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At least 901 records · Page 50Linked to original sources
PubMed2000

Imaging DNA loops induced by restriction endonuclease EcoRII. A single amino acid substitution uncouples target recognition from cooperative DNA interaction and cleavage.

EcoRII is a type IIE restriction endonuclease characterized by a highly cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its DNA recognition site. Transmission electron microscopy provided direct evidence that EcoRII mediates loop formation of linear DNA containing two EcoRII recognition sites. Specific DNA binding of EcoRII revealed a symmetrical DNase I footprint occupying 16-18 bases. Single amino acid replacement of Val(258) by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting properties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. Because the electrophoretic mobility of the mutant enzyme-DNA complexes was significantly higher than that of the wild-type, we investigated if mutant V258N binds as a monomer to the substrate DNA. Analysis of the molecular mass of mutant V258N showed a high percentage of protein monomers in solution. The dissociation constant of mutant V258N confirmed a 350-fold decrease of the enzyme dimerization capability. We conclude that Val(258) is located in a region of EcoRII involved in homodimerization. This is the first report of a specific amino acid replacement in a restriction endonuclease leading to the loss of dimerization and DNA cleavage while retaining specific DNA binding.

Asparagine↗
PubMed1998

Spontaneous loss of viral episomes accompanying Epstein-Barr virus reactivation in a Burkitt's lymphoma cell line.

Life-long viral persistence is a hallmark of human herpesvirus infection. In the Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell line, Mutu, spontaneous loss of all viral episomes accompanied productive viral DNA replication. The molecular configuration of intracellular EBV DNA evolved from monoclonal episomes in cells retaining the original tumor phenotype to predominantly replicating linear DNA and, subsequently, only integrated forms in BL cells that had acquired the lymphoblastoid cell phenotype. Transient appearance of deleted, rearranged WZhet EBV DNA capable of disrupting viral latency, along with the integration of viral DNA into human chromosomes, indicates a genetic instability in the host cell which, if duplicated in vivo, may affect configuration and persistence of the viral genome in expanding malignant cell clones.

Burkitt Lymphoma↗
PubMed1993

Identification of seven putative origins of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus DNA replication.

Seven putative origins of DNA replication (oris) were identified and located on the genome of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV), when an improved infection-dependent replication assay was used. A threefold higher yield of amplified plasmid was achieved when an m.o.i. of 1 was used (instead of 25), and another twofold increase was obtained when the interval between transfection and infection was extended from 5 to 24 h. Six of the putative oris were located in hr regions with homologous sequences. This suggests that all hrs in AcMNPV are bifunctional, i.e. have both ori and enhancer activity for transcription. In addition to the six hrs, the HindIII-K fragment of AcMNPV was also identified to carry a putative ori, although this fragment does not contain an hr region. However, the individual role of these seven oris during viral DNA replication, and whether they are all active simultaneously in vivo, is still unclear. The replication of an ori-containing plasmid starts at the same time (6 h post-infection) and proceeds at the same rate as viral DNA replication. A circular topology of ori-containing plasmids was a prerequisite for replication. Linear DNA, with an ori, did not replicate. Therefore, we suggest a theta structure or a rolling-circle as a model for baculovirus DNA replication.

Animals↗
PubMed1998

Electrotransformation and expression of bacterial genes encoding hygromycin phosphotransferase and beta-galactosidase in the pathogenic fungus Histoplasma capsulatum.

We developed an efficient electrotransformation system for the pathogenic fungus Histoplasma capsulatum and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant Escherichia coli genes, hph and lacZ. Linearized DNA and plasmids containing Histoplasma telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable URA5 marker (native Histoplasma gene or a heterologous Podospora anserina gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the E. coli hph gene flanked by Aspergillus nidulans promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in H. capsulatum, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing Histoplasma URA5 with E. coli lacZ, resulting in expression of functional beta-galactosidase in H. capsulatum. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of H. capsulatum, which have proven useful for examination of targeted gene disruption, regulated gene expression, and potential virulence determinants in this fungus.

Drug Resistance, Microbial↗
PubMed1986

Template requirements for in vivo replication of adenovirus DNA.

