Erratum

Abstract

The condensation and the precipitation of rat liver chromatin upon addition of spermine⁴⁺, spermidine³⁺, hexamminecobalt(III)³⁺ and Mg²⁺ cations have been studied using solubility, fluorescence, circular dichroism, melting curves, electric dichroism and spermidine binding measurements, made on both soluble and precipitated complexes. The soluble complexes obtained with tetra- and trivalent cations were depleted from all histones and enriched in other proteins, particularly high mobility group proteins 1 and 2, which brings about an important enhancement of tryptophan fluorescence without modification of its two lifetimes 5.1 and 1.2 ns. In the precipitates the non-histone proteins are eliminated. Under precipitation by Mg²⁺ ions, the distribution of proteins remains practically unchanged. The electric dichroism and the melting curves indicate that the soluble complexes between polyamines and chromatin undergo important condensation and, at high ratios of cation over phosphate, are constituted by heterogeneous assemblies of non-histone proteins and DNA. On the contrary, the insoluble complexes seem to retain the main features of original chromatin. Precipitation by Mg²⁺ ions reveal much less drastic changes than those produced by polyamines. Precipitation by spermidine occurs when one cation is bound per eight nucleotides, which in addition to the histone positive charges brings about a complete neutralization of chromatin phosphates.     

Comparative study of the condensation of chicken erythrocyte and calf thymus chromatins by di- and multivalent cations

The condensation of chicken erythrocyte (CE) and calf thymus (CT) chromatins upon addition of di- and multivalent cations has been studied using turbidity, precipitation and electric dichroism measurements. For all the cations investigated (Mg2+, Tb3+, Co(NH3)6(3+), spermidine Spd2+ and spermine Sp4+) condensation of CE chromatin occurred before the onset of aggregation, while aggregation of CT chromatin started before condensation with all cations except Mg2+ and Tb3+. Precipitation of CE chromatin required lower di- and multivalent cations concentrations than CT chromatin. The electric dichroism data for both chromatins, at low ionic strength in the absence of di- or multivalent cations, indicated that the nucleoprotein molecules were not totally decondensed but that a "precondensed" state was already present. A positive electric dichroism was observed for the most condensed chromatin fibers, in agreement with the "cross-linker" models. Tb3+ led to less compact condensed particles as judged from the electric dichroism observations, but electron microscopy revealed that "30 nm fibers" were formed. Very little aggregation was produced by Tb3+. On the contrary, spermine produced very large networks of condensed molecules, but large spheroidal particles were also observed. The condensation of CE chromatin happened without changes of solution conductivity upon cation salt addition, regardless of the condensing cation, indicating a cooperative uptake of the ions during this process.     

Comparative Study of the Condensation of Chicken Erythrocyte and Calf Thymus Chromatins by Di- and Multivalent Cations

Abstract

The condensation of chicken erythrocyte (CE) and calf thymus (CT) chromatins upon addition of di- and multivalent cations has been studied using turbidityJulprecipitation and electric dichroism measurements. For all the cations investigated (Mg²⁺, Tb³⁺, Co(NH₃)₆ ³⁺, spermidine Spd²⁺ and spermine Sp⁴⁺) condensation of CE chromatin occurred before the onset of aggregation, while aggregation of CT chromatin started before condensation with all cations except Mg²⁺ and Tb³⁺. Precipitation of CE chromatin required lower di- and multivalent cations concentrations than CT chromatin. The electric dichroism data for both chromatins, at low ionic strength in the absence of di- or multivalent cations, indicated that the nucleoprotein molecules were not totally decondensed but that a “precondensed” state was already present. A positive electric dichroism was observed for the most condensed chromatin fibers, in agreement with the “cross-linker” models. Tb³⁺ led to less compact condensed particles as judged from the electric dichroism observations, but electron microscopy revealed that “30 nm fibers” were formed. Very little aggregation was produced by Tb³⁺. On the contrary, spermine produced very large networks of condensed molecules, but large spheroidal particles were also observed. The condensation of CE chromatin happened without changes of solution conductivity upon cation salt addition, regardless of the condensing cation, indicating a cooperative uptake of the ions during this process.     

