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L de Jong

Publications and source records attributed to L de Jong.

At least 37 records · Page 2Linked to original sources
PubMed1996

Nuclear domains involved in RNA synthesis, RNA processing, and replication.

Two main principles of nuclear organization have been outlined on the basis of contributions by many research groups in recent years. The first principle is that interphase chromosomes occupy discrete territories in the nucleus, with no intermingling of the DNA from different chromosomes. Within a chromosome territory the DNA is organized in chromatin fibers at several levels of folding, that meander through the territory. Transcription and replication take place at the surface of these higher order chromatin fibers, probably on locally unfolded DNA templates. The second principle is that different types of nuclear domains are associated with several specific gene loci. This holds for clusters of interchromatin granules, coiled bodies, RNA 3'-cleavage factor-containing nuclear bodies (cleavage bodies) and probably PML-containing nuclear bodies. These domains may play an important role in the spatial arrangement of genes in the interphase nucleus. Despite these new insights, our knowledge of the function of many nuclear compartments and the molecular interactions responsible for the dynamic organization of a compartmentalized nucleus is still in its infancy.

Animals↗
PubMed1995

Nuclear domains and the nuclear matrix.

This overview describes the spatial distribution of several enzymatic machineries and functions in the interphase nucleus. Three general observations can be made. First, many components of the different nuclear machineries are distributed in the nucleus in a characteristic way for each component. They are often found concentrated in specific domains. Second, nuclear machineries for the synthesis and processing of RNA and DNA are associated with an insoluble nuclear structure, called nuclear matrix. Evidently, handling of DNA and RNA is done by immobilized enzyme systems. Finally, the nucleus seems to be divided in two major compartments. One is occupied by compact chromosomes, the other compartment is the space between the chromosomes. In the latter, transcription takes place at the surface of chromosomal domains and it houses the splicing machinery. The relevance of nuclear organization for efficient gene expression is discussed.

Animals↗
PubMed1995

Localization of the glucocorticoid receptor in discrete clusters in the cell nucleus.

The cell nucleus is highly organized. Many nuclear functions are localized in discrete domains, suggesting that compartmentalization is an important aspect of the regulation and coordination of nuclear functions. We investigated the subnuclear distribution of the glucocorticoid receptor, a hormone-dependent transcription factor. By immunofluorescent labeling and confocal microscopy we found that after stimulation with the agonist dexamethasone the glucocorticoid receptor is concentrated in 1,000-2,000 clusters in the nucleoplasm. This distribution was observed in several cell types and with three different antibodies against the glucocorticoid receptor. A similar subnuclear distribution of glucocorticoid receptors was found after treatment of cells with the antagonist RU486, suggesting that the association of the glucocorticoid receptor in clusters does not require transformation of the receptor to a state that is able to activate transcription. By dual labeling we found that most dexamethasone-induced receptor clusters do not colocalize with sites of pre-mRNA synthesis. We also show that RNA polymerase II is localized in a large number of clusters in the nucleus. Glucocorticoid receptor clusters did not significantly colocalize with these RNA polymerase II clusters or with domains containing the splicing factor SC-35. Taken together, these results suggest that most clustered glucocorticoid receptor molecules are not directly involved in activation of transcription.

Animals↗
PubMed1995

Energy metabolism in young pigs as affected by mixing.

The effect of mixing on energy metabolism was studied in 8-wk-old pigs. In each of two trials, two clusters of 20 pigs (two litters of 10 pigs) were randomly assigned to one of two treatments: control or mixing. Each cluster was housed in two pens. In each trial, after a preliminary period od 2 wk, the two litters within the mixing treatment were mixed at the start of a 2-wk experimental period. During mixing, the five heaviest pigs of each litter were put together in one pen, and the five lightest pigs of each litter were put together in the other pen. In the control treatment, the social structure of both litters in one climatic chamber was not altered. After mixing, a short-term effect on total heat production and activity-related heat production was present. Both were increased (P < .01) only during the 1st h after mixing. Only 57.3% of this increased total heat production was caused by an increased activity. However, no long-term effects of mixing on energy partitioning were present during the total experimental period. The absence of a long-term mixing effect might be caused by the optimal conditions at the moment of mixing. In the preliminary period the transposition of GE into ME increased 1.3% (P < .05), and ME for maintenance decreased 80 kJ.kg(-.75).d(-1) (P < .01) between wk 1 and 2. These large alterations in energy metabolism are probably a carry-over effect of the transportation of the pigs and (or) the changes in housing environment.

Aging↗
PubMed1994

The t(15;17) translocation alters a nuclear body in a retinoic acid-reversible fashion.

