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M O Olson

Publications and source records attributed to M O Olson.

At least 55 records · Page 3Linked to original sources
PubMed1988

cDNA and deduced primary structure of rat protein B23, a nucleolar protein containing highly conserved sequences.

Protein B23 (Mr/pI = 38,000/5.1) is a major RNA-associated nucleolar phosphoprotein which contains highly acidic segments and has a high affinity for silver ions. Using synthetic oligonucleotides as probes cloned cDNAs encoding protein B23 were isolated and characterized. One of the cDNAs, obtained from a rat brain library, contained an insert of 1232 base pairs of DNA encoding a polypeptide of 292 amino acid residues. Segments of the protein sequence were confirmed by partial sequencing of CNBr fragments from rat hepatoma protein B23. The protein contains a methionine-rich amino-terminal sequence and two highly acidic segments in the center of the sequence. The first acidic segment, in which 11 of the 13 residues are acidic, begins at residue 120 and contains a major phosphorylation site. In the second segment (residues 159-187) there are four copies of the sequence Asp-Asp-Glu, and all but two of the 29 residues have acidic side chains. When the sequence of the rat protein was compared with available sequences from other species a high degree of conservation was found; the 77-residue carboxyl-terminal sequence is identical with that of human protein B23 (Chan, P. K., Chan, W.-Y., Yung, B. Y. M., Cook, R. G., Aldrich, M. B., Ku, D., Goldknopf, I. L., and Busch, H. (1986) J. Biol. Chem. 261, 14335-24341), and about 63% of the residues are identical when the rat B23 sequence is compared with protein N038 from Xenopus laevis (Schmidt-Zachmann, M. S., Hügle-Dörr, B., and Franke, W. (1987) EMBO J. 6, 1881-1890). Except for the presence of highly acidic regions no significant similarities were found with protein C23 (nucleolin), the other major nucleolar protein.

Amino Acid Sequence↗
PubMed1988

Immunoglobulin heavy H chain isotypes in a teleost fish.

Three mouse mAb were derived which identified different populations of the approximately 700,000-Da tetrameric Ig of channel catfish (Ictalurus punctatus). Immunoprecipitation analyses using the three mAb in various combinations showed that the catfish Ig population identified by each mAb was antigenically distinct. Each Ig population contained both classes of catfish L chains (F and G). Solid-phase binding assays indicated that the mAbs preferentially identified H chains rather than L chains. Comparative peptide mapping analyses of the H chains defined by each mAb indicated that H chains were different, although each had the same apparent mass (approximately 70,000 Da). This structural distinction was not based upon allotypic variation because the analysis of the serum from individual catfish showed the presence of each H chain in fish examined. Therefore, the different H chains represent different isotypes; each representing approximately 20% of the total serum Ig. There is at least one undefined additional H chain isotype found in the serum Ig population. Partial sequence information was obtained on the first variable framework region (FR1) of two of the three H chain isotypes. These results indicated that the variable region associated with each isotype was heterogeneous; two and sometimes three different residues were represented in the majority of positions. The sequence information also showed that the FR1 of one H chain was distinct from the other; there was less than a 50% match of the primary residues. The analyses suggest that the V region genes which code for the FR1 are differently associated with each H chain isotype. Finally, the relative levels of the three H chain isotypes were monitored during the temporal immune response of individual catfish to the dinitrophenyl hapten. These results indicated that one H chain isotype was preferentially expressed early in the immune response (approximately 3 wk) and remained the predominantly expressed isotype during the humoral immune response.

Amino Acid Sequence↗
PubMed1988

Isolation of proteins for peptide mapping, amino acid analyses, and sequencing using disulfide crosslinked polyacrylamide gels.

Conditions for recovery of small amounts of proteins (1-50 micrograms) from disulfide crosslinked polyacrylamide gels have been examined. Procedures were developed for solubilization and precipitation of Coomassie blue-stained protein bands excised from gels after electrophoretic separations. The precipitated protein was then resolubilized for use in peptide mapping, amino acid analyses, or microsequencing. The amino acid compositions of standard proteins (bovine albumin, ovalbumin, phosphorylase b, and beta-galactosidase) isolated by this method were in good agreement with the values for the corresponding conventionally purified proteins. Sequencing was done with high repetitive yield on samples of 100 pmol or below. The method has been successfully applied to several proteins and protein fragments.

