In the amphibian germinal vesicle small nuclear RNAs (snRNAs) occur in morphologicafly distinct structures called snurposomes. Three types (A, B, and C) have been distinguished on the basis ofcytological appearance and snRNA composition. C snurposomes in Xenopus are spherical bodies ranging in diameter from <1 ,um to about 10 jum. They stain intensely with antibodies against trimethylguanosine and the small nuclear ribonucleoprotein-specific Sm antigen but give weak or negative in situ hybridization reactions for the snRNAs involved in pre-mRNA splicing (Ul, U2, U4, U5, and U6). We show here that C snurposomes in the Xenopus germinal vesicle contain U7 snRNA, an snRNA of low abundance involved in processing the 3' end of histone pre-mRNA. Xenopus U7 is 58 nucleotides long and is capped at the 5' end with trimethylguanosine. C snurposomes are often associated with B snurposomes, which contain the splicing snRNAs but not U7; B and C snurposomes together constitute a morphologically complex structure known as a sphere or sphere organelle. Although most spheres and C snurposomes are extrachromosomal, a few are attached at the histone gene loci on chromosomes 8, 9, and 16. Because they contain U7 snRNA and occur at the sites of histone pre-mRNA synthesis, C snurposomes presumably play a role in processing histone transcripts.The localization of small nuclear ribonucleoproteins (snRNPs) in the amphibian oocyte nucleus, or germinal vesicle (GV), has been studied by in situ nucleic acid hybridization and immunofluorescence (1-5). snRNPs occur on the lampbrush chromosome loops associated with the nascent transcripts and in three morphologically distinct types of extrachromosomal granules that we designate A, B, and C snurposomes. The A snurposomes, so far identified only in GVs of the newt Notophthalmus, contain Ul snRNA and associated proteins. B snurposomes occur in all amphibian species examined, both urodele and anuran. They contain the five snRNAs involved in pre-mRNA splicing (Ul, U2, U4, U5, and U6) plus several snRNP-specific proteins and the SR group of non-snRNP essential splicing factors (6,7). Until now the snRNP composition of C snurposomes has remained problematic. Here we demonstrate by in situ hybridization that C snurposomes in Xenopus GVs contain U7 snRNA. Studies in sea urchins (8) and cultured mammalian cells (9) show that the U7 snRNP is involved in processing the 3' end of histone pre-mRNAs. C snurposomes often have Bs attached to their surface, the combination being referred to as a sphere or sphere organelle in the cytological literature. It has been known for some time that a few of the several dozen spheres in the GV are attached to the lampbrush chromosomes at the histone gene loci (10,11). The presence of U7 snRNA and the close association with the sites of histone pre-mRNA synthesis suggest that spheres and their constitThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 ...

When demembranated sperm nuclei are placed in a Xenopus egg extract, they become surrounded by a nuclear envelope and then swell to form morphologically typical pronuclei. Granules ranging from <1.0 to -3.0 ,um in diameter appear within such nuclei. Bell et al. identified four nucleolar proteins in these "prenucleolar bodies" by immunofluorescent staining (fibrillarin, nucleolin, B23/NO38, 180-kDa nucleolar protein). By in situ hybridization we show that these bodies also contain U3 and U8 small nuclear RNAs (snRNAs), known to be involved in pre-rRNA processing. Moreover, they contain all the snRNAs involved in pre-mRNA splicing (Ul, U2, U4, U5, and U6), as well as U7, which is required for histone pre-mRNA 3' end formation. In addition to the nucleolar antigens previously identified, we demonstrated staining with antibodies against the Sm epitope, trimethylguanosine, and coilin. Because the composition of these prenucleolar bodies is closer to that of coiled bodies than to nucleoli, we propose that they be referred to as coiled bodies. The existence of large coiled bodies in transcriptionally inactive pronuclei suggests that they may play a role in the import, assembly, and storage of RNA processing components but are not themselves sites of processing. In transcriptionally active nuclei coiled bodies could serve as sites for initial preassembly and distribution of snRNP complexes for the three major RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3' end formation.

