Direct covalent mercuration of nucleotides and polynucleotides

Dale, R.M.K.; Martin, Eric W.; Livingston, D. C.; Ward, David C.

doi:10.1021/bi00682a027

Cited by 196 publications

(74 citation statements)

References 23 publications

(23 reference statements)

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“…nevertheless is still completely adsorbed to agarose containing inercapto groups and thus represents a reagent specific for multiple thiol groups. These findings parallel observations made on mercurated polynucleotides and their tliiol complexes [41].…”

Section: Propcrtics O/'thc Sirhstituted Dcutrunrsupporting

confidence: 89%

Reagents Specific for Cell Surface Components

Pitha

1978

European Journal of Biochemistry

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Mercury, diazonium ions and dyes which bind nucleic acids were covalently linked to dextrans using methods that resulted in non-hydrolyzable reagent-dextran bonds without impairing the binding abilities of the reagents. i.e. these dextran derivatives reacted with thiols, phenols/ imidazoles and nucleic acids respectively. Since these dextran derivatives cannot penetrate into cells and since dextran itself does not bind to cells. these compounds represent reagents specific for the cell surface. They may be used both to evaluate cell surface constituents of intact cells and to affect viable cells via an interaction with those constituents. Mercury-dextran was found to bind to cells; the amount of mercury thus attached to the cells was about ten times smaller than when an equivalent concentration of free mercury ions was used. Mercury-dextran, bound to cells after a 30-min exposure at room temperature, was localized on the surface of these cells, as sodium borohydride reduced this complex giving rise to the intact cells, elementary mercury and free dextran which was released into medium. When cells were constantly exposed to the mercury-dextran. its toxic effects were comparable to that of free mercury ions. Diazonium-dextran, which also binds tightly to the cell surface, was also considerably toxic. Dextrans substituted with dyes which bind to nucleic acids were less toxic than the parent dyes themselves; it was shown that the attachment of such a dye to dextran decreased the binding of dye to cells under detection limits.The cell surface plays an important role in the regulation of cell growth and morphology and is a site for the action of some drugs and hormones [l -31. It consists of a lipid bilayer containing protein and carbohydrate complexes [4-71. whose composition varies with different types of cells and changes when cells are transformed [8,9]. A number of organic compounds of small molecular weight have been developed which react predominantly with only one chemical component on the cell surface. The potential of such specific reagents for membrane research has not yet been fully realized as these reagents tend to diffuse into cells and react with intracellular cell components. Diffusion may be partially prevented by the introduction of a negative charge into the reagent, or alternatively the reagent may be generated by a non-permeating system, e.g. radioiodination Neither approach eliminates complications : after radioiodination of lymphocytes as much as one-fifth of the label was found inside the cells [ I l l . In this work I approach the problem by covalently attaching a specific reagent to a soluble macromolecule which can neither penetrate into nor be absorbed by cells. This type of reagent modification should lead to a better quantification of cell surface components as well to the possibility of selectively influencing chemical groups on the surface of living cells and studying the consequences. The basic idea for this work stems from the study of the binding of living cells to proteins or hormon...

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Section: Propcrtics O/'thc Sirhstituted Dcutrunrsupporting

confidence: 89%

Reagents Specific for Cell Surface Components

Pitha

1978

European Journal of Biochemistry

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“…A 1.8-kb Sau3A fragment ,of the sequence, which is organized in long tandem arrays with a copy number of 3000 per haploid genome was cloned in the plasmid vector pUC8. Plasmid DNAs containing these inserts were purified [18] and were either nick-translated with biotin-11-dUTP [19] or mercurated by incubation with mercury acetate [20,21]. In the following we refer to pUC 1.77 as probe lc and to the plasmid containing the 1.8-kb Sau3A fragment as probe 15~.…”

Section: -898333mentioning

confidence: 99%

Double in situ hybridization in combination with digital image analysis: A new approach to study interphase chromosome topography

Emmerich

Loos

Jauch

et al. 1989

Experimental Cell Research

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Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a ZD-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the ZD-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far.