Condensation of chromatin: role of multivalent cations

We have used electric dichroism to investigate the influence of multivalent cations upon the compaction of chicken erythrocyte chromatin from the unfolded, 10-nm fiber to the 30-nm solenoid and subsequent aggregation. The pattern of condensation, which consists of compaction plus aggregation, is found to be strikingly similar for a variety of cations of differing charge, including the physiologically important polyamines spermine and spermidine. With a few exceptions such as Cu2+ and Gd3+, an optimally compacted fiber with reproducible hydrodynamic properties is produced prior to the onset of aggregation. We report the concentrations of di-, tri-, and tetravalent cations required for optimal condensation; in addition, for tri- and tetravalent cations, we were able to estimate the extent of charge neutralization produced by their binding to the optimally compacted fiber. The results show that the multivalent ion concentration required for optimal compaction decreases as cationic charge increases. In addition, the effect of a mixture of dilute mono- and multivalent cations on chromatin condensation is synergistic, rather than competitive as has been found for the multivalent cation induced condensation of DNA or the B----Z conformational transition. A simple calculation indicates that the entropy of ion uptake in chromatin condensation is surprisingly constant for a range of ionic conditions; this factor may be a dominant one in determining the folding equilibrium.     

Polyamine-DNA interactions. Condensation of chromatin and naked DNA

We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin by polyamines, such as spermine, spermidine and its analogs designated by formula NH3+(CH2)iNH2+(CH2)jNH3+, where i = 2,3 and j = 2,3, putrescine, cadaverine and MgCl2. It has been found that the different polyamines affect DNA and chromatin in a similar way. The level of compaction of the chromatin fibers induced by spermine, spermidine and the triamines NH3+(CH2)3NH2+(CH2)3NH3+ and NH3+(CH2)3NH2+(CH2)2NH3+ and MgCl2 is found to be identical. The triamine NH3+(CH2)3NH2+(CH2)2NH3+ and the diamines studied condense neither chromatin nor DNA. This drastic difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations might play essential roles in their interactions with DNA and chromatin. It is shown that a mixture of mono- and multivalent cations affect DNA and chromatin condensation competitively, but not synergistically, as claimed in a recent report by Sen and Crothers (Biochemistry 25, 1495-1503, 1986). We have also estimated the extent of negative charge neutralization produced by some of the polyamines on their binding to chromatin fibers. The stoichiometry of polyamine binding at which condensation of chromatin is completed is found to be two polyamine molecules per DNA turn. The extent of neutralization of the DNA phosphates by the histones in these compact fibers is estimated to be about 55%. The model of polyamine interaction with chromatin is discussed.     

Effects of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions.

Studies are presented on the influence of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions involving both exogenous and endogenous substrates. The activities toward the model acidic protein substrate, dephosphophosvitin, were maximal at 160--200mM-NaCl (or -KCl or -NH4Cl). Under these conditions, spermidine and spermine added in concentrations up to 2mM were essentially without effect. However, without addition of NaCl to the medium, marked stimulation of these reactions was elicited by these polyamines at 1--2mM concentrations. The stimulatory effects were not due to non-specific changes in the ionic strength or to substitution of spermine for Mg2+, as maximal stimulation by 1 mM-spermine was observed only at optimal (2--4mM) Mg2+ concentrations. Qualitatively similar effects of polyamines were observed with enzyme preparations from the prostates of castrated rats, and with chromatin and non-histone-protein preparations from other tissues besides ventral prostate. When phosphorylation of endogenous non-histone proteins of the chromatin was measured, spermine stimulated both the initial rates and the final extent of transphosphorylation, even in the presence of optimal concentration of NaCl. By contrast, spermine or spermidine had no effect on the chromatin- and non-histone-protein-associated protein kinase reactions determined with lysine-rich histones as substrates. Chemically NN-dimethylated dephosphophosvitin was a less active substrate for the chromatin-associated protein kinase, but its phosphorylation was more markedly stimulated by spermine in comparison with unmodified dephosphophosvitin. These observations hint that the polyamine stimulations of the various protein kinase reactions may be due to effects on the conformations of the non-histone protein substrates rather than on the kinases themselves.     