Amino Acid Sequence↗
PubMed1988

Effects of anti-C23 (nucleolin) antibody on transcription of ribosomal DNA in Chironomus salivary gland cells.

Protein C23 (also called nucleolin or 100-kDa nucleolar protein) is a major nucleolar phosphoprotein involved in ribosome biogenesis. To determine the effects of protein C23 on preribosomal RNA (pre-rRNA) synthesis anti-C23 antiserum was microinjected into nuclei of Chironomus tentans salivary glands. Transcription was measured by incubation of the glands with 32P-labeled RNA precorsors followed by microdissection of nucleoli, RNA extraction, and electrophoretic analyses. Injection of the anti-C23 antibody caused a 2- to 3.5-fold stimulation of 32P incorporation into 38S pre-rRNA. No stimulation was observed in salivary glands injected with preimmune serum or antiserum preabsorbed with protein C23. The stimulatory effect was selective for pre-rRNA as indicated by the lack of stimulation of 32P incorporation into extranucleolar RNA. Injection of the antiserum produced little or no effect on pre-RNA processing as measured by the relative amounts of 32P-labeled intermediate cleavage products of pre-rRNA in stimulated versus control glands. When protein extracts of Chironomus tentans salivary gland nuclei were probed on Western blots with anti-C23 antibody the predominant cross-reacting species was a 110-kDa polypeptide which had an electrophoretic mobility similar to that of protein C23. These results suggest that protein C23 not only is involved in ribosome assembly but also plays a role in regulating the transcription of the preribosomal RNA.

Animals↗
PubMed1987

Phosphorylation of nucleolin by a nucleolar type NII protein kinase.

Nucleolin [C23 or 100 kilodaltons (kDa)] is the major nucleolar phosphorylated protein in exponentially growing Chinese hamster ovary cells. A nucleolar cyclic nucleotide independent protein kinase copurified with nucleolin in a complex which could be dissociated by hydroxyapatite chromatography. The kinase was stimulated by spermine and inhibited by heparin and presented most of the properties of nuclear casein kinase NII. Kinetic analyses showed the apparent Km value for nucleolin (7 X 10(-4) mg/mL) to be lower than those for other casein kinase II substrates such as nuclear protein HMG 14 (0.15 mg/mL), topoisomerase I (0.025 mg/mL), or topoisomerase II (0.04 mg/mL). Similarly, Vmax values were higher for nucleolin than for other substrates. Nucleolin thus appears to be a natural preferential substrate of nucleolar casein kinase NII. The kinase phosphorylated nucleolin in vitro at serine residues in a 29-kDa CNBr fragment located near the amino terminus of the molecule. The enzyme labeled typical casein kinase II sites. These sites were found predominantly in two highly acidic tryptic fragments designated A (residues 21-49) and C (residues 180-221) which contained serines having at least two acidic residues on their carboxyl-terminal sides. These results demonstrate the existence in the nucleolus of a type of NII protein kinase that uses a protein involved in ribosome assembly as preferential substrate.

Amino Acid Sequence↗
PubMed1987

RNA binding fragments from nucleolin contain the ribonucleoprotein consensus sequence.

Nucleolin (C23 or 100 kDa) is a major nucleolar phosphoprotein whose primary structure has recently been determined (Lapeyre, B., Bourbon, H., and Amalric, F. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1472-1476) and found to be associated with preribosomal RNA (Herrera, A. H., and Olson, M. O. J. (1986) Biochemistry 25, 6258-6263). To identify the RNA binding region of the molecule, cyanogen bromide fragments were tested for binding of 18 S and 28 S ribosomal RNA by a "Western blotting" technique. Fragments with apparent molecular masses of 13, 33, and 47 kDa bound RNA with no preference for either 18 S or 28 S RNA. By protein sequencing, these fragments were localized in the carboxyl-terminal two-thirds of the molecule. The nucleolin sequence was searched for the ribonucleoprotein consensus sequence found in other RNA binding proteins. Four copies of a closely related 11-residue sequence were found within 80-90 residue repeats in the RNA binding region between residues 285 and 629. These results suggest that a highly conserved structure for the binding of different classes of RNA is utilized by several proteins.

