Isolation of deuterated histones from yeast grown on media dissolved in H2O

We have succeeded in growing Saccharomyces cerevisiae (baker's yeast) on media containing ²H₂O and isolating the core histones highly deuterated in the non-exchangeable positions. The deuterated histones obtained here are of great value for their possible widespread use for structural studies of chromatin.     

IN VIVO METHYLATION AND TURNOVER OF RAT BRAIN HISTONES

Abstract— The turnover of the different histone components from brain nuclei was studied after the administration of l -[³H]lysine and l -[¹⁴C-methyl]methionine to newborn rats. The radioactivities of the different histone subfractions as well as other proteins were determined over a 280-day period. Biphasic type decay curves (³H and ¹⁴C) were obtained for total brain histones and all the subfractions. From 6 to 40 days of age the half life of total brain histones was 19 days. After reaching brain maturity the half life was 132 days. The lysine rich histone (F₁) was found to turnover the fastest of all the histones, having half lives of 13 and 112 days, respectively. The decay curve for the slightly lysine rich histones (F_2a2, F_2b) gave half lives of 25 days up to 40 days of age and 189 days after reaching brain maturity. The arginine rich histones (F_2a1, F₃) gave a half life of 32 days up to 40 days of age, while no turnover was observed after maturity. The turnover rates of the methyl groups and/or methionyl residues did not vary significantly from the turnover rates of the lysyl residues in the F₂ and F₃ histones. The lysine-rich histones did not contain significant amounts of methionyl residues or methyl groups.
Amino acid analysis of the brain histones revealed that about 3·6 per cent of the lysyl residues in the slightly lysine rich histones were methylated, mainly as ε-N-dimethyllysine. About 13 per cent of the lysyl residues in the arginine rich histones were methylated, mainly as ε-N-monomethyllysine and ε-N-dimethyllysine.     

Metabolism of tritiated and deuterated gibberellins A1, A4 and A9 in Sitka spruce (Picea sitchensis) shoots during the period of cone-bud differentiation

A mixture of tritiated and deuterated gibberellins (GAs) was injected into elongating shoots of Sitka spruce [ Picea sitchensis (Bong.) Carr.] grafts grown under environmental conditions that were either inductive (heat and drought, HD) or non-inductive (cool and wet, CW) for flowering. The metabolites were purified by high performance liquid chromatography (HPLC), detected by liquid scintillation counting of aliquots of collected fractions and identified by gas chromatography–mass spectrometry (GC-MS). Deuterated GA₉ was converted to deuterated GA₄, deuterated GA₃₄, and deuterated GA₁ in both treatments. Deuterated GA₄ was metabolized to deuterated GA₃₄ and deuterated GA₁ in the CW material, but only deuterated GA₁ was detected in the HD material. The amount of detected metabolites was higher in the HD material, caused by a higher rate of metabolism and/or smaller losses of the metabolites during sample purification. GA₁ was converted to a polar unidentified metabolite in both treatments, but to a higher degree in the CW treatment.     

CATABOLISM OF PROSTAGLANDIN ENDOPEROXIDES INTO PROSTAGLANDIN E2 AND F2x BY THE RAT BRAIN

Abstract— Tritium labeled prostaglandin (PG) endoperoxides PGG₂ and PGH₂ were rapidly transformed (2 min, 37°C) in good yield (> 50%) by homogenates of whole rat brain into a mixture of products including prostaglandin E₂ and F_2x: under similar conditions (10min. 37°C) tritium labeled arachidonic acid remained essentially unoxidised. The ratio of PGE-like products: PGF_2x formed was approx 0.5 as determined by radio thin layer chromatography. This ratio changed to unity when homogenates of cerebral cortex or cerebral hemispheres were employed. On the other hand cerebellar homogenates formed PGF_2x in much greater amounts. The structures of the products were confirmed by mass spectrometry and were further supported by experiments using octadeuterio-endoperoxides. In the latter experiments the resulting PGE₂ and PGF_2x contained the expected seven and eight deuterium atoms respectively. Evidence for the formation of heptadeuterio PGD₂. heptadeuterio-6-keto-PGF₁, and hexadeuterio 12-hydroxyheptadecatrienoic acid was also obtained by mass spectrometry. These experiments demonstrate for the first time in brain tissue the biosynthesis of labeled prostaglandins from exogenous tritiated and deuterated precursors.     

