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Available data on the occurrence and expression of somatic histone HI during oogenesis and early embryogenesis of Xenopus laevis are contradictory. In particular the reported presence of a large storage pool of histone HIA in oocytes is difficult to reconcile with the high transcriptional activity of all gene classes in this specific cell type. In the present study we have used polyclonal antibodies raised against somatic Xenopus histone HI (HIA and HIA/B) for combined immunoblotting experiments to quantitate HI pools and immunolocalization studies to visualize chromosome- bound HI. Both approaches failed to detect soluble or chromosomal histone HI in vitellogenic oocytes, eggs, and cleavage-stage embryos up to early blastula. In addition, chromatin assembled in Xenopus egg extract was also negative for histone HI as revealed by immunofluorescence microscopy. Lampbrush chromosomes not only lacked histone HI but also the previously identified histone HI-like B4 protein (Smith et al., 1988, Genes Dev. 2,1284-1295). In contrast, chromosomes of eggs and early embryos fluoresced brightly with anti-B4 antibodies. Our results lend further support to the view that histone HI expression is developmentally regulated during Xenopus oogenesis and embryogenesis similar to what is known from other species.
Transcribed nucleolar chomatin, including the spacer regions interspersed between the rRNA genes, is different from the bulk of nontranscribed chromatin in that the DNA of these regions appears to be in an extended (B) conformation when examined by electron microscopy. The possibility that this may reflect artificial unfolding of nucleosomes during incubation in very low salt buffers as routinely used in such spread preparations has been examined by studying the influence of various ion concentrations on nucleolar chromatin structure. Amplified nucleolar chromatin of amphibian oocytes (Xenopus laevis, Pleurodeles waltlii, Triturus cristatus) was spread in various concentrations of NaCl (range 0 to 20 mM). Below 1 mM salt spacer chromatin frequently revealed a variable number of irregularly shaped beads, whereas above this concentration the chromatin axis appeared uniformly smooth. At all salt concentrations studied, however, the length distribution of spacer and gene regions was identical. Preparations fixed with glutaraldehyde instead of formaldehyde, or unftxed preparations, were indistinguishable in this respect. The observations indicate that (i) rDNA spacer regions are not compacted into nucleosomal particles and into supranucleosomal structures when visualized at chromatin stabilizing salt concentrations (e.g., 20 mM NaCl), and (ii) spacer DNA is covered by a uniform layer of proteins of unknown nature which, at very low salt concentrations (below 1 mM NaCl), can artificially give rise to the appearance of small granular particles of approximately nucleosome-like sizes. These particles, however, are different from nucleosomes in that they do not foreshorten the associated spacer DNA. The data support the concept of an altered nucleohistone conformation not only in transcribed chromatin but also in the vicinity of transcriptional events.
High sensitivity immunolocalization of double and single-stranded DNA by a monoclonal antibody
(1987)
A monoclonal antibody (AK 30-10) is described which specifically reacts with DNA both in double and single-stranded forms but not with other molecules and structures, including deoxyribonucleotides and RNAs. When used in immunocytochemical experiments on tissue sections and permeabilized cultured cells, this antibody detects DNA-containing structures, even when the DNA is present in very small amounts. Examples of high resolution detection include the DNA present in amplified extrachromosomal nucleoli, chromomeres of lampbrush chromosomes, mitochondria, chloroplasts and mycoplasmal particles. In immunoelectron microscopy using the immunogold technique, the DNA was localized in distinct substructures such as the "fibrillar centers" of nucleoli and certain stromal centers in chloroplasts. The antibody also reacts with DNA of chromatin of living cells, as shown by microinjection into cultured mitotic cells and into nuclei of amphibian oocytes. The potential value and the limitations of immunocytochemical DNA detection are discussed.
Using a monoclonal antibody (No-194) we have identified, in Xenopus laevis and other amphibia, an acidic protein of M, 40,000 (ribocharin) which is specifically associated with the granular component of the nucleolus and nucleoplasmic 65S particles. These particles contain the nuclear 28S rRNA and apparently represent the precursor to the large ribosomal subunit in nucleocytoplasmic transit. By immunoelectron microscopy ribocharin has been localized in the granular component of the nucleolus and in interchromatin granules. During mitosis ribocharin-containing particles are associated with surfaces of chromosomes and are recollected in the reconstituting nucleoli in late telophase. We suggest that ribocharin is a specific component of precursor particles of the large ribosomal subunit, which dissociates from the 65S particle before passage through the nuclear envelope, and is reutilized in ribosome biogenesis.