Amino Acid Sequence↗
PubMed1986

Association of protein C23 with rapidly labeled nucleolar RNA.

The association of nucleolar phosphoprotein C23 with preribosomal ribonucleoprotein (RNP) particles was examined in Novikoff hepatoma nucleoli. RNA was labeled with [3H]uridine for various times in cell suspensions, and RNP particles were extracted from isolated nucleoli and fractionated by sucrose gradient ultracentrifugation. The majority of protein C23 cosedimented with fractions containing rapidly labeled RNA (RL fraction). To determine whether there was a direct association of RNA with protein C23, the RL fraction was exposed to ultraviolet (UV) light (254 nm) for short periods of time. After 2 min of exposure there was a 50% decrease in C23 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, with no significant further decrease at longer times. When UV-treated fractions were subjected to phenol/chloroform extractions, as much as 30% of the labeled RNA was found in the phenol (protein) layer, indicating that RNA became cross-linked to protein. Similarly, there was an increase in protein C23 extracted into the water layer after irradiation. By SDS-PAGE analyses the cross-linked species migrated more slowly than protein C23, appearing as a smear detected either by [3H]uridine radioactivity or by anti-C23 antibody. With anti-C23 antibodies, up to 25% of the labeled RNA was precipitated from the RL fraction. Dot-blot hybridizations, using cloned rDNA fragments as probes, indicated that the RNA in the RL fraction and the immunoprecipitated RNA contained sequences from 18S and 28S ribosomal RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

A Streptococcus mutans superoxide dismutase that is active with either manganese or iron as a cofactor.

The superoxide dismutase produced by Streptococcus mutans OMZ176 during aerobic growth in a chemically defined medium (modified FMC) that was treated with Chelex 100 (to lower trace metal contamination) and supplemented with high purity manganese was purified (162-fold) by heat treatment, ammonium sulfate precipitation, and chromatofocusing chromatography. The superoxide dismutase produced during aerobic growth in the same medium, but without manganese and supplemented with high purity iron, was similarly purified (220-fold). The molecular masses of each holoenzyme were approximately 43,000 with a subunit mass of 20,700, indicating that the enzymes were dimers of two equally sized subunits. The superoxide dismutase from manganese-grown cells was a manganese enzyme (MnSOD) containing 1.2 atoms of manganese and 0.25 atoms of iron/subunit. The superoxide dismutase from iron-grown cells was an iron enzyme (FeSOD) containing 0.07 atoms of manganese and 0.78 atoms of iron/subunit. The amino acid compositions of the MnSOD and the FeSOD were virtually identical, and their amino-terminal sequences were identical through the first 22 amino acids. Dialysis of the FeSOD with o-phenanthroline and sodium ascorbate generated aposuperoxide dismutase with 94% loss of activity; subsequent dialysis of apoenzyme with either manganese sulfate or ferrous sulfate reconstituted activity (recoveries of 37 and 30%, respectively). Electrophoretic determination of cytoplasmic radioiron distribution indicated that (during aerobic growth) manganese prevented insertion of iron into superoxide dismutase, although the iron levels of at least two other cytoplasmic fractions were not altered by manganese. Therefore, S. mutans used the same aposuperoxide dismutase to form either FeSOD or MnSOD, depending upon which metal was available in the culture medium. Such "cambialistic" enzymes (those capable of making a cofactor substitution) may represent a previously unrecognized family of superoxide dismutases.

Aerobiosis↗
PubMed1986

Protein and cDNA sequence of a glycine-rich, dimethylarginine-containing region located near the carboxyl-terminal end of nucleolin (C23 and 100 kDa).