Unusual conformational behavior of trisaccharides containing N-acetylglucosamine

Protected trisaccharides containing N-acetylglucosamine can adopt unexpected conformations through the formation of hydrogen bonds involving the amide group. This conformational behavior was observed by NMR spectroscopy when three protected trisaccharides were dissolved in deuterated chloroform and to a lesser extent in deuterated dichloromethane. In contrast, NMR spectra of the same analogues acquired in the hydrogen bond-accepting solvents deuterated acetonitrile and dimethylsulfoxide showed that the N-acetylglucosamine residues adopted the expected ⁴C₁ conformation.     

N-(2-Amino-phenyl)-4-(heteroarylmethyl)-benzamides as new histone deacetylase inhibitors

A variety of N-(2-amino-phenyl)-4-(heteroarylmethyl)-benzamides were designed and synthesized. These compounds were shown to inhibit recombinant human HDAC1 with IC₅₀ values in the sub-micromolar range. In human cancer cells growing in culture these compounds induced hyperacetylation of histones, induced the expression of the tumor suppressor protein p21^WAF1/Cip1, and inhibited cellular proliferation. Certain compounds of this class also showed in vivo activity in various human tumor xenograft models in mice.     

Histones and the first cell cycle in maize germination

The timing of the onset of cell division during seed germination in maize and the role of histones for this process have been studied. Embryonic axes of maize seeds ( Zea mays L. hybrid H-30) were incubated in a sterile nutrient medium for different periods of time. For some experiments putrescine was also added. Mesocotyl, root tip and scutellar node were dissected at specific periods after incubation and the mitotic indices were determined in these tissues. Embryonic axes were incubated in the same medium either with [¹⁴C]-lysine or [³²P]-phosphate. The incorporation of either ¹⁴C or ³²P into histones was followed, both in postribosomal supernatant and in nuclei. It was found that during germination, there is specific timing for meristematic cells entering into cell division. Among the tissues tested, the mesocotyl meristem was the first to initiate this process. De novo synthesis of histones was detected as early as after 6 h of imbibition and the rate increased up to 12 h. Putrescine stimulated cell division and phosphorylation of the histones. The implications of these findings are discussed.     

Histones and chromatin structure in hyperthermophilic Archaea

Abstract: HMf is a histone from the hyperthermophile Methanothermus fervidus . It is the archetype and most studied member of a family of archaeal histones that have primary sequences and three-dimensional structures in common with the eukaryal nucleosome core histones and that bind and compact DNA molecules into nucleosome-like structures (NLS). HMf preparations are mixtures of two similar, small (∼7.5 kDa) polypeptides designated HMfA and HMfB that in vivo form both homodimers and heterodimers. HMfA synthesis predominates during exponential growth but the relative amount of HMfB increases as M. fervidus cells enter the stationary growth phase. Analyses of homogeneous preparations of recombinant (r) (HMfA)₂ and (rHMfB)₂ have demonstrated that these proteins have different DNA-binding and compaction properties in vitro, consistent with different roles in vivo for the (HMfA)₂, (HMfB)₂ and HMfA · HMfB dimers, and for the NLS that they form, in regulating gene expression and in genome compaction and stability.     

Ca2+ -dependent protein phosphorylation in germinated pollen of Nicotiana alata, an ornamental tobacco

The 40 000 g supernatant and 40 000 g pellet from extracts of germinated pollen of Nicotiana alata Link et Otto contain protein kinase activity which catalyzes the phosphorylation of histones, casein and a range of endogenous polypeptides. Phosphorylation of certain low-molecular-weight, casein-derived polypeptides is activated at low (12–37 μ M ) and partially inhibited at higher (540 μ M ) concentrations of free Ca²⁺. Histone phosphorylation is largely Ca²⁺-dependent and is activated by 540 μM free Ca²⁺. No activation of protein phosphorylation by micromolar concentrations of calmodulin is found, but phenothiazine-derived calmodulin antagonists markedly stimulate protein phosphorylation.     

Low-Na+Seawater Induces the Acrosome Reaction and Histone Degradation of Starfish Sperm in the Absence of Egg Jelly

Egg jelly induces the degradation of histones as well as the acrosome reaction in the spermatozoa of Asterina pectinifera . Much similar degradation of histones without any apparent morphological changes such as the acrosome reaction was induced in the spermatozoa by merely dispersing them into Na⁺-free seawater. It required external Ca²⁺ much less than the jelly-induced one in normal seawater, and was not susceptible to Ca²⁺-channel antagonists, verapamil and diltiazem. Once spermatozoa were incubated with egg jelly in Ca²⁺-free seawater, they did not undergo the histone degradation even after subsequent addition of Ca²⁺, but Na⁺-free seawater rescued such blockage. Spontaneous acrosome reaction occurred in seawater containing 10–30 mM Na⁺ in a Ca²⁺-dependent manner. This reaction was accompanied by a rapid increase in intracellular pH (pHi) followed by a large pHi decrease. Diltiazem blocked a large decrease in pHi but scarcely inhibited the acrosome reaction induced by low-Na⁺ seawater. Increasing K⁺ inhibited both pHi changes and the acrosome reaction induced by low-Na⁺ seawater. Decreasing pH of seawater also inhibited the pHi changes but did not affect the acrosome reaction. Strontium was also effective to induce a rapid increase, followed by a gradual decrease, in pHi and the acrosome reaction.     

