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D L Spector

Publications and source records attributed to D L Spector.

At least 19 recordsLinked to original sources
PubMed1992

U1 and U2 small nuclear RNAs are present in nuclear speckles.

The localization of U1 and U2 small nuclear RNAs (snRNAs) has been examined by in situ hybridization using 2'-O-alkyl oligonucleotide probes. We have found that these snRNAs, which are essential for pre-mRNA splicing, localize in a speckled distribution, in addition to being present in three of four foci, in HeLa cell nuclei. However, in cells of defined passage, such as Detroit 551 and WI-38 fibroblasts, these snRNAs are concentrated in nuclear speckles, and foci are not observed. The speckled distribution of U1 and U2 snRNAs is coincident with the speckled regions enriched in small nuclear ribonucleoprotein particle (snRNP) proteins and the essential non-snRNP splicing factor SC-35. The localization of these key components of the pre-mRNA splicing machinery to speckled nuclear regions suggests that these regions may be involved in pre-mRNA splicing.

Cell Compartmentation↗
PubMed1992

Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences.

Five distinct patterns of DNA replication have been identified during S-phase in asynchronous and synchronous cultures of mammalian cells by conventional fluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. During early S-phase, replicating DNA (as identified by 5-bromodeoxyuridine incorporation) appears to be distributed at sites throughout the nucleoplasm, excluding the nucleolus. In CHO cells, this pattern of replication peaks at 30 min into S-phase and is consistent with the localization of euchromatin. As S-phase continues, replication of euchromatin decreases and the peripheral regions of heterochromatin begin to replicate. This pattern of replication peaks at 2 h into S-phase. At 5 h, perinucleolar chromatin as well as peripheral areas of heterochromatin peak in replication. 7 h into S-phase interconnecting patches of electron-dense chromatin replicate. At the end of S-phase (9 h), replication occurs at a few large regions of electron-dense chromatin. Similar or identical patterns have been identified in a variety of mammalian cell types. The replication of specific chromosomal regions within the context of the BrdU-labeling patterns has been examined on an hourly basis in synchronized HeLa cells. Double labeling of DNA replication sites and chromosome-specific alpha-satellite DNA sequences indicates that the alpha-satellite DNA replicates during mid S-phase (characterized by the third pattern of replication) in a variety of human cell types. Our data demonstrates that specific DNA sequences replicate at spatially and temporally defined points during the cell cycle and supports a spatially dynamic model of DNA replication.

Animals↗
PubMed1992

Differences in snRNP localization between transformed and nontransformed cells.

We have examined the localization of snRNPs in a variety of mammalian cells and have observed differences in the organization of these factors in transformed cells, immortal cells, and cells of defined passage number. Cells of defined passage number exhibit a speckled staining pattern after immunolabeling with anti-Sm, anti-B'', or anti-m3G antibodies. Furthermore, 2-3% of the cells, in a given population, exhibit labeling of 1 or 2 round coiled bodies in addition to the speckled-labeling pattern. However, transformed cells exhibited 1-4 intensely stained coiled bodies, in 81-99% of the cells, in addition to the speckled-labeling pattern. Immortal cells exhibited 1-4 intensely stained smaller coiled bodies in 4-40% of the cells, in addition to the speckled-labeling pattern. When immortal cells (REF-52) that had been transformed by adenovirus (REF-52Ad5.4) were examined, these cells exhibited an increase in the percentage of cells containing 1 or 2 intensely stained coiled bodies, in addition to the speckled labeling, from 24 to 99%. On the basis of this study, we conclude that the organization of snRNPs within the mammalian cell nucleus is a reflection of the physiology of the cell that may change upon transformation or immortalization.

Animals↗
PubMed1991

Associations between distinct pre-mRNA splicing components and the cell nucleus.

