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S Shuman

Publications and source records attributed to S Shuman.

At least 199 records · Page 11Linked to original sources
PubMed1988

Factor-dependent transcription termination by vaccinia virus RNA polymerase. Evidence that the cis-acting termination signal is in nascent RNA.

Transcription termination in vitro by vaccinia RNA polymerase is dependent on a trans-acting factor, VTF, that is associated with, if not identical to, the vaccinia mRNA capping enzyme. VTF-induced termination occurs approximately 50 nucleotides downstream of a signal sequence TTTTTNT in the non-transcribed templated strand; thus the cognate sequence UUUUUNU is expressed in the nascent RNA. To address the role of the nascent RNA in chain termination, the effects of nucleotide base analog substitutions were studied. Incorporation of bromo- (Br) UMP or iodo- (I) UMP into RNA abrogated factor-dependent termination without preventing the synthesis of read-through transcripts. Substitution of either ITP or 7'-methylguanosine for GTP did not inhibit factor-dependent termination, nor did the substitution of BrCTP or ICTP for CTP. The early transcripts synthesized in vitro were sensitive to RNase T2 but resistant to RNase H, indicating an absence of extensive hybridization of RNA product to the DNA template. Substitution of BrUTP for UTP did not alter the nuclease sensitivity of the transcripts, suggesting that increased stability of RNA:DNA hybrid structures did not account for the analog effects. These results are consistent with a model in which recognition of the primary sequence UUUUUNU in nascent RNA by the polymerase and/or VTF is required for transcription termination.

Base Sequence↗
PubMed1988

Regulation of myelin P0 glycoprotein synthesis in cultured rat Schwann cells and continuous rat PNS cell lines.

We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2'-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyryl cyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.

Animals↗
PubMed1988

A cyclic AMP analogue induces synthesis of a myelin-specific glycoprotein by cultured Schwann cells.

Neonatal rat Schwann cells, cultured with agents which increase intracellular cyclic AMP, were prompted to resume synthesis of a 170,000 Mr glycoprotein which is specific to peripheral nervous system myelin and is herein referred to as P170K. We have shown previously that similar treatment induces the synthesis by Schwann cells of the myelin lipid, galactocerebroside. In contrast to P170K and galactocerebroside, syntheses of P0 and myelin basic protein were not induced. Intracellular cyclic AMP is thus likely to be a participant in the complex system regulating myelination.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Purification and characterization of a transcription termination factor from vaccinia virions.

A DNA-dependent RNA polymerase that transcribes vaccinia virus early genes was partially purified from virus cores by deoxycholate extraction and DEAE-cellulose column chromatography. Accurately initiated and terminated RNAs were synthesized by this enzyme in the presence of a linear duplex DNA template. Glycerol gradient sedimentation resolved the in vitro transcription system into two components: fraction I, a rapidly sedimenting RNA polymerase that initiated transcription at an early promoter but transcribed beyond the in vivo 3' terminus to yield a run-off transcript, and fraction II, a more slowly sedimenting fraction, itself devoid of RNA polymerase, that restored efficient termination when added back to fraction I. The termination factor was heat-labile, resistant to N-ethylmaleimide, and did not exhibit endonucleolytic activity on run-off transcripts. Factor-dependent termination required specific sequence information upstream of the site of termination. The vaccinia termination factor was purified extensively by column chromatography on DEAE-cellulose, heparin-agarose, phosphocellulose, and DNA-agarose, and by velocity sedimentation in a glycerol gradient. At each step, termination factor copurified with the vaccinia mRNA capping enzyme. The preparation was well over 90% pure with respect to the latter enzyme, suggesting that termination activity was tightly associated with, if not intrinsic to, the capping enzyme. Nonetheless, formation of the 5'-cap structure did not appear to be a prerequisite for termination.

Adenine Nucleotides↗
PubMed1987

Complete sequence of the HLA DQ alpha and DQ beta cDNA from a DR5/DQw3 cell line.

