The monoclonal antibody P11 is directed against a 38 000 dalton protein of Drosophila melanogaster. On polytene chromosomes this protein is present in a subset of the RNA polymerase II‐containing loci. Here we show by density centrifugation and enzyme‐linked immunosorbent assay tests that the P11 antigen is part of nuclear ribonucleoprotein (RNP) complexes. Indirect immunofluorescence shows that, after prolonged heat‐shock, the P11 antigen is present only in the heat‐shock puff 93 D. Identical distribution patterns were obtained with another monoclonal antibody, Q18. Unlike P11, this antibody also cross‐reacts with D. hydei and D. virilis polytene chromosomes, where the puffs 48 B and 20 CD, respectively, are the only loci prominently stained after heat‐shock. The small and giant RNP complexes previously described in these puffs were also observed in puff 93 D. Both types of particle contain the P11 antigen as shown by immunoelectron microscopy. We suggest that the P11 antigen is associated with a special class of RNPs which are possibly involved in the storage of primary transcription products inside the nucleus.

Two structurally distinct RNP complexes (MI and MII), each with a sedimentation value of approx. 40S, were isolated from rat liver nuclear extracts by sucrose gradient centrifugation and subsequent native gel electrophoresis of the 40S hnRNP-containing fractions. MII RNP contained the bulk of hnRNA and hnRNP proteins (i.e. the 32-45KD core proteins and polypeptides of 60-80 and 110-130KD). MI RNP was characterized by the exclusive presence of U-snRNAs (U1, U2, U4, U5 and U6), their well known snRNP polypeptides and a number of Sm-associated proteins in the range of 50-210KD. Immunoselection experiments employing a monoclonal antibody with an established specificity for the U2-snRNP-specific B" polypeptide proved that the RNA and protein components characteristic of MI were part of a single multi-snRNP unit. The prominent 200/210KD protein doublet of MI was identified immunochemically as the rat homologue of the yeast PRP8 protein, a known U5-associated splicing component. Based on the major biochemical and immunochemical features of MI and MII RNP complexes, we conclude that MII represents the monomeric 40S hnRNP structure, whereas MI defines a novel multi-snRNP entity.

The structurally related A/B-type core heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptides of 34-39 kDa (A1, A2, B1 and B2) belong to a family of RNA-binding proteins that are major components of 40 S hnRNP complexes. By two-dimensional gel electrophoresis and peptide mapping analysis we compared each member of the A/B-type core proteins in the human and rat liver cells. This comparison revealed the unique presence in rat cells of major protein species, referred to as mBx polypeptides, that appeared as three charge isoforms at a position corresponding to the minor HeLa B1b protein spot. In addition, clear differences in the ratios of the A1 polypeptide to the A1b isoform were observed. The detection, in sera of patients with rheumatic autoimmune diseases, of two novel autoantibody specificities, one recognizing solely B2 protein and the second both the B2 and mBx polypeptides, helped to identify mBx proteins as new A/B-type hnRNP components, immunologically related to B2 protein. A common immunoreactive V8 protease peptide of approx. 17 kDa has been identified in B2 and mBx hnRNP polypeptides. mBx protein species are identified in cells of murine origin, and have a ubiquitous tissue distribution and developmental appearance.

Nine monoclonal antibodies directed against chromosomal proteins of D. melanogaster were used to study, by indirect immunofluorescence, the distribution of their respective antigens on polytene chromosomes following heat shock. This treatment is known to induce a specific set of transcriptionally active puffs with concomitant reduction of transcriptional activity in previously active loci. Our studies revealed wide differences in the distribution of the individual chromosomal proteins under heat shock conditions with regard to pattern and rate of both elimination from the inactivated loci and accumulation in the activated loci.

Rat liver nuclei were extracted with 0.14 M NaCl and the extracts submitted to sucrose gradient fractionation. Aliquots of the nuclear residue remaining after the 0.14 M NaCl extraction were also extracted either with 0.3 M NaCl or 1 M urea, and the extracts similarly submitted to sucrose gradient fractionation. Thereafter, both the presence and relative distribution of individual U-snRNA (U1-U6) species was followed. Results showed an extensive association of all U-snRNAs to RNP structures of greater than or equal to 40 S. However, characteristic differences in the association of mostly U1 and U5--which were the major identifiable species in the extracts--to these structures were observed. Only a small fraction of U1 appeared complexed to less than or equal to 40 S RNP structures, while most of it sedimenting in the greater than 20 S region of the gradient. In contrast, U5-snRNA had a tight and almost exclusive association to 40 S RNP structures. No pool of 10-12 S U5-snRNP complexes was detected. Combined immunoprecipitation and immunoblotting experiments on nuclear 0.14 M NaCl extracts using anti-Sm and/or anti-RNP antisera showed that all snRNA species, whether recovered as 10-12 S complexes or segregated with greater than or equal to 40 S RNP components, existed as snRNP structures bearing at least their Sm-antigenic polypeptides. These and our previous results [Guialis, A., Arvanitopoulou, A, Patrinou-Georgoula, M. and Sekeris, C.E. (1983) FEBS Lett. 151, 127-133], support the existence of snRNP-enriched RNP structures of greater than or equal to 40 S. In such structures the core polypeptides (Mr = 32,000-45,000) of 40 S monoparticles are not obligatory components.