Interactions of some naturally occurring cations with phenylalanine and initiator tRNA from yeast as reflected by their thermal stability

The thermal unfolding of phenylalanine and initiator tRNA from yeast was investigated over a broad range of solution conditions by differential ultraviolet absorption at 260 nm. Under most conditions, the initiator tRNA exhibits two clearly separated transitions in its differential melting curve which were assigned to unfolding of tertiary and secondary structure elements, respectively. The tertiary transition of this tRNA and the overall transition observed for tRNAPhe do not show a maximum in a curve of Tm values plotted as a function of [Na+]. Such a maximum is usually observed for other nucleic acids at about 1 M Na+. In the presence of 5 mM of the divalent cation Mg2+ (or Ca2+), an overall destabilization of the tRNAs is observed when increasing the sodium concentration. The largest fall in Tm (approximately 15 degrees C) is observed for the tertiary transition of the initiator tRNA. Among various cations tested the following efficiency in the overall stabilization of tRNAPhe is observed: spermine greater than spermidine greater than putrescine greater than Na+ (approximately NH4+). Mg2+ is most efficient at concentrations above 5 mM, but below this concentration spermine and spermidine appear to be more efficient. The same hierarchy in stabilizing power of the polyamines and Na+ is observed for both transitions of the initiator tRNA. However, when compared with Mg2+, the polyamines are far less capable of stabilizing the tertiary structure. In contrast, spermine and spermidine are slightly better than Mg2+ in stabilizing the secondary structure. At increasing concentrations of the polyvalent cations (at fixed [Na+] ) the Tm values of the tRNAs attain a constant value.     

Condensation of the chromatin gel by cations

Calf thymus chromatin gel, containing strongly bound nonhistone proteins, was used to study the effect of easily removable and tightly bound cations on the condensation of chromatin. The chromatin volume was found to be linearly dependent on the reciprocal square root of the concentration of easily removable cations (Tris X H+ + Na+ and Mg2+) except for the initial stages of condensation (up to 7-10 mM monovalent and 0.15-0.2 mM divalent cations). The effect of Mg2+ at the initial stage of condensation was not reproduced by Na+ and vice versa. At higher concentrations the effects of Na+ and Mg2+ were additive. The removal of tightly bound divalent cations by a treatment of the chromatin gel with 1,10-phenanthroline led to an approx. 50% increase in the volume of the chromatin gel, which was maintained at each concentration of easily removable cations. The 1,10-phenanthroline-caused decondensation of the chromatin gel was reversed by Ca2+ but not by Mg2+, Zn2+ and Cu2+. The chromatin gel pretreated with Ca2+ was not further decondensed by 1,10-phenanthroline.     

The superstructure of chromatin and its condensation mechanism

Comparison between the internucleosomal distance found by X-ray solution scattering for chicken erythrocyte (23 nm) and sea urchin (30 nm) chromatin indicates that this distance is proportional to the linker length. The diameter of the condensed sea urchin chromatin fibers is about 45 nm which is significantly larger than in chicken erythrocyte chromatin (35 nm). Trivalent cations (Gd, Tb, Cr) and the polyamines spermine and spermidine were found to induce compaction at much lower concentrations than the divalent cations but Gd, Tb and Cr induce aggregation before full compaction of the fibers. The influence of hydrogen bonding is illustrated by comparison of the effects of NaCl, ammonium chloride and alkylammonium chlorides on condensation. Solubility experiments indicate that there is a nearly linear dependence of the Mg^-- concentration at which precipitation occures on chromatin concentration and confirm the differences between cations observed by X-ray scattering.The chicken erythrocyte chromatin samples were further characterized by their reduced electric dichroism. The values found are consistent with the model derived from X-ray scattering and are compared with those reported in the literature.     