The HLA-D region is composed of three subregions termed DR, DQ, and DP. We previously reported the sequence of a DR5 beta I and two DR5 beta III cDNA from the DR5 cell line Swei. We now report on the nucleotide and deduced amino acid sequence of the DQ alpha and DQ beta cDNA from the same DR5 cell line, which also types as DQw3. Comparison with other available DQ sequences indicates that DQ alpha has one region of major variability, whereas DQ beta appears to have four regions of variability. In addition, these comparisons indicate that DQw3 alpha from DR5 is different from DQw3 alpha from DR4, but identical to DQw2 alpha from DR3. In contrast, DQw3 beta from DR5 is very similar to DQw3 beta from DR4. These data indicate that at least for DQw2 and DQw3 it is the DQ beta chain that is responsible for DQ typing. Most sequence differences in DQ alleles can be attributed to point mutations; however, codon additions/deletions in the DQ alpha chain may contribute to variability. In addition, regions of possible gene conversion in the DQ alpha and DQ beta chains is suggested by the presence of a chi-like sequence in each chain. Finally, comparison of available haplotypes suggest recombination events may take place between DQ beta and DQ alpha, between DQ alpha and DR beta I, and between DR beta I and DR beta III.

Amino Acid Sequence↗
PubMed1987

Compression neuropathies.

Nerve compression, frequently from entrapment at specific anatomic locations, is a common clinical entity, especially in patients with predisposing occupations or with certain medical disorders. It should be pointed out that a nerve can be compressed at more than one level. Early recognition and treatment are the most effective means of preventing long-lasting disability, since, despite the capacity for regeneration bestowed on the peripheral nervous system, functions lost as a result of denervation are never fully restored.

Animals↗
PubMed1987

Identification of a vaccinia virus gene encoding a type I DNA topoisomerase.

Vaccinia virus encapsidates a type I DNA topoisomerase (EC 5.99.1.2). The enzyme was purified from virus cores to apparent homogeneity, yielding a protein of Mr 32,000. The amino-terminal sequence of the isolated Mr 32,000 polypeptide was determined and used to map the putative structural gene for the vaccinia topoisomerase to the H7r open reading frame of the vaccinia genome. This gene encodes a 314-amino acid polypeptide containing a region homologous to a region of the type I topoisomerase from the yeast Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1986

Characterization of two distinct DR beta chain alleles at the beta III locus of the DR5 haplotype: beta III alleles are highly conserved.

The HLA-DR beta region of at least two members of the DRw52 (MT2) supertypic group (DR3, DR5, DRw6, and DRw8) has recently been shown to contain three beta-chain genes--beta I, beta II, and beta III, ordered in the direction of transcription. beta III is apparently a duplication of beta I. Both beta I and beta III are expressed, whereas beta II is a pseudogene. We previously reported the sequence of the DR5 beta I cDNA from the homozygous DR5 cell line Swei. We report here the sequence of two different DR5 beta III cDNA from the same cell line. The assignment of the genes of this and other members of the DRw52 supertypic group to specific loci allow comparisons between products of known alleles and between products of distinct loci. beta I allelic products showed approximately 11% divergence in the first domain, whereas beta I and beta III products showed 17% difference. These differences were clustered and found predominantly in the previously described variable regions (amino acid residues 9 through 13, 26 through 38, and 67 through 74). These data, coupled with the finding of shared variable regions among different DR beta chains, suggest gene conversion as a means of generating polymorphism in the beta I alleles. In contrast, the beta III allelic products showed less than 1% amino acid and nucleotide divergence in the first domain. These differences were outside the variable regions, and attributable to single nucleotide changes. The polymorphism at the beta I locus and the conservation at the beta III locus suggest selective pressure conserving the beta III locus and/or generating polymorphism at the beta I locus, and further suggest that gene conversion may be acting unidirectionally from beta III to beta I.

Alleles↗
PubMed1986

Schwann cell responses to cyclic AMP: proliferation, change in shape, and appearance of surface galactocerebroside.

