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R T Simpson

Publications and source records attributed to R T Simpson.

At least 55 records · Page 3Linked to original sources
PubMed1987

Ascorbate potentiates DNA damage by 1-methyl-1-nitrosourea in vivo and generates DNA strand breaks in vitro.

Ascorbic acid (vitamin C) is an important intracellular reducing agent. It also has been suggested to be (i) a protective agent against development of cancer, (ii) a therapeutic agent for malignancies and (iii) a mutagen. We have found that high concentrations of ascorbate leads to DNA damage in several in vivo and in vitro situations. Guinea-pigs receiving oral 1-methyl-1-nitrosourea (MNU) were used as a whole animal model. Administration of sodium ascorbate prior to MNU increased strand breakage in pancreatic DNA. Concentrations of ascorbate greater than 0.5 mM increased the frequency of DNA strand breaks caused by MNU in both L1210 murine leukemia cells and guinea-pig pancreatic cells in tissue culture; ascorbate alone led to DNA strand breaks in the latter cells. Investigations of the mechanism of DNA damage were carried out with purified DNA. Ascorbate produced single- and double-strand breaks in plasmid DNA. Cleavage was catalyzed by copper(II), inhibited by catalase and blocked by the presence of thiols. We conclude that superoxide and hydrogen peroxide produced during the oxidation of ascorbate leads to generation of hydroxyl free radicals that can mediate DNA strand scissions and potentiate the effects of alkylating carcinogens.

Animals↗
PubMed1986

Structure and developmentally regulated expression of a Strongylocentrotus purpuratus collagen gene.

We have isolated and partially characterized an ca. 20-kilobase-pair Strongylocentrotus purpuratus genomic clone, using a mouse alpha 1 (type IV) collagen cDNA probe. A 1-kilobase-pair HindIII fragment of the clone hybridizes strongly to the probe; this has been subcloned and sequenced. It contains 212 base pairs of sequence coding for (Gly-Xaa-Yaa)n (where Xaa and Yaa are different unspecified amino acids), characteristic of all known collagen genes. There is a single point of discontinuity within the repeating pattern in this exon, similar to the genomic structure of mouse type IV collagen. The (Gly-Xaa-Yaa)n-encoding element is flanked by consensus splicing sequences, and the intervening sequences on either side of it have multiple in-phase termination codons. Electron microscopy of R loops between the phage lambda recombinant clone and poly(A)+ RNA reveals multiple short exons, a feature also seen in vertebrate collagen genes. The (Gly-Xaa-Yaa)n protein-encoding sequence hybridizes to a developmentally regulated 9-kilobase mRNA; the message appears during the morula stage, rises sharply in abundance at the blastula stage, and decreases in proportion to total RNA later in development.

Animals↗
PubMed1986

Isolation of an episomal yeast gene and replication origin as chromatin.

A multicopy yeast plasmid containing the TRP1 gene (coding for N-5'-phosphoribosylanthranilate isomerase) and ARS1 (autonomously replicating sequence 1) has been purified as chromatin. Electrophoretic analysis of nucleic acid and proteins and electron microscopy show that the plasmid chromatin is largely free of contaminants. Electron-microscopic and linking-number analyses indicate that the plasmid chromatin contains seven nucleosomes, as predicted by the indirect end-label analyses of Thoma, Bergman, and Simpson [J. Mol. Biol. (1984) 177, 715-733]. Indirect end label mapping of micrococcal nuclease cuts demonstrates that nucleosome positions and nuclease-sensitive regions are not altered by the purification. The plasmid chromatin behaves homogeneously with respect to its elution from nuclei, template activity, and intrinsic buoyant density. Taken together, these observations suggest that different copies of the TRP1ARS1 plasmid do not differ from each other grossly in chromatin structure. We discuss the potential for understanding eukaryotic gene regulation offered by the ability to isolate unique genes as chromatin.

Aldose-Ketose Isomerases↗
PubMed1985

Effects of sequence alterations in a DNA segment containing the 5 S RNA gene from Lytechinus variegatus on positioning of a nucleosome core particle in vitro.

Reassociation of a 260-base pair cloned fragment of Lytechinus variegatus DNA with core histones has been shown to give rise to a uniquely positioned nucleosome (Simpson, R. T., and Stafford, D. W. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 51-55). In an attempt to define the features that dictate the unique positioning of the nucleosome, we have constructed a number of mutants of this DNA sequence. The ability of these mutants to form positioned nucleosomes was analyzed by DNase I digestion of the DNA after reassociation with chicken erythrocyte core histones. While all the mutants were efficiently incorporated into core particles, not all of these modified sequences were capable of forming a positioned nucleosome. Of the 13 mutants examined, 7 fell into a class that gave rise to nucleosomes in which no unique positioning could be demonstrated. While no specific feature of the DNA sequences has been identified as the critical factor in allowing, or dictating, the formation of positioned nucleosomes, our results do indicate that the region 20-30 bases either side of the center of the core particle appears to contain the major elements necessary for positioning. Additionally, these studies clearly show that differences in the digestion of naked and core particle DNA are related to specific interactions of the DNA and histones rather than to an altered specificity of the enzyme induced by the presence of the proteins.

