Cajal bodies and histone locus bodies in Drosophila and Xenopus.

The organization of the cell nucleus into specialized compartments is important for nuclear function. We address the significance of compartmentalization by studying the Cajal body, an evolutionarily conserved nuclear organelle proposed to be involved in such diverse functions as assembly of the spliceosome, assembly of the transcription machinery, and modification of spliceosomal small nuclear RNAs. The Cajal body is typically identified by the presence of coilin, a protein of poorly defined function. Here, we demonstrate that coilin is not a unique Cajal body marker but also occurs in a related yet distinct nuclear organelle known as the histone locus body in both Drosophila and Xenopus. We stress the importance of multiple markers not only for identification of nuclear bodies but also for assessing their functional significance.

A flow cytometry-based assay to assess RSV-specific neutralizing antibody is reproducible, efficient and accurate.

Respiratory syncytial virus (RSV) is an important cause of respiratory infection in people of all ages, and is the leading cause of hospitalization in infants. Although commercially available monoclonal antibody is available for passive prophylaxis of neonates at risk of severe disease, there is no available vaccine to prevent RSV. Measurement of neutralizing activity will be a key endpoint for vaccine evaluation. Assessment of neutralizing antibody against RSV has been limited to traditional plaque reduction, which is time-consuming and inherently operator dependent and highly variable. Here, we describe a flow cytometry-based RSV-specific neutralization assay which is more rapid than traditional methods, highly sensitive and highly reproducible.

A novel karyoskeletal protein: characterization of protein NO145, the major component of nucleolar cortical skeleton in Xenopus oocytes.

The nucleolus is a ubiquitous, mostly spheroidal nuclear structure of all protein-synthesizing cells, with a well-defined functional compartmentalization. Although a number of nonribosomal proteins involved in ribosome formation have been identified, the elements responsible for the shape and internal architecture of nucleoli are still largely unknown. Here, we report the molecular characterization of a novel protein, NO145, which is a major and specific component of a nucleolar cortical skeleton resistant to high salt buffers. The amino acid sequence of this polypeptide with a SDS-PAGE mobility corresponding to M(r) 145,000 has been deduced from a cDNA clone isolated from a Xenopus laevis ovary expression library and defines a polypeptide of 977 amino acids with a calculated mass of 111 kDa, with partial sequence homology to a synaptonemal complex protein, SCP2. Antibodies specific for this protein have allowed its recognition in immunoblots of karyoskeleton-containing fractions of oocytes from different Xenopus species and have revealed its presence in all stages of oogenesis, followed by a specific and rapid degradation during egg formation. Immunolocalization studies at the light and electron microscopic level have shown that protein NO145 is exclusively located in a cage-like cortical structure around the entire nucleolus, consisting of a meshwork of patches and filaments that dissociates upon reduction of divalent cations. We propose that protein NO145 contributes to the assembly of a karyoskeletal structure specific for the nucleolar cortex of the extrachromosomal nucleoli of Xenopus oocytes, and we discuss the possibility that a similar structure is present in other cells and species.

A role for Cajal bodies in assembly of the nuclear transcription machinery.

Cajal bodies (CBs) are small nuclear organelles that contain the three eukaryotic RNA polymerases and a variety of factors involved in transcription and processing of all types of RNA. A number of these factors, as well as subunits of polymerase (pol) II itself, are rapidly and specifically targeted to CBs when injected into the cell. It is suggested that pol I, pol II, and pol III transcription and processing complexes are preassembled in the CBs before transport to the sites of transcription on the chromosomes and in the nucleoli.

Cajal bodies: the first 100 years.

Cajal bodies are small nuclear organelles first described nearly 100 years ago by Ramón y Cajal in vertebrate neural tissues. They have since been found in a variety of animal and plant nuclei, suggesting that they are involved in basic cellular processes. Cajal bodies contain a marker protein of unknown function, p80-coilin, and many components involved in transcription and processing of nuclear RNAs. Among these are the three eukaryotic RNA polymerases and factors required for transcribing and processing their respective nuclear transcripts: mRNA, rRNA, and pol III transcripts. A model is discussed in which Cajal bodies are the sites for preassembly of transcriptosomes, unitary particles involved in transcription and processing of RNA. A parallel is drawn to the nucleolus and the preassembly of ribosomes, which are unitary particles involved in translation of proteins.

RNA polymerase II in Cajal bodies of amphibian oocytes.

