Wilhelm bernhard workshop on the cell nucleus

CARP FISH NUCLEOLAR DYNAMICS

Vera M. I., Krauskopf M., Alvarez M., Molina A., Quezada C., Navarro C. and Pinto R. Laboratorio de Biología Celular y Molecular, Universidad Andrés Bello and MIFAB, Santiago, Chile
One of the most dramatic adaptive responses of the carp to the seasonal environmental changes is the segregation of the nucleolar components during winter, concomitantly with a remarkable decay of rRNA synthesis and processing of their precursors. On the contrary, in summer-adapted fish, the nucleolar structure agrees with an active state of ribosome biogenesis. To understand the molecular mechanisms that trigger this phenomenon, we have being studying some genes and their expression concerning ribosome synthesis, ribosomal protein L41, protein kinase CK2 and nucleolin, among others. Nucleolin is a major substrate of CK2, implicated in different steps of ribosome synthesis. In summer, the mRNA expression of ribosomal protein L41 and CK2ß, as well as the CK2ß protein are higher than in winter-adapted carp. On the contrary, both, the content of nucleolin and its mRNA, showed higher expression levels in tissues from winter-adapted carp. Recently, it has been proposed that nucleolin could repress the rRNA transcription. To evaluate this hypothesis in our seasonal regulated model, we characterized the carp rRNA cistron, nucleolin gene and two different nucleolin cDNAs. In silico analysis of the rRNA intergenic spacer (IGS) revealed a number of sequence repeats most likely involved in transcriptional regulation. In addition, we perform a phylogenetic study of the 18S carp rRNA. Our results suggest at least three genes coding for nucleolin in carp, organized in 16 exons. Introns 7, 9 and 14 code for U82, U20 and U23 snoRNAs, respectively. Throughout in silico modeling we have established secondary structures for all these three snoRNAs and we assessed their seasonal expression. In conclusion, the rRNA transcription and nucleolar gene expression in a eurythermal fish are not constitutive, on the contrary, they are strongly regulated by environmental parameters.

Grants: FONDECYT 1000061, DI/UNAB 93-01 and 94-01


IDENTIFICATION OF PROTEINS INTER-ACTING WITH THE KARYOSKELETAL PROTEIN NO145

Voltmer S., Kneissel S. and Schmidt-Zachmann M.S.

German Cancer Research Center, Heidelberg, Germany
The nucleolus is a large and complex nuclear substructure with a well defined functional organization. Whereas the major function of this membraneless organelle, the ribosome biosynthesis, has been extensively investigated, little is known about the molecular components contributing to the shape and internal architecture of nucleoli.

Recently, we succeeded in the identification of a novel type of karyoskeletal protein and molecular marker for a specific nucleolar substructure, i.e. a relatively thin cortical layer forming a cage-like perinucleolar structure and consisting of a meshwork of patches and filaments. Whereas this protein- termed NO145 - can be classified as a karyoskeletal protein (resistant to high-salt/detergent extraction) it is also sensitive to regulated proteolysis. Remarkably, protein NO145 (originally identified in Xenopus laevis oocytes) is present throughout all stages of oogenesis but is rapidly degraded during meiotic maturation, i.e. egg formation (Kneissel et al. 2001, Mol. Biol. Cell 12, 3904-3918). The major aim of our current studies is the identification of proteins interacting with protein NO145, e.g. by co-immunoprecipitation experiments and yeast two-hybrid screens. We started the project by constructing a Gal 4 AD fusion library using cDNA prepared from X. laevis ovary. The two hybrid screen was performed by co-transformation of this library with full-length protein NO145 used as a bait. We obtained 60 transformants expressing interacting proteins and started to verify these putative interactions. To identify the genes responsible for a positive two-hybrid interaction the corresponding plasmids were isolated, amplified by PCR and finally sequenced. Detailed data base searches resulted in the identification of several known proteins, but also of X. laevis ESTs coding for unknown proteins. Further verifications of these putative interaction partners of NO145 in vitro and in vivo are in progress.

