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D L Spector

Publications and source records attributed to D L Spector.

At least 37 records · Page 2Linked to original sources
PubMed1994

In vivo analysis of the stability and transport of nuclear poly(A)+ RNA.

We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated by detergent and salt extraction as well as DNase I digestion, the majority of the nuclear poly(A)+ RNA was found to remain associated with the nonchromatin RNP-enriched fraction of the nucleus. After inhibition of RNA polymerase II transcription for 5-10 h, a stable population of poly(A)+ RNA remained in the nucleus and was reorganized into fewer and larger interchromatin granule clusters along with pre-mRNA splicing factors. This stable population of nuclear RNA may play an important role in nuclear function. Furthermore, we have observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement. All of the observed nuclear pores contained poly(A)+ RNA staining suggesting that they are all capable of exporting RNA. In addition, we have directly visualized, for the first time in mammalian cells, the transport of poly(A)+ RNA through the nuclear pore complexes.

Animals↗
PubMed1994

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

Animals↗
PubMed1994

Nucleologenesis: U3 snRNA-containing prenucleolar bodies move to sites of active pre-rRNA transcription after mitosis.

We have investigated the distribution of U3 snRNA and rRNA in HeLa cells and normal rat kidney cells during interphase and mitosis. U3 snRNA, known to be involved in pre-rRNA processing, was detected in nucleoli and coiled bodies during interphase, whereas rRNA was distributed in the nucleoli and throughout the cytoplasm. By comparison, ribosomal protein S6 was detected in nucleoli, coiled bodies, and in the cytoplasm. During nucleologenesis, pre-rRNA was observed in newly forming nucleoli during late telophase but not in prenucleolar bodies (PNBs), whereas U3 snRNA was detected in forming nucleoli and PNBs. Similar findings to those reported here for the localization of U3 snRNA have been reported previously for the U3 small nuclear ribonucleoprotein fibrillarin. These results suggest that components involved in pre-rRNA processing localize to discrete PNBs at the end of mitosis. The nucleolus is formed at specific telophase domains (nucleolar organizing regions) and the PNBs, containing factors essential for pre-rRNA processing, are recruited to these sites of rRNA transcription and processing.

Animals↗
PubMed1993

In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism.

We describe the nuclear organization of pre-mRNA processing components in HeLa cells upon adenovirus 2 infection and their relationship to the localization of viral RNA sequences. We observe a redistribution of cellular splicing factors as well as RNA polymerase II and heterogeneous nuclear ribonucleoprotein particle proteins to sites of viral RNA transcription. Similar results were obtained in cells transiently transfected with a plasmid containing a portion of the beta-tropomyosin gene. Our findings demonstrate a very close association between RNA transcripts and transcription and pre-mRNA splicing factors, suggesting that these processes are both temporally and spatially linked in the cell nucleus. Furthermore, these data suggest a recruiting mechanism that regulates the localization of transcription and splicing factors in response to the initiation of active transcription.

Adenoviruses, Human↗
PubMed1993

Nuclear organization of pre-mRNA processing.

Recent studies have suggested that small nuclear ribonucleoprotein particles (snRNPs), non-snRNP splicing factors, and several heterogeneous nuclear RNP proteins change their organization within the cell in response to transcriptional activity. Several of the RNA substrates with which these factors interact have been shown to localize in tracks that are associated with regions in which splicing factors are concentrated (nuclear speckles). It is now thought that pre-mRNA splicing may occur within these tracks.

Animals↗
PubMed1993

Organization of the double-stranded RNA-activated protein kinase DAI and virus-associated VA RNAI in adenovirus-2-infected HeLa cells.

We have examined the cellular distribution of the double-stranded RNA-activated protein kinase DAI in adenovirus 2 (Ad2)-infected and uninfected HeLa cells. In uninfected cells DAI was found to be concentrated in the cytoplasm. In addition, DAI was localized in the nucleoli and diffusely distributed throughout the nucleoplasm. Cells treated with alpha-interferon displayed a similar pattern of distribution for DAI. When RNA polymerase I activity was inhibited by the drug actinomycin D, nucleoli segregated and DAI was found to colocalize with the dense fibrillar region of the nucleoli. During mitosis, the distribution of DAI paralleled that of rRNA. In adenovirus-infected cells the localization of DAI was similar to that in uninfected interphase cells. VA RNAI was detected in Ad2-infected cells by 10-14 hours post-infection as fine dots in the nucleoplasm. By 18-24 hours post-infection, VA RNAI appeared in bigger and more abundant dots in the nucleoplasm and the cytoplasm was intensively labeled. Transient expression of the VA RNAI gene in uninfected cells resulted in a similar localization of the RNA. Our results are consistent with a role for DAI and VA RNAI in protein synthesis and suggest that DAI may play an early role in ribosome biogenesis in the nucleolus in addition to its cytoplasmic role in translation.

Adenoviruses, Human↗
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↗