Structure and dynamic properties of a glucocorticoid receptor-induced chromatin transition.

Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W. D. Pennie, S. John, and G. L. Hager, Mol. Cell. Biol. 18:3633-3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a "hit-and-run" mechanism for GR action in living cells (J. G. McNally, W. G. Müller, D. Walker, and G. L. Hager, Science 287:1262-1264, 2000).

The nucleosomal array: structure/function relationships.

A nucleosomal array consists of core histone octamer-DNA complexes spaced at approximately 200 bp intervals along a DNA molecule. Nucleosomal arrays are the fundamental building block of chromosomal superstructures, the substrate for transcription, and the first nucleoprotein assembly laid down after DNA replication. The development of homogeneous length-defined nucleosomal arrays has led to a greatly improved understanding of nucleosomal array structural dynamics in the solution state. Under physiological salt conditions, a nucleosomal array is in dynamic equilibrium between folded, self-associated and dissociated conformational states. Folding and self-association are both critically dependent on the core histone tail domains, consistent with an essential functional role for the tail domains in the mediation of chromosomal level DNA compaction in the nucleus. Nucleosomal array folding is repressive in transcription in vitro, but can be overcome by compositional (e.g., tail domain acetylation) and configurational (e.g., histone octamer depletion) changes that are correlated with transcriptional activation in vivo. The mechanism of replication-coupled chromatin assembly also appears to be functionally linked to the dynamic properties of nucleosomal arrays. Although once thought to be both structurally and functionally inert, it is now apparent that the nucleosomal array is a key participant in the biological processes that take place within the chromosomal fibers of eukaryotes.

The inverted repeat regions of the simian varicella virus and varicella-zoster virus genomes have a similar genetic organization.

Simian varicella virus (SVV) causes a varicella-like disease in nonhuman primates. The DNA sequence and genetic organization of the inverted repeat region (RS) of the SVV genome was determined. The SVV RS is 7559 bp in size with 56% guanine+cytosine (G+C) content and includes 3 open reading frames (ORFs). The SVV RS1 ORF encodes a 1279 amino acid (aa) protein with 58 and 39% identity to the varicella-zoster virus (VZV) gene 62 and herpes simplex virus type 1 (HSV-1) ICP4 homologs, respectively. The predicted 261 aa SVV RS2 polypeptide possesses 52% identity with the VZV gene 63 homolog and 23% identity with the HSV-1 ICP22. The SVV RS3 encodes a 187 aa polypeptide with 56% and 28% identity to the VZV gene 64 and the HSV-1 US10 homologs, respectively, and includes an atypical zinc finger motif. A G+C-rich 16 base-pair (bp) sequence which is repeated 7 times and a putative SVV origin of replication were identified between the RS1 and RS2 ORFs. Comparison with the VZV RS indicates the SVV and VZV RS regions are similar in size and genetic organization.

Characterization of the expression and immunogenicity of poliovirus replicons that encode simian immunodeficiency virus SIVmac239 Gag or envelope SU proteins.

The effectiveness of the poliovirus vaccines to induce both systemic and mucosal immunity has prompted the development of this virus as a vector in which to express foreign proteins. Our laboratory has previously reported on the construction and characterization of poliovirus genomes that encode HIV-1 proteins (Porter DC, et al.: J Virol 1996;70:2643-2649). To develop this system further, we have constructed poliovirus genomes, referred to as replicons, which encode the SIVmac239 Gag or Env SU in place of the poliovirus capsid gene (P1). Since the replicons do not encode capsid proteins, they are encapsidated into poliovirus by passage with a recombinant vaccinia virus, VVP1, which provides the poliovirus capsid proteins in trans. Using this system, we have derived stocks of the encapsidated replicons which encode the SIVmac239 or Env SU protein. Infection of cells with the replicon that encodes SIVmac239 Gag resulted in the expression of a 55-kDa protein that was released from the infected cells. Analysis of the sedimentation of the released proteins by sucrose density gradient centrifugation revealed that the protein was released from the cell in the form of a virus-like particle. Infection of cells with the replicons encoding the SIVmac239 Env SU resulted in the expression of a 63-kDa protein, corresponding to the molecular mass predicted for the nonglycosylated SIVmac239 SU protein. A second protein with a molecular mass greater than 160 kDa was also immunoprecipitated. After enzymatic deglycosylation, this protein migrated at a molecular mass consistent with that for an Env SU dimer. Analysis of the medium from cells infected with the replicon encoding SIVmac239 Env SU revealed the presence of a protein of molecular mass 85-90 kDa, possibly representing a fragment of the SIVmac239 or Env SU protein. To determine the immunogenicity of the replicons encoding SIVmac239 Gag or Env SU, transgenic mice that express the human receptor for poliovirus, and are thus susceptible to poliovirus, were immunized via the intramuscular route. A serum antibody response to SIV envelope was detected following booster immunization, establishing that the encapsidated replicon was immunogenic. Finally, we demonstrate that the replicons have the capacity to infect peripheral blood mononuclear monocytes/macrophages, suggesting that this cell is a possible target for in vivo infection. The results of our studies, then, lend further support for the development and application of recombinant poliovirus replicons in a vaccine strategy.