Animals↗
PubMed1985

Sequence-specific interaction of histones with the simian virus 40 enhancer region in vitro.

DNA fragments containing either one or both of the 72-base pair (bp) elements which constitute the SV40 enhancer and the three adjacent 21-bp repeats were associated with histone octomers from chicken erythrocytes in vitro. Both fragments formed complexes with electrophoretic mobilities of nucleosomes containing the appropriate length of DNA. Analysis of DNase I cutting of uniquely end-labeled complexes suggests that the fragment containing a single 72-bp element forms a positioned core particle. Control experiments show that positioning is not due to the 21-bp repeats or to end effects. The fragment with a tandem repeat of the 72-bp element also does not associate randomly with histones. The data are consistent with formation of a core particle on one or the other of the repeated enhancer sequences. We discuss possible functional consequences of such nucleosome positioning.

Animals↗
PubMed1985

Transient alterations of the chromatin structure of sea urchin early histone genes during embryogenesis.

We describe features of the chromatin structure of the early histone gene family of Strongylocentrotus purpuratus during development. Before and after the histone genes are transcriptionally active, chromatin structure is quite similar with well-defined spaced nucleosomes and no major 5'-flanking sites hypersensitive to nucleases. During the period when the genes are active, marked changes in chromatin structure occur. Micrococcal nuclease digestion generates monomer nucleosomes and only trace amounts of higher multimers. Regions hypersensitive to an endogenous nuclease and DNAase I appear in the 5'-flanking regions of genes for H2A, H2B and H3. Each region consists of four sites spanning a DNA length of 200-250 base pairs. In each case, one major cutting site is near the TATA box; the bulk of the sensitive region is in the nontranscribed spacer. Other sites, in 3'-flanking regions of the genes, are sensitive to nucleases only when the histone genes are no longer transcribed.

Animals↗
PubMed1985

Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: a model system for study of higher order structure.

We describe a model system for study of chromatin structure at levels above that of the nucleosome. A series of fragments with lengths ranging from 172 to 207 bp tandemly repeated three to greater than 50 times was prepared; each repeat contains the region important in forming a positioned core particle on a sea urchin 5S rRNA gene upon in vitro association with histones. The tandemly repeated sequences can be studied as linear DNA fragments or as relaxed or supercoiled circular molecules. A number of criteria indicate that nucleosomes position correctly on all the tandemly repeated elements. Measurement of the change in linking number per core particle led to a value of -1.0. Both length and repeat number dependent changes in conformation of the nucleoproteins are observed. We discuss the possibility that some ordered higher level chromatin structure can form with DNA and core histones alone.

Animals↗
PubMed1984

Nuclease digestion of circular TRP1ARS1 chromatin reveals positioned nucleosomes separated by nuclease-sensitive regions.

TRP1ARS1 is a circular yeast DNA of 1453 base-pairs that contains the N-5'phosphoribosyl anthranilate isomerase (TRP1) gene and a sequence important for autonomous replication (ARS1). It exists extrachromosomally in 100 to 200 copies/cell and is presumably packed in nucleosomes. TRP1ARS1 has been partially purified as chromatin from lysed spheroplasts of yeast using gel filtration. A structural analysis of mapping micrococcal nuclease and DNAase I cutting sites with an accuracy of +/- 20 base-pairs is presented. Comparison of nuclease cleavage sites in chromatin and in purified DNA reveals that regions which are protected against nuclease attack are not distributed randomly. These regions are big enough to accommodate nucleosome cores. Three nucleosomes are positioned in the so-called ARS sequences, and are stable at low and high levels of digestion. The TRP1 gene region is covered by four nucleosomes, but they are neither randomly arranged nor precisely positioned. They are not stable and rearrange or disintegrate during digestion. The nucleosomal regions are separated by two segments of DNA (A, B), each about 180 base-pairs long, which are very sensitive to DNAase I and micrococcal nuclease and therefore presumably not packed in nucleosomes. Region B is found 5' to the TRP1 gene and might be related to transcription, whereas region A is centered around the termination codon of the TRP1 gene and the putative origin of replication.

Aldose-Ketose Isomerases↗
PubMed1983

Structural features of a phased nucleosome core particle.

