Biomedical subjects

X He

Publications and source records attributed to X He.

At least 19 recordsLinked to original sources

PubMed2000

A NK1.1+ thymocyte-derived TCR beta-chain transgene promotes positive selection of thymic NK1.1+ alpha beta T cells.

As a consequence of the peptide specificity of intrathymic positive selection, mice transgenic for a rearranged TCR beta-chain derived from conventional alphabeta T lymphocytes frequently carry mature T cells with significant skewing in the repertoire of the companion alpha-chain. To assess the generality of such an influence, we generated transgenic (Tg) mice expressing a beta-chain derived from nonclassical, NK1.1+ alphabeta T cells, the thymus-derived, CD1. 1-specific DN32H6 T cell hybridoma. Results of the sequence analysis of genomic DNA from developing DN32H6 beta Tg thymocytes revealed that the frequency of the parental alpha-chain sequence, in this instance the Valpha14-Jalpha281 canonical alpha-chain, is specifically and in a CD1.1-dependent manner, increased in the postselection thymocyte population. In accordance, we found phenotypic and functional evidence for an increased frequency of thymic, but interestingly not peripheral, NK1.1+ alphabeta T cells in DN32H6 beta Tg mice, possibly indicating a thymic determinant-dependent maintenance. Thus, in vivo expression of the rearranged TCR beta-chain from a thymus-derived NK1.1+ Valpha14+ T cell hybridoma promotes positive selection of thymic NK1.1+ alphabeta T cells. These observations indicate that the strong influence of productive beta-chain rearrangements on the TCR sequence and specificity of developing thymocytes, which operates through positive selection on self-determinants, applies to both classical and nonclassical alphabeta T cells and therefore represents a general phenomenon in intrathymic alphabeta T lymphocyte development.

Animals↗

PubMed2000

Determinants of recognition of ligands binding to benzodiazepine receptor/GABA(A) receptors initiating sedation.

Complementary behavioral and computational studies of 21 structurally diverse, gamma-amino butyric acid (GABA)(A) benzodiazepine receptor ligands that influence spontaneous locomotor activity have been performed in this work. This behavioral endpoint is a well-accepted indicator of sedation particularly for GABA(A)/benzodiazepine receptor ligands. The goal of the work presented here is the identification and assessment of the minimum requirements for ligand recognition of GABA(A)/benzodiazepine receptors leading to activity at the sedation endpoint embedded in a common 3D pharmacophore for recognition. Using the experimental results, together with a systematic computational procedure developed in our laboratory, a five-component 3D pharmacophore for recognition of the GABA(A) receptor subtypes associated with the sedative behavioral response has been developed consisting of: two proton-accepting moieties, a hydrophobic region, a ring with polar moieties and an aromatic ring in a common geometric arrangement in all ligands having an effect at the sedation endpoint. To provide further evidence that the 3D pharmacophore developed embodied common requirements for receptor recognition, a pharmacophore analysis was performed for agonists, inverse agonists and antagonists separately. Each of the resulting pharmacophores contained the same five moieties at comparable distances to those found for the pharmacophore generated using all of them together. This result confirms that this pharmacophore constitutes a recognition pharmacophore representing required features in the overlapping portion of their binding sites. The reliability of this 3D pharmacophore was then assessed in several ways. First, it was determined that ligands that had no effect at the sedation endpoint did not comply with the pharmacophore requirements. Second, four benzodiazepine receptor ligands known to have an effect at the sedation endpoint, but not used in the pharmacophore development were found to satisfy the requirements of this pharmacophore. Third, the geometric and chemical requirements embedded in this pharmacophore were used to search 3D databases resulting in the identification of benzodiazepine receptor ligands known to affect sedation, but not included in the pharmacophore development. Finally, a 3D-quantitative structure analysis procedure (QSAR) model was developed based upon the ligands in the training set superimposed at their sedation pharmacophore points. The 3D-QSAR model shows good predictivity for binding of these ligands to receptor subtypes containing alpha1 but not alpha5 subunits. The pharmacophore developed for the sedation endpoint thus provides a predictive binding model for diverse ligand binding to alpha1 containing receptor subtypes.

Animals↗

PubMed2000

Transient sister chromatid separation and elastic deformation of chromosomes during mitosis in budding yeast.