NUCLEAR LOCATION AND DISTRIBUTION OF PHOSPHORYLATED c-Myc IN PACLITAXEL-TREATED CELLS

Bottone M.G.¹, Soldani C.¹, Tognon G.², Gorrini C.², Biggiogera M.^1,2, Pellicciari C.^1,2 and Scovassi A.I.²

¹Dipartimento di Biologia Animale, University of Pavia and ²Istituto di Genetica Molecolare del CNR, Pavia, Italy.
The proto-oncogene c-myc encodes a phospho-protein, which plays a crucial role both in driving cell proliferation and in promoting apoptosis. In this work, using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was compared with that of other ribonucleoprotein (RNP)-containing components (PANA, hnRNP-core proteins, fibrillarin) or RNP-associated nuclear proteins (SC-35 splicing factor). Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures (Soldani et al., 2002). To address the behavior of phosphorylated c-Myc in cells undergoing drug-induced nuclear restructuring, we treated HeLa cells and colon carcinoma cell lines with paclitaxel. This drug affects microtubule stability by binding -tubulin, thus leading to cell accumulation in the G₂/M phase, polyploidization and apoptosis. We demonstrated that after the treatment with paclitaxel, phosphorylated c-Myc undergoes redistribution, becoming diffused in the nucleoplasm. Remarkably, we found that a high c-myc amplification level potentiates paclitaxel cytotoxicity, confers a multinucleated phenotype and promotes apoptosis to a high extent, thus suggesting that c-myc expression level is relevant in modulating the cell responses to paclitaxel (Bottone et al. 2003).

This work was partially supported by Fondo di Ateneo per la Ricerca (FAR 2002, University of Pavia), PRIN 2002 (n. 2002055584) and FIRB Project RBNE0132MY

References :

Soldani C, Bottone MG, Biggiogera M, Alpini C, Scovassi AI, Martin T, Pellicciari C : Nuclear localization of phosphorylated c-Myc protein in human tumor cells. Eur. J. Histochem., 46: 377-380, 2002

Bottone M.G., Soldani C., Tognon G., Gorrini C., Lazzè M.C., Brison O., Ciomei M., Pellicciari C. and Scovassi A.I : Multiple effects of paclitaxel are modulated by a high c-myc amplification level. Exp. Cell Res. (in press)


APOPTOSIS AND PROLIFERATION IN THE GUT AND GILL EPITHILIUM OF Liza aurata FROM POLLUTED BRACKISH WATER

Ferrando S.*, Mauceri A.*, Ferrando T., Fasulo S.* and Tagliafierro G.

Dipartimento di Biologia Sperimentale, Ambientale e Applicata (DIBISAA), University of Genova and *Dipartimento di Biologia Animale ed Ecologia Marina, University of Messina, Italy
Heavy metal pollution is a well known important causative factor of intestinal and branchial damage: The epithelia often detached from the basal lamina and the lamina propria appears looser, hyperaemic and infiltrated with leucocytes. In the present research we investigate the effects of heavy metal exposure on cell apoptosis and proliferation of the gut and gill epithelial in the mugil Liza aurata from the brackish lagoons of Faro and Ganzirri (Messina). As a control, specimens of Liza aurata from the non polluted brackish lagoon of Marinello (Messina) were also studied. Dewaxed sections from paraformaldehyde fixed samples were treated by the TUNEL method to detect apoptotic nuclei and immunostained with anti-proliferating cell nuclear antigen (PCNA) antiserum to detect mitosis undergoing cells. The results obtained in fish from polluted area showed an increased number of apoptotic nuclei, not only located along the intestinal folds, or branchial lamellae, but also at the base, in the proliferative zone. The latter zone is more extented in the gut, interesting both the bottom and the base of intestinal folds. No apototic cells were seen in the gut of control fish, while a few of them were detected at the apex of gill lamellae. Our data put in evidence that in the fishes from natural polluted areas the reparative processes are enhanced for the maintenance of gut correct functions.


