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At least 487 records · Page 27Linked to original sources
PubMed1993

Interaction between the Chlamydia trachomatis histone H1-like protein (Hc1) and DNA.

The gene encoding the Chlamydia trachomatis histone H1-like protein (Hc1) from serovar L2 was cloned into Escherichia coli by use of expression vector pET11d. In this vector, transcription of the gene is under the control of a bacteriophage T7 promoter, and T7 RNA polymerase is inducible in the host. Following induction, the E. coli cells were lysed gently. Gel filtration of the lysate revealed comigration of DNA and Hc1 in the voided volume. Electron microscopy revealed the DNA to be complexed with protein in large aggregates, often in the form of spherical bodies. Purified recombinant Hc1 maintained its DNA-binding capacity and was able at high concentrations to form condensed aggregates with DNA (one molecule of Hc1 per base pair) independently of the form or size of the DNA but with a slight preference for supercoiled DNA. Hc1 alone is thus able to package DNA into condensed spherical bodies.

Base Sequence↗
PubMed2004

Genomic diversity of Burkholderia pseudomallei clinical isolates: subtractive hybridization reveals a Burkholderia mallei-specific prophage in B. pseudomallei 1026b.

Burkholderia pseudomallei is the etiologic agent of the disease melioidosis and is a category B biological threat agent. The genomic sequence of B. pseudomallei K96243 was recently determined, but little is known about the overall genetic diversity of this species. Suppression subtractive hybridization was employed to assess the genetic variability between two distinct clinical isolates of B. pseudomallei, 1026b and K96243. Numerous mobile genetic elements, including a temperate bacteriophage designated phi1026b, were identified among the 1026b-specific suppression subtractive hybridization products. Bacteriophage phi1026b was spontaneously produced by 1026b, and it had a restricted host range, infecting only Burkholderia mallei. It possessed a noncontractile tail, an isometric head, and a linear 54,865-bp genome. The mosaic nature of the phi1026b genome was revealed by comparison with bacteriophage phiE125, a B. mallei-specific bacteriophage produced by Burkholderia thailandensis. The phi1026b genes for DNA packaging, tail morphogenesis, host lysis, integration, and DNA replication were nearly identical to the corresponding genes in phiE125. On the other hand, phi1026b genes involved in head morphogenesis were similar to head morphogenesis genes encoded by Pseudomonas putida and Pseudomonas aeruginosa bacteriophages. Consistent with this observation, immunogold electron microscopy demonstrated that polyclonal antiserum against phiE125 reacted with the tail of phi1026b but not with the head. The results presented here suggest that B. pseudomallei strains are genetically heterogeneous and that bacteriophages are major contributors to the genomic diversity of this species. The bacteriophage characterized in this study may be a useful diagnostic tool for differentiating B. pseudomallei and B. mallei, two closely related biological threat agents.

Bacterial Typing Techniques↗
PubMed2004

Quantification of the DNA cleavage and packaging proteins U(L)15 and U(L)28 in A and B capsids of herpes simplex virus type 1.

The proteins produced by the herpes simplex virus type 1 (HSV-1) genes U(L)15 and U(L)28 are believed to form part of the terminase enzyme, a protein complex essential for the cleavage of newly synthesized, concatameric herpesvirus DNA and the packaging of the resultant genome lengths into preformed capsids. This work describes the purification of recombinant forms of pU(L)15 and pU(L)28, which allowed the calculation of the average number of copies of each protein in A and B capsids and in capsids lacking the putative portal encoded by U(L)6. On average, 1.0 (+/-0.29 [standard deviation]) copies of pU(L)15 and 2.4 (+/-0.97) copies of pU(L)28 were present in B capsids, 1.2 (+/-0.72) copies of pU(L)15 and 1.5 (+/-0.86) copies of pU(L)28 were found in mutant capsids lacking the putative portal protein pU(L)6, and approximately 12.0 (+/-5.63) copies of pU(L)15 and 0.6 (+/-0.32) copies of pU(L)28 were present in each A capsid. These results suggest that the packaging machine is partly comprised of approximately 12 copies of pU(L)15, as found in A capsids, with wild-type B and mutant U(L)6(-) capsids containing an incomplete complement of cleavage and packaging proteins. These results are consistent with observations that B capsids form by default in the absence of packaging machinery in vitro and in vivo. In contrast, A capsids may be the result of initiated but aborted attempts at DNA packaging, resulting in the retention of at least part of the DNA packaging machinery.

