Thermodynamics of nucleosomal core particles

In this paper, a numerically detailed thermodynamic investigation of nucleosomal core particles is presented. The nonlinear Poisson-Boltzmann equation governs the electrostatic properties of both the DNA and histone protein. Brownian dynamics is used as the leading method, in combination with the analysis of the electrical features of the nucleosome. At elevated temperature, the structure of the nucleosome is destabilized by the decrease in electrical interactions of DNA-histone complexes, which can be explained with the EDL theory. Two obvious unwrapping transitions can be found, occurring within the temperature ranges 43-52 and 65-80 degrees C. The first transition is characterized by the melting of DNA terminal domains, and the feature of the second transition is the massive unwrapping of the DNA middle domain. It can be concluded that the nucleosomal DNA consists of two distinct structures, where the DNA terminal domains are less tightly bound to the histone than the DNA middle domain.     

Thermal denaturation of nucleosomal core particles.

Thermal denaturation of very homogeneous preparations of core particles from chicken erythrocyte chromatin is studied by several techniques. The change in absorbance, which is very closely paralleled by changes in heat capacity, which is very closely paralleled by changes in heat capacity, is a biphasic process with inflexions at 60 degrees C and 74 degrees C. In contrast, isolated DNA of the same length denatures in a single transition around 44 degrees C. Monitoring the circular dichroism of the cores during thermal denaturation reveals biphasic changes in the secondary structure of the DNA, preceding the base unstacking by 10 degrees C in the first and 3 degrees C in the second phase. However, measurable alterations in the secondary structure of the histones are confined to the second phase with a melting temperature at 71 degrees C. Increase in the ionic strength of the buffer from 1 mM to 10 mM leads to almost monophasic melting curves as measured by absorbance and CD, while not causing any measurable conformational changes at room temperature. The melting of core particles is interpreted as a denaturation of about 40 base pairs in the first phase, followed by a massive breakdown of the native structure of a tight histone-DNA complex, which frees the remaining 100 base pairs for unstacking.     

Aberrant DNase I digestion kinetics of nucleosomal core particles from sea urchin sperm

The pancreatic deoxyribonuclease (DNase I) digestion rates at the susceptible sites on nucleosomal core particles from blastula, gastrula and sperm cells of the sea urchin, Parechinus angulosus, have been determined. Although there are differences in their isohistone composition, the rates of digestion are similar for both embryonic stages. The rates of digestion for sperm core particles are 3-5 times lower than for embryo core particles at the more, and up to 2.5 times lower at the less susceptible sites. An explanation for these differences could be sought in the sperm isohistones H2B which are characterized by N-terminal extensions of 20-25 amino acid residues.     

Marked differences in the efficiency of expression of distinct alpha beta T cell receptor heterodimers

Ag recognition by most T lymphocytes is mediated by clonally distributed alpha beta heterodimeric receptors. A major fraction of TCR diversity is believed to be due to the random coexpression in individual T cells of the products of independently rearranging alpha- and beta-genes (combinatorial diversity). However, analysis of cell surface receptors on transfected T hybridoma cells synthesizing various sets of alpha- and beta-chains revealed marked differences in the efficiency of expression of certain alpha beta-pairs. Specifically, using the functionally rearranged gene products of the 2B4 cytochrome c specific T hybridoma (V beta 3, V alpha 11.2) and BW5147 parent lymphoma (V beta 1, V alpha BW), a hierarchy of expression efficiency relative to indirectly measured precursor chain levels in the cell was shown to be 2B4 alpha-BW beta greater than 2B4 alpha - 2B4 beta greater than BW alpha - BW beta greater than BW alpha - 2B4 beta. The estimated difference between the best expressed and worst expressed pairs is on the order of 50-fold. For the beta-chain, the primary determinant of expression efficiency with a given alpha-chain appears to be the V segment, as a second V beta 1-chain with distinct D and J regions from BW beta was expressed with the same pattern. These data imply that alpha- and beta-chains do not form well-expressed TCR in a random manner and that limitations on the useful combinatorial association of these chains may significantly affect the functional T cell repertoire.     

