/03/1121, IAA5039103, EU CE ICA1-CT-200070028, AV0Z5039906, MSM111100003 and NIH GM2392.


REPLICATION FACTORIES AND INTRA-S PHASE CHECKPOINT PATHWAYS

Rossi R., Biamonti G and Montecucco A.

Istituto di Genetica Molecolare del CNR, Pavia, Italy
In mammalian cells DNA replication takes place in subnuclear sites called replication factories consisting of cluster of replicons and their associated replication machinery. Each factory undergoes an assembly- disassembly cycle according to a precise space- and time-dependent program, that parallels the replication of different portions of the genome. How this dynamic process is controlled is still elusive. We have recently observed that the dynamics of replication factories is perturbed in response to DNA damage. We have further investigated the fate of the factories in response to DNA damage and replicational stress in human normal cells and cell lines defective in DNA repair and/or checkpoint pathways. We have observed that in normal fibroblasts, etoposide but not UV irradiation or aphidicolin, induces the disassembly of replication factories and the dissociation of PCNA and DNA ligase I from the chromatin fraction. The effect of etoposide is prevented by agents that promote stalling of replication forks and by general inhibitors of protein kinases. Interestingly, disassembly of replication factories and dissociation of PCNA from chromatin do not occur in cells over-expressing a dominant negative version of ATR kinase indicating that the disassembly of the factories induced by etoposide requires a functional ATR. Moreover, UV-irradiation induces the dispersal of the factories in a subset of DNA repair and checkpoint deficient cells such as Bloom Syndrome cells. Our results demonstrate that the stability of replication factories after treatments that induce DNA damage and/or stalling of replication forks is controlled by the intra-S phase checkpoint.


PERSISTENT DNA DECATENATIONS BETWEEN SISTER CHROMATIDS INDUCE CHECKPOINT DELAYS IN G2 AND EARLY MITOSIS

De la Torre C, Moreno Díaz de la Espina S, Clarke DJ* and Giménez-Abián JF.

Centro de Investigaciones Biológicas, CSIC, Madrid, Spain, and *University of Minnesota, Department of Genetics, Cell Biology and Development, Minneapolis, USA.
Chromosomal assembly and condensation in the cell cycle require topoisomerase II α. When inhibiting it, DNA catenations are kept unresolved. We show that topo II tasks are surveyed by checkpoints during G2 and early mitosis, in Allium cepa L. meristem cells. Checkpoints are signal transduction pathways that brake irreversible phase transitions when intracellular conditions make them undesirable, while licensing them when requirements are fulfilled. Progression from reversible to irreversible chromosome condensation, nuclear envelope breakdown and segregation of sister centromeres are the irreversible transitions in early mitosis. When inhibiting topo II with ICRF-193, undue override of these transitions took place after a delay. This override, known as checkpoint adaptation, allows us to define the unfulfilled requirement that activated the checkpoint. The topo II-dependent individual-ization of chromosomes, brought about by the resolution of randomly produced non-replicative catenations, proved to be a prerequisite for the reversible to irreversible chromosome condensation transition, taking place in midprophase. The resolution of replicative catenations linking sister chromatid arms was a prerequisite for the checkpoint that licensed nuclear envelope breakdown, at the onset of prometaphase. The resolution of residual replicative catenations between sister centromeres was required for the licensing of chromosome segregation by the checkpoint known as the spindle checkpoint, which also surveys whether all duplicated centromeres are bound to both spindle poles via microtubules. While the checkpoint controlling the midprophase transition co-surveys unreplicated DNA, unrepaired DNA and cell size, those, if any, co-surveyed by the checkpoint controlling initiation of prometaphase are unknown.

CHROMATIN


DYNAMICS OF NUCLEAR COMPARTMENTS DURING EARLY EMBRYOGENESIS IN THE MOUSE

Martin C.¹, Zatsepina O.³, Migné C.¹, Chebrout M.¹, Beaujean N.¹, Zink D.⁴ and Debey P^1,2.