Animals↗
PubMed2005

Impact of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole riboside and inhibitors of DNA, RNA, and protein synthesis on human cytomegalovirus genome maturation.

Herpesvirus genome maturation is a complex process in which concatemeric DNA molecules are translocated into capsids and cleaved at specific sequences to produce encapsidated-unit genomes. Bacteriophage studies further suggest that important ancillary processes, such as RNA transcription and DNA synthesis, concerned with repeat duplication, recombination, branch resolution, or damage repair may also be involved with the genome maturation process. To gain insight into the biochemical activities needed for herpesvirus genome maturation, 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) was used to allow the accumulation of human cytomegalovirus concatemeric DNA while the formation of new genomes was being blocked. Genome formation was restored upon BDCRB removal, and addition of various inhibitors during this time window permitted evaluation of their effects on genome maturation. Inhibitors of protein synthesis, RNA transcription, and the viral DNA polymerase only modestly reduced genome formation, demonstrating that these activities are not required for genome maturation. In contrast, drugs that inhibit both viral and host DNA polymerases potently blocked genome formation. Radioisotope incorporation in the presence of a viral DNA polymerase inhibitor further suggested that significant host-mediated DNA synthesis occurs throughout the viral genome. These results indicate a role for host DNA polymerases in genome maturation and are consistent with a need for terminal repeat duplication, debranching, or damage repair concomitant with DNA packaging or cleavage. Similarities to previously reported effects of BDCRB on guinea pig cytomegalovirus were also noted; however, BDCRB induced low-level formation of a supergenomic species called monomer+ DNA that is unique to human cytomegalovirus. Analysis of monomer+ DNA suggested a model for its formation in which BDCRB permits limited packaging of concatemeric DNA but induces skipping of cleavage sites.

Benzimidazoles↗
PubMed1998

The gene product of human cytomegalovirus open reading frame UL56 binds the pac motif and has specific nuclease activity.

Using the cis-acting human cytomegalovirus (HCMV) packaging elements (pac 1 and pac 2) as DNA probes, specific DNA-protein complexes were detected by electrophoretic mobility shift assay (EMSA) in both HCMV-infected cell nuclear extracts and recombinant baculovirus-infected cell extracts containing the HCMV p130 (pUL56) protein. DNA-binding proteins, which were common in uninfected and infected cell extracts, were also detected. Mutational analysis showed that only the AT-rich core sequences in these cis-acting motifs, 5'-TAAAAA-3' (pac 1) and 5'-TTTTAT-3' (pac 2), were required for specific DNA-protein complex formation. The specificity of the DNA-protein complexes was confirmed by EMSA competition. Furthermore, a specific endonuclease activity was found to be associated with lysates of baculovirus-infected cells expressing recombinant p130 (rp130). This nuclease activity was time dependent, related to the amount of rp130 in the assay, and ATP independent. Nuclease activity remained associated with rp130 after partial purification by sucrose gradient centrifugation, suggesting that this activity is a property of HCMV p130. We propose a possible involvement of p130 in HCMV DNA packaging.

Animals↗
PubMed1989

DNA loop domains in mammalian spermatozoa.

The highly condensed and tightly packaged DNA of hamster spermatozoa was found to be organized into topologically constrained DNA loop domains attached at their bases to a nuclear matrix. The loop domains of the sperm nuclei differed from somatic cell loop domains from the same animal in two aspects. Sperm loop domains were 60% smaller than somatic cell loop domains, with an average DNA length of 46 +/- 7 kb in sperm as compared with 76 +/- 11 kb in brain. Secondly, unlike virtually all somatic cell DNA known which is negatively supercoiled, sperm DNA was devoid of detectable supercoiling. The presence of the loop domain structure in the highly condensed DNA of mammalian spermatozoa suggests that this motif is a fundamental aspect of eukaryotic DNA organization.

Animals↗
PubMed1994

Expression of P1 DNA in mammalian cells and transgenic mice.

Because the P1 bacteriophage packages DNA inserts of 80-100 kb, which are much larger than inserts of bacteriophage lambda or cosmid vectors, P1 DNA can be used to express large genes in cultured cells and transgenic mice. We obtained a P1 bacteriophage clone with a 79.5-kb insert (p158) that spanned the entire human apolipoprotein (apo-) B gene. We used the insert from p158 to express the human apo-B gene in both cultured rat hepatoma cells and transgenic mice. In this article, we review our apo-B expression studies and discuss the techniques that we have used for these expression studies.