Nuclear bodies (NBs) are ultrastructurally defined granules predominantly found in dividing cells. Here we show that PML, a protein involved in the t(15;17) translocation of acute promyelocytic leukaemia (APL), is specifically bound to a NB. PML and several NB-associated proteins, found as auto-antigens in primary biliary cirrhosis (PBC), are co-localized and co-regulated. The APL-derived PML-RAR alpha fusion protein is shown to be predominantly localized in the cytoplasm, whereas a fraction is nuclear and delocalizes the NB antigens to multiple smaller nuclear clusters devoid of ultrastructural organization. RA administration (which in APL patients induces blast differentiation and consequently complete remissions) causes the re-aggregation of PML and PBC auto-antigens onto the NB, while PML-RAR alpha remains mainly cytoplasmic. Thus, PML-RAR alpha expression leads to a RA-reversible alteration of a nuclear domain. These results shed a new light on the pathogenesis of APL and provide a molecular link between NBs and oncogenesis.

Animals↗
PubMed1994

In vitro splicing of pre-mRNA containing bromouridine.

The artificial UTP-analogue 5-bromouridine 5'-triphosphate (BrUTP) has been used to label pre-mRNA in vitro and in vivo [1,2]. We have investigated the effect of bromouridine (BrU) in pre-mRNA on the efficiency of splicing. An adenovirus major late II construct was used to prepare four different transcripts, each containing a different amount of BrU. These four transcripts were tested in an in vitro splicing assay. We found that splicing is strongly inhibited if all uridines (U) in the transcript were substituted for BrU. Splicing was restored to some extent if 50% of the Us were replaced by BrU. The splicing efficiency returned to an almost normal level if only 1 our of every 10 Us was substituted for BrU. This demonstrates that only a pre-mRNA containing a small amount of BrU can be spliced normally in vitro. Furthermore, these results strongly suggest that some Us in the adenoviral transcript, probably those at the splice sites, cannot be replaced by BrU and are therefore critical in the splicing reaction.

Adenoviridae↗
PubMed1994

RNA polymerase II transcription is concentrated outside replication domains throughout S-phase.

Transcription and replication are, like many other nuclear functions and components, concentrated in nuclear domains. Transcription domains and replication domains may play an important role in the coordination of gene expression and gene duplication in S-phase. We have investigated the spatial relationship between transcription and replication in S-phase nuclei after fluorescent labelling of nascent RNA and nascent DNA, using confocal immunofluorescence microscopy. Permeabilized human bladder carcinoma cells were labelled with 5-bromouridine 5'-triphosphate and digoxigenin-11-deoxyuridine 5'-triphosphate to visualize sites of RNA synthesis and DNA synthesis, respectively. Transcription by RNA polymerase II was localized in several hundreds of domains scattered throughout the nucleoplasm in all stages of S-phase. This distribution resembled that of nascent DNA in early S-phase. In contrast, replication patterns in late S-phase consisted of fewer, larger replication domains. In double-labelling experiments we found that transcription domains did not colocalize with replication domains in late S-phase nuclei. This is in agreement with the notion that late replicating DNA is generally not actively transcribed. Also in early S-phase nuclei, transcription domains and replication domains did not colocalize. We conclude that nuclear domains exist, large enough to be resolved by light microscopy, that are characterized by a high activity of either transcription or replication, but never both at the same time. This probably means that as soon as the DNA in a nuclear domain is being replicated, transcription of that DNA essentially stops until replication in the entire domain is completed.

Carcinoma↗
PubMed1993

Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus.

Several nuclear activities and components are concentrated in discrete nuclear compartments. To understand the functional significance of nuclear compartmentalization, knowledge on the spatial distribution of transcriptionally active chromatin is essential. We have examined the distribution of sites of transcription by RNA polymerase II (RPII) by labeling nascent RNA with 5-bromouridine 5'-triphosphate, in vitro and in vivo. Nascent RPII transcripts were found in over 100 defined areas, scattered throughout the nucleoplasm. No preferential localization was observed in either the nuclear interior or the periphery. Each transcription site may represent the activity of a single gene or, considering the number of active pre-mRNA genes in a cell, of a cluster of active genes. The relation between the distribution of nascent RPII transcripts and that of the essential splicing factor SC-35 was investigated in double labeling experiments. Antibodies against SC-35 recognize a number of well-defined, intensely labeled nuclear domains, in addition to labeling of more diffuse areas between these domains (Spector, D. L., X. -D. Fu, and T. Maniatis. 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:3467-3481). We observe no correlation between intensely labeled SC-35 domains and sites of pre-mRNA synthesis. However, many sites of RPII synthesis colocalize with weakly stained areas. This implies that contranscriptional splicing takes place in these weakly stained areas. These areas may also be sites where splicing is completed posttranscriptionally. Intensely labeled SC-35 domains may function as sites for assembly, storage, or regeneration of splicing components, or as compartments for degradation of introns.