¹ UMR Biologie du Développement de Reproduction, INRA, Jouy-en-Josas, France, ²USM 503, Département Régulation Développement et Diversité Moléculaire, Muséum national d’Histoire naturelle, Paris, France, ³A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia, and ⁴Department Biologie II, LMU München , Germany
The beginning of embryonic life is characterized by major structural remodelling of nuclear compartments, together with the progressive establishment of nuclear activities, namely RNA polymerases II (pol II) and RNA polymerase I (pol I). We have earlier shown that, in the mouse embryo, pol II activity starts at the beginning of the first celle cycle, whereas pol I starts at the end of the second cell cycle (Bouniol et al., 1995; Zatsepina et al., 2002). Using different markers of centromeres and centromeric heterochrochromatin, in combination with labeling of DNA which is replicated in late S phase of the first cell cycle, we analyze the dynamics of chromosomal domains during the first cell cycles. We show a complete redistribution of chromosomal domains, mainly occuring between the first and second, and later between the second and third cell stages, and involving also the nucleolus-like bodies (NLBs). Differential recruitment of the hetero-chromatin proteins HP1 and HP1 occur during the same period. Similar, though not identical, changes occur in somatic cell nuclei transfered to activated oocytes, such as performed in “cloning” experiments.

References:

Bouniol et al : Exp Cell Res 218, 57-62, 1995

Zatsepina et al: Dev. Biol. 253, 66-83, 2002


HISTONE EXPRESSION IN THE GERM LINE OF DROSOPHILA

Hennig W., Becker A., Feng RR, Berger, A., Ye J., Tang X.

DAAD Laboratory, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, P.R. China
The chromatin constitution in male germ cells differs from that of any other cell type. The cell cycle regulated histones, forming the core of the nucleosomes, are replaced by other more basic proteins. The nucleosomal configuration of the DNA is given up and converted in another form of packing which in its molecular details is not understood (Hennig, 2003). In Drosophila it is not even clear which proteins substitute the somatic histones. We have investigated the chromatin constitution (Akhmanova et al. 1997) and suspected that a histone variant, H3.3, is involved in the repackaging. We have studied the regulation of expression of the two histone H3.3 genes with the aid of GFP constructs in the male and female germ line. It emerges that the 3’-UTRs with their different polyadenylation signals are involved in the cell type-specific regulation of the expression of histone H3.3A and H3.3B. We have also expressed human transition proteins TP1 and TP2 as well as protamine 1 and protamine 2 in the male germ line of Drosophila to study their effect on the chromatin constitution and on fertility. The results will be discussed.

References:

Akhmanova A. , K. Miedema, Y. Wang, M. van Bruggen, J.M.H. Berden, E.M.. Moudrianakis, W. Hennig: Chromosoma 106:335-347, 1997

Hennig W.: Chromosoma 111 489-494, 2003


MALE CHROMATIN REMODELING AFTER FERTILIZATION IN SEA URCHINS: THE ROLE OFA THIOL

Concha C¹, Morín V¹., Even Y²., Oliver M.I¹., Gutiérrez S¹., Puchi M¹., Genevière A.M²., and Imschenetzky M¹.

¹Department of Molecular Biology, University of Concepción, Concepción, Chile, and ²Laboratoire Arago, Banyuls-sur-Mer, France.
In sea urchins male chromatin remodeling involves the replacement ofsperm specific histones (SpH) by maternal histones (CS variants) thatoccurs concomitantly with a step wise SpH degradation. At intermediatestages of male chromatin remodeling hybrid nucleosomes formed byphosphorylated Sp-H2B and Sp-H1 and a subset of poly(ADP-ribosylated) CSvariants were found, whereas after amphimixis the chromatin is organizedexclusively by poly(ADP-ribosylated) CS variants. We have investigatedthe events associated to male chromatin remodeling. Among the moleculesthat were accumulated into male pronucleus we have found a thiol-proteaseand the regulators of the S-phase CdK2 and cyclines E and A. The zymogenof this thiol protease was primarily localized in the nuclei ofunfertilized eggs and found to be activated shortly after fertilization. This enzyme selectively degrades the SpH leaving the maternal CS variantsunaffected. The activity of this enzyme was modulated by the post-translational modification status of its substrates: poly(ADPribosylation) and phosphorylation protects modified histones fromdegradation. When the activity of the thiol-protease was inhibited invivo with E 64-d, the typical degradation of SpH was blocked, thefusion of both pronuclei remained unaffected and the initial S phase wasabolished. The thiol-protease inhibitor E.64-d did not modify the initialS phase in eggs activated with Ca(II) ionophore A-23187, indicating that this compound does not affect per se the initial S phase. Consistently, it was found that nuclear accumulation of the criticalS-phase regulators CdK2/cyclines A and E were not modified by E 64-d.Similarly to normal zygotes, CdK2 was active all through the initial cellcycle in zygotes treated with E 64-d. Taken together these resultsindicate that SpH degradation is a fundamental events for thereestablishment of the diploid condition of the zygotes and has an impacton the license to replicate its DNA. 