Cajal bodies (coiled bodies) are nuclear organelles that contain a variety of components required for transcription and processing of RNA. Cajal bodies in amphibian oocytes are stained by mAb H14, which recognizes the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II when the heptapeptide repeat is phosphorylated on serine-5. Oocytes were treated with the transcription inhibitor 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), which prevents phosphorylation of the CTD. Cajal bodies from oocytes that had been treated for 2-3 h with DRB no longer stained with mAb H14, but staining reappeared when the inhibitor was washed out. Epitope-tagged transcripts of two small subunits of polymerase II, RPB6 and RPB9, were injected into the cytoplasm of Xenopus and Triturus oocytes. Newly translated RPB6 and RPB9 were specifically targeted to Cajal bodies within 4 h, and Cajal bodies remained the site of highest concentration of tagged protein during the next 2 days. These data suggest that polymerase subunits pass through the Cajal bodies with a transit time no greater than a few hours. We discuss the possibility that Cajal bodies are sites of assembly or modification of the transcription machinery of the nucleus.

Assembly of the nuclear transcription and processing machinery: Cajal bodies (coiled bodies) and transcriptosomes.

We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem-loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.

Coilin shuttles between the nucleus and cytoplasm in Xenopus oocytes.

Coiled bodies are discrete nuclear organelles often identified by the marker protein p80-coilin. Because coilin is not detected in the cytoplasm by immunofluorescence and Western blotting, it has been considered an exclusively nuclear protein. In the Xenopus germinal vesicle (GV), most coilin actually resides in the nucleoplasm, although it is highly concentrated in 50-100 coiled bodies. When affinity-purified anti-coilin antibodies were injected into the cytoplasm of oocytes, they could be detected in coiled bodies within 2-3 h. Coiled bodies were intensely labeled after 18 h, whereas other nuclear organelles remained negative. Because the nuclear envelope does not allow passive diffusion of immunoglobulins, this observation suggests that anti-coilin antibodies are imported into the nucleus as an antigen-antibody complex with coilin. Newly synthesized coilin is not required, because cycloheximide had no effect on nuclear import and subsequent targeting of the antibodies. Additional experiments with myc-tagged coilin and myc-tagged pyruvate kinase confirmed that coilin is a shuttling protein. The shuttling of Nopp140, NO38/B23, and nucleolin was easily demonstrated by the targeting of their respective antibodies to the nucleoli, whereas anti-SC35 did not enter the germinal vesicle. We suggest that coilin, perhaps in association with Nopp140, may function as part of a transport system between the cytoplasm and the coiled bodies.

The stem-loop binding protein (SLBP1) is present in coiled bodies of the Xenopus germinal vesicle.

The stem-loop binding protein (SLBP1) binds the 3' stem-loop of histone pre-mRNA and is required for efficient processing of histone transcripts in the nucleus. We examined the localization of SLBP1 in the germinal vesicle of Xenopus laevis oocytes. In spread preparations of germinal vesicle contents, an anti-SLBP1 antibody stained coiled bodies and specific chromosomal loci, including terminal granules, axial granules, and some loops. After injection of myc-tagged SLBP1 transcripts into the oocyte cytoplasm, newly translated myc-SLBP1 protein was detectable in coiled bodies within 4 h and in terminal and axial granules by 8 h. To identify the region(s) of SLBP1 necessary for subnuclear localization, we subcloned various parts of the SLBP1 cDNA and injected transcripts of these into the cytoplasm of oocytes. We determined that 113 amino acids at the carboxy terminus of SLBP1 are sufficient for coiled body localization and that disruption of a previously defined RNA-binding domain did not alter this localization. Coiled bodies also contain the U7 small nuclear ribonucleoprotein particle (snRNP), which participates in cleavage of the 3' end of histone pre-mRNA. The colocalization of SLBP1 and the U7 snRNP in the coiled body suggests coordinated control of their functions, perhaps through a larger histone-processing particle. Some coiled bodies are attached to the lampbrush chromosomes at the histone gene loci, consistent with the view that coiled bodies in the oocyte recruit histone-processing factors to the sites of histone pre-mRNA transcription. The non-histone chromosomal sites at which SLBP1 is found include the genes coding for 5 S rRNA, U1 snRNA, and U2 snRNA, suggesting a wider role for SLBP1 in the biosynthesis of small non-spliced RNAs.

Construction and characterization of hexon-chimeric adenoviruses: specification of adenovirus serotype.