Interaction of polyamines with chromatin and DNA: formation of compact structures

We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin induced by spermine, triamines NH3+(CH2)iNH+(CH2)jNH3+, designated as much less than i, j much greater than: much less than 3, 4 much greater than (spermidine), much less than 3, 3 much greater than, much less than 2, 3 much greater than, much less than 2, 2 much greater than; the diamines putrescine and cadaverine and MgCl2. It is found that the different polyamines affected DNA and chromatin in a similar way. The degree of compaction of the chromatin fibers induced by spermine, triamines except much less than 2, 2 much greater than and Mg2+ has been found to be identical. The triamine much less than 2, 2 much greater than and the diamines studied do not condense either chromatin of DNA. Such a big difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations are important for their interactions with DNA and chromatin. The stoichiometry of polyamine binding to chromatin at which condensation occurred is found to be 2 polyamine molecules per DNA helical turn. Polyamines are supposed to bind to the exposed sites of core DNA every 10 b.p. The extent of DNA phosphate neutralization by the histones is estimated to be about 55%. It has been shown that a mixture of mono- and multivalent cations affected DNA and chromatin condensation competitively and not synergistically, as claimed in a recent report by Sen and Crothers.     

Effect of polyamines on phosphorylation of non-histone chromatin proteins from hog liver.

The effects of polyamines on the $\text{in vitro}$ phosphorylation of non-histone chromatin proteins from hog liver has been found to be dose dependent. Maximal increase occurred at 0.2 mM spermine and 2 mM spermidine, respectively. These results suggest that spermine and spermidine may have a regulating function for phosphorylation of non-histone chromatin proteins in hog liver.     

Coprecipitation of topoisomerase activity with simian virus 40 nucleoprotein complexes by divalent cations

Simian virus 40 (SV40) nucleoprotein complexes extracted from nuclei of infected monkey cells (CV1) were precipitated with Ca2+, Mg2+, and Mn2+ divalent cations. Most of the viral chromatin but only a fraction of the proteins in the crude nuclear extracts were recovered after precipitation with 10 mM MgCl2. At this optimal concentration, DNA topoisomerase activity (nicking closing enzyme) coprecipitated with the SV40 nucleoprotein complexes.     

An electro-optical study of the mechanisms of DNA condensation induced by spermine

Condensation of DNA by spermine has been studied by electric dichroism, electric birefringence and rotational relaxation times at 1 mM ionic strength. Using Manning's theory, we found that condensation occurs for a fraction of neutralized phosphate charges (r) equal to 0.90, in good agreement with previous studies using spermidine, synthetic polyamines and trivalent cations (e.g. Co(NH3)36 +, Tb3 +). Our results are compatible with the presence in solution of torus-shaped condensed structures in a narrow range of spermine concentration; further addition of the polyamine produced precipitation due to the self-aggregation of several toroids. For spermine concentrations lower than that required for collapse, important changes of the orientation mechanism in the electric field and of DNA stiffness were observed. Whereas free DNA was mainly oriented by a fast-induced polarizability mechanism, DNA-spermine complexes displayed an important permanent dipole component, in the spermine concentration range where extension of the DNA molecules was present. The birefringence relaxation times suggested that, in the first step, the stiffness of the DNA molecules increased, and then, at higher spermine concentration, bending of the DNA molecules occurred so that condensation into toroidal particles became possible.     

Exchange of H1 histone depends on aggregation of chromatin, not simply on ionic strength

Chromatin solubility was observed at several concentrations of various cations. Spermine and spermidine precipitated (50%) chromatin at about 0.2 mM, Ca2+ and Mg2+ at about 1-2 mM, and Na+ at about 100 mM. Further increases in cation concentration induced more aggregation, but eventually excess cation increased chromatin solubility so that 50% solubility was observed again at 60 mM Mg2+ and 180 mM Na+. H1 histone was 50% released by 80 mM MgCl2 or 425 mM NaCl. Combinations of MgCl2 and NaCl showed that Mg2+ and Na+ are synergistic in the induction of aggregation in lower concentrations (less than 2 mM) of Mg2+ but antagonistic at higher concentrations, and a similar effect of NaCl on spermidine-induced precipitation was shown below and above about 0.2 mM spermidine. At 5 mM, MgCl2 proved capable of precipitating chromatin depleted of H1 histone, but no concentration of NaCl was capable of doing so. These phenomena can be rationalized by supposing that neutralization of chromatin by any cation (including H1 histone) favors aggregation and also that cross-linking of chromatin fibers by multivalent cations (including H1 histone) is also critically important. The exchange of H1 histone between chromatin fragments was tested in various concentrations of different salts. H1 exchange was correlated with chromatin aggregation rather than with ionic strength and thus appears to depend on fiber to fiber contact. Under conditions where H1 exchanges between chromatin fibers that are permitted to make contact with each other, no H1 exchange occurred between chromatin inside the nucleus and chromatin outside, even though H1 histone is capable of passage through the nuclear membrane.     

