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The suprachromosomal organization of DNA as a morphogenetic element in plant development
229
"Trinoculaire" de Microscopies Electroniques, Lausanne, juin 1995 THE SUPRACHROMOSOMAL ORGANIZATION OF DNA AS A MORPHOGENETIC ELEMENT IN PLANT DEVELOPMENT _Messen_.ssKathy, Bral Martine, Vermijlen David and Van Oostveldt Patrick Laboratory of Biochemistry and Molecular Cytology, Faculty of Agricultural and Applied Biological Sciences, University of Gent, Belgium The suprachromosomal organization of DNA in cell nuclei is examined by the use of (whole mount) fluorescence in situ hybridization (FISH), fluorescence immunocytochemistry (FICC) and confocal scanning laser microscopy (CSLM). The distribution of ribosomal DNA (5.8S, 18S, 25S and 5S rDNA) and a chromosome specific highly repetitive sequence (a 500bp repeated sequence) were examined in the chromatin of diploid (2n) and tetraploid (4n) nuclei of different tissues of Arabidopsis thaliana (Bauwens et at. (1991), Chromosoma, 101, 41-48). Qualitative and quantitative analysis of the images recorded with a Bio-Rad MRC-600 confocal scanning laser microscope atler FISH with the 45S rDNA probe on diploid nuclei showed two larger and two smaller sites. Some of the nuclei exhibited more than the four expected spots, possibly due to polyploidy or differential amplification of the rDNA. Many of the interphase nuclei had a reduced number of hybridization spots, which could be the result of a (non random) chromosomal association. Some research was also done on 5S rDNA, where the diploid nuclei showed mostly two rDNA-spots and the tetraploid nuclei mostly four labeled sites. All this previous work was done on squashpreparations of four (2n) or five days (4n) old seedlings. Whole mount in situ hybridization (Bauwens et al. (1994), Plant Journal, 6, 123-131) was used to examine whether the suprachromosomal organization was tissue specific or not. Preliminary tests indicate that there exists such a correlation e.g. The hybridization of the 500bp repeated sequence is quite specific. In the root these spots seem to be oriented according to a line running parallel to the newly formed cell wall and therefore are oriented in a tissue specific manner. Another part of the research exits in optimizing protocols for the (whole mount) immunolocalization of tubulines in Arabidopsis thaliana. Our goal is to combine the immunolocalization of tubulines with (whole mount) in situ hybridization and to determine in this way the relation between the suprachromosomal organization and cell division.
DNA CONDENSED BY H I HISTONES FORMS SMALL PITCH C H O L E S T E R I C LIQUID CRYSTALLINE PHASES
LOCALIZATION OF m R N A s A N D PROTEINS OF P14 A N D P20 IN RAT PITUITARY B. RQN$IN *, I.M BOUTIN +, B. GRANDCLEMENT * C. BRISSON *, G. MOREL * • CNRS URA 1459, Institut Pasteur de Lyon, Avenue Tony Gamier, 69355 Lyon, France Tel : 72 72 67 97, Fax : 78 61 13 45 + Centre de recherche - Hftel-Dieu de Montrral, Qurbec, Canada The s t e r o i d / t h y r o i d receptor superfamily have proteins which are able to b i n d a ligand, and generate a specific sight of D N A recognition, that induces activation or inhibition of the genes. In this f a m i l y the t r a n s c r i p t i o n factor called "Chicken O v a l b u m i n U p s t r e a m Promotor-Transcription Factor" (COUP-TF) belongs to a protein family called: "Orphan receptor" because, u p to date, no specific ligand was identified to bind with them. Two genes: APO AI regulatory protein-1 (ARP-I) and erb A-related gene (ear-3) coding respectively for P20 and P14 proteins have been localized in anterior pituitary of a cycling and pregnan female rat in order to u n d e r s t a n d their effect on h o r m o n gene in pituitary. T h e i r localization was done by two techniques. Firstly, the localization of these p r o t e i n s u s i n g i m m u n o c y t o l o g y was c o n f i n e d to the lactotrophs and somatotrophs cells. P14 and P20 immunoreactivities are present in the cytoplasm and in the nucleus of these ceils, but the signal is higher in the cells of pregnan females than in cycling females, These o b s e r v a t i o n s are similar for the two proteins. Secondary the mRNA localization of P14 and P20 was done using in situ h y b r i d i z a t i o n w i t h specific e l g o n u c l e o t i d e s labeled w i t h digoxygenin. The in situ hybridization signal is p r e s e n t in the nucleus and in the cytoplasm of the same cells. But, the m R N A expression was higher in cycling females for P14, t h a n p r e g n a n female and inversely for P20, These results suggest that P14 and P20 are inhibitors of PRL gene. Moreover, d o p a m i n e is a m o d u l a t o r of COUP-TF a n d in the same way, the regulator of PRL secretion inducing a tonic inhibition of its gene.
ELECTRON MICROSCOPICAL TRANSCRIBING CHROMATIN.