This study has used the strategy of gene replacement to characterize the contribution of the adenovirus (Ad) capsid protein hexon to serotype definition. By replacing the Ad type 5 (Ad5) hexon gene with sequences from Ad2, we have changed the type specificity of the chimeric virus. The type-determining epitopes are primarily associated with loop 1 of hexon and, to a much lesser degree, with loop 2. In spite of the serotype distinctiveness of the chimeric hexon viruses, epitope similarity between the vectors resulted in a low level of cross-reactive neutralizing antibody, which in combination with activated cellular and innate arms of the immune system is sufficient to suppress gene transduction following readministration in vivo.

Coilin can form a complex with the U7 small nuclear ribonucleoprotein.

Coiled bodies (CBs) in the amphibian oocyte nucleus are spherical structures up to 10 microm or more in diameter, much larger than their somatic counterparts, which rarely exceed 1 microm. Oocyte CBs may have smaller granules attached to their surface or embedded within them, which are identical in structure and composition to the many hundreds of B-snurposomes found free in the nucleoplasm. The matrix of the CBs contains the diagnostic protein p80-coilin, which is colocalized with the U7 small nuclear ribonucleoprotein (snRNP), whereas the attached and embedded B-snurposomes contain splicing snRNPs. A few of the 50-100 CBs in the oocyte nucleus are attached to lampbrush chromosomes at the histone gene loci. By coimmunoprecipitation we show that coilin and the U7 snRNP can form a weak but specific complex in the nucleoplasm, which is dependent on the special U7 Sm-binding site. Under the same conditions coilin does not associate with the U1 and U2 snRNPs. Coilin is a nucleic acid-binding protein, as shown by its interaction with single-stranded DNA and with poly r(U) and poly r(G). We suggest that an important function of coilin is to form a transient complex with the U7 snRNP and accompany it to the CBs. In the case of CBs attached to chromosomes at the histone gene loci, the U7 snRNP is thus brought close to the actual site of histone pre-mRNA transcription.

Assembly of lampbrush chromosomes from sperm chromatin.

We have examined the behavior of demembranated sperm heads when injected into the germinal vesicle (GV) of amphibian oocytes. Xenopus sperm heads injected into Xenopus GVs swelled immediately and within hours began to stain with an antibody against RNA polymerase II (Pol II). Over time each sperm head became a loose mass of chromosome-like threads, which by 24-48 h resolved into individually recognizable lampbrush chromosomes (LBCs). Although LBCs derived from sperm are unreplicated single chromatids, their morphology and immunofluorescent staining properties were strikingly similar to those of the endogenous lampbrush bivalents. They displayed typical transcriptionally active loops extending from an axis of condensed chromomeres, as well as locus-specific "landmarks. " Experiments with [3H]GTP and actinomycin D demonstrated that transcription was not necessary for the initial swelling of the sperm heads and acquisition of Pol II but was required for maintenance of the lampbrush loops. Splicing was not required at any stage during formation of sperm LBCs. When Xenopus sperm heads were injected into GVs of the newt Notophthalmus, the resulting sperm LBCs displayed very long loops with pronounced Pol II axes, like those of the endogenous newt LBCs; as expected, they stained with antibodies against newt-specific proteins. Other heterologous injections, including sperm heads of the frog Rana pipiens and the zebrafish Danio rerio in Xenopus GVs, confirm that LBCs can be derived from taxonomically distant organisms. The GV system should help identify both cis- and trans-acting factors needed to convert condensed chromatin into transcriptionally active LBCs. It may also be useful in producing cytologically analyzable chromosomes from organisms whose oocytes do not go through a typical lampbrush phase or cannot be manipulated by current techniques.

Tumor necrosis factor alpha plays a central role in immune-mediated clearance of adenoviral vectors.

Adenovirus (Ad) gene transfer vectors are rapidly cleared from infected hepatocytes in mice. To determine which effector mechanisms are responsible for elimination of the Ad vectors, we infected mice that were genetically compromised in immune effector pathways [perforin, Fas, or tumor necrosis factor alpha (TNF-alpha)] with the Ad vector, Ad5-chloramphenicol acetyl transferase (CAT). Mice were sacrificed at 7-60 days postinfection, and the levels of CAT expression in the liver determined by a quantitative enzymatic assay. When the livers of infected mice were harvested 28 days postinfection, the levels of CAT expression revealed that the effectors most important for the elimination of the Ad vector were TNF-alpha > Fas > perforin. TNF-alpha did not have a curative effect on infected hepatocytes, as the administration of TNF-alpha to infected severe combined immunodeficient mice or to infected cultures in vitro had no specific effect on virus persistence. However, TNF-alpha-deficient mice demonstrated a striking reduction in the leukocytic infiltration early on in the infection, suggesting that TNF-alpha deficiency resulted in impaired recruitment of inflammatory cells to the site of inflammation. In addition, the TNF-deficient mice had a significantly reduced humoral immune response to virus infection. These results demonstrate a dominant role of TNF-alpha in elimination of Ad gene transfer vectors. This result is particularly important because viral proteins that disable TNF-alpha function have been removed from most Ad vectors, rendering them highly susceptible to TNF-alpha-mediated elimination.