MODULATION OF NUCLEAR POLY(ADP-RIBOSYLATION) IN APOPTOTIC CELLS

Soldani C.¹, Bottone M.G¹, Biggiogera M.^1,2, Pellicciari C.^1,2, Denegri M²., and Scovassi A.I.²

¹Dipartimento Biologia Animale, University of Pavia and ²Istituto di Genetica Molecolare del CNR, Pavia, Italy
Poly(ADP-ribosylation) is a post-translational modification of proteins playing a crucial role in many processes. The best known poly(ADP-ribosylating) enzime, PARP-1, is a DNA nick sensor and uses NAD⁺ to form polymers of ADP-ribose, which are further degraded by the enzyme poly(ADP-ribose) glycohydrolase (PARG). During apoptosis, PARP-1 is responsible for an early and transient poly(ADP-ribose) synthesis, and is cleaved by caspases. PARP-1 proteolysis produces an 89 kDa C-terminal fragment, with a reduced catalytic activity, and a 24 kDa N-terminal peptide, which retains the DNA binding domains. We monitored the fate of p89 and the synthesis of polymers of ADP-ribose in HeLa cells driven to apoptosis by actinomycin D and etoposide. Tricolor fluorescence techniques were used to detect DNA, poly(ADP-ribose), and phosphatidylserine residues at the cell surface. The polymer synthesis proved to occur at early apoptotic stages, whereas the poly(ADP-ribose) was no longer detectable in late-apoptotic cells. To correlate the absence of polymer accumulation with PARG activity, we analyzed the localization of this enzyme and we demonstrated its presence in the nucleus of early apoptotic cells. The p89 PARP-1 fragment, which was found in the nucleoplasm of early apoptotic cells, migrated from the nucleus into the cytoplasm of cells with advanced chromatin condensation and DNA fragmentation. This demonstrates that p89 is among the several nuclear proteins which are extruded from the nucleus, reach the cytoplasm and are finally released inside apoptotic bodies. Collectively, our data indicate that poly(ADP-ribose) synthesis by activated PARP-1 occurs early during the apoptotic process, as a cellular emergency reaction, and that PARP-1 proteolysis is concomitant with PARP-1 activation. The extrusion of p89 from the nucleus in the late steps of apoptosis could allow a better understanding of the role of nuclear proteins as autoimmunity inducers.


FLUORESCENCE ENERGY TRANSFER FOR STUDYING CHROMATIN ARRANGEMENT IN G₀/G₁ TRANSITION AND APOPTOSIS

Croce AC.¹, Lussignoli S. ¹, Bottone M.G. ², Pellicciari C. ^{1, 2}, Barni S. ^{1, 2}, Bottiroli G. <sup>1

1</sup>Istituto di Genetica Molecolare del CNR, Sezione Istochimica e Citometria, and ²Dipartimento di Biologia Animale, University of Pavia, Italy
Fluorescence Resonance Energy Transfer (FRET) is a photo-physical process by which a fluorophore (the donor, D) in an excited state transfers its energy to a neighbouring fluorophore (the acceptor, A) through a non-radiative dipole-dipole interaction. FRET occurs in a spatial range of about 120 Å, and its efficiency depends on both distance and reciprocal orientation of the dye molecules. Provided that the dyes specifically bind the target biomolecules, FRET analysis can give information on the supramolecular arrangements of the biological structures. FRET process was exploited to investigate conformational changes of chromatin in cells under different functional activity: cultured human fibroblasts in G₀ (quiescent) or G₁ (activation of genes which are specific for cell cycle progression) phases; cultured rat thymocytes, under normal conditions or undergoing spontaneous or drug induced apoptosis (chromatin condensation, DNA cleavage). Hoechst 33258 (a non-intercalating, AT specific dye) and Propidium Iodide (an intercalating, base unspecific dye) were selected as a D/A couple due to their very suitable excitation/emission spectral properties. FRET efficiency (which was defined through the enhancement of the acceptor sensitised emission) was measured in single cells by microspectrofluorometry, and proved to be higher in G₁ than in G₀ fibroblasts. The increase of FRET efficiency was already appreciable at short time after cell cycle stimulation, when changes in the proliferation marker expression statin and Ki67 are still absent. FRET imaging on single nuclei showed a different spatial distribution of FRET efficiency between G₀ and G₁ fibroblasts. In thymocytes the induction of apoptosis resulted in an increase in FRET efficiency, that preceded the occurrence of cell morphology alterations. Due to its high sensitivity, FRET analysis provides a suitable tool for detecting very early structural changes of chromatin in different physiological expression of the cell.


H1 SUBTYPE COMPOSITION AND AFFINITY FOR CHROMATIN IN SITU IN NUCLEATED MATURE ERYTHROCYTES

Koutzamani E.¹, Loborg H.¹, Sarg B.², Lindner H.H.², and Rundquist I¹.