Polyclonal rabbit antisera that detect the Vpr protein of SIVSM and SIVMAC on immunoblots of purified virions.

Antisera suitable for detection of SIVSM or SIVMAC Vpr proteins on Western blots of purified virions are currently not available. We have expressed the Vpr protein of SIVSMPBj1.9 in a gst-based prokaryotic expression system and used it to raise polyclonal antisera in rabbits. Two immune sera were obtained that specifically recognized both cell- and virion-associated Vpr protein on immunoblots of three different SIV isolates (SIVSMPBj1.9, SIVMACBK28, and SIVMAC239). Because Vpr is believed to play an important role in HIV/SIV replication and pathogenesis, these reagents will allow the extension of functional analyses of this protein to a broader spectrum of viruses. Both antisera and the gst-Vpr expression plasmid have been submitted to the NIAID AIDS Research and Reagent Program and are available to interested investigators.

Attenuation and activation characteristics of steel and tungsten and the suitability of these materials for use in a fast neutron multileaf collimator.

A computer controlled multileaf collimator (MLC) is being designed to replace the multirod collimator (MRC) at present used to shape the d(48.5) + Be neutron beam from the Harper Hospital superconducting cyclotron. The computer controlled MLC will improve efficiency and allow for the future development of intensity modulated radiation therapy with neutrons. The existing MRC uses tungsten rods, while the new MLC will use steel as the leaf material. In the current study the attenuation and activation characteristics of steel are compared with those of tungsten to ensure that (a) the attenuation achieved in the MLC is at least equivalent to that of the existing MRC, and (b) that the activation of the steel will not result in a significant change in the activation levels within the treatment room. The latter point is important since personnel exposure (particularly to the radiation therapy technologists) from induced radioactivity must be minimized. Measurement of the neutron beam attenuation in a broad beam geometry showed that a 30 cm thick steel leaf yielded 2.5% transmission. This compared favorably with the 4% transmission obtained with the existing MRC. Irradiation of steel and tungsten samples at different depths in a 30 cm steel block indicated that the activation of steel should be no worse than that of tungsten.

DNA sequence and genetic organization of the unique short (US) region of the simian varicella virus genome.

Simian varicella virus (SVV) infection of nonhuman primates is a model for the study of human varicella zoster virus (VZV) infections. The DNA sequence of the entire SVV unique short (US) region and adjacent flanking sequences of the inverted repeats were determined. The US region is 4904 bp in size and has a 60.9% A + T base composition. Four potential open reading frames (ORFs), designated SVUS 1, SVUS 2, SVUS 3, and SVUS 4, were identified and found to be remarkably similar in size, genetic content, and transcriptional orientation to their respective VZV US counterparts; ORF 65, ORF 66 (US PK), ORF 67 (gpIV), and ORF 68 (gpI). The SVUS 1 ORF encodes a putative 9 kDa homolog of the herpes simplex virus type-1 (HSV-1) US9 tegument phosphoprotein. The SVUS 2 ORF encodes a predicted 345 amino acid polypeptide that contains a number of sequence domains conserved in cellular and viral serine/threonine (S/T) protein kinases and exhibits extensive homology with previously reported alphaherpesviral US S/T PKs, including VZV ORF 66, HSV-1 US3, pseudorabies virus (PRV) PK, and equine herpesvirus (EHV-1) ORF 69. The SVUS 3 and SVUS 4 ORFs exhibit features characteristic of membrane glycoproteins: an amino terminal signal sequence, potential N-linked glycosylation sites, and a large hydrophobic transmembrane domain. The predicted 353 amino acid protein encoded by SVUS 3 ORF is homologous to the VZV gpIV (ORF 67), HSV-1 gI (US7), PRV gp63, and EHV-1 gI (ORF 73) gene products. The SVUS 4 ORF encodes a putative 604 amino acid polypeptide which exhibits extensive homology with VZV gpI and more limited homology with HSV-1 gE (US8), PRV gpI, and EHV gE (ORF 74). This report describes the initial characterization of individual SVV genes and further defines the evolutionary relationships between SVV, VZV, and other alphaherpesviruses.