SUMMARYSystemic lupus erythematosus (SLE) is characterized by the existence of a heterogeneous group of autoantibodies directed against nuclear intact structures, such as nucleosomes and small nuclear ribonucleoproteins (snRNPs). Autoantibodies against snRNPs are of special interest because they are detectable in the majority of SLE patients. Although the B-cell antigenic determinants have been well characterized, very limited data have been reported in regard to the T-cell epitopes of snRNPs. Furthermore, several studies have demonstrated that determination of the auto-T-cell epitopes recognized by freshly isolated T cells is dif®cult from unprimed lupus mice when self-antigen-pulsed B cells or macrophages are used as antigen-presenting cells (APCs) in vitro. In the present study, we showed a novel approach for determining the auto-T-cell epitopes, using bone marrow-derived dendritic cells (BMDCs) pulsed with the murine U1A protein ± an immunodominant antigen of the U1 snRNPs ± which is capable of activating freshly isolated T cells from unprimed (NZBrNZW) F 1 (BWF 1 ) mice in vitro. The T-cell epitope area was found to be located at the C-terminus of U1A, overlapping the T-cell epitope of human U1A that has been reported in human SLE. Identi®cation of the autoreactive T-cell epitope(s) in snRNPs will help to elucidate how reciprocal T±B determinant spreading of snRNPs emerges in lupus. The results presented here also indicate that it is feasible to use this approach to further explore strategies to design immunotherapy for patients with lupus.

Finding an interactive ligand-receptor pair is crucial to many applications, including the development of monoclonal antibodies. Biopanning, a commonly used technique for affinity screening, involves a series of washing steps and is lengthy and tedious. Here we present an approach termed continuous microfluidic assortment of interactive ligands, or CMAIL, for the screening and sorting of antigen-binding single-chain variable antibody fragments (scFv) displayed on bacteriophages (phages). Phages carrying native negative charges on their coat proteins were electrophoresed through a hydrogel matrix functionalized with target antigens under two alternating orthogonal electric fields. During the weak horizontal electric field phase, phages were differentially swept laterally depending on their affinity for the antigen, and all phages were electrophoresed down to be collected during the strong vertical electric field phase. Phages of different affinity were spatially separated, allowing the continuous operation. More than 105 CFU (colony forming unit) antigen-interacting phages were isolated with ~100% specificity from a phage library containing 3 × 109 individual members within 40 minutes of sorting using CMAIL. CMAIL is rapid, sensitive, specific, and does not employ washing, elution or magnetic beads. In conclusion, we have developed an efficient and cost-effective method for isolating and sorting affinity reagents involving phage display.

In the GV of amphibian oocytes, the splicing snRNPs (U1, U2, U4, U5, and U6) occur on most of the lampbrush chromosome loops in association with the nascent transcripts. They also occur in thousands of small extrachromosomal bodies (1-4 microns in diameter) designated B snurposomes. U7 snRNA, which is involved in processing the 3' end of histone pre-mRNAs, occurs in a few dozen extrachromosomal bodies (1-20 microns in diameter) called C snurposomes. C snurposomes often have B snurposomes attached to their surface and B-like inclusions; these compound structures are known as spheres or sphere organelles. One or two sphere organelles are attached to the lampbrush chromosomes at the histone gene loci. Two snRNAs (or snoRNAs) known to be involved in pre-rRNA processing (U3 and U8) occur in the 1000 or so extrachromosomal nucleoli of the GV. We looked for a snurposome that might contain U3 and U8 but not rDNA or rRNA. We were unable to find such a snurposome, but we did identify a hitherto unrecognized population of minute nucleoli in the size range of B snurposomes. Prenucleolar bodies in telophase/early interphase nuclei meet the definition of a pre-rRNA snurposome in that they contain U3 snoRNA and fibrillarin (and probably other processing components) but lack rDNA and do not synthesize rRNA. The structures previously identified as prenucleolar bodies in pronuclei formed in vitro in Xenopus egg extracts share many components with coiled bodies from HeLa nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)