A Monoclonal Antibody That Recognizes a Phosphorylated Epitope Stains Lampbmsh Cl'n'omosome Loops and Small Granules in the Amphibian Germinal Vesicle

An mAb library was produced against proteins from the germinal vesicle (GV) of the frog Xenopus/aev/s; mAb 104 was selected from this library on the basis of its immunofluorescent staining of lampbrush chromosome loops. Chromosomes from several species of frogs and salamanders stained equally well. The antibody also stained the surface of numerous small granules in the GV nucleoplasm. The interior of the same granules was stained by antibodies against small nuclear ribonucleoproteins (snRNPs). mAb 104 also stained somatic nuclei from many vertebrate and invertebrate species, usually in a finely punctate pattern similar to that described for anti-snRNP and other antinuclear antibodies. The staining of somatic nuclei was much stronger during the mitotic stages than during interphase. Immunoblot analysis showed that mAb 104 recognizes a phosphorylated epitope. AMPBRUSH chromosomes from amphibian oocytes provide a useful system for studying proteins associated with nascent RNA transcripts. Each chromosome has a central axis consisting of transcriptionally inactive chromatin (chromomeres) from which loops of active chromatin extend laterally. The bulk of a loop consists of nascent transcripts with associated ribonucleoprotein (RNP)~ still attached to the DNA template. The RNP matrix of a loop is so abundant that individual transcription units are readily visible by light optical microscopy (Scheer et al., 1976; Gall et al., 1983). Some loops consist of a single transcription unit, whereas many contain two or more units in various orientations. Antibodies have been used to identify proteins associated with the nascent transcripts on lampbrush loops (Scott and Sommerville, 1974; Sommerville et al., 1978; Martin and Okamura, 1981; Lacroix et al., 1985; Roth and Gall, 1987; Gall and Callan, 1989; Pifiol-Roma et al., 1989). The majority of loops are morphologically similar and contain a set of common proteins, including both heterogeneous nuclear RNPs (hnRNPs) and small nuclear RNPs (snRNPs). A few "landmark" loops are morphologically distinct (reviewed in Callan, 1986), and in several cases their protein composition is known to be unusual. We have produced a number of mAbs from mice injected with germinal vesicle (GV) proteins from the frog Xenopus laevis, and the newt Notophthalmus viridescens. Several of these bind strongly to lampbrush chromosome loops (Roth 1. Abbreviations used in this paper: GV, germinal vesicle; hn, heterogeneous nuclear; RNP, ribonucleoprotein; sn, small nuclear. and Gall, 1987) or to other intranuclear structures. Most are relatively species-specific; for instance, most mAbs raised against Xenopus react with Xenopua and other anurans (e.g., Rana) but not with urodeles or other vertebrates. One mAb, designated 104, has proved of unusual interest because it cross-reacts with a wide variety of vertebrate and invertebrate species. In immunofluorescence assays mAb 104 stains most larnpbrush loops and numerous small granules in the GV nucleoplasm. In somatic cells it stains similar but smaller nuclear granules in a strongly cell cycle-dependent manner. Immunoblot analysis shows that the epitope recognized by the antibody contains a phosphate residue. Here we describe our studies on mAb 104, with emphasis on its reaction with lampbrush loops and its usefulness in defining a population of intranuclear granules. Materials and Methods