Transcriptional analysis of two simian varicella virus glycoprotein genes which are homologous to varicella-zoster virus gpI (gE) and gpIV (gI).

Simian varicella virus (SVV) causes a natural, varicella-like disease in nonhuman primates. The unique short region of the SVV genome contains four open reading frames (ORFs), two of which encode glycoproteins that exhibit extensive homology with varicella-zoster virus (VZV) gpIV (gI) and gpI (gE). Northern hybridization, primer extension, and RNase protection analyses were employed to define precisely the transcripts mapping to the SVV gpIV and gpI genes. A total of five transcripts composing two coterminal families of RNAs were mapped to the SVV gpIV and gpI ORF region. Based on transcriptional mapping and previous DNA sequence analysis, two transcripts 1.3 and 2.2 kb in size were assigned to the SVV gpIV and gpI genes, respectively. The transcriptional patterns described in this study for the SVV gpIV and gpI ORFs are analogous to those previously reported for the homologous glycoproteins genes encoding the herpes simplex virus type 1 Us7 (gI) and Us8 (gE) and VZV gpIV and gpI genes. In addition, the transcriptional start site for the VZV gpI RNA was determined. DNA alignments of the promoter regions for the SVV and VZV gpIV and gpI genes revealed a number of cis-acting elements which are conserved between the two viruses. The characterization of SVV glycoprotein genes will facilitate future studies to define their role in SVV pathogenesis and immunity and assist in the construction of recombinant vaccines which could be evaluated in the simian varicella model.

Simian varicella virus: characterization of virion and infected cell polypeptides and the antigenic cross-reactivity with varicella-zoster virus.

Simian varicella virus (SVV) causes a varicella-like disease in non-human primates. In this study, SVV virions were purified from SVV-infected BSC-1 cells by zonal and differential gradient centrifugation and the virion polypeptide composition was analysed by SDS-PAGE. SVV virions had a buoyant density of 1.21 g/ml, identical to the value obtained for varicella-zoster virus (VZV) virions purified by the same method. Electron microscopy of the concentrated SVV virions revealed characteristic herpesvirus morphology. SVV virions consisted of at least 30 polypeptide species ranging from 16K to greater than 200K. The electrophoretic profiles of radiolabelled SVV and VZV virion polypeptides were very similar. Immunoprecipitations of solubilized SVV-infected cell preparations using SVV immune sera revealed at least 18 viral polypeptides with an Mr range of 12K to 142K and six glycoproteins ranging from 46K to 115K. In addition, extensive cross-reactivity between SVV and VZV proteins and glycoproteins was demonstrated by immunoprecipitation with heterologous immune sera. The high degree of antigenic relatedness between SVV and VZV provides further support for simian varicella as a model for VZV infections.

Core histone tail domains mediate oligonucleosome folding and nucleosomal DNA organization through distinct molecular mechanisms.

Defined oligonucleosome model systems have been used to investigate the molecular mechanisms through which the core histone tail domains modulate chromatin structure. In low salt conditions, the tail domains function at the nucleosome level to facilitate proper organization of nucleosomal DNA, i.e. wrapping of DNA around the histone octamer. Mg2+ ions can substitute for the tail domains to yield a trypsinized oligonucleosome structure that is indistinguishable from that of an intact nucleosomal array in low salt. However, Mg(2+)-dependent formation of highly folded oligonucleosome structures absolutely requires the histone tail domains, and is associated with rearrangement of the tails to a non-nucleosomal location. We conclude that the tail domains mediate oligonucleosome folding and nucleosomal DNA organization through fundamentally different molecular mechanisms.

Active dielectric film laser.

A laser configuration of an optical amplifier that consists of an active dielectric thin film is described. Both the spatial and the temporal mode structure are studied. Analytical and numerical formulations are used to describe the effects of this laser configuration, and experiments are performed to verify the predicted results. It is predicted and observed that the resonator beam width is dominated by the active region width of the film, not by the resonator-mirror curvature.

Quantitative analysis of macromolecular conformational changes using agarose gel electrophoresis: application to chromatin folding.