Polyamines in bacteriophage R17 and its RNA.

Bacteriophage R17 and its RNA were found to contain significant amounts of spermidine but not of putrescine. When isolated at 0.01 M KCl, up to 1,000 molecules of spermidine were associated with the virion. The phage RNA isolated with phenol plus sodium lauryl sulfate contained approximately 70 to 90 molecules of spermidine. The association appeared to be ionic because the bound spermidine could be dissociated by KCl, MgCl2, or both. Effects of polyamines on in vitro translation were studied using both poly(U) and phage R17-RNA as mRNA. Addition of spermidine to the system at suboptimal concentrations of Mg2+ resulted in marked stimulations of the rate of protein synthesis. Putrescine alone had no effect but stimulated the incorporation in the presence of suboptimal concentrations of spermidine plus Mg2+. The isolated amino acid-incorporating system contained suboptimal soluble and bound polyamines. A comparison of incorporation was made in this system using R17-RNA with and without bound spermidine. No effects of these bound cations were detected on the rate or extent of incorporation of valine. The ratio of incorporation of histidine (present in non-coat proteins) to valine (total protein) revealed little difference as a functions of cation in the system or a function of the spermidine present in R17-RNA.     

Divalent cation-induced changes in conformation of protein kinase C

Using physical techniques, circular dichroism and intrinsic and extrinsic fluorescence, the binding of divalent cations to soluble protein kinase C and their effects on protein conformation were analyzed. The enzyme copurifies with a significant concentration of endogenous Ca2+ as measured by atomic absorption spectrophotometry, however, this Ca2+ was insufficient to support enzyme activity. Intrinsic tryptophan fluorescence quenching occurred upon addition to the soluble enzyme of the divalent cations, Zn2+, Mg2+, Ca2+ or Mn2+, which was irreversible and unaffected by monovalent cations (0.5 M NaCl). Far ultraviolet (200-250 nm) circular dichroism spectra provided estimations of secondary structure and demonstrated that the purified enzyme is rich in alpha-helices (42%) suggesting a rather rigid structure. At Ca2+ or Mg2+ concentrations similar to those used for fluorescence quenching, the enzyme undergoes a conformational transition (42-24% alpha-helix, 31-54% random structures) with no significant change in beta-sheet structures (22-26%). Maximal effects on 1 microM enzyme were obtained at 200 microM Ca2+ or 100 microM Mg2+, the divalent cation binding having a higher affinity for Mg2+ than for Ca2+. The Ca2(+)-induced transition was time-dependent, while Mg2+ effects were immediate. In addition, there was no observed energy transfer for protein kinase C with the fluorescent Ca2(+)-binding site probe, terbium(III). This study suggests that divalent cation-induced changes in soluble protein kinase C structure may be an important step in in vitro analyses that has not yet been detected by standard biochemical enzymatic assays.     

Aggregation of nucleosomes by divalent cations.

Conditions of precipitation of nucleosome core particles (NCP) by divalent cations (Ca(2+) and Mg(2+)) have been explored over a large range of nucleosome and cation concentrations. Precipitation of NCP occurs for a threshold of divalent cation concentration, and redissolution is observed for further addition of salt. The phase diagram looks similar to those obtained with DNA and synthetic polyelectrolytes in the presence of multivalent cations, which supports the idea that NCP/NCP interactions are driven by cation condensation. In the phase separation domain the effective charge of the aggregates was determined by measurements of their electrophoretic mobility. Aggregates formed in the presence of divalent cations (Mg(2+)) remain negatively charged over the whole concentration range. They turn positively charged when aggregation is induced by trivalent (spermidine) or tetravalent (spermine) cations. The higher the valency of the counterions, the more significant is the reversal of the effective charge of the aggregates. The sign of the effective charge has no influence on the aspect of the phase diagram. We discuss the possible reasons for this charge reversal in the light of actual theoretical approaches.     