The adenovirus (Ad) DNA origin of replication was defined through an analysis of the DNA sequences necessary for the replication of plasmid DNAs with purified viral and cellular proteins. Results from several laboratories have shown that the origin consists of two functionally distinct domains: a 10-base-pair sequence present in the inverted terminal repetition (ITR) of all human serotypes and an adjacent sequence constituting the binding site for a cellular protein, nuclear factor I. To determine whether the same nucleotide sequences are necessary for origin function in vivo, we developed an assay for the replication of plasmid DNAs transfected into Ad5-infected cells. The assay is similar to that described by Hay et al. (J. Mol. Biol. 175:493-510, 1984). With this assay, plasmid DNA replication is dependent upon prior infection of cells with virus and only occurs with linear DNA molecules containing viral terminal sequences at each end. Replicated DNA is resistant to digestion with lambda-exonuclease, suggesting that a protein is covalently bound at both termini. A plasmid containing only the first 67 base pairs of the Ad2 ITR replicates as well as plasmids containing the entire ITR. Deletions or point mutations which reduce the binding of nuclear factor I to DNA in vitro reduce the efficiency of plasmid replication in vivo. A point mutation within the 10-base-pair conserved sequence has a similar effect upon replication. These results suggest that the two sequence domains of the Ad origin identified by in vitro studies are in fact important for viral DNA replication in infected cells. In addition, we found that two separate point mutations which lie outside these two sequence domains, and which have little or no effect upon DNA replication in vitro, also reduce the apparent efficiency of plasmid replication in vivo. Thus, there may be elements of the Ad DNA origin of replication which have not yet been identified by in vitro studies.

Adenoviruses, Human↗
PubMed1987

Repair of UV-induced lesions in Xenopus laevis oocytes.

We characterized a DNA repair system in frog oocytes by comicroinjection of UV-irradiated pBR322 DNA and radiolabeled nucleotides. Repair synthesis was monitored by incorporation of label into recovered pBR322 DNA and by a novel method in which the removal of UV photoproducts was determined from the shift of DNA topoisomers that occurs during gel electrophoresis upon repair of these lesions. We investigated the effects of several drugs in the oocyte system and found that although novobiocin, an inhibitor of topoisomerase II, was an effective inhibitor of repair, VM-26, another inhibitor of topoisomerase II, was not. In addition, the topoisomerase I inhibitor camptothecin had no effect on repair in this system. Finally, circular DNA (either supercoiled or nicked circular) was repaired at least 50 times more rapidly than linear DNA.

Animals↗
PubMed1988

DNA supercoiling facilitates formation of the transcription initiation complex on the fibroin gene promoter.

Transcription of the Bombyx mori fibroin gene in a posterior silk gland extract can be separated into three functional steps on the basis of sensitivity to Sarkosyl: 1) formation of an initiation complex, which is blocked by 0.025% Sarkosyl; 2) conversion of the initiation complex to an elongation complex, a step sensitive to 0.05% Sarkosyl; 3) the subsequent elongation of RNA chain which occurs in the presence of 0.05% Sarkosyl. Whereas the last two steps are rapid and unaffected by template topology, the first step is slow and affected by DNA conformation. In the posterior silk gland extract, closed circular DNA forms a superhelical state and supports more rapid assembly of the initiation complex than linear DNA does. Both DNA supercoiling and rapid assembly of the initiation complex require ATP and are abolished by the addition of a topoisomerase II inhibitor VP16. These results suggest that DNA supercoiling enhances the fibroin gene transcription by facilitating formation of the initiation complex.

Animals↗
PubMed2002

Non-viral gene delivery to atelectatic and ventilated lungs.