The accurate segregation of chromosomes at mitosis requires that all pairs of chromatids bind correctly to microtubules prior to the dissolution of sister cohesion and the initiation of anaphase. By analyzing the motion of GFP-tagged S. cerevisiae chromosomes, we show that kinetochore-microtubule attachments impose sufficient tension on sisters during prometaphase to transiently separate centromeric chromatin toward opposite sides of the spindle. Transient separations of 2-10 min duration occur in the absence of cohesin proteolysis, are characterized by independent motion of the sisters along the spindle, and are followed by the apparent reestablishment of sister linkages. The existence of transient sister separation in yeast explains the unusual bilobed localization of kinetochore proteins and supports an alternative model for spindle structure. By analogy with animal cells, we propose that yeast centromeric chromatin acts as a tensiometer.

Chromatids↗

PubMed2000

Development of a scintillation proximity assay for beta-ketoacyl-acyl carrier protein synthase III.

Assays of beta-ketoacyl-acyl carrier protein synthases III (KASIII; FabH), a key enzyme initiating bacterial type II fatty acid biosynthesis, usually involve incubation of radiolabeled acetyl-coenzyme A and malonyl-acyl carrier protein (MACP). The radiolabeled acetoacetyl-ACP product is precipitated and separated from the substrate before quantitation. We have developed a scintillation proximity assay (SPA) where use of biotinylated MACP (BMACP) allows the generation of a biotinylated acetoacetyl-ACP. This product, when captured by the streptavidin-coated scintillant-impregnated microspheres, generates an SPA signal. A BMACP K(m) of 7.1 microM was determined using this SPA with the Streptomyces glaucescens FabH. A similar MACP K(m) (6 microM) was determined in a precipitation assay, demonstrating that BMACP is an effective substrate for FabH. IC(50) values of 15.2 microM (SPA) and 24.8 microM were obtained with iodoacetamide and the S. glaucescens FabH. Comparable IC(50) values of 160 microM (SPA) and 125 microM were also obtained with the antibiotic thiolactomycin and the Escherichia coli FabH. These observations demonstrate that FabH inhibitors can be readily detected using a SPA with BMACP and that the effectiveness of inhibitors in the SPA is comparable to that obtained using MACP and a standard TCA precipitation assay. A FabH SPA adaptable to high-throughput screening should facilitate the discovery of potential novel antibiotics.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed2000

Intracellular Ca(2+) and Ca(2+)/calmodulin-dependent kinase II mediate acute potentiation of neurotransmitter release by neurotrophin-3.

Neurotrophins have been shown to acutely modulate synaptic transmission in a variety of systems, but the underlying signaling mechanisms remain unclear. Here we provide evidence for an unusual mechanism that mediates synaptic potentiation at the neuromuscular junction (NMJ) induced by neurotrophin-3 (NT3), using Xenopus nerve-muscle co-culture. Unlike brain-derived neurotrophic factor (BDNF), which requires Ca(2+) influx for its acute effect, NT3 rapidly enhances spontaneous transmitter release at the developing NMJ even when Ca(2+) influx is completely blocked, suggesting that the NT3 effect is independent of extracellular Ca(2+). Depletion of intracellular Ca(2+) stores, or blockade of inositol 1, 4, 5-trisphosphate (IP3) or ryanodine receptors, prevents the NT3-induced synaptic potentiation. Blockade of IP3 receptors can not prevent BDNF-induced potentiation, suggesting that BDNF and NT3 use different mechanisms to potentiate transmitter release. Inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) completely blocks the acute effect of NT3. Furthermore, the NT3-induced potentiation requires a continuous activation of CaMKII, because application of the CaMKII inhibitor KN62 reverses the previously established NT3 effect. Thus, NT3 potentiates neurotransmitter secretion by stimulating Ca(2+) release from intracellular stores through IP3 and/or ryanodine receptors, leading to an activation of CaMKII.

Animals↗

PubMed2000

Cloning and characterization of human Lnk, an adaptor protein with pleckstrin homology and Src homology 2 domains that can inhibit T cell activation.