A non-radioactive in situ hybridization method is described for the localization of transcription units of defined genes to lateral loops of Xenopus laevis lampbrush chromosomes. Two Xenopus cONA probes were used encoding the nucleolar protein N038/ B23 and cytokeratin 1(8). Both proteins are known to be synthesized in Xenopus oocytes, and Northern blot analysis revealed the presence of the corresponding mRNAs in different oogenic stages. The probes were enzymatically labeled with biotin-dCTP and hybridized to lampbrush chromosomes. The sites of hybridization were detected either by indirect immunofluorescence microscopy using rabbit antibodies against biotin and fluorescein-conjugated antirabbit IgG or enzymatically using peroxidase-conjugated streptavi din. The probe encoding the nucleolar protein hybridized to two sets of lateral loops on different bivalents, the cytokeratin probe to at least four. Our finding that each probe hybridized to more than one chromosomal locus may reflect the tetraploid nature of the Xenopus laevis genome or results from cross-hybridization to other transcriptionally active members of the N038/ B23-nucleoplasmin or the cytokeratin-Iamin gene families. The method described should facilitate further in situ hybridization studies with appropriate genomic clones in order to map specific DNA sequences to defined loop regions and to come to a better understanding of the relationship between loop organization and gene transcription unit.
Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtKz cells with WGA or antibody PIt and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1 . Although PtKz cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome- associated components.
In order to investigate the DNA localization within Ehrlich tumor cell nucleoli during mitosis, two recent immunocytochemical methods using either an anti-DNA or an anti-bromodeoxyuridine (BrdU) monoclonal antibody have been applied. In both cases, the immunogold labeling has been performed on ultrathin sections of cells embedded either in Lowicryl K4M or in Epon, respectively. Identical results are observed with both immunocytochemical approaches. In the interphase nucleolus, besides the labeling of the perinucleolar chromatin shell and of its intranucleolar invaginations which penetrate into the nucleolar body and often terminate at the fibrillar centers, a few gold particles are also preferentially found towards the peripheral region of the fibrillar centers. In contrast, the dense fibrillar component and the granular component are never labeled. During mitosis, the fibrillar centers persist at the chromosomal nucleolus organizing regions (NOR's) and can be selectively stained by the silver method. However, these metaphase fibrillar centers are no longer decorated by the DNA- or BrdU antibodies. These results indicate that until the end of prophase, rRNA genes are present inside the fibrillar center material, disappear during metaphase and reappear in reconstituting nucleoli during telophase. Thus, fibrillar centers appear to represent structures sui generis, which are populated by rRNA genes only when the nucleolus is functionally active. In segregated nucleoli after actinomycin D treatment, the DNA labeling is exclusively restricted to the perinucleolar chromatin blocks. These findings also suggest that the DNA content of the fibrillar center material varies according to the rRNA transcription level of the cells. The results are discussed in the light of the present knowledge of the functional organization of the nucleolus.
After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtKz) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed. These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent "nucleolar segregation". The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase.
The ultrastructure of twO kinds of transcription ally active chromatin, the lampbrush chromosome loops and the nucleoli from amphibian oocytes and primary nuclei of the green alga Acetabularia, has been examined after manual isolation and dispersion in low salt media of slightly alkaline pH using various electron microscopic staining techniques (positive staining, metal shadowing, negative staining, preparation on positively charged films, etc.) and compared with the appearance of chromatin from various somatic cells (hen erythrocytes, rat hepatocytes, ClIltured murine sarcoma cells) prepared in parallel. While typical nucleosomes were revealed with all the techniques for chromatin from the latter three cell system, no nucleosomes were identified in either the lampbrush chromosome structures or the nucleolar chromatin. Nucleosomal arrays were absent not only in maximally fibril-covered matrix units but also in fibril-free regions between transcriptional complexes, including the apparent spacer intercepts between different transcriptional units. Moreover, comparisons of the length of the repeating units of rDNA in the transcribed state with those determined in the isolated rDNA and with the lengths of the first stable product of rDNA transcription, the pre-rRNA, demonstrated that the transcribed rDNA was not significantly shortened and/or condensed but rather extended in the transcriptional units. Distinct granules of about nucleosomal size which were sometimes found in apparent spacer regions as well as within matrix units of reduced fibril density were shown not to represent nucleosomes since their number per spacer unit was not inversely correlated with the length of the specific unit and also on the basis of their resistance to treatment with the detergent Sarkosyl NL-30. It is possible to structurally distinguish between transcriptionally active chromatin in which the DNA is extended in a non-nucleosomal form of chromatin and condensed, inactive chromatin within the typical nucleosomal package. The characteristic extended structure of transcriptionally active chromatin is found not only in the transcribed genes but also in non-transcribed regions within or between ("spacer") transcriptional units as well as in transcriptional units that are untranscribed amidst transcribed ones and/or have been inactivated for relatively short time. It is hypothesized that activation of transcription involves a transition from a nucleosomal to an extended chromatin organisation and that this structural transition is not specific for single "activated" genes but may involve larger chromatin regions, including adjacent untranscribed intercepts.