Animals↗
PubMed2006

From structure of the complex to understanding of the biology.

The most extensive structural information on viruses relates to apparently icosahedral virions and is based on X-ray crystallography and on cryo-electron microscopy (cryo-EM) single-particle reconstructions. Both techniques lean heavily on imposing icosahedral symmetry, thereby obscuring any deviation from the assumed symmetry. However, tailed bacteriophages have icosahedral or prolate icosahedral heads that have one obvious unique vertex where the genome can enter for DNA packaging and exit when infecting a host cell. The presence of the tail allows cryo-EM reconstructions in which the special vertex is used to orient the head in a unique manner. Some very large dsDNA icosahedral viruses also develop special vertices thought to be required for infecting host cells. Similarly, preliminary cryo-EM data for the small ssDNA canine parvovirus complexed with receptor suggests that these viruses, previously considered to be accurately icosahedral, might have some asymmetric properties that generate one preferred receptor-binding site on the viral surface. Comparisons are made between rhinoviruses that bind receptor molecules uniformly to all 60 equivalent binding sites, canine parvovirus, which appears to have a preferred receptor-binding site, and bacteriophage T4, which gains major biological advantages on account of its unique vertex and tail organelle.

Bacteriophages↗
PubMed1999

Geometry of phage head construction.

The process of phage capsid assembly is reviewed, with particular attention to the probable role of curvature in helping to determine head size and shape. Both measures of curvature (mean curvature and Gaussian curvature, explained in Appendix I), should act best when the assembling shell is spherical, which could account for procapsids having this shape. Procapsids are also relatively thick, which should help head size determination by the mean curvature. The accessory role of inner and outer scaffolds in size determination and head nucleation is also reviewed. Nucleation failure generates various malformations, including non-closure, but the most common is the tube or polyhead, where the subunits' inherent curvature is expressed as a constant mean curvature. This induces lattice distortions that only partly understood. An extra tubular section in normal heads leads to the prolate shape, with a more complex and variable geometry. Newly assembled procapsids are both enlarged and toughened by the head transformation. In the procapsid the Gaussian curvature is uniformly distributed. But toughening tends to equalize bond lengths, so all the Gaussian curvature gets concentrated in the vertices, being zero elsewhere. This explains head angularization. Because of this change in Gaussian curvature, the regular subunit packing in the polyhedral head cannot be mapped onto the procapsid. This explains part of the hexon distortions found in this region. The implications of translocase-induced DNA twist, end rotation and the coiling of packaged DNA, are discussed. The symmetry mismatches between the head, connector and tail are discussed in relation to the possible alpha-helical structures of their DNA channels.

Bacteriophages↗
PubMed1992

Nucleotide variation and molecular structure of the heterochromatic repetitive AluI DNA in the brine shrimp Artemia franciscana.

It has been suggested that DNA bending could play a role in the regulation of gene expression, chromosome segregation, specific recombination and/or DNA packaging. We have previously demonstrated that an AluI DNA family of repeats is the major component of constitutive heterochromatin in the brine shrimp A. franciscana. By the analysis of cloned oligomeric (monomer to hexamer) heterochromatic fragments we verified that the repetitive AluI DNA shows a stable curvature that determines a solenoidal geometry to the double helix. This particular structure could be of relevant importance in conferring the characteristic heterochromatic condensation. In this paper we evaluate how the point mutations that occurred during the evolution of the AluI sequence of A. franciscana could influence the sequence-dependent tridimensional conformation. The obtained data underline that, in spite of the high sequence mutation frequency (10%) of the repetitive DNA, the general structure of the heterochromatic DNA is not greatly influenced, but rather there is a substantial variation of the copy number of the repetitive AluI fragment. This variation could be responsible for the hypothetical function of the constitutive heterochromatin.

Animals↗
PubMed2003

Origin and evolution of polydnaviruses by symbiogenesis of insect DNA viruses in endoparasitic wasps.