Grants: Universidad de Concepción (DIUC) 200.031.088-10, FONDECYT 1011073,  and ECOS France /CONICYT Chile


EXPRESSION OF HUMAN METHYLASES IN THE MALE GERM LINE OF DROSOPHILA

Weyrich A., Dong M., Ge Y., Xu G., W. Hennig

Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, P.R. China
Human de novo DNA methylases Dnmt3a and Dnmt3b were combined with a testis-specific promoter in P element transformation vectors and transformed into Drosophila melanogaster. We recovered 7 or 6 transformant lines, respectively, and investigated the expression of the enzymes. We will report the results on Dnmt3b. The enzyme is expressed in testes as can be demonstrated in Western blots and by immuno-histology. Spermatogenesis was normal in all strains and no effects on fertility were observed. These results are of particular interest as it has recently been shown by Maggert and Golic (2002) that the Y chromosome of Drosophila is subject to imprinting. On the other hand, Weissmann et al. (2003) demonstrated lethality of the Dnmt3a in Drosophila tansformants. Our observations suggest that DNA methylation may be a normal event in the male germ line of Drosophila.

References:

K.A. Maggert, K.G. Golic: Genetics 162:1245-1258 (2003)

F. Weissmann, I. Muyrers-Chen, T. Musch, D. Stach, M. Wiessler, R. Paro, F. Lyko: Mol Cell Biol 23:2577-2586 (2003)


DYNAMICS OF HETEROCHROMATIN PROTEINS MEASURED BY FLUORESCENCE CORRELATION SPECTROSCOPY IN LIVING CELLS

Schmiedeberg L., Weißhart K. and Hemmerich P.

Institute for Molecular Biotechnology, Jena, Germany
Heterochromatin is a major compartment of the mammalian cell nucleus that represents densely packed and transcriptionally inactive DNA. Heterochromatin protein 1 (HP1) is involved in packaging and maintaining the heterochromatin structure. HP1 proteins belong to the class of non-histone proteins, that bind to modified residues of histones as predicted by the histone code hypothesis. The methylation of lysine 9 of histone H3 is prerequisite for binding of HP1 and maintainance of the repressive state of heterochromatin. In order to investigate the binding properties and the dynamic behaviour of HP1 proteins in living cells, we created HEp-2 cell lines stably expressing GFP-HP1 fusion proteins and determined their dynamic behaviour by Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS). Results will be presented which compare the two technologies.


THE NON-HISTONE CHROMOSOMAL PROTEIN HMGB1 AS A MODULATOR OF CHROMATIN STRUCTURE

Polyanichko A., Chikhirzhina E., Skvortsov A., Kostyleva E., Vorob’ev V.

Institute of Cytology of the Russian Academy of Sciences, St.-Petersburg, Russia
The structure and function of the non-histone chromosomal protein HMGB1 as a modulator of chromatin structure have been studied. Interaction of the protein HMGB1 with DNA has been investigated using hydrodynamic and optical methods including complementary methods of circular dichroism and absorption spectroscopy in UV and IR regions. The role of different domains of the HMGB1 molecule in interaction with DNA has been analyzed by comparison of the full-length native HMGB1 molecule and the recombinant HMGB1-(A+B) molecule with a deleted negatively charged C-terminal domain of the HMGB1 molecule. It was shown that interaction of the HMGB1 with DNA proceeds in two stages. In the first stage, at the low protein/DNA ratios, interaction of the HMGB1 is defined mainly by the activity of the DNA binding domain, this interaction being slightly weakened by the influence of the acidic C-terminal domain of HMGB1. Interaction of the HMGB1 with DNA results in changes in the conformation of the components of the complex. The alpha-helicity of the DNA binding domains increases in HMGB1 and local structural changes in DNA occur which induce the DNA condensation. At the rise of the protein/DNA ratio and with the increase of ionic strength the cooperative phase in HMGB1-DNA interaction begins which is connected with protein-protein interactions between the HMGB1 molecules. The unordered negatively charged C-terminal tail of the HMGB1 plays an essential role in the formation of the complexes. Recombinant protein HMGB1-(A+B) lacking the C-terminal domain forms with DNA the complexes with a highly ordered structure, which reveal anomalously high optical activity. The data obtained show that the negatively charged C-terminal domain of the HMGB1 can perform in chromatin the function of modulator of the both protein-protein and protein-DNA interactions.