Lnk was originally cloned from a rat lymph node cDNA library and shown to participate in T cell signaling. Human Lnk (hLnk) was cloned by screening a Jurkat cell cDNA library. hLnk has a calculated molecular mass of 63 kDa, and its deduced amino acid sequence indicates the presence of an N-terminal proline-rich region, a pleckstrin homology domain, and a Src homology 2 domain. When expressed in COS cells, hLnk migrates with an apparent molecular mass of 75 kDa. Confocal fluorescence microscope analysis indicates that in COS cells transfected with an expression vector encoding a chimeric Lnk-green fluorescent protein, hLnk is found at the juxtanuclear compartment and also appears to be localized at the plasma membrane. Lnk is tyrosine-phosphorylated by p56lck. Following phosphorylation, p56lck binds to tyrosine-phosphorylated hLnk through its Src homology 2 domain. In COS cells cotransfected with hLnk, p56lck, and CD8-zeta, hLnk associated with tyrosine-phosphorylated TCR zeta-chain through its Src homology 2 domain. The overexpression of Lnk in Jurkat cells led to an inhibition of anti-CD3 mediated NF-AT-Luc activation. Our study reveals a potentially new mechanism of T cell-negative regulation.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Mechanism of activation of the chloroplast ATP synthase. A kinetic study of the thiol modulation of isolated ATPase and membrane-bound ATP synthase from spinach by Eschericia coli thioredoxin.

The mechanism of thiol modulation of the chloroplast ATP synthase by Escherichia coli thioredoxin was investigated in the isolated ATPase subcomplex and in the ATP synthase complex reconstituted in bacteriorhodopsin proteoliposomes. Thiol modulation was resolved kinetically by continuously monitoring ATP hydrolysis by the isolated subcomplex and ATP synthesis by proteoliposomes. The binding rate constant of reduced thioredoxin to the oxidized ATPase subcomplex devoid of its epsilon subunit could be determined. It did not depend on the catalytic turnover. Reciprocically, the catalytic turnover did not seem to depend on thioredoxin binding. Thiol modulation by Trx of the epsilon-bearing ATPase subcomplex was slow and favored the release of epsilon. The rate constant of thioredoxin binding to the membrane-bound ATP synthase increased with the protonmotive force. It was lower in the presence of ADP than in its absence, revealing a specific effect of the ATP synthase turnover on thioredoxin-gamma subunit interaction. These findings, and more especially the comparisons between the isolated ATPase subcomplex and the ATP synthase complex, can be interpreted in the frame of the rotational catalysis hypothesis. Finally, thiol modulation changed the catalytic properties of the ATP synthase, the kinetics of which became non-Michaelian. This questions the common view about the nature of changes induced by ATP synthase thiol modulation.

Adenosine Triphosphatases↗

PubMed2000

Different Interactions between Isomeric Tetrakis-(N-Hexadecylpyridiniumyl) Porphyrins and CdS Nanoparticles.

Using porphyrin amphiphiles TC(16)PyP(2), TC(16)PyP(3), and TC(16)PyP(4) as photosensitizers, the interaction between amphiphilic porphyrins and colloidal CdS nanoparticles was studied by observing their absorption spectra, fluorescence spectra, and fluorescence lifetimes. The experimental results reveal that upon addition of CdS nanoparticles to a TC(16)PyP(3) or TC(16)PyP(4) solution, TC(16)PyP(3) or TC(16)PyP(4) is adsorbed onto the surface of the colloidal nanoparticles due to electrostatic action. The absorption spectra display the characteristic absorption of metalloporphyrin. Moreover, this adsorption also leads to red-shifted fluorescence spectra and the quenching of fluorescence emission. These changes are related to the formation of complexes. Nearly 90% of the fluorescence emission of 5x10(-6) mol/L TC(16)PyP(4) can be quenched with 6.8x10(-4) mol/L CdS colloid nanoparticles. Only 60% of the fluorescence emission of 5x10(-6) mol/L TC(16)PyP(3) can be quenched with 6.8x10(-4) mol/L CdS nanoparticles. The fluorescence quenching is attributable mainly to static quenching. According to the fluorescence quenching curves, the apparent association constants of TC(16)PyP(4) and TC(16)PyP(3) with colloidal CdS nanoparticles are 1.42x10(3) (mol/L)(-1) and 6.76x10(2) (mol/L)(-1), respectively. However, TC(16)PyP(2) does not adsorb onto the surface of colloid nanoparticles due to its larger steric hindrance; its absorption and fluorescence spectra are unchanged. Copyright 2000 Academic Press.

Journal Article↗

PubMed2000

Transcriptional activation of the rice tungro bacilliform virus gene is critically dependent on an activator element located immediately upstream of the TATA box.