¹Department of Biomedicine and Surgery, Division of Cell Biology, Faculty of Health Sciences, Linköpings Universitet, Sweden, and ²Department of Medical Chemistry and Biochemistry, University of Innsbruck, Austria
The linker histones, commonly referred to as H1, are involved in the formation and maintenance of the higher order structure of the chromatin fiber and most likely also in epigenetic modulation of gene expression. This family consists of seven subtypes of which the highly specialized isoforms H5 and H1° accumulate in some terminally differentiated cells. In avian and amphibian erythrocytes, expressing H5 and H1° respectively, these proteins have been linked to cessation of cell proliferation and condensation of chromatin. The aim of this study was to analyse the affinity of linker histones for chromatin in these cell types, having similar biological features, but containing different subsets of linker histones. Using 4´6-diamidino-2-phenylindole (DAPI) as an indirect cyto-chemical probe for linker histone affinity in situ, we detected a substantial difference in the association of linker histones for chromatin between these two cell types. Linker histones in chicken erythrocytes were more strongly bound to chromatin than H1 in frog erythrocytes. Linker histone dissociation from chromatin was also analysed using electrophoretic and chromatrographic methods. The results showed that H5 dissociated at higher ionic strength than H1°, indicating a stronger affinity for chromatin. We also found that the amount of H5 in chicken erythrocyte nuclei was about 60% of the total amount of linker histones. In frog erythrocytes, H1° represented about 15% of the total amount of linker histones. Taken together, our results show that linker histones have a substantially higher affinity for chromatin in chicken erythrocytes than in frog erythrocytes. This difference can be explained by the high content of arginine-rich H5 in chicken erythrocytes. Our results also show a lack of correlation between linker histone affinity and chromatin condensation.


CORRELATION BETWEEN GENE POSITIONING AND GENE EXPRESSION

Philimonenko A. A.¹, Janáček J. ², Weipoltshammer K.³, Schöfer C.³, and Hozák P.<sup>1

1</sup>Institute of Experimental Medicine and ²Department of Biomathematics, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; ³Institute for Histology and Embryology, University of Vienna, Austria
We have investigated the spatial relationship of transcriptionally active or inactive genes within chromosome territories in the interphase nucleus of human fibroblasts. As a model we used paternally imprinted genes located within the 15q11-q13 region of chromosome 15 (SNRPN locus), deletion of which leads to Prader-Willi syndrome. For the simultaneous detection of genes and chromosome territories, we used in situ hybridization with commercially available DNA probes to SNRPN locus, and to the q arm of human chromosome 15. A computer program was written which allows the detection of the border of chromosome territory, and the distance between the border and the center of mass (labelling) of the gene. The analysis demonstrated no significant differences in distances from gene to chromosome territory border between active and inactive genes. Significant portion of genes were found located outside of 15q territory – however, only if the allele were transcriptionally active. In agreement with previously obtained data, we also observed a distinct substructure in interphase chromosome territories. Strongly labeled chromosomal subdomains were surrounded by less intensely labeled areas. Using a tresholding of digital images of human fibroblast nuclei we discriminated these sub-chromosomal domains and evaluated the distances between their borders and SNRPN loci. The SNRPN gene loci were located preferentially at the borders of chromosomal subdomains. These data support the idea that genes are preferentially located at the outer side of domains with condensed chromatin, but no strict correlation was found between active and inactive genes within the entire chromosomal domain. The only striking difference was the presence of ~ 7% of active genes at relatively large distances from the chromosomal territories.
CLUSTERING OF TELOMERES IN Saccharomyces cerevisiae

Straatman K.R., Sheehy M.M., Koziol M., Cotton V.E. and Louis E.J.

Department of Genetics, University of Leicester, UK
The ends of the chromosomes, the telomeres, are important for capping the ends of the DNA molecule to prevent the chromosomes from fusing together. Several groups have shown by fluorescent in situ hybridization (FISH) or by labelling of telomere associated proteins that the telomeres of the yeast Saccharomyces cerevisiae are organized in several clusters at the nuclear periphery. We are interested in how this organization of telomeres is maintained and which telomeres are together in a cluster. We are also interested in whether telomeres stay together in a cluster or move between clusters. Live imaging has shown that telomeres are very dynamic structures and move several hundreds of nanometres per second. Mitotic recombination data suggests which telomeres might be clustered together. At the sequence level, several telomeres share homologous sequences in their subtelomeric regions, suggesting that they might be in the same cluster, at least part of the time during the cell cycle. These sequence alignments are in agreement with the genetic data. We are now trying to visualize this localization of the telomeres using FISH. To investigate if the sequence of a telomere determines in which cluster it is, a telomeric translocation between telomere XL and XVR was made and we are in the process of analysing these mutants genetically and physically.