SC-35 is a non-snRNP spliceosome component that is specifically recognized by the anti-spliceosome monoclonal antibody alpha SC-35. In this paper we provide direct evidence that SC-35 is an essential splicing factor and we examine the immunolocalization of SC-35 by confocal laser scanning microscopy and by electron microscopy. We have found that the speckled staining pattern observed by fluorescence microscopy corresponds to structures previously designated as interchromatin granules and perichromatin fibrils. Although snRNP antigens are also concentrated in these nuclear regions, we show that the two types of spliceosome components are localized through different molecular interactions: The distribution of SC-35 was not affected by treatment with DNase I or RNase A, or when the cells were heat shocked. In contrast, snRNP antigens become diffusely distributed after RNase A digestion or heat shock. Examination of cells at different stages of mitosis revealed that the SC-35 speckled staining pattern is lost during prophase and speckles containing SC-35 begin to reform in the cytoplasm of anaphase cells. In contrast, snRNP antigens do not associate with speckled regions until late in telophase. These studies reveal a dynamic pattern of assembly and disassembly of the splicing factor SC-35 into discrete nuclear structures that colocalize with interchromatin granules and perichromatin fibrils. These subnuclear regions may therefore be nuclear organelles involved in the assembly of spliceosomes, or splicing itself.

Antigens↗
PubMed1991

Immunocytochemical localization of casein kinase II during interphase and mitosis.

We have developed specific antibodies to synthetic peptide antigens that react with the individual subunits of casein kinase II (CKII). Using these antibodies, we studied the localization of CKII in asynchronous HeLa cells by immunofluorescence and immunoelectron microscopy. Further studies were done on HeLa cells arrested at the G1/S transition by hydroxyurea treatment. Our results indicate that the CKII alpha and beta subunits are localized in the cytoplasm during interphase and are distributed throughout the cell during mitosis. Further electron microscopic investigation revealed that CKII alpha subunit is associated with spindle fibers during metaphase and anaphase. In contrast, the CKII alpha' subunit is localized in the nucleus during G1 and in the cytoplasm during S. Taken together, our results suggest that CKII may play significant roles in cell division control by shifting its localization between the cytoplasm and nucleus.

Amino Acid Sequence↗
PubMed1991

Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors.

We have used in situ hybridization and immunocytochemistry to compare the nuclear localization of a specific nascent pre-mRNA and the essential non-snRNP splicing factor SC-35. Nascent c-fos transcripts were detected in serum-induced mouse fibroblasts by in situ hybridization with genomic c-fos probes. Prior to serum induction no c-fos RNA is detected, but these transcripts localize to two dots in the interphase nucleus after induction. The time course of appearance of the dots correlates with the previously determined time course of transcriptional activation of the gene. Upon further analysis by confocal laser scanning microscopy, we have determined that the dots extend through the depth of the nucleus, forming paths. By using high-voltage electron microscopy, we have found that the c-fos path extends out and comes into direct contact with the nuclear envelope. We have also compared the localization of c-fos transcripts with the speckled nuclear regions that are enriched in snRNPs and the non-snRNP splicing factor SC-35. Direct observations of three-dimensional rotations have revealed a close association between the c-fos transcripts and the nuclear speckles. This study demonstrates a direct link between specific nascent RNA transcripts and nuclear speckles that are enriched in pre-mRNA splicing factors.

3T3 Cells↗
PubMed1990

Higher order nuclear organization: three-dimensional distribution of small nuclear ribonucleoprotein particles.

The structural and functional organization of the cell nucleus has been investigated using three-dimensional reconstruction, immunoelectron microscopy, and high-resolution in situ autoradiography. Nuclear regions enriched in small nuclear ribonucleoprotein particles (snRNPs) form a reticular network within the nucleoplasm that extends between the nucleolar surface and the nuclear envelope. The snRNPs occupy approximately 18% of the volume of CHOC 400 cell nuclei. The in situ sites of DNA replication and transcription are complementary to, rather than coincident with, the nuclear regions concentrated in snRNPs. Based on these data a three-dimensional model of the organization of the mammalian cell nucleus is presented.

Animals↗
PubMed1990

Differential distribution of factors involved in pre-mRNA processing in the yeast cell nucleus.