ESR characteristics of Photosystem I in deuterium oxide: Further evidence that electron acceptor A1 is a quinone

The appearance of ESR signals from Photosystem I (PS I) electron acceptors A₁ and A₀ in water or deuterium oxide suspension was followed using a low-temperature photoaccumulation technique. In deuterated samples the A₁ signal was narrowed by a factor of 0.66 compared with the control. This effect was fully reversible upon resuspension of treated samples in H₂O. The narrow ESR signal from deuterated A₁ had similar power saturation characteristics to the normal signal; however, a signal from a second component resolved by deuteration was saturated at higher microwave powers than the control. The power saturation behaviour of A⁻₁ in un-modified reaction centres indicated that it is an anionic semiquinone in a ‘protic’ environment. Deuteration reversibly modified the relative extents of reduction of iron sulphur electron acceptors A and B such that centre B became the more stable electron acceptor. The g-value and line-width of iron sulphur centre X was not modified by deuteration although it appeared to become more efficiently reduced. These results are discussed in the light of current evidence from optical, electron spin polarisation and extraction experiments that suggest that A₁ is a quinone, probably vitamin K-1.     

In vitro exchange of nucleosomal histones H2a and H2b

We have asked whether exogenous, radiolabeled histones can exchange with nucleosomal histones in an in vitro system. Using two different electrophoretic techniques, we were able to separate the histones contained in nucleosomes from those histones which were simply bound to the surface of the chromatin. Fluorography was used to determine which of the exogenous histones exchange with the nucleosomal histones. We observed substantial exchange of histones H1, H2a, and H2b when the chromatin and exogenous histones were incubated under approximately physiological conditions. We have also observed a small amount of exchange of H2a and H2b, as well as a substantial exchange of H1, from one chromatin fragment to another. Other conditions affecting the exchange of histones H2a and H2b are also reported.     

A pH-dependent interaction between histones H2A and H2B involving secondary and tertiary folding.

It has been shown by high-resolution proton magnetic resonance (PMR) spectroscopy and circular dichroism (CD) that an H2A/H2B histone complex exists after salt extraction of these histones from chromatin and that this complex can be fully renatured from both urea-denatured acid-extracted and from urea-denatured salt-extracted histones. The histone complex is shown to involve specific secondary and tertiary structure. Formation of this complex is observed to be critically dependent on pH, occurring at and above pH 5. It cannot be induced below pH 5 by increase in ionic strength. From CD spectra the H2A/H2B complex is shown to contain about 37% alpha helix but no beta structure, the latter being confirmed by infrared spectroscopy in the 6-mum region. The PMR spectra show that the structured region includes most of the aromatic residues of both histones, at least two histidine residues of H2B and probably histidines 31 and 82 of histone H2A. The secondary structure of histones H2A and H2B is predicted using the Chou and Fasman procedure and comparisons are made between the predictions for histones of different species. These results in conjunction with the experimental evidence lead to the conclusion that at least residues 31-95 of H2A and residues 37-114 of H2B, i.e. the more apolar regions of the molecules, are involved in the tertiary structure of the H2A/H2B complex.     

Nucleosome assembly and epigenetic inheritance

In eukaryotic cells, histones are packaged into octameric core particles with DNA wrapping around to form nucleosomes, which are the basic units of chromatin (Kornberg and Thomas, 1974). Multicellular organisms utilise chromatin marks to translate one single genome into hundreds of epigenomes for their corresponding cell types. Inheritance of epigenetic status is critical for the maintenance of gene expression profile during mitotic cell divisions (Allis et al., 2006). During S phase, canonical histones are deposited onto DNA in a replication-coupled manner (Allis et al., 2006). To understand how dividing cells overcome the dilution of epigenetic marks after chromatin duplication, DNA replication coupled (RC) nucleosome assembly has been of great interest. In this review, we focus on the potential influence of RC nucleosome assembly processes on the maintenance of epigenetic status.     