Surface galactocerebroside (galC) was induced on cultured Schwann cells by two analogues of cyclic adenosine 3',5'-monophosphate (cAMP), dibutyryl cAMP and 8-bromo cAMP (as previously reported by Sobue and Pleasure) and also by forskolin, a potent adenylate cyclase activator. These reagents also induced a morphological transition of many of the Schwann cells, from an elongated spindle shape to flattened cells extending fenestrated cytoplasmic sheets. Surface galC and these changes in Schwann cell shape were not elicited by raising the extracellular cAMP concentration, nor by many compounds known to promote the differentiation of other cell types, suggesting that intracellular cAMP is the unique signal for their induction. The cAMP analogues also induced Schwann cell proliferation (as previously reported by Raff et al.), as did forskolin. The concentrations of cAMP analogues and forskolin eliciting largest increases in numbers of Schwann cells in the cultures were 10-fold lower than the concentrations required for optimal induction of Schwann cell surface galC.

Animals↗
PubMed1986

Immunochemical characterization of peripheral nervous system myelin 170,000-Mr glycoprotein.

A recently described 170,000-Mr glycoprotein, specific to peripheral nervous system (PNS) myelin, was purified from rat PNS myelin by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to immunize guinea pigs and rabbits. The resultant antisera proved specific for 170,000-Mr glycoprotein by enzyme-linked immunosorbent assay, by immunoprecipitation of the appropriate peptide from solubilized PNS myelin, and by immunoblot analysis of rat PNS myelin. The anti-rat 170,000-Mr glycoprotein antisera cross-reacted with proteins of similar molecular weight in human and bovine PNS myelin, but such proteins were not detected in human or rat CNS myelin or other rat tissues. The 170,000-Mr glycoprotein was also detected by this immunoblot procedure in recently isolated rat Schwann cells but not in those kept in culture for greater than or equal to 3 days. By indirect immunofluorescent microscopy, anti-rat 170,000-Mr glycoprotein antibody bound to rat PNS myelin sheaths but not to other rat tissues. Together, these studies indicate the 170,000-Mr glycoprotein is specific to PNS myelin of several species and that a neuronal influence may be required for its expression by Schwann cells.

Animals↗
PubMed1985

Tissue culture studies of Schwann cell proliferation and differentiation.

Neonatal rat sciatic nerve Schwann cells in monolayer culture are stimulated to proliferate and to express a lipid and a protein characteristic of myelin by agents which raise intracellular cyclic adenosine 3',5'-monophosphate. Both glial growth factor and axolemmal fragments increase the rate of mitosis of cultured rat and human Schwann cells. Rat Schwann cell mitosis is enhanced by a soluble factor produced by concanavalin A-stimulated blood mononuclear cells and inhibited by lead salts. Schwann-like cells cultured from human dermal and plexiform neurofibromas resemble normal human Schwann cells in phenotype and response to mitogens.

Animals↗
PubMed1983

Peripheral nervous system myelin and Schwann cell glycoproteins: identification by lectin binding and partial purification of a peripheral nervous system myelin-specific 170,000 molecular weight glycoprotein.

Radioiodinated lectins were used to detect glycoproteins of peripheral nervous system (PNS) myelin (rat, human, bovine) and cultured rat Schwann cells. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and transferred to nitrocellulose filters. The filters were overlaid with radioiodinated lectins of known saccharide affinities. These included concanavalin A, Helix pomatia, Limulus polyphemus, Maclura pomifera, peanut, soybean, Ulex europaeus, and wheat germ agglutinins. Inclusion of the appropriate monosaccharide in the overlay solution (0.2 M) inhibited lectin binding to the nitrocellulose-fixed proteins. Fluorography permitted identification of 26 myelin glycoproteins and many more in Schwann cells. All lectins labeled a band present in myelin, but not Schwann cells, corresponding to the major PNS myelin protein, P0. Our attention focused on a high-molecular-weight myelin glycoprotein [apparent molecular weight (Mr) 170,000], which appeared abundant by Coomassie Blue staining and which was heavily labeled by all lectins except concanavalin A. A protein with approximately this Mr and lectin-binding pattern was present in human and bovine PNS myelin as well, but not detected in rat Schwann cells, CNS myelin, liver and fibroblast homogenates, or cultured bovine oligodendroglia. Hence this 170,000 Mr glycoprotein is apparently unique to PNS myelin.

Animals↗
PubMed1982

RNA capping by HeLa cell RNA guanylyltransferase. Characterization of a covalent protein-guanylate intermediate.