To investigate the transcriptional mechanisms of rice tungro bacilliform virus, we have systematically analyzed an activator element located immediately upstream of the TATA box in the rice tungro bacilliform virus promoter and its cognate trans-acting factors. Using electrophoretic mobility shift assays, we showed that rice nuclear proteins bind to the activator element, forming multiple specific DNA-protein complexes via protein-protein interactions. Copper-phenanthroline footprinting and DNA methylation interference analysis indicated that multiple DNA-protein complexes share a common binding site located between positions -60 to -39, and the proteins contact the activator element in the major groove. DNA UV cross-linking assays further showed that two nuclear proteins (36 and 33 kDa), found in rice cell suspension and shoot nuclear extracts, and one (27 kDa), present in root nuclear extracts, bind to this activator element. In protoplasts derived from a rice (Oryza sativa) suspension culture, the activator element is a prerequisite for promoter activity and its function is critically dependent on its position relative to the TATA box. Thus, transcriptional activation may function via interactions with the basal transcriptional machinery, and we propose that this activation is mediated by protein-protein interactions in a position-dependent mechanism.

Badnavirus↗

PubMed2000

Probing the ground state structure of the green fluorescent protein chromophore using Raman spectroscopy.

We present Raman spectra, obtained using 752 nm excitation, on wild-type GFP and the S65T mutant of this intrinsically fluorescent protein together with data on a model chromophore, ethyl 4-(4-hydroxyphenyl)methylidene-2-methyl-5-oxoimidazolacetate . In the pH range 1-14, the model compound has two macroscopic pK(a)s of 1.8 and 8.2 attributed to ionization of the imidazolinone ring nitrogen and the phenolic hydroxyl group, respectively. Comparison of the model chromophore with the chromophore in wild-type GFP and the S65T mutant reveals that the cationic form, with both the imidazolinone ring nitrogen and the phenolic oxygen protonated, is not present in these particular GFP proteins. Our results do not provide any evidence for the zwitterionic form of the chromophore, with the phenolic group deprotonated and the imidazolinone ring nitrogen protonated, being present in the GFP proteins. In addition, since the position of the Raman bands is a property exclusively of the ground state structure, the data enable us to investigate how protein-chromophore interactions affect the ground state structure of the chromophore without contributions from excited state effects. It is found that the ground state structure of the anionic form of the chromophore, which is most relevant to the fluorescent properties, is strongly dependent on the chromophore environment whereas the neutral form seems to be insensitive. A linear correlation between the absorption properties and the ground state structure is demonstrated by plotting the absorption maxima versus the wavenumber of a Raman band found in the range 1610-1655 cm(-1).

Acetates↗

PubMed2000

Expression of the RNA-binding protein TIAR is increased in neurons after ischemic cerebral injury.

T-cell restricted intracellular antigen-related protein (TIAR) is an RNA recognition motif-type RNA-binding protein that has been implicated in the apoptotic death of T-lymphocytes and retinal pigment epithelial cells. Western blots prepared with a monoclonal antibody against TIAR showed expression in normal rat hippocampus, and induction by 15 min of global cerebral ischemia. This increased expression was evident at 8 hr after ischemia and maximal at 24 hr, whereas expression at 72 hr was reduced below basal levels. Expression of TIAR protein was also increased in parietal cortex 6 and 24 hr after 90 min of focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, as well as in cultured cortical neurons and astroglia after exposure to hypoxia in vitro. Immunocytochemistry showed that increased expression of TIAR occurred mainly in the CA1 sector of hippocampus 24 hr after global ischemia, and in cortical and striatal neurons 24 hr after 20 or 90 min of focal ischemia. Double-labeling studies showed that TIAR protein expression was co-localized with DNA damage in neuronal cells. The findings suggest that TIAR may be involved in neuronal cell death after cerebral ischemic injury.

Animals↗

PubMed2000

Sodium binding site of factor Xa: role of sodium in the prothrombinase complex.

