Biomedical subjects

T M Fletcher

Publications and source records attributed to T M Fletcher.

At least 19 recordsLinked to original sources

PubMed1998

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.

Animals↗

PubMed1998

Effect of DNA secondary structure on human telomerase activity.

Telomeres are specialized DNA-protein complexes located at the chromosome ends. The guanine-rich telomeric sequences have the ability to form G-quadruplex structures under physiological ionic conditions in vitro. Human telomeres are maintained through addition of TTAGGG repeats by the enzyme telomerase. To determine a correlation between DNA secondary structure and human telomerase, telomerase activity in the presence of various metal cations was monitored. Telomerase synthesized a larger proportion of products corresponding to four, five, eight, and nine full repeats of TTAGGG in 100 mM K+ and to a lesser extent in 100 mM Na+ when a d(TTAGGG)3 input primer was used. A more even product distribution was observed when the reaction mixture contained no added Na+ or K+. Increasing concentrations of Cs+ resulted in a loss of processivity but not in the distinct manner observed in K+. When the input primer contained 7-deaza-dG, the product distribution resembled that of reactions without K+ even in the presence of 100 mM K+. Native polyacrylamide gel electrophoresis indicated that d(TTAGGG)4, d(TTAGGG)5, d(TTAGGG)8, and d(TTAGGG)9 formed compact structures in the presence of K+. The oligonucleotide d(TTAGGG)4 had a UV spectrum characteristic of that of the G-quadruplex only in the presence of K+ and Na+. A reasonable explanation for these results is that four, five, eight, and nine repeats of TTAGGG form DNA secondary structures which promote dissociation of the primer from telomerase. This suggests that telomerase activity in cells can be modulated by the secondary structure of the DNA template. These findings are of probable relevance to the concept of telomerase as a therapeutic target for drug design.

Cesium↗

PubMed1997

Complementation of integrase function in HIV-1 virions.

Proviral integration is essential for HIV-1 replication and represents an important potential target for antiviral drug design. Although much is known about the integration process from studies of purified integrase (IN) protein and synthetic target DNA, provirus formation in virally infected cells remains incompletely understood since reconstituted in vitro assays do not fully reproduce in vivo integration events. We have developed a novel experimental system in which IN-mutant HIV-1 molecular clones are complemented in trans by Vpr-IN fusion proteins, thereby enabling the study of IN function in replicating viruses. Using this approach we found that (i) Vpr-linked IN is efficiently packaged into virions independent of the Gag-Pol polyprotein, (ii) fusion proteins containing a natural RT/IN processing site are cleaved by the viral protease and (iii) only the cleaved IN protein complements IN-defective HIV-1 efficiently. Vpr-mediated packaging restored IN function to a wide variety of IN-deficient HIV-1 strains including zinc finger, catalytic core and C-terminal domain mutants as well as viruses from which IN was completely deleted. Furthermore, trans complemented IN protein mediated a bona fide integration reaction, as demonstrated by the precise processing of proviral ends (5'-TG...CA-3') and the generation of an HIV-1-specific (5 bp) duplication of adjoining host sequences. Intragenic complementation between IN mutants defective in different protein domains was also observed, thereby providing the first evidence for IN multimerization in vivo.

Amino Acid Sequence↗

PubMed1997

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.

Animals↗

PubMed1997

Analytical ultracentrifugation and agarose gel electrophoresis as tools for studying chromatin folding in solution.

Analytical ultracentrifugation and agarose gel electrophoresis each can be used to accurately quantify changes in structure that accompany chromatin folding in solution. Analytical ultracentrifugation directly measures the extent of compaction of each species present in a chromatin sample under a wide range of solution conditions. Agarose gel electrophoresis yields information about changes in the average surface charge density, size and/or shape, and conformational flexibility during chromatin folding. When used together, these methodologies are particularly powerful. Protocols for the characterization of chromatin folding by analytical ultracentrifugation and agarose gel electrophoresis are described. Discussion focuses on analysis and interpretation of experimental chromatin folding data.