Use of stable isotopes to identify benzoate as a metabolite of benzene degradation in a sulphidogenic consortium

An enriched sulphidogenic consortium capable of mineralizing benzene was used to study the metabolic pathway of anaerobic benzene degradation. Benzoate was detected in active cultures and benzene was confirmed to be the source of this benzoate by the addition of deuterated benzene (D6) and subsequent detection of deuterated benzoate (D5) in active cultures but not in autoclaved controls. Benzoate was utilized by this culture at 1/12 the rate of benzene, while its presence did not inhibit benzene utilization. The benzene utilization rate was reduced, however, in the presence of 2-fluorobenzoate. When the culture was supplemented with [¹³C]-bicarbonate, the carboxyl group on benzoate was not labelled with [¹³C]-carbon, suggesting that this transformation relies on a more complex set of reactions than simple addition of carbonate.     

Phylogenetic analysis of the core histones H2A, H2B, H3, and H4.

Despite the ubiquity of histones in eukaryotes and their important role in determining the structure and function of chromatin, no detailed studies of the evolution of the histones have been reported. We have constructed phylogenetic trees for the core histones H2A, H2B, H3, and H4. Histones which form dimers (H2A/H2B and H3/H4) have very similar trees and appear to have co-evolved, with the exception of the divergent sea urchin testis H2Bs, for which no corresponding divergent H2As have been identified. The trees for H2A and H2B also support the theory that animals and fungi have a common ancestor. H3 and H4 are 10-fold less divergent than H2A and H2B. Three evolutionary histories are observed for histone variants. H2A.F/Z-type variants arose once early in evolution, while H2A.X variants arose separately, during the evolution of multicellular animals. H3.3-type variants have arisen in multiple independent events.     

The histone H3/H4.N1 complex supplemented with histone H2A-H2B dimers and DNA topoisomerase I forms nucleosomes on circular DNA under physiological conditions

We have fractionated the whole cell extract of Xenopus oocytes (oocyte S-150) and isolated the endogenous components required for DNA supercoiling and nucleosome formation. Histone H2B and the three oocyte-specific H2A proteins were purified as free histones. Histones H3 and H4 were purified 100-fold in a complex with the acidic protein N1. In the presence of DNA topoisomerase I or II, histone H3/H4.N1 complexes supercoil DNA in a reaction that is inhibited by Mg2+, and this inhibition is relieved by NTPs. The supercoiling reaction induced by H3/H4.N1 complexes is enhanced by free histone H2A-H2B dimers, which by themselves do not supercoil DNA. Nuclease digestions and protein analyses indicate that H3/H4.N1 complexes form subnucleosomal particles containing histones H3 and H4. Nucleosomes containing 146-base pair DNA and the four histones are formed when histones H2A and H2B complement the reaction.     

Exchange of histones H1, H2A, and H2B in vivo

We have asked whether histones synthesized in the absence of DNA synthesis can exchange into nucleosomal structures. DNA synthesis was inhibited by incubating hepatoma tissue culture cells in medium containing 5.0 mM hydroxyurea for 40 min. During the final 20 min, the cells were pulsed with [3H]lysine to radiolabel the histones (all five histones are substantially labeled under these conditions). By two electrophoretic techniques, we demonstrate that histones H1, H2A, and H2B synthesized in the presence of hydroxyurea do not merely associate with the surface of the chromatin but instead exchange with preexisting histones so that for the latter two histones there is incorporation into nucleosome structures. On the other hand, H3 and H4 synthesized during this same time period appear to be only weakly bound, if at all, to chromatin. These two histones have been isolated from postnuclear washes and purified. Some possible implications of in vivo exchange are discussed.     

Metabolism of tritiated and deuterated gibberellin A9 in Norway spruce (Picea abies) shoots during the period of cone-bud differentiation

A mixture of tritiated and deuterated gibberellin A₉ (GA₉) was injected into elongating shoots of Norway spruce [ Picea abies (L.) Karst.] grafts grown under environmental conditions that were either inductive (heat and drought, HD) or noninductive (cool and wet, CW) for flowering. The shoots were divided into needles and shoot stems. The metabolites were purified by high performance liquid chromatography (HPLC), detected by liquid scintillation counting of aliquots of collected fractions and identified by gas chromatography-mass spectrometry (GC-MS). Deuterated GA₉ was converted to deuterated GA₄ in both treatments. The major metabolite in the CW-treated material was GA₅₁. The HD-treated material did not convert GA₉ to GA₅₁, but a cellulase-hydrolysable GA₉-conjugate was formed. The same metabolites were found in the shoot stems, though in smaller amounts. The amounts of detected metabolites were higher in the HD material, caused by a higher rate of metabolism and/or smaller losses of the metabolites during sample purification. The estimated amounts of endogenous GAs show that the HD-treated material contained higher amounts of GA₉ but no differences in the amounts of GA₄ were found.