Quantitative analysis of chromatin electrophoretic mobility (mu) in agarose gels provides a measure of three structural parameters: average surface electrical charge density, which is proportional to the gel-free mu (mu 0), effective radius (Re), and particle deformability [Fletcher, T. M., Krishnan, U., Serwer, P., & Hansen, J. C. (1994) Biochemistry 33, 2226-2233]. To determine whether the intramolecular conformational changes associated with salt-dependent chromatin folding influence these electrophoretic parameters, defined oligonucleosomes were reconstituted from monodisperse tandemly repeated 5S DNA and varying amounts of histone octamers. These oligonucleosomes were subjected to both quantitative agarose gel electrophoresis and analytical velocity ultracentrifugation in buffers containing 0-2 mM MgCl2. Ionic conditions that caused a 40% increase in the oligonucleosome sedimentation coefficient (s20,w) also caused both a 30% decrease in Re and a 60% decrease in the magnitude of the mu 0. Furthermore, the Mg(2+)-dependent changes in s20,w, Re, and mu 0 each exhibited the same nonlinear dependence on the degree of nucleosome saturation of the DNA. These data demonstrate that quantitative agarose gel electrophoresis can be used to detect and characterize the process of chromatin folding. In addition, they suggest that this approach can be used for characterization of the conformational dynamics of many other types of macromolecular assemblies, including those systems that are not yet amenable for study by more traditional quantitative biophysical techniques.

Human telomerase inhibition by 7-deaza-2'-deoxypurine nucleoside triphosphates.

Telomeres play an important role in chromosome organization and stability. Human telomerase is a terminal transferase that adds TTAGGG units onto the telomere end. In general, telomerase activity is not detected in normal somatic cells but is present in immortalized cells. Consequently, telomerase might be a selective target for cancer chemotherapy. Using cell-free biochemical telomerase assay, we have found that 7-deaza-2'-deoxyguanosine-5'-triphosphate (7-deaza-dGTP) and 7-deaza-2'-deoxyadenosine-5'-triphosphate (7-deaza-dATP) were potent telomerase inhibitors. The concentrations of inhibitors in which 50% of the telomerase activity was inhibited (IC50 values) were 11 and 8 microM for 7-deaza-dGTP and 7-deaza-dATP, respectively. Additional studies show that both 7-deaza-dGTP and 7-deaza-dATP were also incorporated into telomeric DNA by telomerase. However, incorporation of 7-deaza-dATP or 7-deaza-dGTP results in a telomeric ladder that is prematurely shortened. No difference in the number or position of pause sites were observed when 7-deaza-dATP was compared to dATP as substrates. On the other hand, both a shift and an increase in pause sites was observed when dGTP was replaced by 7-deaza-dGTP. Incorporation of 7-deaza nucleotides by telomerase may be used as a tool for the study of telomerase mechanism and function. In addition, this may be a novel approach in the design of new telomerase inhibitors.

Enhanced transcription factor access to arrays of histone H3/H4 tetramer.DNA complexes in vitro: implications for replication and transcription.

Defined model systems consisting of physiologically spaced arrays of H3/H4 tetramer.5S rDNA complexes have been assembled in vitro from pure components. Analytical hydrodynamic and electrophoretic studies have revealed that the structural features of H3/H4 tetramer arrays closely resemble those of naked DNA. The reptation in agarose gels of H3/H4 tetramer arrays is essentially indistinguishable from naked DNA, the gel-free mobility of H3/H4 tetramer arrays relative to naked DNA is reduced by only 6% compared with 20% for nucleosomal arrays, and H3/H4 tetramer arrays are incapable of folding under ionic conditions where nucleosomal arrays are extensively folded. We further show that the cognate binding sites for transcription factor TFIIIA are significantly more accessible when the rDNA is complexed with H3/H4 tetramers than with histone octamers. These results suggest that the processes of DNA replication and transcription have evolved to exploit the unique structural properties of H3/H4 tetramer arrays.

Enzymatic activity of endogenous telomerase associated with intact nuclei from human leukemia CEM cells.

Telomerase, a telomere-specific DNA polymerase and novel target for chemotherapeutic intervention, is found in many types of cancers. Telomerase activity is typically assayed using an exogenous primer and cellular extracts as the source of enzyme. Since the nuclear organization might affect telomerase function, we developed a system in which telomerase in intact nuclei catalyzes primer extension. Telomerase activity in isotonically isolated nuclei from human CEM cells shows low processivity (addition of up to four TTAGGG repeats). In contrast, telomerase activity which leaks into a 500 g postnuclear supernatant and the activity in a CHAPS extract are highly processive. The nucleotide inhibitor, 7-deaza-dGTP, seems to be more inhibitory against the nuclei-associated enzyme compared to telomerase from cytoplasmic extracts. However, 7-deaza-dATP and ddGTP are less inhibitory against nuclei-associated telomerase. The results suggest that the association of telomerase with the nuclear chromatin affects telomerase activity. Examination of telomerase activity in a more natural nuclear environment may shed new light on the telomerase function and provide a useful system for the evaluation of new telomerase inhibitors.