BACKGROUND: Three different nonviral vectors and naked DNA were evaluated for in vivo transfer of plasmid DNA to rat lungs through airways in either atelectatic or ventilated lungs. METHODS: The F344 rats underwent instillation of 300 microg DNA (pCIluc, luciferase) to the left lung. Naked DNA, linear polyethylenimine, branched polyethylenimine, and lipid GL-67 (in either atelectatic or ventilated lungs) were assessed (n = 5 per group). After 24 hours, left lung PaO2 (mm Hg) and luciferase activity (RLU/mg) were measured. The median (range) was given, and the analysis of variance was applied, followed by the planned comparison on log-transformed data. RESULTS: In atelectatic lungs, lipid GL-67 was best (927 [330 to 4112] RLU/mg; p < 0.001 versus other groups of atelectatic lung; p < 0.001 versus all other groups), but highest luciferase activity in all groups was measured in ventilated lungs using linear polyethylenimine (1,240 [922 to 2519] RLU/mg; p < 0.001 versus other groups of ventilated lung; p < 0.001 versus all other groups). In comparison with naked DNA, all nonviral vector systems significantly impaired PaO2 24 hours after airway transfection (p < 0.001; naked DNA versus all other groups). Regardless of transfection technique, PaO2 was worst in lungs transfected by linear polyethylenimine. CONCLUSIONS: Highest transfection was achieved with GL-67 in atelectatic lungs and with linear polyethylenimine in ventilated lungs. All gene delivery systems impaired gas exchange of the transduced lung in comparison with naked DNA.

Animals↗
PubMed1981

Enzymatic properties of the bacteriophage phi X174 A protein on superhelical phi X174 DNA: a model for the termination of the rolling circle DNA replication.

Incubation of phi X174 replication form I DNA with the A* protein of phi X174 in the presence of MN2+ results in the formation of three different types of DNA molecules: open circular form DNA (RFII), linear form DNA (RFIII) and the relaxed covalently closed form DNA (RFIV). The RFII and RFIII DNAs are shown to be A* protein-DNA complexes by electron microscopy using the protein labeling technique of Wu and Davidson (1). The linear double-stranded RFIII DNA molecule carries at one end a covalently attached A* protein whereas at the other end of the molecule the single-stranded termini are covalently linked to each other. The structure of the RFIII DNA shows its way of formation. The described properties of the A* protein indicate the way the larger A protein functions in the termination step of the rolling-circle type of phi X174 DNA replication.

Bacteriophage phi X 174↗
PubMed2000

Analysis of proteins encoded in the bipartite genome of a new type of parvo-like virus isolated from silkworm - structural protein with DNA polymerase motif.

Bombyx mori densonucleosis virus type 2 (BmDNV-2) is a small, spherical virus containing two complementary single-stranded linear DNA molecules (VD1, VD2). BmDNV-2 is a new type of virus with a unique, yet unspecified replication mechanism which is different from that of parvoviruses (Bando, H., Choi, H., Ito, Y., Nakagaki, M. , Kawase, S., 1992. Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence, Arch. Virol. 124, 187-193; Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M. , Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Hayakawa, T., Asano, S., Sahara, K., Iizuka, T., Bando, H., 1997. Detection of replicative intermediate with closed terminus of Bombyx densonucleosis virus. Arch. Virol. 142, 1-7). Recent analyses on the genomic information of BmDNV-2 identified open reading frames which code for three tentative nonstructural proteins and four (VP1 to 4) of the six known structural proteins (Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M., Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Nakagaki et al., in preparation). In this report we demonstrate that the two largest ORFs, VD1-ORF1 and VD2-ORF1, code for the two remaining structural proteins. In addition, computer-assisted analysis revealed that the structural protein encoded in VD1-ORF1 contains sequences conserved among various DNA polymerases, and showed an evolutionary relationship with the DNA polymerases involved in protein-primed replication.

Amino Acid Motifs↗
PubMed1981

Electron microscopic mapping of wheat germ RNA polymerase II binding sites on cloned CaMV DNA.

The binding sites of wheat germ RNA polymerase II were mapped on the cloned CaMV genome by observation of enzyme-linear DNA complexes by electron microscopy. Twelve sites are observed. Three of them are relatively stable in the presence of heparin and are found at positions 8-9, 21-23, and 41-44 map units on the physical map of the genome. These positions correspond to AT-rich regions of the viral genome which contain potential promoter sites. These results are discussed with reference to current information on the structure and expression of the CaMV genome.

Binding Sites↗
PubMed1979

The nucleotide sequence of the right-hand terminus of adenovirus type 5 DNA: implications for the mechanism of DNA replication.