Cell Nucleolus↗
PubMed1992

Binding of matrix attachment regions to lamin B1.

Eukaryotic chromatin is organized into topologically constrained loops that are attached to the nuclear matrix. The regions of DNA that interact with the matrix are called matrix attachment regions (MARs). We studied the spatial distribution of MAR-binding sites in the nuclear matrix from rat liver cells, following a combined biochemical and ultrastructural approach. We found that MAR-binding sites are distributed equally over the internal fibrogranular network and the peripheral nuclear lamina. Internal and peripheral binding sites have similar binding characteristics: both sets of binding sites show specific and saturable binding of MARs from different organisms. By means of a DNA-binding protein blot assay and in vitro binding studies, we identified lamin B1 as a MAR-binding protein, which provides evidence for a specific interaction of DNA with the nuclear lamina.

Animals↗
PubMed1992

Heat shock-induced redistribution of a 160-kDa nuclear matrix protein.

In this paper we describe a 160-kDa protein (p160) which is present in the nuclear matrix of rat, mouse, and human cells. Biochemical and ultrastructural analysis shows that p160 is associated with the internal matrix and is not present in the lamina-pore complex. Immunoelectron microscopy shows that the protein is part of the extranucleolar, fibrogranular network of the nuclear matrix. During an in vivo 42 degrees C heat treatment of HeLa cells, A431 human epidermoid cells, and T24 human bladder carcinoma cells, p160 transiently formed large clusters inside the nucleus. These p160 clusters are associated with the nuclear matrix network, as judged by immunolabeling on isolated nuclear matrices. The percentage of cells showing p160 clusters increased proportionally with longer heat treatments, reaching a maximum after a period of 3 h. At this time 70 +/- 5% of the cells displayed these clusters. Clustering decreased after longer heat treatments and the anti-p160 staining pattern became diffuse granular again. Other nuclear components, such as the A1 antigen of hnRNP (ribonucleoprotein), the Sm antigen of snRNPs, and lamins A and C, did not cluster during the 42 degrees C treatment, indicating that this reallocation is characteristic for the p160 matrix protein. These results demonstrate that p160 is an internal nuclear matrix element with a dynamic spatial distribution.

Animals↗
PubMed1992

Stabilization of the nuclear matrix by disulfide bridges: identification of matrix polypeptides that form disulfides.

The molecular structure of the nuclear matrix is still poorly understood. We have tried to assess which proteins are important structural elements by examining the process of stabilization of the nuclear matrix by sodium tetrathionate. Sodium tetrathionate stabilizes the nuclear matrix by oxidizing sulfhydryl groups to disulfides. We show that tetrathionate-stabilized matrices are disassembled in buffers containing SDS, indicating that the stabilized nuclear matrix is not a continuous network of cross-linked proteins. Using monobromobimane, a thiol-specific fluorescent reagent, we show that many protein thiols in the stabilized matrix are oxidized. By chromatography on activated thiol-Sepharose we estimated that about 50% of the matrix proteins had oxidized sulfhydryl groups. The protein composition of the material bound to activated thiol-Sepharose was similar to that of the not-bound material. A few proteins are highly enriched in the fraction that was bound to the column. This indicates that many matrix protein species are partially oxidized and that some proteins are completely oxidized. The oxidized protein thiols are found in relatively large complexes as determined by SDS gel-electrophoresis under nonreducing conditions. These results are interpreted in terms of protein-protein interactions in the matrix. The possible role of thiols and disulfides in the in vivo organization of the nucleus is discussed.

Animals↗
PubMed1992

A monoclonal antibody recognizing nuclear matrix-associated nuclear bodies.

We have isolated a monoclonal antibody, 5E10, that labels discrete spots in the interphase nucleus. By immunoblotting mAb 5E10 recognized predominantly a 126 kDa polypeptide with an isoelectric point of 5.5. Indirect immunofluorescence showed that mAb 5E10 labeled spots in many cell lines and tissues from rat or human origin, but not in cells from mouse, chicken, African green monkey, or the lower eukaryotes Saccharomyces and Dictyostelium. In the human bladder carcinoma cell line T24 the number of nuclear spots were found to be 21 +/- 10 (n = 132). In many cells spots were found also in the cytoplasm. In a small fraction of T24 cells the mAb revealed thread-like structures in addition to spots. Throughout mitosis the antigen was found to be clustered in the cytoplasm, not associated with metaphase chromosomes. The spherical structures that contain the antigen were tightly bound to the nuclear matrix. Immunogold labeling with mAb 5E10 showed that the antigen is localized in 0.3 microns diameter spherical, electron-dense structures, reminiscent of nuclear bodies. Double-labeling experiments showed that these spots do not colocalize with U1 snRNPs and centromeres. The spots did colocalize with nuclear speckles recognized by a primary biliary cirrhosis autoimmune serum, which is thought to recognize nuclear bodies. On the basis of these observations we conclude that mAb 5E10 recognizes discrete nuclear substructures, most likely nuclear bodies.