RNA capping by partially purified HeLa cell GTP:RNA guanylyltransferase has been shown to occur in the following sequence of two partial reactions involving a covalent protein-guanylate intermediate: (i) E(P68) + GTP in equilibrium E(P68-GMP) + PPi (ii) E(P68-GMP) + ppRNA in equilibrium GpppRNA + E(P68) Initially, the enzyme reacts with GTP in the absence of an RNA cap acceptor to form a covalent protein-guanylate complex. This complex consists of a GMP residue linked via a phosphoamide bond to a Mr = 68,000 protein. The enzyme then transfers the guanylate residue from the Mr = 68,000 polypeptide to the 5' end of diphosphate-terminated poly(a) to yield the capped derivative GpppA(pA)n. Both partial reactions have been shown to be reversible. In the reverse of Reaction i, E(P68--GMP) reacts with PPi to regenerate GTP. In the reverse of Reaction ii, the enzyme catalyzes the transfer of the 5'-GMP from capped RNA to the Mr = 68,000 protein to form protein-guanylate complex. A divalent cation is required for both partial reactions. The Mr = 68,000 protein is presumed to be a subunit of the HeLa guanylyltransferase. This interpretation is consistent with the sedimentation coefficient of 4.2 S of the native enzyme. Preliminary studies of RNA guanylyltransferase from mouse myeloma tumors suggest a similar mechanism of transguanylylation involving a Mr = 68,000 protein-guanylate complex. These data, in conjunction with previous studies of vaccinia virus guanylyltransferase (Shuman, S., and Hurwitz, J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 187-191) suggests that covalent GMP-enzyme intermediates may be a general feature of the RNA capping reaction.

Animals↗
PubMed1981

Mechanism of mRNA capping by vaccinia virus guanylyltransferase: characterization of an enzyme--guanylate intermediate.

Vaccinia virus RNA guanylyltransferase catalyzes the transfer of GMP from GTP to the 5'-triphosphate or diphosphate terminus of RNA to generate the cap structure G(5')ppp(5')N-. The guanylylation reaction consists of a series of at least two partial reactions: (i) GTP + E in equilibrium E-pG + PPi, (ii) E-pG + (p)ppNpNpN- leads to GpppNpNpN- + E. Inthe first of these, GTP reacts with capping enzyme in the absence of an RNA acceptor to form a covalent enzyme-guanylate intermediate. The GMP is linked to the Mr 95,000 subunit of the capping enzyme via a phosphoamide bond, as judged by the acid-labile, alkali-stable nature of the bond and by the susceptibility of the linkage to cleavage by hydroxylamine at pH 4.75. The isolated enzyme-guanylate complex is able to transfer the guanylate moiety to triphosphate-terminated poly(A) to yield the 5' cap structure GpppA or to pyrophosphate to regenerate GTP. Both partial reactions of transguanylylation require a divalent cation.

Guanosine Monophosphate↗
PubMed1980

Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme).

A core-associated enzyme, which catalyzes a nucleotide-pyrophosphate exchange with GTP, has been purified from vaccinia virions. The enzyme requires MgCl2 for activity, has an alkaline pH optimum, and specifically utilizes GTP as the exchanging nucleotide. The enzyme does not catalyze exchange of GMP with GTP. The GTP-PPi exchange enzyme co-purifies with vaccinia capping enzyme (RNA guanylyltransferase and RNA (guanine-7-)methyltransferase) through successive chromatography steps on DEAE-cellulose, DNA-cellulose, and phosphocellulose. GTP-PPi exchange and capping activities remain physically associated during sedimentation in a glycerol gradient. Under high salt conditions (1 M NaCl), GTP-PPi exchange, capping, and methylating activities co-sediment with an RNA triphosphatase activity and a nucleoside triphosphate phosphohydrolase activity as a 6.5 S multifunctional enzyme complex which contains two major polypeptides of 96,000 and 26,000 molecular weight. The characteristics of the various enzymatic reactions catalyzed by this complex are described. The GTP-PPi exchange reaction of vaccinia guanylyltransferase affords a simple, sensitive assay for capping enzyme function. The relevance of the GTP-PPi exchange reaction to the mechanism of transguanylylation is considered.

Cations, Divalent↗