MICROSPECKLES, AN UNUSUAL NUCLEAR DOMAIN OF QUIESCENT (G₀ AND G₀₂) ONION CELLS, ARE TRANSIENT STORAGE SITES FOR snRNPs

Cui, P and Moreno Díaz de la Espina, S

Nuclear Matrix Laboratory, CIB CSIC, Madrid, Spain
Antibodies against U2B”, the sDMA epitope of Sm proteins and coilin, and p105, a protein of interchromatin granules (IGs), were used to investigate the nuclear distribution of the splicing factors in onion meristematic root cells in different physiological conditions. In steady-state proliferating cells, the spliceosomal components were distributed into a diffuse nucleoplasmic network, similar to that of IGs, and numerous Cajal bodies (CBs) by confocal microscopy. These two domains are the counterpart of the perichromatin fibrils and granules, IGs and CBs observed by EM after EDTA and bismuth oxynitrate stainings, and immunogold labelling with the same antibodies. The dormant cells of the root blastema in bulbs are stopped in both pre-replicative (G₀) and post replicative (G₂) states and after water activation they must undergo dramatic metabolic changes for progression through the first post-quiescence cell cycle and later cell division. They have a nuclear distribution of the proteins in small CBs and in an unusual nuclear domain, the micro-speckles, corresponding to storage sites for RNPs, which were rapidly mobilised after water imbibition, and showed a different ultrastructure from that of CBs and the interchromatin RNP network at the EM. The spliceosomal proteins relocated to a diffuse nucleoplasmic network and CBs when the cells were released from dormancy by water soaking and they re-started their proliferative activity. Inhibition of RNA synthesis by DRB treatment in proliferating cells demonstrated that the micro-speckles were not the morphological expression of a transcription block. Fractionation and confocal microscopy studies showed a differential association of the splicing factors with the nuclear matrix depending not only on the protein, but also on nuclear activity. Our results suggest a reversible relocation of the spliceosomal proteins between different sub-nuclear domains in physiological conditions.
SEQUENCE VARIATIONS IN HUMAN HISTONE H1.2 AND H1.4 SUBTYPES

Erikson-Gréen A.¹, Sarg B.², Söderkvist P.¹, Rundquist I.¹, and Lindner H.H.<sup>2

1</sup>Department of Biomedicine and Surgery, Division of Cell Biology, Faculty of Health Sciences, Linköpings universitet, Sweden and ²Department of Medical Chemistry and Biochemistry, University of Innsbruck, Austria
Histone H1 binds to the nucleosome at the entry and exit point of the DNA from the nucleosome core, as well as to linker DNA. The human histone H1 family consists of seven subtypes, H1.1-H1.5, H1 and H1t, all consisting of a highly conserved globular domain and less conserved N- and C-terminal tails of high positive charge. In this study, a hydrophilic-interaction liquid chromatographic method was used for detecting sequence variations within the subtypes. Two sequence variants of wild type H1 sequences were found. In K562 erythroleukemic cells, alanine in position 17 of H1.2 was substituted by valine, and in Raji B-lymphoblastoid cells lysine in position 173 of H1.4 was replaced by arginine. DNA sequencing of the corresponding gene segments confirmed these findings. In K562 cells, a homozygous GCC>GTC shift at codon 18 was detected, causing the H1.2 Ala17Val sequence variant, since the initial methionine is removed in H1 histones. In Raji cells, a heterozygous AAA>AGA codon change at position 174 in H1.4 was found, giving rise to the Lys173Arg substitution. The allele frequency of these sequence variants in a normal population was determined. An RFLP-assay was developed for detection of the H1.2 GCC>GTC shift at codon 18, and the allele frequency was determined to be 6.8%, indicating that this is a relatively frequent polymorphism. The AAA>AGA codon change at position 174 in H1.4 was detected using denaturing HPLC. This sequence variation was only found in Raji cells and it was not present in a normal population, or in six other cell lines derived from individuals suffering from Burkitt’s lymphoma. The significance of these sequence variants is unclear, but binding characteristics of the deviating histones H1.2 and H1.4 may be altered, and thereby processes like chromatin higher order organization and epigenetic control of gene regulation can be affected.