Chromatin↗

PubMed1996

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.

Cells, Cultured↗

PubMed1996

Nuclear import and cell cycle arrest functions of the HIV-1 Vpr protein are encoded by two separate genes in HIV-2/SIV(SM).

The vpr genes of human and simian immunodeficiency viruses (HIV/SIV) encode proteins which are packaged in the virus particle. HIV-1 Vpr has been shown to mediate the nuclear import of viral reverse transcription complexes in non-dividing target cells (e.g. terminally differentiated macrophages), and to alter the cell cycle and proliferation status of the infected host cell. Members of the HIV-2/SIV(SM) group encode, in addition to Vpr, a related protein called Vpx. Because these two proteins share considerable sequence similarity, it has been assumed that they also exhibit similar functions. Here, we report that the functions of Vpr and Vpx are distinct and non-redundant, although both proteins are components of the HIV-2/SIV(SM) virion and reverse transcription complex. Characterizing SIV(SM) proviruses defective in one or both genes, we found that Vpx is both necessary and sufficient for the nuclear import of the viral reverse transcription complex. In contrast, Vpr, but not Vpx, inhibited the progression of infected host cells from the G2 to the M phase of the cell cycle. Thus, two independent functions of the HIV-1 Vpr protein are encoded by separate genes in HIV-2/SIV(SM). This segregation is consistent with the conservation of these genes in HIV-2/SIV(SM) evolution, and underscores the importance of both nuclear transport and cell cycle arrest functions in primate lentivirus biology.

Animals↗

PubMed1996

Reversible oligonucleosome self-association: dependence on divalent cations and core histone tail domains.

Regularly spaced nucleosomal arrays equilibrate between unfolded and highly folded conformations in <2 mM MgCl2, and self-associate above 2 mM MgCl2 [Schwarz, P. M., & Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. Here we use analytical and differential sedimentation techniques to characterize the molecular mechanism and determinants of oligonucleosome self-association. Divalent cations induce self-association of intact nucleosomal arrays by binding to oligonucleosomal DNA and neutralizing its negative charge. Neither linker histones nor H2A/H2B dimers are required for Mg2+ - dependent self-association. However, divalent cations are unable to induce self-association of trypsinized nucleosomal arrays lacking their N- and C-terminal core histone tail domains. This suggests that the H3/H4 tail domains directly mediate oligonucleosome self-association through a non-Coulombic-based mechanism. Self-association occurs independently of whether the oligonucleosome monomers are folded or unfolded. The first step in the self-association pathway is strongly cooperative and produces a soluble association intermediate that sediments approximately 10 times faster than the oligonucleosome monomers. The size of the oligonucleosome polymers increases rapidly as a consequence of small increases in the divalent cation concentration, eventually producing polymeric species that sediment at >> 10 000 S. Importantly, all steps in the self-association pathway are freely reversible upon removal of the divalent cations. Taken together, these data indicate that short oligonucleosome fragments composed of only core histone octamers and DNA possess all of the structural features required to achieve chromosome-level DNA compaction. These findings provide a molecular basis for explaining many of the recently uncovered structural features of interphase and metaphase chromosomal fibers.

Animals↗

PubMed1996

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.

Animals↗

PubMed1995

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.

Animals↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1995

Simian varicella virus antibody response in experimental infection of African green monkeys.

The humoral immune response to simian varicella virus (SVV) was investigated following primary and secondary experimental infection of African green monkeys. Neutralization and immunoprecipitation assays were used to determine antibody titers to SVV throughout the course of infection. The immune response to specific viral polypeptides was analyzed by immunoprecipitation analysis. The results demonstrate that the simian varicella model offers a useful approach to investigate immune mechanisms in human varicella zoster virus (VZV) infections.

Animals↗

PubMed1994

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.

Base Sequence↗

PubMed1994

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.

Animals↗

PubMed1994

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)

Animals↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

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.

Animals↗