The nucleotide sequence of the right-hand terminal 3% of adenovirus type 5 (Ad5) DNA has been determined, using the chemical degradation technique developed by Maxam and Gilbert (1977). This region of the genome comprises the 1003 basepair long HindIII-I fragment and the first 75 nucleotides of the adjacent HindIII-F fragment, extending from the right-hand terminus to the sequences from which the main body of the mRNA of early region 4 is transcribed. One of the origins of adenovirus DNA replication is located within this part of the genome. The sequencing results are discussed in relation to several models proposed for the mechanism of replication of linear DNA molecules, which invariably depend on the presence of specific arrangements of nucleotides at the termini of those linear DNAs.

Adenoviridae↗
PubMed1993

Rapid sequence evolution of the mammalian sex-determining gene SRY.

In mammals, induction of male sex determination requires the Y-chromosome gene SRY. SRY encodes a protein with a central 'high mobility group' domain (HMG box) of about 78 amino acids. HMG boxes are found in a wide variety of proteins that bind to DNA with high affinity but differing degrees of sequence specificity. The human SRY protein binds to linear DNA with sequence specificity and to cruciform DNA structures without sequence specificity. The DNA-binding activity of the SRY protein resides in the HMG box and mutations in this region are associated with sex reversal in XY females. No function has been ascribed to the portions of the SRY protein outside the HMG box. SRY belongs to a family of genes that are related by sequence homology within the DNA-binding domain: the genes most similar to SRY (> 60%) have been named SOX genes (SRY box genes). None of the known SOX genes is homologous to SRY outside the HMG-box region. Although SRY is an important developmental regulator, its sequence is poorly conserved between species apart from the HMG-box domain. Here we investigate the coding sequence of SRY in primates and find that evolution has been rapid in the regions flanking the conserved domain. The high degree of sequence divergence and the frequency of non-synonymous mutations suggest either that the majority of the coding sequence has no functional significance or that directional selection has occurred.

Amino Acid Sequence↗
PubMed2001

DNA repair of a single UV photoproduct in a designed nucleosome.

Eukaryotic DNA repair enzymes must interact with the architectural hierarchy of chromatin. The challenge of finding damaged DNA complexed with histone proteins in nucleosomes is complicated by the need to maintain local chromatin structures involved in regulating other DNA processing events. The heterogeneity of lesions induced by DNA-damaging agents has led us to design homogeneously damaged substrates to directly compare repair of naked DNA with that of nucleosomes. Here we report that nucleotide excision repair in Xenopus nuclear extracts can effectively repair a single UV radiation photoproduct located 5 bases from the dyad center of a positioned nucleosome, although the nucleosome is repaired at about half the rate at which the naked DNA fragment is. Extract repair within the nucleosome is >50-fold more rapid than either enzymatic photoreversal or endonuclease cleavage of the lesion in vitro. Furthermore, nucleosome formation occurs (after repair) only on damaged naked DNA (165-bp fragments) during a 1-h incubation in these extracts, even in the presence of a large excess of undamaged DNA. This is an example of selective nucleosome assembly by Xenopus nuclear extracts on a short linear DNA fragment containing a DNA lesion.

Animals↗
PubMed1983

Site-specific recombination of yeast 2-micron DNA in vitro.

Most strains of the yeast Saccharomyces cerevisiae harbor several copies of a 2-micron plasmid circle DNA termed "2 micron." This circular plasmid contains two 599-base-pair precise inverted repeats across which a site-specific inversion event occurs in vivo. This inversion is promoted by a plasmid-encoded function called "FLP." We have cloned the FLP gene of 2-micron DNA under control of a strong yeast promoter and transformed yeast cells with a plasmid containing the cloned FLP gene. Cell-free extracts from such a transformant promote highly efficient inversion of 2-micron DNA in vitro. The reaction requires a cation and works efficiently on supercoiled, relaxed circular, or linear DNA. The FLP activity bears certain similarities to the cre protein, a site-specific recombinase encoded by bacteriophage P1.

Base Sequence↗
PubMed1994

P nucleotide insertions and the resolution of hairpin DNA structures in mammalian cells.

