Identification of N(G)-methylarginine residues in human heterogeneous RNP protein A1: Phe/Gly-Gly-Gly-Arg-Gly-Gly-Gly/Phe is a preferred recognition motif.

Three sites of N(G),N(G)-arginine methylation have been located at residues 205, 217, and 224 in the glycine-rich, COOH-terminal one-third of the HeLa A1 heterogeneous ribonucleoprotein. Together with the previously determined dimethylated arginine at position 193 [Williams et al., (1985) Proc.

Antibodies to the 45 kDa DEK nuclear antigen in pauciarticular onset juvenile rheumatoid arthritis and iridocyclitis: selective association with MHC gene.

Objective: To study the frequency of autoantibodies to the 45 kDa DEK nuclear antigen, a putative oncoprotein, in a sample of patients with juvenile rheumatoid arthritis (JRA), and to make correlations with disease subtype and complications such as iridocyclitis. Class I and Class II HLA associations with reactivity to the antigen were also sought.

Methods: Sera from 146 HLA typed patients with JRA representing all subtypes were analyzed for reactivity with the 45 kDa DEK protein by immunoblotting.

A novel autoantibody to the putative oncoprotein DEK in pauciarticular onset juvenile rheumatoid arthritis.

Objective: To define the frequency of a novel autoantibody reactive with a 45 kDa protein in children with juvenile rheumatoid arthritis (JRA). This protein is expressed by the putative oncogene DEK associated with a subtype of acute myeloid leukemia.

Methods: The sera of 158 children with JRA were analyzed for the presence of anti-DEK by immunoblotting using purified DEK protein and compared with sera of 109 children with other rheumatic diseases and 25 healthy controls with no connective tissue disease.

The putative oncoprotein DEK, part of a chimera protein associated with acute myeloid leukaemia, is an autoantigen in juvenile rheumatoid arthritis.

The 45-kD autoantigen associated with juvenile rheumatoid arthritis (JRA) has been isolated from HeLa cell nuclei and purified about 2500-fold to near homogeneity in a five-step chromatographic procedure. Purification of the antigen was monitored by immunoblot assays using a nearly monospecific anti-45-kD serum from a child with JRA. Tryptic peptide mapping and partial amino acid sequencing of the purified 45-kD antigen demonstrated its identity with the DEK protein.

Cloning and sequence analysis of a human type A/B hnRNP protein.

A cDNA encoding a 284 residue long type A/B hnRNP protein has been cloned. This protein, previously referred to as type C [(1987) J. Biol.

Autoantibodies to DNA topoisomerase II in juvenile rheumatoid arthritis.

Sera from 58 children with juvenile rheumatoid arthritis were examined for the presence of antibodies to DNA topoisomerase II. Eight sera were reactive in immunoblotting with purified human topoisomerase II and a protein encoded by a cloned cDNA expressed in Escherichia coli which represents the carboxy-terminal domain of the human enzyme. In addition, the sera detect topoisomerase II in mitotic chromosomes and chromosome scaffolds.

Antinuclear antibody profile in juvenile rheumatoid arthritis.

Immunoblot positive sera from children with juvenile rheumatoid arthritis detected from 1 to greater than or equal to 10 proteins in HeLa nuclear sonicates. Thirty percent of the sera reacted with histone H1. Antibodies to at least 1 of 6 most frequently detected nonhistone proteins were present in 85% of the sera.

Purification and RNA binding properties of a C-type hnRNP protein from HeLa cells.

A protein of the C group, most likely C3 (Mr approximately 42,000, pI approximately 6, corresponding to IEF 48m,n of the HeLa protein catalogue (Celis, J. E., Bravo, R.

Purification and domain structure of core hnRNP proteins A1 and A2 and their relationship to single-stranded DNA-binding proteins.

Protein A1 (Mr approximately 32,000), a major glycine-rich protein of heterogeneous nuclear ribonucleoproteins (hnRNP), was purified to near homogeneity under nondenaturing conditions from HeLa cells. Limited proteolysis of the native protein yields a trypsin-resistant N-terminal nucleic acid-binding domain about 195 amino acids long which has a primary structure nearly identical to that of the 195-amino acid-long single-stranded DNA (ssDNA)-binding protein UP1 (Mr 22,162) from calf thymus (Williams, K.R.

Antibodies to hnRNP core proteins inhibit in vitro splicing of human beta-globin pre-mRNA.

In vitro splicing of human beta-globin pre-mRNA can be fully inhibited by treatment of the splicing extract with polyclonal antibodies against hnRNP core proteins prior to the addition of pre-mRNA. Inhibition of the first step in the splicing pathway, cleavage at the 5' splice site and lariat formation, requires more antibodies than inhibition of the second step, cleavage at the 3' splice site and exon ligation. The anti-hnRNP antibodies can also inhibit the splicing reaction after the formation of the active nucleoprotein splicing complex which is known to occur during the initial lag period.

Cloning of cDNA sequences for an Artemia salina hnRNP protein: evidence for conservation through evolution.

A cDNA clone was isolated for Artemia salina protein HD40, a component of heterogenous nuclear ribonucleoproteins. Enriched Artemia 15S poly(A)+ RNA was used as a template and double-stranded cDNA sequences were inserted into the Pst I restriction endonuclease site of E. coli plasmid pBR322.

Isolation of a minor species of actin from the nuclei of Acanthamoeba castellanii.