The yeast cell nucleus has previously been shown to be divided into two regions by a variety of microscopic approaches. We used antibodies specific for the 2,2,7-trimethylguanosine cap structure of small nuclear ribonucleic acids (snRNAs) and for a protein component of small nuclear ribonucleoprotein particles to identify the distribution of small nuclear ribonucleoprotein particles within the yeast cell nucleus. These studies were performed with the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae. By using immunofluorescence microscopy and immunoelectron microscopy, most of the abundant snRNAs were localized to the portion of the nucleus which has heretofore been referred to as the nucleolus. This distribution of snRNAs is different from that found in mammalian cells and suggests that the nucleolar portion of the yeast nucleus contains functional domains in addition to those associated with RNA polymerase I activity.

Antibodies↗
PubMed1989

Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.

Cell Fractionation↗
PubMed1987

The v- and c-myc oncogene proteins colocalize in situ with small nuclear ribonucleoprotein particles.

The intranuclear distribution of the v-myc and c-myc oncogene proteins were studied by immunofluorescence and immunoelectron microscopy. The nuclear distribution pattern of these proteins is shown to be identical to the distribution of small nuclear ribonucleoprotein particles (snRNPs). Colocalization was observed in cells expressing either the v- or c-myc proteins or in cells microinjected with the recombinant human c-myc protein. Immunolocalization studies revealed the v-myc protein and snRNPs to be concentrated within a nuclear network which excludes the nucleolus, nuclear pore-lamina complex, and portions of the nucleoplasm which contain the bulk of DNA. These results identify a nuclear region enriched in the myc-oncogene protein and snRNPs and raise the possibility that these nuclear constituents may function in related processes.

Animals↗
PubMed1986

Macromolecular domains containing nuclear protein p107 and U-snRNP protein p28: further evidence for an in situ nuclear matrix.

Polyclonal antibodies have been produced which react with a nuclear protein having a molecular weight of 107kD and a pI of 8.7-8.8 (designated p107). This protein is shown to be a component of the residual ribonucleoprotein (RNP) network of the nuclear matrix. P107 localized exclusively to the nuclear interior but not within nucleolar or chromatin domains. We have taken advantage of this unique probe to examine whether the RNP network of the isolated nuclear matrix has a physical counterpart in situ. We show that RNA, p107, divalent cations and the 28 kD Sm antigen of U-snRNPs are components of in situ macromolecular assemblies. While the morphology and intranuclear distribution of these assemblies are insensitive to the removal of chromatin, they are markedly altered by degradation of RNA. Digestion in situ of RNA in the presence of EDTA followed by extraction with high ionic strength buffers solubilized the components of these assemblies. Electron microscopic and immunobiochemical data are presented which support the concept that the residual RNP network of the nuclear matrix is an isolate of a pre-existing structure, and that perturbations in this internal network can be created by RNA degradation, depletion of essential metal ions and proteolysis.

Animals↗
PubMed1986

Redistribution of U-snRNPs during mitosis.

In this study we examined the distribution of U-snRNPs during mitosis using a monoclonal antibody which recognizes the 28 kD protein associated with U-snRNP particles. During interphase U-snRNPs are concentrated within discrete intranuclear protein clusters. The onset of mitosis is accompanied by a breakdown of these nuclear protein clusters and U-snRNPs are redistributed between the condensing chromosomes. During metaphase and anaphase, two populations of U-snRNPs can be identified: those associated with the chromosome surface and a 'free' form distributed throughout the cytoplasm. During telophase, the reformation of nuclear protein clusters appears to be associated with the chromosome surface and coincides with chromatin decondensation. These studies suggest that the interphase assembly of nuclear domains containing U-snRNPs is sensitive to the transcriptionally active or decondensed state of chromatin.

Anaphase↗
PubMed1986

Nonhistone protein BA is a glutathione S-transferase localized to interchromatinic regions of the cell nucleus.