By a combination of protein chemistry and recombinant DNA methods a glycine-rich region was found to be located near the carboxyl terminus of the nucleolar specific phosphoprotein, nucleolin, from Novikoff hepatoma (protein C23) and Chinese hamster ovary cells (100-kDa nucleolar protein). A sequence of 192 amino acid residues was derived from partial sequences of cyanogen bromide and N-bromosuccinimide fragments of protein C23 and deduced protein sequence from Chinese hamster ovary cell 100-kDa cDNA sequences. The 66 residues sequenced by protein methods were identical to the corresponding residues deduced by DNA sequencing. The multiple residues of NG,NG-dimethylarginine (DMA) contained in the nucleolin polypeptide were found to be limited to a segment of less than 10 kDa near the carboxyl-terminal end of the protein. This segment also contained internally repeated sequences (e.g. 7 copies of the sequence Gly-Gly-Arg-Gly-Gly were found) which were unrelated to sequences closer to the amino-terminal end. Most arginine residues in this region were surrounded by 2 or 3 glycine residues and were relatively close in sequence to phenylalanine residues.

Amino Acid Sequence↗
PubMed1986

Preribosomal ribonucleoprotein particles are a major component of a nucleolar matrix fraction.

Biochemical and morphological studies were performed on Novikoff hepatoma ascites cell nucleolar matrix fractions prepared by deoxyribonuclease I digestion and high-molarity salt extractions essentially according to a published method [Berezney, R., & Buchholz, L. A. (1981) Exp. Cell Res. 20, 4995-5002]. The nucleolar matrix fraction was enriched in polypeptides of molecular mass of 28, 37.5, 40, 70, 72, 110 (protein C23), and 160 kDa, compared to the nuclear fraction in which polypeptides of molecular mass of 31, 33.5, 43.5, 46, 50, 56, and 59 kDa were predominant. About one-fourth of the protein, half of the RNA, and less than 4% of the DNA originally present in the nucleoli remained in the matrix fraction. Addition of single agents such as ethylenediaminetetraacetic acid, ribonuclease A, or mercaptoethanol during preparation had no significant effect on the polypeptide composition of the nucleolar matrix fraction. However, the combination of mercaptoethanol and ribonuclease A caused most of the RNA and protein to be removed, including protein C23 and the 160-kDa polypeptide, with polypeptides in the range of Mr 30 000-50 000 remaining. Electron microscopy of nucleolar matrix fractions revealed the presence of particles similar in size to the granular elements of nucleoli. However, when ribonuclease A and mercaptoethanol were included in the procedure, only amorphous material remained. Many proteins of nucleolar preribosomal RNP particles were also associated with the nucleolar matrix fraction. RNA from the nucleolar matrix fraction was enriched in sequences from 18S and 28S ribosomal RNA. These results indicate that preribosomal RNP particles are major constituents of a nucleolar matrix fraction prepared by the deoxyribonuclease I-high-molarity salt method.

Animals↗
PubMed1984

Immunoglobulin light chain classes in a teleost fish.

Analysis of predominant, approximately 700,000 dalton serum antibody (Ab) of the channel catfish by SDS-PAGE revealed only one molecular mass species of heavy (H) chain (approximately 70,000 daltons), whereas a marked heterogeneity in the relative mobilities of the light (L) chains was evident. Three molecular mass L chain variants of approximately 26,000, approximately 24,000, and approximately 22,000 daltons were observed. The relationships between the L chain variants were not clear until mouse monoclonal Ab (mAb) reactive with catfish immunoglobulin (Ig) were developed. Two of these mAb, designated 3F12 and 1G7, were found to independently recognize different populations of catfish Ig. In addition, when these two mAb were used in combination they immunoprecipitated greater than 95% of specifically purified catfish anti-DNP or anti-fluorescein Ab. The L chains of catfish Ig recovered from immunoabsorbent affinity matrices conjugated with either mAb 3F12 or 1G7 were found to be clearly distinct. Monoclonal Ab 3F12 reacted with a subpopulation of catfish Ig that contained the approximately 24,000 and approximately 22,000 dalton L chain variants, whereas mAb 1G7 reacted with another subpopulation of catfish Ig that contained only the approximately 26,000 dalton L chain variant. Solid phase plate binding assays with the use of mildly reduced catfish Ig H and L chains showed that both of these mAb preferentially reacted with catfish L chains. Subsequent analysis of the two antigenically distinct L chain populations by peptide mapping procedures demonstrated that these L chains were structurally different. Furthermore, analysis of the serum from individual catfish showed the presence of both L chain types in each of 25 catfish examined. These studies strongly suggest that distinct L chain classes have evolved in fish.

