The Core Histone-binding Region of the Murine Cytomegalovirus 89K Immediate Early Protein

The gene regulatory immediate early protein, pp89, of murine cytomegalovirus interacts with both DNA-associated and isolated histones in vitro. We characterized the histone-binding region of pp89 and its cellular localization during cell division to examine the possible interaction between pp89 and chromatin. pp89 expressed constitutively in cell line BALB/c 3T3 IE1 does not interact with condensed chromatin. As observed in infected cells, pp89 is localized within the nucleus of cells during interphase but spreads throughout the cell plasma following degradation of the nuclear membrane during early mitosis. In late telophase, pp89 is reorganized within the nucleus. Analysis of pp89 deletion mutants and of fragments generated by cleavage at pH 2·5 revealed that the regions responsible for association with histone are located between amino acids 71 and 415, and are not identical with the domain that shows homology to histone H2B or the highly acidic carboxy-terminal region. A potential gene-activating role of the high affinity of pp89 for isolated histones and the low affinity for DNA-associated histones is discussed

An acidic region of the 89K murine cytomegalovirus immediate early protein interacts with DNA

The product of the ie 1 gene, the regulatory immediate early protein pp89 of murine cytomegalovirus (MCMV), interacts with core histones, which can mediate the association of pp89 with DNA. We report the capacity of pp89 to interact directly with DNA in the absence of cellular proteins. After separation of proteins by SDS–PAGe, pp89 bound ds- and ssDNA, with a preference for ssDNA. Binding to specific DNA sequences in the MCMV genome was not detected. The DNA-binding region of pp89 was located to amino acids 438 to 534 by analysis of deletion mutants expressed as -galactosidase or TrpE fusion proteins. This region is identical to the highly acidic C-terminal region spanning amino acids 424 to 532. The human cytomegalovirus IE1 protein, which contains a similar extended C-terminal acidic region, does not react with DNA under the same experimental conditions

Thermochemistry of small cationic iron-sulfur clusters

Journal ArticleThe kinetic energy dependences of the reactions of Fen 1 with COS (n=2-6) and CS2 (n=2-5) are studied in a guided-ion beam tandem mass-spectrometer. The main products arise from sulfur transfer and subsequent losses of Fe atoms. In the case of CS2 , this reactant also formally replaces one Fe atom of the cluster to form Fen21CS2 1 with losses of further Fe atoms at elevated energies. In addition, the kinetic energy dependences of the reactions of FenS1 (n=2-4) with Xe and CS2 are studied. The former system yields collision-induced dissociations, whereas the latter reagent effects sulfur transfer accompanied by subsequent losses of Fe atoms. Analyses of the cross sections for endothermic reactions yield the bond energies D0(Fen1-S), n=2-5, D0(SFen+1 1-Fe), n=2-5, D0(SFen+-S), n=1-3, and D0(S2Fen+ 1-Fe), n=2, 3, as well as the ionization energy IE(Fe2S2). These values are derived with explicit consideration of the lifetimes of the energized reaction intermediates. The binding between sulfur and the cluster core strengthens as the cluster size increases, which is rationalized by simple structural arguments

Interaction of the 89K murine cytomegalovirus immediate-early protein with core histones

The conditions that permit the interaction of immediate-early proteins of murine cytornegalovirus (MCMV) with DNA were studied. Chromatography of extracts from infected cells on MCMV DNA cellulose and calf thymus DNA cellulose showed that pp89, the regulatory major immediate-early protein, interacts with DNA and dissociates at salt concentrations between 0.3 and 0.6 M NaCl. pp76, a cleavage product of pp89, and additional minor ie1 proteins eluted already at low ionic strength. Cellular DNA-binding factors were required for association of pp89 with DNA. These factors were identified as core histones. Chromatography of IE proteins on histone-Sepharose in the absence of DNA revealed a high-binding affinity that was resistant to 2 M NaCl. These results suggest that pp89 has no direct DNA-binding activity. A role for an amino acid sequence homology in the N-terminal region of pp89 with histone H2B in the pp89-histone-DNA Interaction is discussed

The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription

To study trans-activation of gene expression by murine cytomegalovirus (MCMV) immediate-early (IE) proteins, the IE coding region 1 (ie1), which encodes the 89,000-Mr IE phosphoprotein (pp89), was stably introduced into L cells. A cell line was selected and characterized that efficiently expressed the authentic viral protein. The pp89 that was constitutively expressed in L cells stimulated the expression of transfected recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of viral promoters. The regulatory function of the ie1 product was confirmed by transient expression assays in which MCMV IE genes were cotransfected into L cells together with recombinant constructs of the CAT gene. For CAT activation by the ie1 product, a promoter region was required, but there was no preferential activation of a herpes simplex virus type 1 delayed-early promoter. All plasmid constructs that contained the intact coding sequences for pp89 induced gene expression in trans. The MCMV enhancer region was not essential for the expression of a functional IE gene product, and testing of the cis-regulatory activity of the MCMV enhancer revealed a low activity in L cells. Another region transcribed at IE times of infection, IE coding region 2, was unable to induce CAT expression and also did not augment the functional activity of ie1 after cotransfection

Presentation of CMV immediate-early antigen to cytolytic T lymphocytes is selectively prevented by viral genes expressed in the early phase