During oviposition, many endoparasitic wasps inject virus-like particles into their insect hosts that enable these parasitoids to evade or directly suppress their hosts' immune system, especially encapsulation by hemocytes. These particles are defined as virions that belong to viruses of the two genera that comprise the family Polydnaviridae, bracoviruses (genus Bracovirus) transmitted by braconid wasps, and ichnoviruses (genus Ichnovirus) transmitted by ichneumonid wasps. Structurally, bracovirus virions resemble nudivirus and baculovirus virions (family Baculoviridae), and ichnovirus virions resemble those of ascoviruses (family Ascoviridae). Whereas nudiviruses, baculoviruses and ascoviruses replicate their DNA and produce progeny virions, polydnavirus DNA is integrated into and replicated from the wasp genome, which also directs virion synthesis. The structural similarity of polydnavirus virions to those of viruses that attack the wasps' lepidopteran hosts, along with polydnavirus transmission and replication biology, suggest that these viruses evolved from insect DNA viruses by symbiogenesis, the same process by which mitochondia and chloroplasts evolved from bacteria. Molecular evidence supporting this hypothesis comes from similarities among structural proteins of ascoviruses and the Campoletis sonorensis ichnovirus. Implications of this hypothesis are that polydnaviruses evolved from viruses, but are no longer viruses, and that DNA packaged into polydnavirus virions is not viral genomic DNA per se, but rather wasp genomic DNA consisting primarily of wasp genes and non-coding DNA. Thus, we suggest that a better understanding of polydnaviruses would result by viewing these not as viruses, but rather as a wasp organelle system that evolved to shuttle wasp genes and proteins into hosts to evade and suppress their immune response.

Amino Acid Sequence↗
PubMed1982

Organization of herpes simplex virus type 1 deoxyribonucleic acid during replication probed in living cells with 4,5',8-trimethylpsoralen.

The structure of herpes simplex virus type 1 (HSV-1) DNA in the nuclei of living infected cells was studied with the DNA photoaffinity probe 4,5',8-trimethylpsoralen. The rate of photobinding to HSV-1 DNA was compared to that of a suitable internal control at different times during infection. The rates of photobinding to DNA packaged in virions, capsids, and prereplicative and postreplicative DNA were characteristically different. By 4 h after infection, after the initiation of DNA replication, the rate of photobinding to HSV-1 DNA increased 4 times relative to the rate of binding to the host DNA. The enhanced rate of photobinding to HSV-1 DNA was maintained at all later times during infection and was not affected when frequent single-strand breaks were introduced in HSV-1 DNA by gamma irradiation of infected cells. The results suggest that the bulk of the replicating herpes DNA is free of torsional tension and that the differing rates of photobinding are attributable to changes in accessibility of the HSV-1 DNA. The results are compatible with previous proposals, based on in vitro studies, that intranuclear HSV-1 DNA is primarily free of nucleosomal organization and suggest that there are few, if any, unrestrained DNA supercoils averaged over the entire HSV-1 genome.

Affinity Labels↗
PubMed1997

The type IC hsd loci of the enterobacteria are flanked by DNA with high homology to the phage P1 genome: implications for the evolution and spread of DNA restriction systems.

EcoR124l, EcoDXXl and Ecoprrl are the known members of the type IC family of DNA restriction and modification systems. The first three are carried on large, conjugative plasmids, while Ecoprrl is chromosomally encoded. The enzymes are coded by three genes, hsdR, hsdM and hsdS. Analysis of the DNA sequences upstream and downstream of the type IC hsd loci shows that all are highly homologous to each other and also to sequences present in the bacteriophage P1 genome. The upstream sequences include functional phd and doc genes, which encode an addiction system that stabilizes the P1 prophage state, and extend to and beyond pac, the site at which phage DNA packaging begins. Downstream of the hsd loci, P1 DNA sequences begin at exactly the same place for all of the systems. For EcoDXXl and Ecoprrl the P1 homology extends for thousands of base pairs while for EcoR124l and IS1 insertion and an associated deletion have removed most of the P1-homologous sequences. The significance of these results for the evolution of DNA restriction and modification systems is discussed.

Bacteriophage P1↗
PubMed1984

Coupling with packaging explains apparent nonreciprocality of Chi-stimulated recombination of bacteriophage lambda by RecA and RecBC functions.