DYNAMICS OF PML DURING HYPOTONIC SHOCK–INDUCED FORMATION OF MICRO-NUCLEI.

Kurchashova S. Yu., Kireyev I. I., *Gulak P. V., Polyakov V. Yu.

A.N. Belozersky Institute of Physico–Chemical Biology, Moscow State University and *Insitute of Biology of Gene, Moscow, Russia.
The promyelocytic leukemia nuclear bodies (PML NBs) are nuclear multiprotein structures that are tightly bound to the nuclear matrix.. Cells typically contain 10–30 PML NBs per nucleus with diameters 0.2–1 m, their number and size change during the cell cycle. Although the functions of PML and PML NBs remain largely unknown, evidence is accumulating for a role of PML in transcription regulation (Fuchsova et al., 2002). The PML protein is necessary for the proper formation of PML NBs and a defining marker for this structure. We have studied the role of individual chromosomes in postmitotic reconstruction of PML NBs. An experimental model of micronuclei formation offers several advantages for the study of nuclear domain assembly at the end of mitosis. The process of nuclear domain formation occurs more slowly than in intact cells and the micronuclei formed around individual chromosomes allow us to estimate the participation of the individual chromosomes in the formation of nuclear domains. Here, we demonstrate that PML protein is specifically associated with certain chromosomes in Hela and PK cells during micronuclei formation. The formation of micronuclei around individual chromosomes was induced by hypotonic treatment and transfer of cells to the culture medium The interaction between chromosomes and PML starts during hypotonic treatment and further persists upon subsequent transfer of cells to the culture medium. PML localization was observed as a spot associated with some chromosomes and also diffusely distributed in the cytoplasm. Immunofluorescent localization of PML protein in micronuclei revealed characteristic pattern of small discrete foci. The integrity of these foci was dependent on the association with RNA but not with DNA as shown by DNase and RNase treatment of cells. Based on these data we propose that PML specifically associates with certain chromosomes, and this formation does not require spatial association of chromosomes inside one nucleus.

SEPARATION OF NUCLEAR PROTEIN COMPLEXES BY BLUE NATIVE POLY-ACRYLAMIDE GEL ELECTROPHORESIS

Nováková Z.¹, Hodný Z.¹, Man P.² and Hozák P.<sup>1

1</sup>Department of Cell Ultrastructure and Molecular Biology, Institute of Experimental Medicine, and ²Mass Spectrometry Laboratory, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
The nucleus of eukaryotic cell is functionally and structurally compartmentalized. To understand the structure and function of individual nuclear domains, the knowledge about the protein composition of complexes building up these structures is needed. We explored therefore the potential of previously developed method for separation of membrane protein complexes in native state, the blue native poly-acrylamide gel electrophoresis (BN-PAGE), for separation of large nuclear protein complexes coupled with mass spectrometry analysis. In this study we demonstrate that modifications in solubilization of nuclear complexes are prerequisite for efficient separation by BN-PAGE. A solution for another problem, the high protein background disabling the subsequent mass spectrometric identification of protein complex composition (due to uncomplete separation of very basic nuclear proteins such as histones), was also sought. The usefulness of the method in nuclear research is demonstrated by 1), immunological detection of RNA polymerase I and II complexes and other nuclear protein complexes separated by one-dimensional blue native electrophoresis, and 2), mass spectrometric identification of subunit composition of U1snRNP particle after two-dimensional combination of colorless and blue native PAGE.