Two lines of evidence point to a hairpin DNA intermediate in V(D)J joining (V, variable; D, diversity; J, joining) [Lieber, M.R. (1991) FASEB J. 4, 2934-2944]. One is the presence of P nucleotide insertions (short inverted-repeat sequence) in V(D)J junctions [Lafaille, J. J., DeCloux, A., Bonneville, M., Takagaki, Y. & Tonegawa, S. (1989) Cell 59, 859-870]; a second is the detection of site-specifically broken DNA molecules with covalently closed (hairpin) termini in thymus DNA [Roth, D. B., Menetski, J. P., Nakajima, P., Bosma, M. J. & Gellert, M. (1989) Cell 70, 983-991]. However, P nucleotide insertions could be generated in ways not involving a hairpin structure, and because physical evidence for hairpin-ended DNA fragments has been obtained only with mutant mice, there is some uncertainty regarding the role of hairpin molecules in the normal V(D)J joining pathway. To determine whether mammalian cells are capable of metabolizing this odd type of DNA terminus and whether, in doing so, junctions with P insertions are in fact created, a linear DNA molecule with a hairpin closure at each end was transfected into several murine cell lines. The hairpin-ended molecules were recircularized, and the junctions exhibited P insertions at a high frequency. This result directly links the presence of P insertions to a hairpin precursor, providing strong evidence for the notion that a hairpin DNA intermediate exists in V(D)J recombination. A comparison of hairpin end joining in various cells, including those derived from mice with the severe combined immunodeficiency (scid) mutation, is presented.

3T3 Cells↗
PubMed2002

Novel integrating adenoviral/retroviral hybrid vector for gene therapy.

A hybrid adenoviral vector system was designed to incorporate an excisable retroviral cassette that can be stably integrated into the host cell genome. The vector contains the terminal sequences of two Moloney murine leukemia virus retroviral long terminal repeats (LTRs), fused to form a junction fragment, and is flanked by two loxP recognition sequences. Cre recombinase-directed excision liberates a circular, double-stranded DNA molecule containing the LTR junction fragment. Despite the natural intermediate for retroviral integrase being a linear DNA molecule, we show that, in the presence of Cre and retroviral Gag and Pol, the excised circle can be integrated into the target cell genome through both specific integrase (Int)-directed mechanisms and by a random integration process. The loxP cassette, carrying in addition a selectable marker gene, was incorporated into the E1-deleted region of an adenoviral vector. Infection of cells expressing Cre, Gag, and Pol generated clones that survived long term in drug selection (>3 months). Int-mediated integration was demonstrated in seven of nine clones by sequencing of the integration sites. In addition, the introduction of the loxP cassette into 293 cells coexpressing Cre and Int alone in the absence of other Gag and Pol proteins was sufficient to catalyze the integration mechanism. These experiments demonstrate that it is possible to generate high-titer adenovirus-mediated delivery of a C-type retroviral provirus that can subsequently undergo retroviral Int-mediated integration into dividing and nondividing cells.

Adenoviruses, Human↗
PubMed1988

Alteration of bacterial DNA structure, gene expression, and plasmid encoded antibiotic resistance following exposure to enoxacin.

Enoxacin inhibits growth of Escherichia coli K12 strains primarily by binding to the GyrA subunit of DNA gyrase (topoisomerase II); strains with gyrA, but not gyrB, mutations are less susceptible to the bactericidal effects of this agent. In sensitive strains, enoxacin completely inhibits DNA synthesis within 5 min and produces drug-gyrase-DNA complexes at numerous sites throughout the E. coli chromosome, as shown by the formation of linear DNA molecules after detergent treatment. Enoxacin, even at subminimal inhibitory concentrations, induces the bacterial SOS system, even in partially resistant gyrA strains. This drug also inhibits the induced expression of the lacZ encoded beta-galactosidase, regardless of whether this gene is located on the chromosome, a low copy number F' plasmid or high copy number Col E1 related plasmids. This inhibition of gene expression at subminimal inhibitory concentrations is likely to be a factor, in addition to gyrase inhibition, in the elimination of Col E1 plasmids and to the reduction in R plasmid conjugal transfer. Enoxacin enhances the bactericidal effects of kanamycin in both in-vitro and in-vivo models, suggesting that this quinolone may be effective in the treatment of infections due to strains resistant to antibacterials as a consequence of plasmid encoded resistance determinants.

Anti-Bacterial Agents↗