The fine structural distribution of nuclear ribonucleoproteins (RNP)' has been extensively studied during the last two decades by techniques of ultrastructural cytochemistry and autoradiography (for review see reference 10). These investigations have allowed the characterization of nucleoplasmic structural components such as perichromatin granules, interchromatin granules, and coiled bodies. In addition, the introduction of a new differential staining technique for nuclear nucleoproteins (4) enabled Monneron and Bernard (21) to describe perichromatin fibrils, nuclear RNP constituents that could not be visualized by standard double fixation and double staining procedures . These latter structures have been shown to represent the in situ morphological counterpart of newly synthesized heterogeneous nuclear (hn)RNP (2, 11), and kinetic analysis of the distribution of radioactive RNA demonstrated that these constituents are formed within areas at the periphery of condensed chromatin, and that at least a part of them then migrates towards the interchromatin space (11, 24; for review see reference 6). Similar experiments revealed only a low level of [3H]uridine labeling in the clusters of interchromatin granules, even after prolonged periods of chase (7,8), and suggested the presence of rather stable RNA species within these nuclear components.Although the above methodological approaches provide good evidence about the RNA nature of various nuclear structural constituents, we have no direct information about 'Abbreviation used in this paper: RNP, ribonucleoproteins ; sn, small nuclear RNA; hn, heterogeneous nuclear RNA. ABSTRACT The ultrastructural distribution of nuclear ribonucleoproteins (RNP) has been investigated by incubation of thin sections of mouse or rat liver, embedded in Lowicryl K4M or prepared by cryoultramicrotomy, with antibodies specific for RNP . The antibodies were localized by means of a protein A-colloidal gold complex . Anti-small nuclear (sn)RNP antibodies, specific for determinants of the nucleoplasmic snRNP species containing U t , U, U4, U5, and U6 RNAs, were found associated preferentially with perichromatin fibrils, interchromatin granules, and coiled bodies . This indicates an early association of snRNP with structural constituents containing newly synthesized heterogeneous nuclear RNA . It also suggests a possible structural role of some snRNPs in nuclear architecture .Antibodies against the core proteins of heterogeneous nuclear RNP particles associate preferentially with the border regions of condensed chromatin, and in particular with perichromatin fibrils and some perichromatin granules. These results are discussed in view of recent knowledge about the possible role of nucleoplasmic RNP-containing components in the functions of the cell nucleus .

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active premRNA transcriptional and processing events.

Human and pig cDNAs for a novel stomach protein, the product of a gene expressed at high levels specifically in cells of the antrum mucosa, have been characterized. The general exon/intron structure of the genomic DNA is conserved in humans and mice. The predicted protein sequences of the human and mouse mRNAs contain 185 and 184 amino acids, respectively. The protein isolated from pig antral extracts has an NH2 terminus consistent with cleavage of a 20-amino acid signal peptide. Human cDNA was expressed in E. coli to generate a protein antigen for antibody production. The antibodies detected polypeptides of approximately 18 kDa in antrum extracts from all mammalian species tested. Immunocytochemistry located antrum mucosal protein (AMP)-18 to surface mucosal cells of the mouse antrum and, specifically, to secretion granules, suggesting that it is cosecreted with mucins. Antrum extracts and recombinant human AMP-18 exhibit growth-promoting activity on epithelial cells that can be blocked by the specific antisera. We suggest that AMP-18 is a "gastrokine" that maintains the integrity of the gastric mucosal epithelium.

Antrum mucosal protein (AMP)-18 is a novel 18-kDa protein synthesized by cells of the gastric antrum mucosa. The protein is present in secretion granules of murine gastric antrum epithelial cells and is a component of canine antrum mucus, suggesting that it is secreted into the viscoelastic gel layer on the mucosal surface. Release of the protein appears to be regulated because forskolin decreased the amount of immunoreactive AMP-18 in primary cultures of canine antrum mucosal epithelial cells, and indomethacin gavaged into the stomach of mice reduced AMP-18 content in antrum mucosal tissue before inducing histological injury. A functional domain of the protein was identified by preparing peptides derived from the center of human AMP-18. A 21-mer peptide stimulated growth of gastric and intestinal epithelial cells, but not fibroblasts, and increased restitution of scrape-wounded gastric epithelial monolayers. These functions of AMP-18 suggest that its release onto the apical cell surface is regulated and that the protein and/or peptide fragments may protect the antral mucosa and promote healing by facilitating restitution and proliferation after injury.

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