The regulation of antigen processing and presentation to MHC class I-restricted cytolytic T lymphocytes was studied in cells infected with murine cytomegalovirus. Recognition by cytolytic T lymphocytes of the phosphoprotein pp89, the immunodominant viral antigen expressed in the immediate-early phase of infection, was selectively prevented during the subsequent expression of viral early genes. The surface expression of MHC class I glycoproteins and their capacity to present externally added pp89-derived antigenic peptides were not affected. Because recognition of several other antigens occurred during the early phase, a general failure in processing and presentation was excluded. Since neither rate of synthesis, amount, stability, nor nuclear transport of pp89 was modified, the failure in recognition indicates a selective interference with pp89 antigen processing and presentation

Exposing elusive cationic magnesium-chloro aggregates in aluminate complexes through donor control

The cationic magnesium moiety of magnesium organohalo aluminate complexes, relevant to rechargeable Mg battery electrolytes, typically takes the thermodynamically favourable dinuclear [Mg2Cl3]+ form in the solid-state. We now report that judicious choice of Lewis donor allows the deliberate synthesis and isolation of the hitherto only postulated mononuclear [MgCl]+ and trinuclear [Mg3Cl5]+ modifications, forming a comparable series with a common aluminate anion [(Dipp)(Me3Si)NAlCl3]. By pre-forming the Al-N bond prior to introduction of the Mg source, a consistently reproducible protocol is reported. Usage of the green solvent 2-methyltetrahydrofuran in place of THF in the context of Mg/Al battery electrolyte type complexes is also promoted

Doppler-free ion imaging of hydrogen molecules produced in bimolecular reactions

Abstract We report on the use of two-color Doppler-free [(1 + 1 0 ) + 1/1 0 ] resonance enhanced multiphoton ionization (REMPI) for threedimensional imaging of state-selected product molecules from bimolecular reactions. We demonstrate the viability of this method by measuring differential cross sections for the reaction H + D 2 ! D + HD(v 0 = 1,j 0 = 1,5,8) at 1.7 eV collision energy. We achieve higher resolution allowing us to observe oscillations that were not resolved by previous experiments; these oscillations agree closely with quantum mechanical calculations

Structural and reactivity insights in Mg–Zn hybrid chemistry : Zn–I exchange and Pd-catalysed cross-coupling applications of aromatic substrates

Expanding the synthetic potential of Mg-Zn hybrid organyl reagents (generated via transmetallation reactions), this study uncovers a versatile approach, involving a sequence of direct Zn-I exchange and Pd catalysed cross-coupling reactions which grants access to a wide range of asymmetric bis(aryls). By combining X-ray crystallography with ESI-MS and non-deuterium NMR spectroscopic studies, new light is shed on the heterobimetallic constitution of the intriguing organometallic species [(THF)4MgCl 2Zn(tBu)Cl] (1) and [{Mg2Cl3(THF) 6}+{ZntBu3}-] (2), formed through transmetallation of tBuMgCl with n equivalent amounts of ZnCl2 (n = 1 and 3 respectively). Operating by cooperative effects, alkyl-rich hybrid 2 can effectively promote direct Zn-I exchange reactions with aromatic halides in short periods of time at room temperature in THF solution. The structural elucidation of key organometallic intermediates involved in some of these Zn-I exchanges, provides new reactivity insights into how these bimetallic systems operate. Thus, while the reaction of 2 with 3 equivalents of 2-iodoanisole (3b) gives magnesium dizincate [{Mg(THF)6}2+{Zn(o-C 6H4-OMe)3}2 -] (4) which demonstrates the 3-fold activation of the tBu groups attached to Zn in 2, using 2-iodobenzonitrile (3i), only two tBu groups react with the substrate, affording [(THF)4MgCl(NC-o-C6H4)ZnI(o-C 6H4-CN)(THF)] (7). In 7 Mg and Zn are connected by an aryl bridge, suggesting that the formation of contacted ion-pair hybrids may have a deactivating effect on the outcome of the Zn-I exchange process. A wide range of homoleptic tris(aryl) zincate intermediates have been prepared in situ and used as precursors in Pd catalysed cross-coupling reactions, affording bis(aryls) 6a-s in excellent yields under mild reaction conditions without the need of any additive or polar cosolvent such as NMP or DMI

Organozinc pivalate reagents : segregation, solubility, stabilisation and structural insights

The pivalates RZnOPiv⋅Mg(OPiv)X⋅n LiCl (OPiv=pivalate; R=aryl; X=Cl, Br, I) stand out amongst salt-supported organometallic reagents, because apart from their effectiveness in Negishi cross-coupling reactions, they show more resistance to attack by moist air than conventional organometallic compounds. Herein a combination of synthesis, coupling applications, X-ray crystallographic studies, NMR (including DOSY) studies, and ESI mass spectrometric studies provide details of these pivalate reagents in their own right. A p-tolyl case system shows that in [D8]THF solution these reagents exist as separated Me(p-C6H4)ZnCl and Mg(OPiv)2 species. Air exposure tests and X-ray crystallographic studies indicate that Mg(OPiv)2 enhances the air stability of aryl zinc species by sequestering H2O contaminants. Coupling reactions of Me(p-C6H4)ZnX (where X=different salts) with 4-bromoanisole highlight the importance of the presence of Mg(OPiv)2. Insight into the role of LiCl in these multicomponent mixtures is provided by the molecular structure of [(THF)2Li2(Cl)2(OPiv)2Zn]