ANALYSIS
OF
LIVOLANT Franc oise. GARCIA Nicolas, PELTA Juan, Laboratoire de Physique des Solides, Bdt 510 Universitd Paris-Sud, 91405 Orsay Cedex, France.
In the cell, histone H1 is involved in the condensation of the chromatin fiber and also in the regulation of the transcriptional activity of the DNA molecule. In vitro, H1 is known to induce the condensation of DNA in the form of microaggregates characterized by a chiral supramolecular organization of DNA (~-type sprectrum). For a review of these different aspects, see Zlatanova and Yaneva (1991) DNA and Cell Biology 10, 239248 In order to determine the structure of HI-DNA complexes, the precipitation of short DNA fragments was obtained by the addition of H1 histones in TE buffer in the presence of various NaC1 concentrations. Condensed DNA was collected by centrifugation and analyzed in polarizing microscopy and in freeze-fracture electron microscopy. HI-condensed DNA forms a cholesteric liquid crystalline phase with polygonal textures. This phase is characterized by a helical pitch much smaller (0.4 to t I.tm) than the pitch of the histone free DNA cholesteric phase (about 2.5 gm). Double spirals and herring-bone patterns are frequently observed on the replicas. They correspond to different orientations of the fracture plane relative to the focal lines which are numerous in this mesophase. At high magnification, the series of nested arches which characterize a cholesteric structure are not always observed but instead can be seen frequently a regular packing of granular structures that we assume to correspond to H1-DNA complexes. Their struture remains to be analyzed more precisely by X-ray diffraction.
ten Heggeler-Bordier Beatrice and Wahli Walter. Institut de Biologie Animale, Universite de Lausanne, Bfitiment de Biologie, 1015 Lausanne, Switzerland.
Electron microscopy was used to monitor the fate of reconstituted nucleosomes during in vitro transcription by the T7 RNA polymerase and RNA polymerase II from rat liver nuclear extract. With the purified T7 RNA polymerase the nucteosomes disappeared in the transcribed region of the pSG5 template but in the in vitro transcription of a vitellogenin gene from Xenopous Laevis with RNA polymerase II in a rat liver nuclear extract, the nucleosomes persisted on the template. Nucleosomes also disappeared on a 20-fold repeat of the 5S rRNA gene of the sea urchin Lytechinus variegatus cloned downstream of the T7 promoter of the plasmid BS/KS-. When nuclear extract was added to the in vitro transcription reaction with the T7 RNA polymerase and the 20-fold repeat of the 5S rRNA gene, the nucleosomes did also stay on the template. These results suggests that a factor in the nuclear extract is responsable for the maintenance of the nucleosomes during in vitro transcription.
"Trinoculaire" de Microscopies Electroniques, Lausanne, juin 1995 THE SUPRACHROMOSOMAL ORGANIZATION OF DNA AS A MORPHOGENETIC ELEMENT IN PLANT DEVELOPMENT _Messen_.ssKathy, Bral Martine, Vermijlen David and Van Oostveldt Patrick Laboratory of Biochemistry and Molecular Cytology, Faculty of Agricultural and Applied Biological Sciences, University of Gent, Belgium The suprachromosomal organization of DNA in cell nuclei is examined by the use of (whole mount) fluorescence in situ hybridization (FISH), fluorescence immunocytochemistry (FICC) and confocal scanning laser microscopy (CSLM). The distribution of ribosomal DNA (5.8S, 18S, 25S and 5S rDNA) and a chromosome specific highly repetitive sequence (a 500bp repeated sequence) were examined in the chromatin of diploid (2n) and tetraploid (4n) nuclei of different tissues of Arabidopsis thaliana (Bauwens et at. (1991), Chromosoma, 101, 41-48). Qualitative and quantitative analysis of the images recorded with a Bio-Rad MRC-600 confocal scanning laser microscope atler FISH with the 45S rDNA probe on diploid nuclei showed two larger and two smaller sites. Some of the nuclei exhibited more than the four expected spots, possibly due to polyploidy or differential amplification of the rDNA. Many of the interphase nuclei had a reduced number of hybridization spots, which could be the result of a (non random) chromosomal association. Some research was also done on 5S rDNA, where the diploid nuclei showed mostly two rDNA-spots and the tetraploid nuclei mostly four labeled sites. All this previous work was done on squashpreparations of four (2n) or five days (4n) old seedlings. Whole mount in situ hybridization (Bauwens et al. (1994), Plant Journal, 6, 123-131) was used to examine whether the suprachromosomal organization was tissue specific or not. Preliminary tests indicate that there exists such a correlation e.g. The hybridization of the 500bp repeated sequence is quite specific. In the root these spots seem to be oriented according to a line running parallel to the newly formed cell wall and therefore are oriented in a tissue specific manner. Another part of the research exits in optimizing protocols for the (whole mount) immunolocalization of tubulines in Arabidopsis thaliana. Our goal is to combine the immunolocalization of tubulines with (whole mount) in situ hybridization and to determine in this way the relation between the suprachromosomal organization and cell division.