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“…Newly synthesized RNA made by isolated nuclei in vitro in the presence of a mercurinucleotide (5,6) and an appropriate mercaptan was purified free of endogenous RNA by affinity chromatography (7) * To whom all correspondence should be addressed. 2475 with 1.5% (vol/vol) dimethyl sulfoxide as previously described (4).…”

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confidence: 99%

Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: direct evidence for increased transcription during erythroid differentiation.

Orkin¹,

Swerdlow²

1977

Proc. Natl. Acad. Sci. U.S.A.

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Murine erythroleukemic cells accumulate cytoplasmic globin mRNA during differentiation induced in tissue culture by dimethyl sulfoxide. Cellular accumulation of globin RNA may reflect transcriptional activation of the globin genes and/or posttranscriptional stabilization of globin RNA during differentiation. To evaluate possible transcriptional controls directly, globin RNA synthesis by isolated erythroleukemic cell nuclei was studied.Conditions were established for optimal nuclear RNA synthesis in vitro in the presence of a mercurinucleotide (Hg-CTP Isolation of Nuclei. Washed cell pellets were suspended in 0.3 M sucrose (Schwarz/Mann ultrapure) containing 2 mM Mg(OAc)2, 3 mM CaCI2, 10 mM Tris-HCl at pH 8.0, and 2 mM 2-mercaptoethanol. After addition of Triton X-100 to 0.25%, the cell suspension was stirred on a vortex mixer for 30 sec. The crude nuclei were gently sedimented at 1000 X g for 5 min at 40, and then resuspended in the same buffer minus Triton X-100. An equal volume of 2 M sucrose containing 5 mM Mg(OAc)2, 10 mM Tris at pH 8.0, and 2 mM mercaptoethanol was immediately added, and the mixture was homogenized with five strokes of a tight-fitting Teflon-glass homogenizer. Purified nuclei were sedimented through a cushion of 2 M sucrose containing 2 mM Mg(OAc)2, 3 mM CaCl2, and 10 mM Tris at pH 8.0 by centrifugation at 25,000 rpm in a Beckman SW-40 rotor for 45 min at 40. The nuclear pellet was resuspended in 25% (vol/vol) glycerol/5 mM Mg(OAc)2/50 mM Tris at pH 8.0/2 mM mercaptoethanol at 1 to 2 X 108 nuclei per ml, and frozen in liquid nitrogen.RNA Synthesis by Isolated Nuclei. Conditions for RNA synthesis were modified slightly from those described for other systems (8, 9). Each reaction mixture contained 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) at pH 8.0, 5 mM Mg(OAc)2, 1 mM MnCl2, 150 mM KCI, 12.5% glycerol, 4 mM phosphoenolpyruvate, pyruvate kinase (3 units/ml), 0.185 mM of three ribonucleoside triphosphates, 0.037 mM of the fourth ribonucleoside triphosphate labeled with 3H, and approximately 5 X 107 rapidly thawed nuclei per ml. Mercurinucleotide, either Hg-CTP or Hg-UTP (P.L. Biochemicals), replaced the corresponding unmodified nucleotide at the same concentration in the reaction mixture when RNA containing mercury (Hg-RNA) was prepared. Mercaptans, as noted below, were present at 10 mM final concentration in the reaction mixture. The reaction mixture was incubated at 230 with intermittent, gentle shaking and aliquots were withdrawn at appropriate times, spotted on Whatman 3MM paper, and processed as described by Ernest et al. (9) to monitor RNA synthesis. No adjustment was made for background cpm at time zero.Preparative Isolation of RNA after Nuclear Synthesis. After incubation, nuclei were diluted 10-fold with 20 mM Tris at pH 7.5/50 mM NaCl/1 mM EDTA/1% sodium dodecyl sulfate and extracted vigorously in a vortex mixer with an equal volume of 600 phenol previously equilibrated with Tris buffer. The aqueous phase was reextracted three times with chloroform/is...

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Direct covalent mercuration of nucleotides and polynucleotides

Cited by 196 publications

References 23 publications

Reagents Specific for Cell Surface Components

Reagents Specific for Cell Surface Components

Double in situ hybridization in combination with digital image analysis: A new approach to study interphase chromosome topography

Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: direct evidence for increased transcription during erythroid differentiation.

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