Effect of Actinomycin D on the Expression of Nuclear Antigen SS-B/La (original) (raw)
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cDNA cloning of a human autoimmune nuclear ribonucleoprotein antigen
Proceedings of the National Academy of Sciences, 1985
Sera from patients with systemic lupus erythematosus and other autoimmune disorders contain antibodies against nuclear proteins. One such autoantibody system, known as Sm, reacts with antigens associated with small nuclear RNA molecules. In this paper we report the use of Sm autoantibodies to isolate a cDNA clone for the mRNA of one of these nuclear antigens. A HeLa cell cDNA library was screened by message selection followed by autoantibody reaction of cell-free translation products. This led to the identification of a cDNA clone, p281, containing sequences complementary to mRNA for an Sm autoantibody-reactive, 11,000 Mr protein. This cloned Sm antigen comigrated with the small nuclear RNA-associated protein known as "E" and reacted with four out of four Sm autoantibodies that precipitate E protein from total mRNA translation products. RNA gel blot hybridization with clone p281 DNA revealed a poly(A)+ mRNA of -600 nucleotides in human and marmoset (New World primate) cells. Southern blot hybridization of HeLa cell and human
Proceedings of the National Academy of Sciences of the United States of America, 1989
The Sm snRNPs play a central role in the processing of pre-mRNA. Anti-Sm antibodies, the diagnostic hallmark of systemic lupus erythematosus, target the B/B and D polypeptides of these snRNPs. We have used patient autoantibodies to clone a cDNA from a human fibroblast cDNA library that encodes the full length of a polypeptide identical with, or closely related to, polypeptide B. This cDNA is comprised of 1139 bases and contains an open reading frame of 855 nucleotides that is capable of encoding 285 amino acids. The first 223 amino acids at the NH2 terminus exhibit nearly complete homology with polypeptide N, a newly recognized brain-and heart-specific component of Sm snRNPs. The derived amino acid sequence for B differs from that of the N polypeptide primarily by a 50-amino acid insert 12 residues upstream from the homologous COOH termini of these polypeptides. The structural differences in these cDNAs for B and N may regulate tissue-specific alternative splicing mechanisms for mRNA. In addition, these clones make it possible to map in fine detail the most characteristic autoimmune responses of systemic lupus erythematosus.
Fractionation of HeLa cell nuclear extracts reveals minor small nuclear ribonucleoprotein particles
Proceedings of the National Academy of Sciences, 1987
Upon chromatographic fractionation of HeLa cell nuclear extracts, small RNAs of 145 and 66/65 nucleotides, respectively, were detected that are distinct from the abundant small RNAs present in the extract. These RNAs are precipitated by antibodies directed against the trimethylguanosine cap structure, characteristic for small nuclear RNAs (snRNAs) of the U type. The RNAs of 145 and 66/65 nucleotides appear to be associated with at least one of the proteins common to the major small nuclear ribonucleoprotein particles Ul to U6, since they are specifically bound by anti-Sm antibodies. These criteria characterize the RNAs that are 145 and 66/65 nucleotides in length as U-type snRNAs. Upon gel filtration, the RNAs are found within particles of molecular weights-150,000 and 115,000, respectively. The RNA of 145 nucleotides represents a different minor snRNA, designated U11, whereas the RNA of 66/65 nucleotides may correspond to either mammalian U7 or UlO RNA. Nuclei of metazoan cells contain a set of small RNAs, the U-type small nuclear RNAs (snRNAs), which are complexed with several distinct proteins to form small nuclear ribonucleoprotein particles (snRNPs) (see refs. 1 and 2 for recent reviews). The snRNAs U1-U6 are extremely abundant in metazoans (about 105_106 copies per cell) and, with the exception of U6 RNA, contain a characteristic 2,2,7-trimethylguanosine (M3G) cap structure at their 5' termini. U6 RNA is packaged into one snRNP with U4 RNA, whereas U1, U2, and U5 RNAs are organized into monomeric particles (3-5). These snRNPs, except for U3, are immunoprecipitated by antibodies from patients suffering from connective tissue diseases. Anti-Sm antibodies recognize common proteins present in all particles identified so far, whereas proteins characteristic for U1 or U2 snRNP are recognized either by patient sera or by monoclonal antibodies raised against purified U1 snRNP (reviewed in ref. 1). In addition, the particles can be precipitated by antibodies directed against the m3G cap structure (6-8).