NUCLEAR PROTEINS, MATRIX AND BODIES

DEVELOPMENTAL CHANGES IN THE DISTRIBUTION OF TIGHT DNA-PROTEIN COMPLEXES ALONG BARLEY CHROMOSOMES REVEALED BY MAPPED MOLECULAR MARKERS

¹Sjakste T., ²Röder M.

¹Institute of Biology, Salaspils, Latvia; ²Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Gatersleben, Germany
Rearrangements in DNA packaging as in DNA interactions with functional protein groups reflect the switches in gene expression during plant development. PCR-amplification of mapped microsatellite markers and RFLP-STS markers was successfully used to label DNA fragments in chromatin profiling experiments. Three methods of fractionation of DNA complexes with functional protein groups were used: isolation of the nuclear matrix, isolation of DNA complexes with tightly bound proteins (TBPs), and nucleoprotein-celite (NPC) chromatography. The presence of specific DNA fragments in various fractions obtained by the different isolation procedures was monitored by PCR-amplification of the 22 and 24 markers previously mapped along barley chromosomes 1H and 7H. In young leaves amplification of all markers was found in the nuclear matrix. Five markers on 1H and six markers on 7H were specific for nuclear matrix fraction in young leaves and for insoluble chromatin fraction in senescent leaves. Probably, these markers are located closely to origins of replication in young leaves. In old leaves these DNA attachment points are degraded. Specific patterns of TBPs were revealed in all types of tissues studied. The first leaf development was followed by a release of most parts of the chromosomes from the complexes with TBPs. On the contrary, senescence of the leaf was followed by an increase in DNA sites involved in interactions with TBPs. The distribution of TBPs appeared to be organ specific. In seeds the PCR-amplification of most markers was evenly distributed between all fractions of NPC-chromatography, some of them between the chromatin and tight DNA-NM complexes. During germination and leaf development the pattern changed and some markers became highly specific for the DNA-NM fraction. All methods revealed the tissue specific developmental reorganization of tight DNA protein complexes along the chromosomes. Apparently, different methods detected interactions of different protein groups with DNA.

Polymorphism of nuclear multi-functional protein XPD and its influence on DNA repair

Rzeszowska-Wolny J.¹, Pietrowska M.¹, Polanska J.³, Palyvoda O.¹, Jaworska J.¹, Butkiewicz D.², Hancock R⁴.

¹Department of Experimental and Clinical Radiobiology; ²Department of Tumor Biology, M. Sklodowska-Curie Memorial Institute, Centre of Oncology, and ³Institute of Automation, Technical University, Gliwice, Poland; ⁴Laval University Cancer Research Centre, Quebec, Canada
The protein XPD, a 5’-3’ helicase and a component of the basal transcription factor TFIIH, is also involved in DNA repair. Repair mechanisms maintain genomic stability by detecting and correcting damaged DNA sequences and by signaling cell death when DNA repair fails. All living cells contain repair mechanisms specialized in recognition and removal of different types of DNA damage; oxidative damage is generally removed by base excision repair (BER), bulky DNA adducts by nucleotide excision repair (NER), mismatched bases by mismatch repair, and strand breaks by non-homologous end joining or homologous recombination. XPD takes part in the NER pathway. The repair pathways and their proteins are conserved in different species; however recently it was shown that polymorphic variants of some genes coding for repair proteins are present in human populations and can influence repair capacity and cancer risk of carriers. In this report we show that polymorphism of the XPD protein can cause the engagement of NER in removal of radiation-induced oxidative damage from DNA. Individuals differ in their reaction to the same dose of genotoxic agents and cultured cells from different donors show differences in DNA repair kinetics and efficiency. In studies performed in vitro on lymphocytes of 80 individuals, we assessed DNA damage and repair induced by gamma-irradiation. Cells homozygous for the XPD allele containing Asn in codon 312 showed significantly more efficient repair of radiation-induced DNA damage than homozygotes with Asp in this position. Our results suggest that NER takes part in repair of ionizing radiation-induced DNA damage and also that polymorphism in genes coding for proteins of this pathway can influence an individual’s response to ionizing radiation.

Supported by State Committee for Scientific Research (KBN, Poland) Grant 4 P05A 015 19 and Canadian Institutes of Health Research grant MOP-14351


NUCLEAR STRESS BODIES REVEAL A LINK BETWEEN CHROMATIN ORGANIZATION AND DISTRIBUTION OF RNA PROCESSING FACTORS.

Biamonti G., Rizzi N., Chiodi I., Corioni M., Denegri M., Cobianchi F., Riva S.