The nature of residue 225 on a consensus loop in serine proteases determines whether a protease can bind Na(+). Serine proteases with a Pro at this position are unable to bind Na(+), but those with a Tyr or Phe can bind Na(+). Factor Xa (FXa), the serine protease of the prothrombinase complex, contains a Tyr at this position. Na(+) is also known to stimulate the amidolytic activity of FXa toward cleavage of small synthetic substrates, but the role of Na(+) in the prothrombinase complex has not been investigated. In this study, we engineered a Gla-domainless form of FX (GDFX) in which residue Tyr(225) was replaced with a Pro. We found that Na(+) stimulated the cleavage rate of chromogenic substrates by FXa or GDFXa approximately 8-24-fold with apparent dissociation constants [K(d(app))] of 37 and 182 mM in the presence and absence of Ca(2+), respectively. In contrast, Na(+) minimally affected the cleavage rate of these substrates by the mutant, and no K(d(app)) for Na(+) binding to the mutant could be estimated. Unlike the wild-type enzyme, the reactivity of the mutant with antithrombin was independent of Na(+) and impaired approximately 32-fold. Ca(2+) improved the reactivity of the mutant with antithrombin approximately 5-fold. Affinity of the mutant for binding to factor Va was weakened and its ability to activate prothrombin was severely impaired. Further studies with the wild-type prothrombinase complex revealed that FXa binds to factor Va with a similar K(d(app)) of 1. 1-1.8 nM in the presence of Na(+), K(+), Li(+), Ch(+), and Tris(+) and that the catalytic efficiency of prothrombinase is enhanced less than 1.5-fold by the specific effect of Na(+) in the reaction buffer. These results suggest that (1) the loop including residue 225 (225-loop) is a Na(+) binding site in FXa, (2) the Na(+)- and Ca(2+)-binding loops of FXa are allosterically linked, and (3) the Tyr conformer of the 225-loop is critical for factor Xa function; however, both Na(+)-bound and Na(+)-free forms of factor Xa in the prothrombinase complex can efficiently activate prothrombin.

Animals↗

PubMed2000

Pharmacophore/receptor models for GABA(A)/BzR subtypes (alpha1beta3gamma2, alpha5beta3gamma2, and alpha6beta3gamma2) via a comprehensive ligand-mapping approach.

Pharmacophore/receptor models for three recombinant GABA(A)/BzR subtypes (alpha1beta3gamma2, alpha5beta3gamma2, and alpha6beta3gamma2) have been established via an SAR ligand-mapping approach. This study was based on the affinities of 151 BzR ligands at five distinct (alpha1-3,5,6beta3gamma2) recombinant GABA(A)/BzR receptor subtypes from at least nine different structural families. Examination of the included volumes of the alpha1-, alpha5-, and alpha6-containing subtypes indicated that region L(2) for the alpha5-containing subtype appeared to be larger in size than the analogous region of the other receptor subtypes. Region L(Di), in contrast, appeared to be larger in the alpha1 subtype than in the other two subtypes. Moreover, region L(3) in the alpha6 subtype is either very small or nonexistent in this diazepam-insensitive subtype (see Figure 16 for details) as compared to the other subtypes. Use of the pharmacophore/receptor models for these subtypes has resulted in the design of novel BzR ligands (see 27) selective for the alpha5beta3gamma2 receptor subtype. alpha5-Selective ligand 27 when injected directly into the hippocampus did enhance memory in one paradigm (Bailey et al., unpublished observations); however, systemic administration of either 9 or 27 into animals did not provide an observable enhancement. This result is in complete agreement with the observation of Liu (1996). It has been shown (Liu, 1996; Wisden et al., 1992) that in the central nervous system of the rat (as well as monkeys and pigeons) there are several native subtypes of the GABA(A) receptor which exhibit different functions, regional distributions, and neuronal locations. Although 27 binds more potently at alpha5beta3gamma2 receptor subtypes and is clearly an inverse agonist (Liu et al., 1996; Liu, 1996), it is possible that this ligand acts as an agonist at one or more subtypes. Liu (1996) clearly showed that a number of imidazobenzodiazepines were negative modulators at one subtype and agonists at another. Therefore, selectivity for a particular subtype at this point is not sufficient to rule out some physiological effect at other GABA(A)/BzR subtypes. The inability of 27 to potentiate memory when given systemically is again in support of this hypothesis, especially since alpha1beta2gamma2 subtypes are distributed throughout the brain (Wisden et al., 1992). A drug delivered systemically is far more likely to interact with all subtypes than one delivered to a specific brain region. This observation (systemic vs intrahippocampal) provides further support for the design of more subtype-specific ligands at the BzR to accurately define their pharmacology, one key to the design of new drugs with fewer side effects.

Animals↗

PubMed2000

Growth of Qinghai Tibetans living at three different high altitudes.