DNA CONDENSED BY H I HISTONES FORMS SMALL PITCH C H O L E S T E R I C LIQUID CRYSTALLINE PHASES
LOCALIZATION OF m R N A s A N D PROTEINS OF P14 A N D P20 IN RAT PITUITARY B. RQN$IN *, I.M BOUTIN +, B. GRANDCLEMENT * C. BRISSON *, G. MOREL * • CNRS URA 1459, Institut Pasteur de Lyon, Avenue Tony Gamier, 69355 Lyon, France Tel : 72 72 67 97, Fax : 78 61 13 45 + Centre de recherche - Hftel-Dieu de Montrral, Qurbec, Canada The s t e r o i d / t h y r o i d receptor superfamily have proteins which are able to b i n d a ligand, and generate a specific sight of D N A recognition, that induces activation or inhibition of the genes. In this f a m i l y the t r a n s c r i p t i o n factor called "Chicken O v a l b u m i n U p s t r e a m Promotor-Transcription Factor" (COUP-TF) belongs to a protein family called: "Orphan receptor" because, u p to date, no specific ligand was identified to bind with them. Two genes: APO AI regulatory protein-1 (ARP-I) and erb A-related gene (ear-3) coding respectively for P20 and P14 proteins have been localized in anterior pituitary of a cycling and pregnan female rat in order to u n d e r s t a n d their effect on h o r m o n gene in pituitary. T h e i r localization was done by two techniques. Firstly, the localization of these p r o t e i n s u s i n g i m m u n o c y t o l o g y was c o n f i n e d to the lactotrophs and somatotrophs cells. P14 and P20 immunoreactivities are present in the cytoplasm and in the nucleus of these ceils, but the signal is higher in the cells of pregnan females than in cycling females, These o b s e r v a t i o n s are similar for the two proteins. Secondary the mRNA localization of P14 and P20 was done using in situ h y b r i d i z a t i o n w i t h specific e l g o n u c l e o t i d e s labeled w i t h digoxygenin. The in situ hybridization signal is p r e s e n t in the nucleus and in the cytoplasm of the same cells. But, the m R N A expression was higher in cycling females for P14, t h a n p r e g n a n female and inversely for P20, These results suggest that P14 and P20 are inhibitors of PRL gene. Moreover, d o p a m i n e is a m o d u l a t o r of COUP-TF a n d in the same way, the regulator of PRL secretion inducing a tonic inhibition of its gene.
ELECTRON MICROSCOPICAL TRANSCRIBING CHROMATIN.
ANALYSIS
OF
LIVOLANT Franc oise. GARCIA Nicolas, PELTA Juan, Laboratoire de Physique des Solides, Bdt 510 Universitd Paris-Sud, 91405 Orsay Cedex, France.
In the cell, histone H1 is involved in the condensation of the chromatin fiber and also in the regulation of the transcriptional activity of the DNA molecule. In vitro, H1 is known to induce the condensation of DNA in the form of microaggregates characterized by a chiral supramolecular organization of DNA (~-type sprectrum). For a review of these different aspects, see Zlatanova and Yaneva (1991) DNA and Cell Biology 10, 239248 In order to determine the structure of HI-DNA complexes, the precipitation of short DNA fragments was obtained by the addition of H1 histones in TE buffer in the presence of various NaC1 concentrations. Condensed DNA was collected by centrifugation and analyzed in polarizing microscopy and in freeze-fracture electron microscopy. HI-condensed DNA forms a cholesteric liquid crystalline phase with polygonal textures. This phase is characterized by a helical pitch much smaller (0.4 to t I.tm) than the pitch of the histone free DNA cholesteric phase (about 2.5 gm). Double spirals and herring-bone patterns are frequently observed on the replicas. They correspond to different orientations of the fracture plane relative to the focal lines which are numerous in this mesophase. At high magnification, the series of nested arches which characterize a cholesteric structure are not always observed but instead can be seen frequently a regular packing of granular structures that we assume to correspond to H1-DNA complexes. Their struture remains to be analyzed more precisely by X-ray diffraction.
ten Heggeler-Bordier Beatrice and Wahli Walter. Institut de Biologie Animale, Universite de Lausanne, Bfitiment de Biologie, 1015 Lausanne, Switzerland.
Electron microscopy was used to monitor the fate of reconstituted nucleosomes during in vitro transcription by the T7 RNA polymerase and RNA polymerase II from rat liver nuclear extract. With the purified T7 RNA polymerase the nucteosomes disappeared in the transcribed region of the pSG5 template but in the in vitro transcription of a vitellogenin gene from Xenopous Laevis with RNA polymerase II in a rat liver nuclear extract, the nucleosomes persisted on the template. Nucleosomes also disappeared on a 20-fold repeat of the 5S rRNA gene of the sea urchin Lytechinus variegatus cloned downstream of the T7 promoter of the plasmid BS/KS-. When nuclear extract was added to the in vitro transcription reaction with the T7 RNA polymerase and the 20-fold repeat of the 5S rRNA gene, the nucleosomes did also stay on the template. These results suggests that a factor in the nuclear extract is responsable for the maintenance of the nucleosomes during in vitro transcription.