The simian varicella virus and varicella zoster virus genomes are similar in size and structure.

Simian varicella virus (SVV) DNA was purified from viral nucleocapsids and the molecular structure of the SVV genome was determined. SVV DNA was analyzed by agarose gel electrophoresis of BamHI, BglII, EcoRI, and PstI restriction endonuclease digests. SVV and varicella zoster virus (VZV) DNAs were demonstrated to have distinct restriction endonuclease profiles. Summation of the sizes of individual restriction endonuclease fragments indicate the size of SVV DNA is congruent to 121 kilobase pairs (kbp) or congruent to 76.8 megadaltons (Md). Electron microscopy, lambda exonuclease analysis, and Southern blot DNA hybridizations were utilized to determine the molecular structure of the SVV genome and to construct restriction endonuclease maps. The results indicate that SVV DNA consists of a long component (L, congruent to 100 kbp) covalently linked to a short component (S, congruent to 20 kbp) which is composed of a unique short sequence (Us, 5.3 +/- 0.7 kbp) bracketed by inverted repeat sequences (TRs and IRs, congruent to 7.2 kbp). The presence of 0.5 M PstI restriction endonuclease fragments indicates that the S component may invert relative to the L component and that the genome exists in two major isomeric forms. The findings demonstrate that the SVV and VZV genomes are similar in size and structure.

Characterization and mapping of simian varicella virus transcripts.

The size and genomic location of viral transcripts expressed in simian varicella virus (SVV)-infected Vero cells were determined. Total cellular RNA and polyadenylated RNA were isolated from SVV-infected and mock-infected Vero cells. Viral transcripts were detected by Northern blot hybridization analysis using overlapping SVV DNA probes representative of the entire SVV genome. The results indicated that all regions of the SVV genome are transcribed during SVV infection in vitro. At least 53 distinct viral RNA species ranging in size from 9.2 to 0.8 kb were detected. DNA probes derived from the SVV DNA long (L) and short (S) components hybridized to 44 RNAs (9.2 to 0.8 kb) and nine RNAs (4.9 to 0.8 kb), respectively. A transcript map of the SVV genome was constructed. The comparison made between the transcript maps of SVV and varicella-zoster virus (VZV) provides further support that the SVV and VZV genomes have an analogous gene organization.

Quantitative agarose gel electrophoresis of chromatin: nucleosome-dependent changes in charge, sharp, and deformability at low ionic strength.

The surface electrical charge density and the deformability of nucleosomal arrays have been characterized by quantitative agarose gel electrophoresis. Monodisperse linear DNA (2.5-3.3 kbp) was reconstituted with histone octamers into either saturated (approximately 1 nucleosome/200-bp DNA) or subsaturated (< 1 nucleosome/200-bp DNA) nucleosomal arrays. The electrophoretic mobility (mu) of both nucleosome-free DNA and nucleosomal arrays was determined at low ionic strength in 0.2-3.0% agarose gels. A semilogarithmic plot of mu vs gel concentration was linear for DNA and convex for saturated nucleosomal arrays. By extrapolating the mu to 0% agarose, the magnitude of the gel-free mu of saturated nucleosomal arrays was found to be approximately 20% lower than that of nucleosome-free DNA molecules. This difference is explained by the net neutralization of approximately 85 DNA negative charges by each histone octamer. By using an internal standard to measure the effective pore size (Pe) of the agarose gel, the effective radius (R) of DNA and nucleosomal arrays was determined at each agarose concentration. In the more dilute gels (Pe > or = 400 nm), the differences between the effective R values of DNA, subsaturated nucleosomal arrays, and saturated nucleosomal arrays are consistent with the differences in their hydrodynamic shapes as measured by analytical velocity centrifugation. However, as Pe decreases, the effective R of both nucleosome-free DNA and subsaturated nucleosomal arrays decreases significantly. This is in contrast to the effective R of saturated nucleosomal arrays, which remains constant at all Pe.(ABSTRACT TRUNCATED AT 250 WORDS)