Different mechanism for in vitro formation of nucleosome core particles

The interaction of different histone oligomers with nucleosomes has been investigated by using nondenaturing gel electrophoresis. In the presence of 0.2 M NaCl, the addition of the pairs H2A,H2B or H3,H4 or the four core histones to nucleosome core particles produces a decrease in the intensity of the core particle band and the appearance of aggregated material at the top of the gel, indicating that all these histone oligomers are able to associate with nucleosomes. Equivalent results were obtained by using oligonucleosome core particles. Additional electrophoretic results, together with second-dimension analysis of histone composition and fluorescence and solubility studies, indicate that H2A,H2B, H3,H4, and the four core histones can migrate spontaneously from the aggregated nucleosomes containing excess histones to free core DNA. In all cases the estimated yield of histone transfer is very high. Furthermore, the results obtained from electron microscopy, solubility, and supercoiling assays demonstrate the transfer of excess histones from oligonucleosomes to free circular DNA. However, the extent of solubilization obtained in this case is lower than that observed with core DNA as histone acceptor. Our results demonstrate that nucleosome core particles can be formed in 0.2 M NaCl by the following mechanisms: (1) transfer of excess core histones from oligonucleosomes of free DNA, (2) transfer to excess H2A,H2B and H3,H4 associated separately with oligonucleosomes to free DNA, (3) transfer to excess H2A,H2B initially associated with oligonucleosomes to DNA, followed by the reaction of the resulting DNA-(H2A,H2B) complex with oligonucleosomes containing excess H3,H4, and (4) a two-step transfer reaction similar to that indicated in (3), in which excess histones H3,H4 are transferred to DNA before the reaction with oligonucleosomes containing excess H2A,H2B. The possible biological implications of these spontaneous reactions are discussed in the context of the present knowledge of the nucleosome function.     

Preparation and structural characterization of nucleosomal core particles lacking one H2A.H2B dimer

Nucleosomal core particles lacking one H2A.H2B dimer, (H2A.H2B)1 (H3.H4)2/DNA (146 bp), have been prepared by treatment of nucleosomal cores with dimethylmaleic anhydride, a reversible reagent for protein amino groups. The preparative procedure is simple, produces quantitative conversion of nucleosomal cores into dimer-deficient cores without formation of other subnucleosomal particles, and can be applied to the preparation of different H2A.H2B-deficient mono and oligonucleosomal particles. The structural properties of the dimer-deficient cores and complete nucleosomal cores reconstituted from the deficient particles and H2A.H2B dimers have been studied by DNase I digestion, thermal denaturation and circular dichroism.     

Reconstitution of mononucleosomes: characterization of distinct particles that differ in the position of the histone core

Reconstitution of mononucleosomes from DNA and core histones was carried out to study the positioning of histone octamers on the DNA. Using random DNA molecules in the 200 to 250 bp size range we found that the reconstitution products consisted of a mixture of three different types of particles that could be separated by low ionic strength gel electrophoresis. In one particle, DNA was complexed with histones along its entire length indicating the binding of more than one histone octamer. The second particle contained only one histone core that was always associated, however, with the terminal 145 bp of the DNA regardless of its sequence which can be ascribed to a DNA end effect. Only the third particle consisted of histone octamers bound at internal positions of the DNA and is therefore the only particle suitable for investigating the influence of the DNA sequence on the positioning of the histone cores. A defined 154 bp pBR 322 restriction fragment that contains three BspRI restriction sites was also reconstituted with core histones. The accessibility of these sites to BspRI was measured in order to delineate the utility of restriction nucleases as probes for the structure of chromatin. Two sites located close to the center of the DNA were less susceptible by at least a factor of 1000 as compared to free DNA while the susceptibility of the third site in the terminal section of the DNA decreased about 50 fold after reconstitution.     

Two distinct segments of the hepatitis B virus surface antigen contribute synergistically to its association with the viral core particles

The long surface antigen polypeptide (L-HBsAg) of hepatitis B virus (HBV) is believed to mediate contact between the virus envelope and nucleocapsid protein (HBcAg). The N and C termini of L-HBsAg were shortened progressively in order to define the minimum contiguous sequence of amino acids that contains the residues necessary for association with HBcAg. The resulting mutants were expressed in rabbit reticulocyte lysates and their interaction with HBcAg was examined with an immunoprecipitation assay and an equilibrium binding assay in solution to give relative dissociation constants. Binding of HBcAg particles by L-HBsAg displayed two widely differing dissociation constants, indicating two distinct binding sites between the molecules. The two distinct sites, one located between residues 24 and 191 and the other between residues 191 and 322 of L-HBsAg, contribute synergistically to high-affinity binding to HBcAg, but disruption of either of these segments resulted in a much weaker interaction showing only one dissociation constant. Inhibition of the interaction by peptides that bind to the tips of the nucleocapsid spikes differentiated contacts in HBcAg for the two binding domains in L-HBsAg and implied that the amino-terminal binding domain contacts the tips of the HBcAg spikes. Analysis of specific single amino acid mutants of L-HBsAg showed that Arg92 played an important role in the interaction.     