Proceedings of the National Academy of Sciences of the United States of America, 1981
At least three separate regions of the Epstein-Barr virus (EBV) genome encode RNA in a cellline that is growth transformed and nonpermissively infected with EBV. Six polyadenylylated cytoplasmic RNAs have been identified from these three re-.ons. An abundant RNA 3.0-3.1 kilobases (kb) long is encoded by DNA of the internal reiteration, IR, and DNA that maps at 25.7-30 me adaltons. A second, abundant, 2.9-kb RNA is primarily encoded by DNA at 110-03 megadaltons but probably has a 3' end to the left of 110 megadaltons. A third, abundant, 3.7-kb RNA is largely encoded by DNA at 63-66 megadaltons and has a 5' end to the left of 63 megadaltons. A less-abundant 1.5-kb RNA is also encoded by IR. The least-abundant polyadenylylated RNAs identified are 2.3 and 2.0 kb. These RNAs have 3' ends mapping at 5-7 megadaltons and 5' ends mapping to the right of 7 megadaltons. The data suggest that there may be two additional polyadenylylated cytoplasmic RNAs, a 3-kb RNA mapping at 26.2-30 megadaltons and a minor RNA mapping at 102-110 megadaltons. An abundant 0.16-kb non lyadenylylated RNA is also present in the cytoplasm of IB-4 cells. This RNA precipitates from the cytoplasm in the presence of high concentrations of magnesium, indicating that it is complexed with protein or polyribosomes.
Antibodies to double-stranded RNA: Specificity and serum nucleases
Journal of Immunological Methods, 1975
An antiserum was prepared in rabbits to the synthetic double-stranded ribonucleic acid (ds RNA) poly rI:rC. Using a liquid-phase radioimmunoassay, the antiserum cross-reacted with a natural ds RNA isolated from the cytoplasmic polyhedrosis virus of the silkworm, binding 95% of the RNA at a 1 : 20 serum dilution. Preliminary tests of the specificity of the antiserum showed that it did not bind single-stranded RNA (ss RNA) or deoxyribonucleic acid (DNA), but also revealed that the serum contained an enzyme activity which degraded ss RNA into acid-insoluble fragments. It was therefore possible that the failure to bind ss RNA resulted from the degradation of the antigen rather than from an absence of cross-reacting antibodies. However, when the serum ribonuclease activity was inhibited by macaloid, the antiserum still did not bind the ss RNA antigen. This demonstrated that the antibodies to ds RNA did not cross-react with ss RNA. The existence of serum enzymes capable of degrading nucleic acid antigens emphasizes the need for caution in assessing the specificity of such antisera.
Clinical & Experimental Immunology
Nuclear ribonuclear protein (nRNP) and Sm were extracted from a 30-60% ammonium sulphate fraction of rabbit thymus extract by affinity chromatography. Immunoadsorbent columns were prepared from IgG extracted from an anti-nRNP serum, an anti-Sm serum and two monoclonal autoantibodies derived from a MRL/lpr mouse. All four immunoadsorbents isolated both nRNP and Sm antigens indicating that they exist as the 'nRNP/Sm complex' A species of Sm which did not bind to the anti-nRNP column and which was subsequently purified with the anti-Sm column was termed 'free Sm'. The Sm in the complex and free Sm were immunologically identical on immunodiffusion and gave similar polypeptide bands on polyacrylamide gel electrophoresis. However, they differed in that complexed Sm was sensitive to heat and RNAase. These studies provide direct evidence of a physical association between the nRNP and Sm antigens and indicate an additional molecular species of Sm whose resistance to RNAase ma...
Clinical & Experimental Immunology, 2008
In systemic autoimmunity, the human B cell response to the La (SS-B) autoantigen is polyclonal and directed to both conserved and human-specific epitopes. This study has further characterized the B cell epitope(s) present within the conserved central region of the La protein, LaC (amino acids 11 1-242) containing the RNA recognition motif (RRM, aa 11 1-187). Ten overlapping and non-overlapping protein fragments spanning LaC were expressed in bacteria as NH2-terminal fusions with glutathione-S-transferase. The fusion proteins were tested by ELISA for reactivity with a panel of human anti-La sera in order to define the nature of the epitopes. Ninety-two percent of patient sera containing anti-La antibodies reacted with the region of La containing the RRM. Fine mapping of this reactivity using deletion mutants indicated that the deletion of 19 amino acids from either the NH2-terminal or COOH-terminal region of the RRM was associated with loss of antibody reactivity, suggesting that the immunodominant epitope expressed in this region is discontinuous. Autoantibodies affinity-purified from the La RRM fragment to remove other specificities immunoprecipitated newly synthesized native La (SS-B)/Ro (SS-A) complexes, providing additional evidence that autoantibodies were recognizing a conformational epitope. The findings indicate that the human autoantibody response to La involves recognition of a conformational determinant involving the conserved RRM region without necessarily interfering with the RNA-dependent association of the La/Ro ribonucleoprotein.