Actin was extracted from isolated nuclei of Acanthamoeba castellanii and purified to homogeneity under nondenaturing conditions by diethylaminoethylcellulose and Sephadex G-100 chromatography. The pure protein has the same molecular weight as cytoplasmic Acanthamoeba actin and a very similar amino acid composition. Isoelectrofocusing shows that nuclear actin is slightly more acidic than the major cytoplasmic species, and comparative analysis of peptides from tryptic and cyanogen bromide digests shows that both actins are very similar but not chemically identical.

Model investigations on the structure of heterogeneous nuclear ribonucleoproteins.

Protein HD40 , an RNA-helix destabilizing protein (Mr 40 000) is the major component of 30S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from Artemia salina. The physical properties and the amino acid composition of HD40 are analogous to those of a group of well conserved core hnRNP proteins of higher eukaryotes. HD40 binds to and disrupts the secondary structure of single-stranded polynucleotides and forms extended nucleoprotein filaments at a stoichiometry of one protein per 12-15 nucleotides.

Structure of complexes between a major protein of heterogeneous nuclear ribonucleoprotein particles and polyribonucleotides.

We have investigated the structure of complexes formed between a series of poly(A)n (n = 30 to 480) and HD40 (helix-destabilizing protein, molecular weight of 40,000), the major protein component of 30 S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from the brine shrimp Artemia salina. Protein HD40 is similar to corresponding hnRNP proteins from higher eukaryotes and the complexes it forms with single-stranded nucleic acids are strikingly similar to the native "beads-on-a-string" structure of hnRNP. Using analytical ultracentrifugation and electron microscopy we find: (1) complexes formed between HD40 and long ribohomopolymers also have a beads-on-a-string structure, showing that the ability to form this structure is an inherent property of HD40, and is not dependent on any structural features of natural RNA; (2) complexes between HD40 and poly(A)160 form disks that are about 3 nm high by 18 nm in diameter and contain 20 HD40 molecules; (3) complexes of HD40 with poly(A)n with fewer than 160 nucleotides form sectors of a disk: 40 nucleotides give rise to a quarter of a disk, 80 nucleotides, half a disk, etc.

The effects of bisulfite on growth and macromolecular synthesis in Escherichia coli.

Bisulfite reversibly inhibits the growth of a variety of microorganisms and has been used as a preservative in foods and beverages for that reason. We have now measured macromolecule synthesis in Escherichia coli K12 after bisulfite treatment. RNA synthesis, the synthesis of total protein, and of an inducible enzyme, beta-galactosidase, stopped almost immediately upon addition of 2 mM (or higher concentrations) of bisulfite.

Reversal of growth inhibition in Escherichia coli by bisulfite.

Bisulfite inhibits growth, protein synthesis and RNA synthesis in micro-organisms. These inhibitory effects, in Escherichia coli K12, were diminished when certain amino acids were added to the minimal growth medium. The effects of individual amino acids were additive, synergistic and independent of their concentration.

Nucleic acid binding properties of major proteins from the heterogeneous nuclear ribonucleoproteins of wheat.

Glycine-rich core hnRNP proteins purified from wheat bind tightly to single-stranded but not to double-stranded nucleic acids with a preference for natural RNA over single-stranded DNA. Binding results in i) a progressive disruption of the residual secondary structure of the polynucleotide and the formation of an extended nucleoprotein filament until a protein to polynucleotide weight ratio of about 5:1 is attained. As more protein is added, this is followed by ii) the formation of globular structures along the polynucleotide chain with a concomitant reduction in the contour length of the nucleoprotein complex.

A RNA helix-destabilizing protein is a major component of Artemia salina nuclear ribonucleoproteins.

A major component of 30S heterogeneous nuclear ribonucleoprotein (hnRNP) particles from Artemia salina is HD40, a protein that has been characterized as a RNA helix-destabilizing protein [Marvil, D. K., Nowak, L.

A single-stranded nucleic acid-binding protein from Artemia salina. II. Interaction with nucleic acids.

A helix-destabilizing protein, HD40 (Mr 40,000), isolated from the cytoplasm of Artemia salina (Marvil, D.K., Nowak, L.

A single-stranded nucleic acid-binding protein from Artemia salina. I. Purification and characterization.

A protein that binds tightly to single-stranded but not to double-strained nucleic acids has been purified to homogeneity from a high salt wash of ribosomes from cryptobiotic Artemia saline gastrulae. The protein, designated HD40 to indicate a helix-destabilizing protein with a molecular weight of 40,000, is present in the high-salt ribosomal wash at a level of about 2 molecules per 80 S ribosome. The protein is monomeric at salt concentrations from 0.

Nucleic acid binding and unfolding properties of ribosomal protein S1 and the derivatives S1-F1 and m1-S1.

The nucleic acid binding and unwinding properties of wild-type Escherichia coli ribosomal protein S1 have been compared to those of a mutant form and a large trypsin-resistant fragment, both reported recently [J. Mol. Biol.

Interactions of 4', 6-diamidine-2-phenylindole with synthetic polynucleotides.

4', 6-Diamidine-2-phenylindole forms fluorescent complexes with synthetic DNA duplexes containing AT, AU and IC base pairs; no fluorescent complexes were observed with duplexes containing GC base pairs or with duplexes containing a single AT base pair sandwiched between GC pairs. The binding site size is one molecule of dye per 3 base pairs. The intrinsic binding constants are higher for alternating sequence duplexes than for the corresponding homopolymer pairs.