Animals↗
PubMed1983

Interaction of nucleolar phosphoprotein C23 with cloned segments of rat ribosomal deoxyribonucleic acid.

Protein C23, a predominant nucleolar phosphoprotein and a putative nucleolus organizer protein, was analyzed for its general DNA binding characteristics and for its selectivity in binding plasmid DNAs containing cloned fragments of the genes that code for ribosomal RNA (rDNA). By use of nitrocellulose filter disk assays, the protein bound saturably to nuclear DNA with a relatively high affinity. Binding was maximal at low ionic strength (0-0.1 M KCl) with progressively decreasing binding at or above 0.2 M. In competition assays protein C23 showed a marked preference for linear single-stranded vs. double-stranded DNA and little or no affinity for ribosomal RNA. The relative affinities of rDNA sequences for protein C23 were determined with cloned fragments spanning 15.8 kilobases (kb) of DNA starting approximately 3.7 kb upstream from the initiation site for 45S preribosomal RNA to near the 3' end of the sequence coding for 28S RNA. Of the five linearized plasmids tested, only one (pKW1) was an effective competitor for 32P-labeled nuclear DNA. As measured by the concentration of competing DNA required to achieve 50% competition, pKW1 was approximately 20-fold more effective than the second best competitor. The DNA insert in pKW1 is a 3.5-kb sequence which is located in the nontranscribed spacer region less than 0.5 kb upstream from the initiation site for 45S preribosomal RNA. These results suggest that protein C23 has a preference for binding DNA sequences in the nontranscribed spacer of rDNA.

Animals↗
PubMed1981

Functional and structural studies on a tryptic fragment of eucaryotic elongation factor Tu from rabbit reticulocytes.

Treatment of eucaryotic elongation factor Tu (eEF-Tu; Mr 53 000) with trypsin results in cleavage of the factor at at least two sites, one and probably both of which are located near the amino-terminal end of the polypeptide chain. The products after exposure of eEF-Tu to trypsin for 2 h is a single polypeptide of 43 000 daltons (eEF-Tut) and as yet unidentified polypeptides of Mr less than or equal to 5000. The presence of high glycerol concentrations of GDP in the reaction mixture markedly retards the rate of tryptic cleavage, while GTP has little effect. When eEF-Tu is bound to eucaryotic elongation factor Ts in an eEF-T complex, it is much more resistant to the action of trypsin. The loss of factor activity during tryptic digestion (as measured by its ability to bind aminoacyl-tRNA to 80S (ribosomes) is much slower than the rate of eEF-Tut formation, and 2-h digests containing only eEF-Tut are about 30% as active as the native enzyme. However, no ribosome-dependent activity is detectable after purification of eEF-Tut by ion-exchange chromatography, followed by gel filtration. Purified eEF-Tut binds guanine nucleotides, although with diminished activity compared with that of eEF-Tu. Amino-terminal sequence analyses of eEF-Tut reveal a striking sequence homology with the functionally related factor from Escherichia coli (EF-Tu). The first four residues of eEF-Tut, Gly-Ile-Thr-Ile, are identical with the first four residues of a 37 000-dalton tryptic fragment of E. coli EF-Tu, and other homologies are evident in the first twelve amino-terminal residues of the corresponding tryptic fragments.

Amino Acid Sequence↗
PubMed1981

Sequence of a phosphorylation site in nucleolar protein B23.

The sites of phosphorylation in protein B23, a silver-staining preribosomal ribonucleoprotein particle protein, were analyzed by tryptic peptide mapping. Three 32P peptides were found using in vitro labeling of nucleoli. An additional unlabeled phosphopeptide was identified by amino acid analysis. The sequence of the latter was Asp-Thr(P)-Pro-Ala-Lys. These results suggest that protein B23 contains one class of site labeled rapidly in vitro and another type of site phosphorylated only in vivo.

Adenosine Triphosphate↗