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J Paoletti

Publications and source records attributed to J Paoletti.

50 records · Page 3Linked to original sources
PubMed1979

Relaxation of chromatin structure induced by ethidium binding. Involvement of the intercalation process.

In this paper we study the effects of the binding of ethidium on the structure of chromatin, using micrococcal nuclease as a structural probe. This binding induces two structural changes of chromatin either isolated or in the nuclei. (a) An unfolding of the overall structure which results in an activation of the rate of degradation by the nuclease. (b) A disorganisation of the core particle structure which has the effect of unwrapping the DNA from the histone core, this disruption can go on so far as to leave only 90 base pairs. By comparing the bindings of ethidium and tetramethylethidium, we conclude that the first type of structural change is due to an electrostatic effect and does not depend upon intercalation. On the other hand, the second one is due to the intercalation process and to the change of topological constraints on the DNA that such a process involves.

Animals↗
PubMed1977

The structure of chromatin: interaction of ethidium bromide with native and denatured chromatin.

The binding of ethidium bromide, as monitored by fluorescence enhancement, to chromatin prepared by nuclease digestion has been compared with the binding of the dye to sheared chromatin. The nuclease preparation (native chromatin) is characterized by a high affinity region of the Scatchard plot (r = 0-0.025, K1 = 1 X 10(6) M-1), a transition (r = 0.025-0.05), and a low affinity region (r = 0.05-0.12, K2 = 3 X 10(5) M-1). The final amount of ethidium bromide bound per base is 0.12 as compared with 0.20 for free DNA. Sheared chromatin has the two regions of high and low affinity (K1 = 2 X 10(6) M-1, K2 = 5 X 10(5) M-1) as originally shown by Angerer and Moudrianakis (1972), but the transition is much reduced or absent. Binding of the dye to native chromatin is independent of salt at concentrations ranging from 0.2 mM EDTA to 10 mM Tris-Cl, 10 mM NaCl, 0.2 mM EDTA, while sheared chromatin and DNA both bind ethidium bromide electrostatically as well as by intercalation at the low salt concentration, leading to extensive energy transfer. Thus the phosphate groups in native chromatin are unavailable to external cations even at very low salt. Polarization of fluorescence of ethidium bromide intercalated into native chromatin at low r is very high, indicating a highly rigid structure. As r approaches 0.02, there is a very rapid depolarization; at r = 0.03, the polarization is no greater than that of the dye intercalated into DNA. Depolarization is not due to energy transfer. The Scatchard plot derived for the bulk preparation of native chromatin is very similar to the one derived for the monomer nu body. These results indicate that the DNA in native chromatin is in a very rigid form, with its phosphate anions neutralized by structural components, not by free salt. Ethidium bromide intercalation appears partially to disrupt this structure, perhaps by unwinding, leading to slight changes in its properties.

Binding Sites↗
PubMed1975

Separation of lymphocyte chromatin into template-active fractions with specificity for eukaryotic RNA polymerase II or prokaryotic RNA polymerase.

When chromatin prepared from WI-L2 lymphocytes by low salt extraction and shearing is centrifuged on a glycerol gradient, one area of the gradient yields chromatin enriched in template activity for Escherichia coli DNA-dependent RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase) as compared to Saccharomyces cerevisiae RNA polymerase II (or B). Another area yields chromatin preferred by the eukaryotic enzyme. Kinetic studies indicate that the differences in activity cannot be explained by differences in affinity of the enzymes for the various templates. The DNA isolated from either fraction has a molecular weight of 8.5 X 106. The "yeast active" fraction seems enriched in proteins. Mixing experiments indicate that the yeast enzyme does not alter the template in such a way as to improve it for the bacterial enzyme.

Cell Line↗
PubMedDate not supplied

An ellipticine derivative (oxazolopyridocarbazolium) 3' linked to tetrathymidylate stacks intramolecularly with the nearest thymine at low concentration and head-to-tail intermolecularly at high concentration.

The solution conformation of a tetrathymidylate linked through an ester bond to an ellipticine derivative oxazolopyridocarbazolium (OPC) at the 3' position was investigated using one- and two-dimensional nmr experiments. Since the total electric charge of the OPC ring may influence self-association, we first determined the pKa of the oxazole cyclic acidic function. Nuclear Overhauser effect spectroscopy experiments showed that, at low concentration, the OPC stacks intramolecularly with the nearest thymine at the 3' end. At highest concentration, however, the OPC rings are self-associated. The stacking constant was calculated using 1H chemical shift dilution experiment. The conformational model suggested by P-nmr was tested by molecular mechanics computations.

Alkaloids↗
PubMedDate not supplied

Effects of the 6-alkyl substitution of ellipticine on the uptake by NIH-3T3 cells and on the cytotoxicity.

New ellipticine derivatives of the 2-methyl ellipticinium (NME) series, i.e. 2,6-dimethylellipticinium (6-Me-NME) and 2-methyl-6-n-propylellipticinium (6-Pr-NME), have been studied as cytotoxic compounds. Their uptake by NIH-3T3 cells and efflux have been measured by a sensitive and specific high performance liquid chromatographic assay. These compounds are equitoxic if we compare their cytotoxicity by two methods: growth inhibition and cloning efficiency. However, they accumulate in NIH-3T3 cells at different steady state levels and the efflux rates are not similar. This raises the question of the mode of action of these drugs.

Alkaloids↗