Chi (chi, 5'-GCTGGTGG) is a recombinator in RecA- and RecBC-mediated recombination in Escherichia coli. In vegetative recombination between two bacteriophage lambda strains, one with and the other without Chi (a+ chi +b- X a- chi 0b+), the chi-containing recombinant (a- chi +b-) is less abundant than the non-chi-containing recombinant (a+ chi 0b+). Previously this was taken was evidence for nonreciprocality of chi-stimulated exchange. This inequality, however, is now seen to result from an event at cos (lambda's packaging origin) that both activates Chi and initiates DNA packaging. An event at rightward cos leads to activation of leftward chi on the same chromosome for an exchange to its left. From the resulting circulating dimer (--cos-a+- chi 0-b+-cos-a-- chi +-b- --), the cos that activated chi is more likely to be used for rightward packaging initiation than is the cos from the other parent. Consistent with this coupling model is "biased packaging" in lambda carrying two cos sites per monomer genome. When their maturation is dependent on dimerization by chi-stimulated exchange, the phage particles result more often from packaging from the cos that activates chi than from packaging from the other cos. Since Chi activation and packaging can be uncoupled, we infer that some early and reversible step in packaging activates chi. A strong candidate for this step is a double-strand break at cos that provides an oriented entry site for a recombinase.

Bacterial Proteins↗
PubMed1978

New late gene, dar, involved in the replication of bacteriophage T4 DNA. III. DNA replicative intermediates of T4 dar and a gene 59 mutant suppressed by dar.

A mutation in the dar gene of phage T4 restored the arrested DNA synthesis caused by the gene 59 mutation. We have studied the DNA replicative intermediates in cells infected with a dar mutant and a dar-amC5 (gene 59) mutant by velocity sedimentation in neutral and alkaline sucrose gradients. In T4 dar-infected cells, compared to the wild type, three kinds of abnormalities were observed in DNA replication (i) There were unusually rapidly sedimenting intermediates (800S). (ii) When centrifuged in alkaline gradients, there was less single-stranded DNA exceeding 1 phage unit. (iii) The rate of repair of DNA intermediates was slower. It has been proposed by others that the 200S DNA replicative intermediates are required for DNA packaging, but our results showed that the 800S DNA of dar does not have to be converted into the 200S form to undergo conversion to mature viral DNA. Therefore, 200S DNA may not be an obligatory intermediate for mature viral DNA formation. In amC5 (gene 59)-infected cells, the DNA was completely converted 2 to 3 min after intiation of replication to the biologically inactive 63S DNA, and DNA synthesis was concomitantly arrested. However, in dar-am-C5 (gene 59)-infected cells, the formation of abnormal 63S DNA did not occur and 200S DNA appeared instead. An endonucleolytic activity, normally associated with the cell membrane and capable of making double-stranded cuts, was found in the cytoplasm of T4 dar-infected cells. Because the total activity of this endonuclease is the same for both wild-type T4D and the dar mutant, it seems unlikely that the dar protein has endonucleolytic activity itself. However, the finding does explain the abnormal sedimentation of dar DNA intermediates (800S) as well as the proposed suppression mechanism of the gene 59 mutation.

Centrifugation, Density Gradient↗
PubMed2007

Structural and thermodynamic principles of viral packaging.

Packaging of genetic material inside a capsid is one of the major processes in the lifecycle of bacteriophages. To establish the basic principles of packing double-stranded DNA into a phage, we present a low-resolution model of bacteriophage varphi29 and report simulations of DNA packaging. The simulations show excellent agreement with available experimental data, including the forces of packaging and the average structures seen in cryo-electron microscopy. The conformation of DNA inside the bacteriophage is primarily determined by the shape of the capsid and the elastic properties of DNA, but the energetics of packaging are dominated by electrostatic repulsions and the large entropic penalty associated with DNA confinement. In this slightly elongated capsid, the DNA assumes a folded toroidal conformation, rather than a coaxial spool. The model can be used to study packaging of other bacteriophages with different shapes under a range of environmental conditions.

Bacillus Phages↗
PubMed1999

Physical and functional interactions between the herpes simplex virus UL15 and UL28 DNA cleavage and packaging proteins.

Herpes simplex virus (HSV) DNA is cleaved from concatemers and packaged into capsids in infected cell nuclei. This process requires seven viral proteins, including UL15 and UL28. UL15 expressed alone displays a nuclear localization, while UL28 remains cytoplasmic. Coexpression with UL15 enables UL28 to enter nuclei, suggesting an interaction between the two proteins. Additionally, UL28 copurified with UL15 from HSV-infected cells after ion-exchange and DNA affinity chromatography, and the complex sedimented as a 1:1 heterodimer upon sucrose gradient centrifugation. These findings are evidence of a physical interaction of UL15 and UL28 and a functional role for UL15 in directing UL28 to the nucleus.

Animals↗