"Micronucleoli" in the Xenopus germinal vesicle.

The germinal vesicle of the Xenopus oocyte contains 1500 or more extrachromosomal nucleoli that are assembled on amplified copies of the rRNA genes. Many of these nucleoli have diameters of 10-15 micron, but some are much smaller, ranging down to 1 micron or less. Morphologically the smaller nucleoli or "micronucleoli" resemble the similarly sized B snurposomes, but they can be recognized with appropriate antibody probes (e.g., anti-nucleolin and anti-fibrillarin). We describe here a sensitive fluorescent staining technique that uses avidin and propidium iodide to visualize the rDNA in the amplified nucleoli. Many large nucleoli stain about as brightly as haploid yeast nuclei on the same slides. They presumably contain about 12 Mb of DNA, equivalent to 900 rDNA repeats. The smallest micronucleoli display only a tiny dot of stain, which must correspond to relatively few rDNA repeats.

Coiled bodies without coilin.

Nuclei assembled in vitro in Xenopus egg extract contain coiled bodies that have components from three different RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3'-end formation. In addition, they contain SPH-1, the Xenopus homologue of p80-coilin, a protein characteristic of coiled bodies. To determine whether coilin is an essential structural component of the coiled body, we removed it from the egg extract by immunoprecipitation. We showed that nuclei with bodies morphologically identical to coiled bodies (at the light microscope level) formed in such coilin-depleted extract. As expected, these bodies did not stain with antibodies against coilin. Moreover, they failed to stain with an antibody against the Sm proteins, although Sm proteins associated with snRNAs were still present in the extract. Staining of the coilin- and Sm-depleted coiled bodies was normal with antibodies against two nucleolar proteins, fibrillarin and nucleolin. Similar results were observed when Sm proteins were depleted from egg extract: staining of the coiled bodies with antibodies against the Sm proteins and coilin was markedly reduced but bright nucleolin and fibrillarin staining remained. These immunodepletion experiments demonstrate an interdependence between coilin and Sm snRNPs and suggest that neither is essential for assembly of nucleolar components in coiled bodies. We propose that coiled bodies are structurally heterogeneous organelles in which the components of the three RNA processing pathways may occur in separate compartments.

The Sm binding site targets U7 snRNA to coiled bodies (spheres) of amphibian oocytes.

Using cytoplasmic and nuclear injection assays, we show that U7 snRNA constructs are targeted rapidly and specifically to the coiled bodies (spheres) in the germinal vesicle (GV) of the amphibian oocyte, including those coiled bodies attached to the lampbrush chromosomes at the histone gene loci. Because the U7 snRNP is required for removing the 3' end of histone pre-mRNA, we suggest that a major function of coiled bodies is to recruit U7 snRNPs to the histone gene loci, before they associate with the pre-mRNA. Targeting to coiled bodies requires the specific U7 Sm binding site; replacement of the U7 Sm site by that of U2 snRNA reduces this targeting dramatically. No other part of the molecule is required, and the U7 Sm binding site alone is sufficient to direct nuclear import of an unrelated RNA sequence and its specific targeting to coiled bodies. Injected U7 constructs displace the endogenous U7 in the coiled bodies, the amount of injected U7 that ends up in coiled bodies being roughly equal to the amount of endogenous U7 snRNA.

A zinc-binding domain is required for targeting the maternal nuclear protein PwA33 to lampbrush chromosome loops.

In oocytes of the newt Pleurodeles waltl, the maternal nuclear protein PwA33 occurs on the lampbrush chromosomes and in some nucleoplasmic particles of the germinal vesicle. PwA33 is a modular protein and we used site-directed mutagenesis to alter the sequences encoding two metal-binding regions, the C3HC4 (or RING finger) and B-box motifs. Several mutant clones were generated and their synthetic transcripts were injected into Pleurodeles oocytes for in vivo analysis. In the oocyte, all translation products localized in the germinal vesicle. Proteins encoded by RING finger mutant clones were distributed in a pattern identical to that of the wild type protein, but when His266 of the B-box was mutated, PwA33 failed to localize in the lampbrush chromosomes and the nucleoplasmic particles. Using an in vitro colorimetric assay, we demonstrated that PwA33 is a zinc-binding protein and that mutations in the RING finger and B-Box altered its metal-binding properties. The RING finger motif bound two Zn2+ ions and the binding ratios of several mutants were consistent with the tertiary structure recently proposed for this motif. The B-box coordinated one Zn2+ and this binding was inhibited by the His266 mutation. The failure of the His266 mutation to bind zinc and to localize properly within the germinal vesicle suggests that an intact B-box is required for normal functioning of the PwA33 protein in the oocyte.