A DNA-binding nonhistone protein, protein BA, was previously demonstrated to co-localize with U-snRNPs within discrete nuclear domains (Bennett, F. C., and L. C. Yeoman, 1985, Exp. Cell Res., 157:379-386). To further define the association of protein BA and U-snRNPs within these discrete nuclear domains, cells were fractionated in situ and the localization of the antigens determined by double-labeled immunofluorescence. Protein BA was extracted from the nucleus with the 2.0 M NaCl soluble chromatin fraction, while U-snRNPs were only partially extracted from the 2.0 M NaCl-resistant nuclear structures. U-snRNPs were extracted from the residual nuclear material by combined DNase I/RNase A digestions. Using an indirect immunoperoxidase technique and electron microscopy, protein BA was localized to interchromatinic regions of the cell nucleus. Protein BA was noted to share a number of chemical and physical properties with a family of cytoplasmic enzymes, the glutathione S-transferases. Comparison of the published amino acid composition of protein BA and glutathione S-transferases showed marked similarities. Nonhistone protein BA isolated from saline-EDTA nuclear extracts exhibited glutathione S-transferase activity with a variety of substrates. Substrate specificity and subunit analysis by SDS polyacrylamide gel electrophoresis revealed that it was a mixture of several glutathione S-transferase isoenzymes. Protein BA isolated from rat liver chromatin was shown by immunoblotting and peptide mapping techniques to be two glutathione S-transferase isoenzymes composed of the Yb and Yb' subunits. Glutathione S-transferase Yb subunits were demonstrated to be both nuclear and cytoplasmic proteins by indirect immunolocalization on rat liver cryosections. The identification of protein BA as glutathione S-transferase suggests that this family of multifunctional enzymes may play an important role in those nuclear domains containing U-snRNPs.

Amino Acids↗
PubMed1985

Alterations in chromatin conformation are accompanied by reorganization of nonchromatin domains that contain U-snRNP protein p28 and nuclear protein p107.

The intranuclear distribution of nuclear matrix-associated protein p107 and the 28-kD Sm antigen of U-snRNPs have been studied using double-label immunofluorescence and immunoperoxidase electron microscopy. In interphase nuclei of HeLa cells, Novikoff hepatoma cells, and rat kangaroo kidney cells, p107 was confined to discrete interchromatin domains. The domains had an irregular contour, with an average diameter of 1-1.5 micron. Each domain appeared to be composed of interconnected granules. The Sm antigen colocalized and appeared concentrated in these domains but also showed some general nucleoplasmic distribution. During mitosis, the interchromatin domains disassembled such that the Sm portion redistributed to the perichromosomal and spindle regions and the p107 component redistributed throughout the mitotic cytoplasm. During anaphase, p107 assembled into discrete clusters throughout the mitotic cytoplasm. The Sm antigen was not a component of these clusters. Double-label immunofluorescence with anti-p107 and the anti-DNA tight-binding protein, AhNa1, showed that the extranuclear p107 domains assumed an interchromatin localization only after the chromosomes had decondensed. The correlation between chromosome decondensation and the occurrence of p107 within interchromatin domains was also observed during chicken erythrocyte nuclear reactivation. We propose that the discrete interchromatin domains that contain p107 and p28 may be important for processing and splicing of RNA and that their structural assembly within nuclei is sensitive to the presence of the transcriptionally active conformation of chromatin.

Animals↗
PubMed1984

Silver staining, immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23.

Nucleolar organizer region (NOR)-specific silver staining and immunolocalization of nucleolar phosphoproteins B23 and C23 were compared in Novikoff hepatoma ascites cells. Silver staining and protein C23 immunostaining were both localized in the fibrillar shell surrounding the fibrillar center and in the fibrillar center. During mitosis, silver staining and protein C23 were localized at the NORs. Therefore, protein C23 and the silver-staining protein both seem to be associated with rDNA-containing structures (Mirre and Stahl 1981). A comparison of toluidine blue staining specific for RNA and B23 immunostaining demonstrated that protein B23 was associated with RNA-containing regions of the nucleolus and was absent from the fibrillar centers. Localization of these proteins and their functions are discussed in relation to the organization of the nucleolus.

Animals↗
PubMed1984

Colocalization of U1 and U2 small nuclear RNPs by immunocytochemistry.

The in situ localization of U1 and U2 snRNPs was examined, using autoantibodies directed against each of these snRNPs, by immunofluorescence and immunoelectron microscopy. This study has shown U1 and U2 snRNPs to colocalize in the nuclei of PtK2 cells. Thirty to fifty immunostained clusters were observed per interphase nucleus. This study suggests these nuclear protein clusters to be the sites of RNA processing.

Animals↗