Strain differences of the ability to hydroxylate methotrexate in rats

Converting activity of methotrexate (MTX) to 7-hydroxymethotrexate (7-OH-MTX) was examined using eight strains of rats. Marked variability of the activity was found in liver cytosols from the rats. The highest activity was observed with Sea:SD rats, followed by LEW/Sea and Jcl:Wistar rats. The lowest activity was observed with WKA/Sea rats. The difference in the activity between Sea:SD and WKA/Sea strains was 104-fold. The variation was correlated to the strain difference of benzaldehyde oxidase activity in the rats. The cytosolic 7-hydroxylase activities in other tissues of Sea:SD rats were much higher than those of WKA/Sea, similarly to the case in liver. The liver microsomes of Sea:SD rats exhibited no 7-hydroxylase activity toward MTX even in the presence of NADPH. The cytosolic 7-hydroxylating activity of the livers of Sea:SD rats was inhibited by menadione, β-estradiol, chlorpromazine and disulfiram, inhibitors of aldehyde oxidase, but not oxypurinol, an inhibitor of xanthine oxidase. The purified aldehyde oxidase from the livers of Sea:SD rats exhibited a significant 7-hydroxylating activity toward MTX. However, xanthine oxidase had no ability to hydroxylate MTX. These facts suggest that MTX hydroxylating activity in rats is predominantly due to aldehyde oxidase, and the strain differences are due to the variations of the flavoenzyme level.     

Src-/- fibroblasts are defective in their ability to disassemble focal adhesions in response to phorbol ester/hyaluronan treatment

Exogenous hyaluronan promotes a rapid recruitment of Src to lamellae of mutant active H-ras transformed fibroblasts and an Src- and RHAMM (CD168)-dependent increase in random motility. These responses are accompanied by a loss of vinculin-positive lamellae focal adhesions. Nontransformed immortalized wild-type fibroblasts (WT) do not increase random motility in response to hyaluronan alone, but do increase motility in response to a combination of PMA treatment followed by hyaluronan. PMA treatment alone increases the number of lamellae/cell, percentage of cells with lamellae and number of focal adhesions/lamellae. Subsequent addition of hyaluronan does not affect the number of lamellae/cell but reduces both the number of focal adhesion/lamellae and the percentage of cells forming focal adhesion-positive lamellae. These effects are prevented by blocking RHAMM antibodies and mimicked by agonist RHAMM antibodies. Src-/- fibroblasts exhibit a limited response to PMA but do not increase motility or disassemble focal adhesions in response to a subsequent addition of HA. Rescue of Src-/- fibroblasts with either SrcA or c-Src restores response to close to WT levels. These results suggest that Src activity is uniquely required for both PMA and PMA-induced hyaluronan regulation of random motility and focal adhesion turnover.     

Regulation of nucleosomal core histone variant levels in differentiating murine erythroleukemia cells

During hexamethylenebis(acetamide)-induced terminal differentiation of murine erythroleukemia (MEL) cells in vitro, the histone variant proportions undergo changes similar to those observed in vivo in terminally differentiating cells of the young mouse. Thus, there is a rapid increase in the relative amounts of the variants H2A.1 and H2B.2 in parallel with the increase in the number of hemoglobin-producing cells and the sharp decrease in the growth rate. We show that the changes in variant proportions are not associated with slower growth per se but are most likely due to differential changes in the rates of variant synthesis as a result of commitment to terminal differentiation. In addition, we observed an inducer-specific increase in the rate of synthesis and the relative amount of the minor H2A variant 4, well before hemoglobin accumulation. We also present evidence that H2A and H2B histones are synthesized and incorporated into chromatin at a significant rate even when DNA synthesis is inhibited, suggesting turnover of these histones. H2A and H2B turnover can be detected directly even in exponentially growing cells. H2A.1 and H2B.2 have higher turnover rates than H2A.2 and H2B.1, respectively, in exponentially growing cells, a difference which is even more pronounced in induced cells. The magnitude of the differential turnover is not sufficient to account for the changes in the histone variant proportions in the short life of induced MEL cells but could explain the slow accumulation of H2A.2, H2B.1, and H3.3 in nondividing adult tissues of the mouse.     

Phenotypically similar but ecologically distinct: differences in competitive ability and predation risk among amphipods

Nature is often more diverse than expected with multiple species appearing to occupy the same niche. This observation is especially perplexing when the co‐occurring species are cryptic (i.e. only distinguishable via molecular markers), because phenotypic similarity is expected to correspond with strong niche overlap. One way that phenotypically similar species can coexist is if fine‐scale phenotypic differences affect how species interact with other members of the community that ultimately results in performance tradeoffs. An alternative explanation for co‐occurrence is that phenotypic similarity leads to ecological equivalence allowing species to co‐occur for long periods. We tested whether three phenotypically similar amphipod species that co‐occur exhibit performance tradeoffs that may allow them to stably coexist in lakes. We found that despite their similarity the three species differed in how well they performed in competition with each other and their ability to avoid predation by fish and invertebrate predators. In some species comparisons, performance tradeoffs were apparent with species that perform well against heterospecifics performing poorly against predators and vice versa. We also found evidence for direct antagonistic interactions among amphipod species, in the form of wounding, which may play a role in structuring amphipod assemblages. Finally, the two species with the most similar phenotypes showed comparable responses to competitors and predators, which suggests that long‐term co‐occurrence via ecological equivalence may also be important in this system. Collectively, our results suggest that a mix of performance tradeoffs and ecological equivalence may allow for higher diversity than expected in amphipod assemblages.