Presence of species-specific, monomorphic antigens on sheep and goat binucleate cells.

Immunocytochemical assays for sheep and goat species-specific, monomorphic antigens were developed utilizing polyclonal antisera from sheep and goats immunized by interspecific pregnancy. The assays were applied to cell isolates from sheep and goat fetal cotyledons collected from allogeneic pregnancies at Days 35, 40 and 120 of gestation. The isolates contained 7 to 48% binucleate cells (BNC). Using these assays, the sheep-specific antigen was detected on sheep cotyledonary cell isolates on all days of gestation tested (P < 0.001); the assay also detected the antigen on the BNC subset of the cotyledonary cell isolate population (P < 0.001). The caprine-specific antigen was shown to be present on cotyledonary cell isolates (P < 0.05), although the presence of the antigen could not be demonstrated with statistical confidence on goat BNC due to insufficient numbers of discernible cells. Binucleate cells contribute to the formation of the syncytial layer of the placenta by fusing with maternal epithelial cells and with the syncytium. The species-specific antigen (or antigens) is present on BNC at the appropriate time of gestation at which it (they) could play a role in the humoral immune response to interspecific and hybrid pregnancies observed in ewes and does.

Is the sphere organelle/coiled body a universal nuclear component?

We present evidence for the essential homology of four nuclear organelles that have previously been described under four different names: coiled bodies in mammalian somatic nuclei, prenucleolar bodies in nuclei assembled in vitro in Xenopus egg extract, sphere organelles in amphibian germinal vesicles (GVs), and Binnenkörper in insect GVs. Each of these organelles contains coilin or a coilin-related protein plus a variety of small nuclear ribonucleoproteins. We suggest that the sphere organelle/coiled body is a "universal" nuclear component in the sense that it is involved in common nuclear processes and hence will be found in one form or another in most eukaryotic cells. We postulate that it functions in the assembly and sorting of snRNP complexes for three RNA processing pathways: pre-mRNA splicing, rRNA processing, and histone pre-mRNA 3' end formation. Specifically, the sphere organelle/coiled body may be the initial site for assembly of processing complexes, which are then sorted to other places in the nucleus, where the actual RNA processing takes place.

Human p80-coilin is targeted to sphere organelles in the amphibian germinal vesicle.

Cultured vertebrate cells often display one or more coiled bodies in their nuclei. These are spherical structures approximately 0.5-1.0 micron in diameter that contain high concentrations of small nuclear ribonucleoproteins (snRNPs); they are distinct from nuclear speckles and nucleoli, the other major sites of snRNP concentration. Coiled bodies in human cells contain a unique protein, p80-coilin, that has an M(r) = 80 kDa. Cloned p80-coilin cDNA encodes 576 amino acids with a calculated molecular weight of 62.6 kDa. To determine which of several snRNP-containing structures in the amphibian germinal vesicle (GV) might be the homologue of coiled bodies, we injected myc-tagged transcripts of full-length human p80-coilin into the cytoplasm of Xenopus oocytes and followed the fate of the translated proteins with an antibody specific for the tag. Western blots of GV proteins showed rapid appearance of both full-length and truncated p80-coilin in the nucleus. Immunofluorescent staining of spread GV contents demonstrated specific uptake of p80-coilin by the sphere organelle within 1 h after injection. Similar experiments were performed with a series of deletion constructs that lacked progressively longer segments from the carboxy terminus. A construct that contained only the first 102 amino acids (18% of the molecule) was specifically targeted to the sphere organelle. Conversely, a construct lacking the first 92 amino acids failed to localize, although it was imported into the GV. Thus, a relatively short region at the amino terminus of human p80-coilin is both necessary and sufficient for localization in the sphere organelle. Sphere organelles in the GV and coiled bodies in somatic nuclei are clearly related in composition. We suggest that they are homologous organelles with similar functions in preassembly and sorting of RNA processing components. Differences in their composition suggest functional specialization in the two cell types.