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The spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 share eight proteins B', B, D1, D2, D3, E, F and G which form the structural core of the snRNPs. This class of common proteins plays an essential role in the biogenesis of the snRNPs. In addition, these proteins represent the major targets for the so-called anti-Sm auto-antibodies which are diagnostic for systemic lupus erythematosus (SLE). We have characterized the proteins F and G from HeLa cells by cDNA cloning, and, thus, all human Sm protein sequences are now available for comparison. Similar to the D, B/B' and E proteins, the F and G proteins do not possess any of the known RNA binding motifs, suggesting that other types of RNA-protein interactions occur in the snRNP core. Strikingly, the eight human Sm proteins possess mutual homology in two regions, 32 and 14 amino acids long, that we term Sm motifs 1 and 2. The Sm motifs are evolutionarily highly conserved in all of the putative homologues of the human Sm proteins identified in the data base. These results suggest that the Sm proteins may have arisen from a single common ancestor. Several hypothetical proteins, mainly of plant origin, that clearly contain the conserved Sm motifs but exhibit only comparatively low overall homology to one of the human Sm proteins, were identified in the data base. This suggests that the Sm motifs may also be shared by non-spliceosomal proteins. Further, we provide experimental evidence that the Sm motifs are involved, at least in part, in Sm protein-protein interactions. Specifically, we show by co-immunoprecipitation analyses of in vitro translated B' and D3 that the Sm motifs are essential for complex formation between B' and D3. Our finding that the Sm proteins share conserved sequence motifs may help to explain the frequent occurrence in patient sera of anti-Sm antibodies that cross-react with multiple Sm proteins and may ultimately further our understanding of how the snRNPs act as auto-antigens and immunogens in SLE.

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Electrophoresis of the mixture of proteins from purified snRNPs U1, U2, U4/U6 and U5 on SDS-polyacrylamide gels that had been allowed to polymerise in the presence of high TEMED concentrations have revealed the presence of proteins in the snRNPs that previously had eluded detection. The most striking case is that of protein D, heretofore generally observed as a single broad band; in high-TEMED gels, this splits into three clearly-separated bands, identified as three distinct proteins. We have denoted these proteins D1 (16 kDa), D2 (16.5 kDa) and D3 (18 kDa). Chemical and immunological studies have shown that D1 is identical with the common snRNP protein D, whose structure was recently resolved by cDNA cloning (Rokeach et al. (1988), Proc. Natl. Acad. Sci. USA, 85, 4832-4836) and that D2 and D3 are clearly distinct from D1 and very probably from each other. In addition to D1, proteins D2 and D3 are present in purified U1, U2, U4/U6 and U5 snRNPs isolated from HeLa cells, so these also belong to the group of common snRNP proteins. They are also found in snRNPs isolated from mouse cells, indicating that the role of these proteins in the structure and/or function of UsnRNPs has been conserved in evolution. Interestingly, patients with systemic lupus erythematosus produce populations of anti-Sm autoantibodies that react differentially with the D proteins; some recognise all of them and others only a subset. The high-TEMED gels allow improved resolution not only of the D proteins, but also of some of the U5-specific proteins contained in 20S U5 snRNPs, in particular the 15-kDa protein. In addition, under these conditions, the common G protein, previously observed as a single band, appears as a doublet. Whether the additional band represents a distinct common snRNP protein or a post-translationally modified version of G is not yet known.

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Surface proteins of different Salmonella R mutants were labeled selectively by treating live bacteria with cycloheptaamylose-dansylchloride. The labeled proteins were extracted from the cells with 6 M urea and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From the urea extract a 55-kilodalton protein common to numerous Salmonella strains could be isolated by ion-exchange chromatography and gel filtration free of lipopolysaccharide. Immunization of rabbits with isolated protein led to the formation of specific antibodies. Such antiprotein antisera could be employed in Western blots for the specific identification of the 55-kilodalton protein in bacterial extracts containing mixtures of different Salmonella proteins. The importance of this antigen is emphasized by antisera against acetone-killed Salmonella bacteria, showing a preferential interaction with the 55-kilodalton protein in Western blots. Active immunization of mice with the 55-kilodalton protein afforded significant protection against experimental infection with S. typhimurium.

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The reversible crosslinked protein complex L13-L21 was isolated from Escherichia coli ribosomes in milligram amounts. Tight couples (70 S) were crosslinked with the bifunctional reagent diepoxybutane. The reacted ribosomes were separated into their subunits by sucrose gradient centrifugation in the presence of 1 mM magnesium. The crosslinked subunits were analyzed by symmetrical two-dimensional gel electrophoresis. One crosslinked protein complex detected within the large subunit was purified by salt extraction, acidic acid extraction, and ion-exchange chromatography. Two-dimensional gel electrophoresis and immunological results established L13 and L21 as the components of this crosslink.

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The complete covalent structure of ribosomal protein S1 of Escherichia coli has been determined and predictions made of its secondary structure. Protein S1 (E. coli MRE 600) is a single-chain, acidic protein with 557 amino acid residues of the composition Asp43, Asn23, Thr25, Ser25, Glu60, Gln14, Pro10, Gly48, Ala48, Val67, Met6, Ile30, Leu45, Tyr6, Phe17, His8, Lys43, Arg30, Trp7, Cys2 and an Mr of 61159. The two -SH groups of S1 are located in the central region of the chain at positions 292 and 349, the latter being the reactive group whose modification results in the reported loss of the nucleic-acid-unfolding ability of S1. The central region also contains the majority of the tryptophan, histidine and methionine residues of S1 and is predicted to have a secondary structure dominated by beta-sheets and turns. A direct proof for the location of the nucleic-acid-binding domain of S1 in the central region has recently been obtained [Subramanian et al. (1981) Eur. J. Biochem. 119, 245-249]. The N-terminal region of S1, which contains the ribosome-binding domain has a relatively high predicted alpha-helix content and no preponderance of basic amino acids. The facile trypsin-sensitive site in S1 is located at Arg-171, approximately at the border between the N-terminal and central regions. The acidic and basic amino acids of S1 (32.8% of all residues) are distributed throughout the chain, often in small clusters of between two and six residues. The amino acid sequence of S1 contains three 24-residue stretches with strong internal homology. Two of the stretches are located in the central, RNA-binding region, suggesting a possible role in the RNA-binding and helix-destabilizing functions of S1. A fragment of Mr 10(4) from the central region of S1 gives an anomalously high apparent Mr by dodecylsulfate gel electrophoresis, indicating a stable structural element therein and accounting for the apparent high Mr of S1 as determined by gel electrophoresis.

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The primary structure of proteins S1, the largest protein component of the Escherichia coli ribosome, has been elucidated by determining the amino acid sequence of the protein (from E. coli MRE600) and the nucleotide sequence of the S1 gene (rpsA, of a K-12 strain). The two methods gave results in perfect agreement except of two positions where possible strain specific differences were found. Protein S1 (MRE600) is composed of 557 amino acid residues (no modified amino acids were detected) and has Mr 61,159. The DNA sequence for protein S1 (K-12) suggests 556 amino acid residues. A computer survey of the sequence revealed three regions in S1 with a high degree of internal homology. The ribosome binding domain of S1 (NH2 terminus) does not show any preponderance of basic amino acids. The two cysteine and the majority of tryptophan residues of S1 as well as two od the three homologous regions were located in its middle region which contains the nucleic acid binding domain. The pattern of degenerate codon usage in the S1 gene is nonrandom and similar to that reported for other ribosomal protein genes.

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