NUCLEAR DISTRIBUTION OF MFP1-LIKE PROTEINS IN RELATION TO CELL ACTIVITY IN ONION

Samaniego, R and Moreno Díaz de la Espina, S

Nuclear Matrix Laboratory, CIB CSIC, Madrid, Spain
MFP1 is a protein of the plant nuclear matrix with both a long coiled-coil domain and a C-terminal DNA binding sub-domain. Their orthologs have been sequenced in Arabidopsis thaliana, tomato, tobacco and rice, but its functionality is currently unknown. We have used two domain-specific sera against LeMFP1 (288) and AtMFP1 (OSU 91) to look for proteins sharing antigenic determinants in onion. Both sera recognised a 78 kD protein, that is removed from nuclei after DNA digestion and high salt extraction. A second protein 90 kD and basic pI (8.5-9.4) was exclusively detected by serum 288, and presents different phosphorylation states that determine its binding to the NM. Both proteins are ubiquitous nuclear components in meristematic (either proliferative or quiescent) and differentiated cells, and show a dynamic pattern of nuclear distribution. The 78 kD protein distributes in small foci displaying a punctated nuclear pattern, that re-distributes during S-phase in hydroxyurea synchronised cells, and appear conserved in differentiated cells of roots and leaves. It localizes to nucleoli only in dormant and differentiated cells. The 90 kD protein accumulates in large nuclear foci associated to the NM, which are more abundant in proliferating cells. The small and large foci observed by confocal microscopy are the counterpart of the clusters of gold particles at the chromatin peripheries, and of dense nuclear bodies, respectively. These bodies are different from CBs and other nuclear bodies so far described in plants. On view of these data and also of previous results from our group and others, the relationships of the onion proteins with the sequenced homologues of tomato and Arabidopsis, and also the functionality of these proteins in the plant nucleus are discussed.

THE NUCLEAR MATRIX PROTEIN PSMA6 GENE INTRONIC MICROSATELLITE ALLELE IS ASSOCIATED WITH TYPE II DIABETES MELLITUS

Sjakste N¹, Kalis M¹, Pirags V¹, Lazdins M¹, Groop L<sup>2

1</sup>University of Latvia, Riga, Latvia and ²Department of Endocrinology, Lund University, University Hospital Malmö, Sweden
Proteasomes are ubiquitous particles with multicatalytic proteinase activity aimed on degradation of ubiquinated proteins including transcription factors, oncogene products, etc. Alterations of the proteasome functions and structure lead to numerous pathologies. Proteasomes compose an integral part of the nuclear matrix, where PSMA 6 protein is found among other proteasomal proteins. This study was aimed on a search for correlation of the TG dinucleotide repeat polymorpfism (HSMS006) in the 6th intron of the human proteasome core particle PSMA6 gene and Type II diabetes mellitus. Polymorphism studies of the microsatellite tract length were performed in non-insulin dependent diabetes mellitus patients (91 individuals from Botnia (Sweden) and 66 from Latvia) compared to a control group (88 age-matched individuals with normal insulin sensitivity from Botnia and 33 from Latvia) using PCR and DNA fragment length determination. In the control groups we have revealed 10 alleles presented by 13 to 25 TG repeats, alleles with 18 and 21 repeats being the most frequent. Allele distribution in Botnic and Latvian populations were similar. In Type 2 diabetes patients from both populations the (TG)22 allele was two times more frequent as compared to the corresponding control group. It appears that polymorphism of the TG repeat in the 6th intron of the human proteasome core particle PSMA6gene is correlated to Type II diabetes mellitus morbidity.

INTERFERON-ALPHA INDUCTION OF PML NUCLEAR BODIES IS INHIBITED BY TRICHOSTATIN A

Vlasakova J., Hodny Z., and Hozak P.

Department of Cell Ultrastructure and Molecular Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
The nucleus of the eukaryotic cell is a complex organelle compartmentalized into structural and functional domains. One of these nuclear domains are matrix-associated multi-protein complexes, promyeolocytic leukemia nuclear bodies (PML NBs). The gene product, PML protein, is essential for proper formation and integrity of PML NBs. Although the biochemical function of PML and PML NBs remain still unclear, there is an evidence for its contribution in response to a variety of cell states like in cancer, apoptosis, and viral infection. It is known that type I and II interferons (IFNs) dramatically increase the transcription of the PML gene through an IFN-stimulated response element present in the PML gene promoter. Additionally, IFNs increases expression of other structural components of PML NBs - Sp100 and ISG20. This can contribute to the observed increase in the number of PML NBs. In this study we have shown that the cell response to interferon is strongly reduced in cells treated with trichostatin A, an inhibitor of protein deacetylases. This indicates that the protein hyperacetylation interferes with some stage of PML NBs assembly. The effect of trichostatin A on the expression of structural components of PML NBs and de novo formation of PML NBs after their dispersion by Cd²⁺ ions or by heat shock is also presented.