This study compares the stature, weight, skinfolds, upper arm muscle area, and chest dimensions of Tibetan children, adolescents, and young adults who were born and raised, or who had lived from infancy, at 3,200 m, 3,800 m, and 4,300 m in Qinghai Province, People's Republic of China. While the individuals measured in Qinghai are among the tallest and heaviest Tibetans reported in the literature, they are nevertheless smaller and lighter than well-off children living at low altitude. The pattern of size variation among Tibetan males and females measured at the three high altitudes, along with evidence of a secular trend at 4,300 m, suggests that nutrition may significantly effect growth at high altitude. Only minor differences in thorax dimensions exist between Tibetan males and females measured at 3,200 m and 3,800 m. However, Tibetan males at 4,300 m possess slightly narrower and deeper chests (during and after adolescence) than males at 3,200 m and 3,800 m. Since individuals from 3,800 m and 4,300 m belong to the same local populations, this characteristic is unlikely to be genetically determined. However, it may be related to differences in the degree of hypoxia or to the influences of other environmental conditions.

Adolescent↗

PubMed2000

Mass spectrometric study of six cyclic esters

A series of cyclic esters, which are optically active as a consequence of their helical structures, were synthesized to investigate the relationships between their structures and their optical activities. This paper reports the electron impact fragmentation mechanisms of these six cyclic esters. Accurate mass measurements and mass analyzed ion kinetic energy spectrometry confirmed fragmentation patterns. The stability of the fragment ions has a great influence on the fragmentation pathways, but no correlation with the optical activity was found.

Journal Article↗

PubMed2000

Increased expression of apoptosis-linked gene 2 (ALG2) in the rat brain after temporary focal cerebral ischemia.

Calcium is an important mediator of programmed cell death induced by transient cerebral ischemia, and calcium-binding proteins have been implicated in calcium-regulated signal transduction. Apoptosis-linked gene 2 is a calcium-binding protein required for cell death induced by different apoptotic stimuli. By Western blot analysis, we found that apoptosis-linked gene 2 protein was expressed in normal brains, and that expression increased in ischemic brains after 20 or 90 min of transient focal cerebral ischemia. Immunocytochemistry showed increased apoptosis-linked gene 2 protein expression in frontal cortex, a region where neurons underwent ischemic stress but still survived, after 20 or 90 min of focal cerebral ischemia. Apoptosis-linked gene 2 protein was also up-regulated in the ischemic border-zone of parietal cortex 24h after 20 min of focal ischemia, and was remarkably over-expressed in the caudate-putamen and parietal cortex, (where cells are destined to die) 24h after 90 min of ischemia. The expression pattern of apoptosis-linked gene 2 protein was similar to that of deoxyribonucleic acid damage detected by Klenow labeling assay. Our results suggest that apoptosis-linked gene 2 may be involved in the regulation of cell death after transient focal cerebral ischemia.

Animals↗

PubMed2000

High-performance liquid chromatography-electrospray ionization-mass spectrometry study of ginkgolic acid in the leaves and fruits of the ginkgo tree (Ginkgo biloba).

A method is developed for qualitative analysis of ginkgolic acids in the leaves and fruits of Ginkgo biloba by high-performance liquid chromatography (HPLC)-electrospray ionization-mass spectrometry technique. Negative ionization mode is successful in obtaining a very abundant deprotonated molecule [M - H]-. The mass detection sensitivity is higher than ultraviolet detection but relies heavily on the concentration of acetic acid in the HPLC eluent, which consists of acetonitrile-water-acetic acid. The method is also very specific for the analysis of ginkgolic acid with no interferences from the sample matrix.

Acetic Acid↗

PubMed2000

Unwinding of a herpes simplex virus type 1 origin of replication (Ori(S)) by a complex of the viral origin binding protein and the single-stranded DNA binding protein.

A herpes simplex virus type 1 (HSV-1) Ori(S) analogue in which the A+T sequence linking the box I and II elements was replaced by two single-stranded oligo(dT)s is unwound by the UL9 protein-ICP8 complex. Unwinding of wild-type Ori(S) by the UL9 protein-ICP8 complex was also observed under conditions which destabilize the A+T sequence. These experiments support a model for the unwinding of Ori(S) in which destabilization of the A+T sequence can generate a single-stranded DNA binding site for ICP8, which then associates with the UL9 protein bound to boxes I and II to promote the bidirectional unwinding of Ori(S).

Base Sequence↗