Animals↗
PubMed1991

The kinetics of the hydroxylation of procollagen by prolyl 4-hydroxylase. Proposal for a processive mechanism of binding of the dimeric hydroxylating enzyme in relation to the high kcat/Km ratio and a conformational requirement for hydroxylation of -X-Pro-Gly- sequences.

Prolyl 4-hydroxylase modifies only approx. 5% of the hydroxylatable prolyl residues in procollagen at a relatively high rate, after which the rate of further hydroxylation rapidly decreases. This suggests that the probability to exist in a defined hydroxylation-committed conformation differs between the numerous -X-Pro-Gly- sequences in the substrate. The enzyme reaction is characterized by the unusually high kcat/Km ratio of 3 x 10(9) M-1 s-1. To explain these kinetic features, an extremely high second-order rate constant for the association of enzyme and the subset of rapidly hydroxylated prolyl residues has to be assumed. A two-step mechanism is proposed in which diffusional constraints on the rate of association of prolyl 4-hydroxylase with hydroxylatable prolyl residues can be overcome. Upon encountering a random coil pro-alpha chain, the dimeric enzyme is first 'aspecifically' bound, followed by rapid transfers between different segments of the flexible peptide substrate via fast transitions between 'aspecific' single and double bound intermediate states. The rate of the second step, the productive (specific) binding of hydroxylation-committed -X-Pro-Gly- sequence to the active site, can be enhanced significantly by such an, in essence, 'one-dimensional' search. This processive mechanisms of binding does not necessarily imply many hydroxylation reactions during one encounter between enzyme and a peptide with several substrate sites as suggested previously in a slightly different model (De Waal, A. and De Jong, L. (1988) Biochemistry 27, 150-155).

Amino Acid Sequence↗
PubMed1991

The nucleus: a black box being opened.

Until recently our knowledge about the structural and functional organization of the cell nucleus was very limited. Recent technical developments in the field of ultrastructural analysis, combined with ongoing research on the properties of the nuclear matrix, give new insight into how the nucleus is structured. Two types of observations shape our ideas about nuclear organization. First, most nuclear functions (replication, transcription, RNA processing, and RNA transport) are highly localized within the nucleus, rather than diffusely distributed. Moreover, they are associated with the nuclear matrix. Second, chromatin is organized in discrete loops, bordered by nuclear matrix attachment sequences (MARs). Each loop may contain one or several genes. The arrangement of chromatin in loops has profound consequences for the regulation of gene expression.

Animals↗
PubMed1990

Identification of a 43-kDa nuclear antigen associated with proliferation by monoclonal antibody K 112.

Monoclonal antibody (MAb) K 112 was generated after a single intrasplenic immunization with a recurrent laryngeal squamous-cell carcinoma. The antibody detects a 43-kDa nuclear antigen, with a pI of 5.4, which is expressed only in cycling cells. Expression is typically seen in a granular pattern excluding the nucleoli. During mitosis the bulk of the antigen is diffusely distributed in the cytoplasm. Identical reactivity was observed for tissues or cells of all mammalian species tested. These data indicate that MAb K 112 recognizes a protein belonging to the class of cell-cycle-related nuclear antigen molecules.

Animals↗
PubMed1990

The nuclear matrix from cells of different origin. Evidence for a common set of matrix proteins.

We compared the protein composition of the nuclear matrix isolated from several murine embryonal carcinoma cells and mature tissues by two-dimensional gel electrophoresis. Two nuclear matrix fractions were investigated: the "peripheral" nuclear matrix (matrix proteins that remain insoluble after reduction), and the "internal" nuclear matrix (matrix proteins released by reduction). The two subfractions have completely different protein compositions. Although numerous differences in nuclear matrix protein composition among different cell types were observed, a limited set of polypeptides common to all mouse cell types was identified. A majority of these common proteins was also present in cells from other mammalian species (i.e. rat and human). For this set of proteins, we coin the term "minimal matrix." As expected, lamin B, known to be expressed throughout differentiation, is part of the common set of peripheral nuclear matrix proteins. Lamins A and C are not because these proteins were absent from undifferentiated embryonal carcinoma cells. Since these common nuclear matrix proteins occur in all mammalian nuclear matrices analyzed so far, it is likely that they have a basic role in nuclear organization and function.

Animals↗