Chicken erythrocyte inner histones associate with a cloned 260-base-pair (bp) segment of Lytechinus variegatus DNA in a unique location. The fragment contains a 120-bp segment encoding 5S rRNA, a 90-bp flanking sequence to the 5' side of the transcribed segment, and a 50-bp downstream flanking sequence. Association of DNA, uniquely labeled at one end or the other and at either the 3' or the 5' terminus of a given strand, with histones at 0.1 M ionic strength leads to formation of a compact complex which sediments at about 13 S. Analysis of cutting of the complex by DNase I shows that protection from the nuclease is confined to a region beginning 20 bp from the left end of the segment and extending to about 165 bp from the left end. Within the protected region, the two DNA strands differ in their susceptibilities to the nuclease, the precise location of nuclease cutting sites and the spacing between these sites, and the relative susceptibilities of specific cutting locations. It seems that information present in DNA and the histone octomer is sufficient to create a precisely phased nucleosome in which interactions of the two DNA strands with histones are not the same. The structure of this unique nucleosome is not predicted by the intellectual model based on studies of mixed populations of nucleosome core particles.

Animals↗
PubMed1981

Modulation of nucleosome structure by histone subtypes in sea urchin embryos.

Switches of the types of histones synthesized and incorporated into chromatin occur during sea urchin embryogenesis. In an attempt to define the possible effects of these variant histones on chromatin structure, I have isolated and characterized nucleosome core particles from Strongylocentrotus purpuratus blastula (nearly 100% early histones) and pluteus (75% late histones). Both particles contain 146-base-pair lengths of DNA wrapped around an octamer of H2A, H2B, H3, and H4. Although sharing these similarities with the canonical core particle, the nucleosome structures have certain features that differ from those of typical adult tissues. Both the reversible and the irreversible conformational transitions occurring on heating core particles are destabilized in the embryonic particles vs. "typical" core particles. The blastula core particle unfolds more easily than pluteus (or other) nucleosomes under the stress of low ionic strength. The rate of DNase I digestion of pluteus core particles is about half that of particles from blastula; certain cutting sites differ in their susceptibility between the two embryonic particles and between these two and the canonical core particle. The data demonstrate that the variant histones synthesized during early embryogenesis have demonstrable effects on chromatin structure, even at this basic level.

Animals↗
PubMed1980

Structure of sea urchin sperm chromatin core particle.

Sea urchin sperm chromatin contains forms of H1, H2A, and H2B which differ from those present in adult tissues. We have delineated some effects of the variant H2A and H2B on chromatin by study of the structure of the core particle from Strongylocentrotus purpuratus sperm. The particle contains 145 base pairs of DNA and equal amounts of the four smaller histones. It sediments at 11 S and has a circular dichroism spectrum similar to that of particles containing more typical histones. The sperm core particle undergoes a shape change at low ionic strength, as observed for chicken erythrocyte particles. In contrast to these similarities, the melting profile of the sperm particle is quite different from that of erythrocyte; both the reversible transition and the irreversible denaturation of the core particle occur at higher temperatures. The sperm core particle is digested by DNase I more slowly than the core particle from chicken erythrocyte. Cutting site maps for the sperm core particle reveal the same basic organization of DNA by these histones; however, certain features of the map differ significantly from that for chicken erythrocyte, demonstrating a modulation of the canonical core particle structure by the unusual histones present in sea urchin sperm.

Amino Acids↗
PubMed1980

Conformation of 145 base pair length poly (dG-dC) . poly (dG-dC) in solution and in association with histones.

We have studied the conformation of poly (dG-dC) . poly (dG-dC) in three conditions; i) associated with histones octamers, ii) alone at ionic strength 0.1, and ii) in solutions of over 2.5 M NaCl. The circular dichroism spectrum for the polymer bound to histones differs from that for the free polymer; the difference spectrum is similar to those for native and poly (dA-dT) . poly (dA-dT) core particles. Under the first two conditions, the 31P NMR spectrum is symmetric with line widths of 91 and 41 Hz, respectively, at 109.3 MHz. In high salt, two 31P peaks of equal intensity are observed, confirming recent results of Patel et al. (1) and indicating an alternating geometry for the phosphodiester backbone. Using this highly homogeneous DNA, we confirm that the Pohl-Jovin transition (2) is an intramolecular rearrangement, not requiring complete strand separation.

Circular Dichroism↗
PubMed1979

Mechanism of a reversible, thermally induced conformational change in chromatin core particles.

The well separated transitions observed on heating synthetic core particles containing poly(dA-dT).poly(dA-dT) have allowed physicochemical studies to define the mechanism of the first, reversible transition. The shape of the particle, assessed by sedimentation velocity measurements, becomes extended at temperatures about 10 degrees C below the optical melting temperature; this change is unaffected by cross-linking the histones into a stable octamer. A portion of the DNA undergoes a transition to a structure more like that of protein-free poly(dA-dT).poly(dA-dT), as judged by circular dichroism measurements, also at temperatures below those necessary for optical melting. Digestion of 5'-32P-labeled particles with S1 nuclease at a temperature partly through the first optical transition demonstrates that the initial phase in melting core particles is separation of about 20 base pairs of DNA at each end of the particle segment from the histone core. Implications of this mechanism for transcription of histone-bound DNA segments in chromatin are discussed.

Chromatin↗