DNA denaturation in situ. Effect of divalent cations and alcohols

Heat denaturation profiles of rat thymus DNA, in intact cells, reveal the presence of two main DNA fractions differing in sensitivities to heat. The thermosensitive DNA fraction shows certain properties similar to those of free DNA: its stability to heat is decreased by alcohols and is increased in the presence of the divalent cations Ca2+, Mn2+, or Mg2+ at concentrations of 0.1-1.0 mM. Unlike free DNA, however, this fraction denatures over a wide range of temperature, and is heterogeneous, consisting of at least two subfractions with different melting points. The thermoresistant DNA fraction shows lowered stability to heat in the presence of Ca2+, Mn2+, or Mg2+ and increased stability in the presence of alcohols. It denatures within a relatively narrow range of temperature, consists of at least three subfractions, and, most likely, represents DNA masked by histones. The effect of Ca2+, Mn2+, or Mg2+ in lowering the melting point of the thermoresistant DNA fraction is seen at cation concentrations comparable to those required to maintain gross chromatin structure in cell nuclei or to support superhelical DNA conformation in isolated chromatin (0.5-1.0 mM). It is probable that factors involved in the maintenance of gross chromatin organization in situ and/or related to DNA superhelicity also have a role in modulating DNA-histone interactions, and that DNA-protein interactions as revealed by conventional methods using isolated chromatin may be different from those revealed when gross chromatin morphology remains intact.     

Cations and the accessibility of chromatin to nucleases.

When rat liver nuclei prepared with polyamines as stabilising cations are digested with DNAase II, release of both inactive chromatin and Mg-soluble, active chromatin is greatly reduced, in comparison to digestion of liver nuclei prepared with Mg2+ as stabilising cation. Chromatin release from polyamine stabilised nuclei is also inhibited relative to Mg-stabilised nuclei following digestion with micrococcal nuclease under two very different cation conditions. Nuclei prepared with polyamines and monovalent ions as stabilising cations exhibit properties intermediate between these two extremes with both nucleases. These effects are due to residual binding of polyamines to chromatin, which is thus maintained in a condensed state, inaccessible to nucleases. Since polyamine binding is not easily reversed, concentrations of polyamines and other cations must be rigidly controlled in experiments on chromatin structure if artefacts are to be avoided. The significance of these findings to the nature and properties of active chromatin within the intact nucleus is considered.     

Glycerolipid biosynthesis in rat adipose tissue. 11. Effects of polyamines on Mg2+-dependent phosphatidate phosphohydrolase

The effect of polyamines (spermine, spermidine and putrescine) on the Mg2+-dependent phosphatidate phosphohydrolase was investigated. Phosphatidate phosphohydrolase activity was measured in the presence of aqueous dispersed phosphatidate as substrate, and the release of inorganic phosphate was taken as a measure of phosphatidate phosphohydrolase activity. In the presence of various polyamines there was activation of the Mg2+-dependent phosphatidate phosphohydrolase activity. Under this condition, the Km of enzyme towards phosphatidase decreased from 1.6 x 10(-4) to 9.8 x 10(-5) M and the Mg2+ requirement decreased from 5 to 0.5 mM. These polyvalent cations did not replace Mg2+, but potentiate the phosphohydrolase activity in the presence of Mg2+. The activation of Mg2+-dependent phosphatidate phosphohydrolase activity by polyamines was observed in the presence of 3-sn-phosphatidylcholine, suggesting that these modulators of phosphatidate phosphohydrolase activity may be acting through different mechanisms. These studies demonstrate that polyamines may be important regulators of Mg2+-dependent phosphatidate phosphohydrolase activity in adipose tissue.