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At least 19 recordsLinked to original sources
PubMed2003

Earrings and padlocks for the double helix: topological labeling of duplex DNA.

Concatenation of hybridization probe with DNA target is crucial for highly localized detection of targeted sequences and might also be used in various gene-therapy applications. Several approaches based on the attachment of a circular oligonucleotide to designated DNA sites have been proposed. Recently, earring-like probes provide a true topological linkage between a probe and the target, thus allowing the DNA labeling by essentially immobile tags. The latest development in this direction takes advantage of oligonucleotide uptake by supercoiled DNA and is an important step forward.

DNA↗
PubMed1984

DNA binding protein from ovaries of the frog, Xenopus laevis which promotes concatenation of linear DNA.

A soluble extract of Xenopus laevis ovaries catalyzed ATP-dependent concatenation of linear duplex DNA molecules. DNA ligase and a unique X. laevis DNA binding protein were required for the formation of concatemers. A linear DNA concatenation system was reconstituted using T4 DNA ligase and homogeneous X. laevis DNA binding protein. This system catalyzed intermolecular ligation of DNA molecules into linear concatemers of up to ten or more times monomer length.

Adenosine Triphosphate↗
PubMed1991

DNA footprint enhancement using tandem binding sites.

A concatenated DNA fragment containing a five-repeat binding site was used for DNase I footprinting. Under the same conditions, the tandem repeat assay greatly enhanced the DNA footprint as compared with a native DNA sequence with only one binding site. This technique provides an approach for improving poor DNA footprints.

Base Sequence↗
PubMed2004

The adenovirus E4orf6 protein inhibits DNA double strand break repair and radiosensitizes human tumor cells in an E1B-55K-independent manner.

The adenoviral protein E4orf6 has been shown to inhibit both in vitro V(D)J recombination and adenoviral DNA concatenation, two processes that rely on cellular DNA double strand break repair (DSBR) proteins. Most of the known activities of E4orf6 during adenoviral infection require its interaction with another adenoviral protein, E1B-55K. Here we report that E4orf6, stably expressed in RKO human colorectal carcinoma cells or transiently expressed by adenoviral vector in U251 human glioblastoma cells, inhibits DSBR and induces significant radiosensitization in the absence of E1B-55K. Expression of a mutant form of E4orf6 (L245P) failed to radiosensitize RKO cells. E4orf6 reduced DSBR capacity in transfected and infected cells, as measured by sublethal DNA damage repair assay and phosphorylated H2AX (gamma-H2AX) levels, respectively. Consistent with the inhibitory effect of E4orf6 on DSBR, expression of wild-type but not mutant E4orf6 reduced recovery of a transfected, replicating reporter plasmid (pSP189) in 293 cells but did not increase the mutation frequency measured in the reporter plasmid. The kinase activity of DNA-PKcs (the DNA-dependent protein kinase catalytic subunit) toward heterologous substrates was not affected by expression of E4orf6; however, autophosphorylation of DNA-PKcs at Thr-2609 following ionizing radiation was prolonged in the presence of E4orf6 when compared with control-infected cells. Our results demonstrate for the first time that E4orf6 expression hinders the cellular DNA repair process in mammalian cells in the absence of E1B-55K or other adenoviral genes and suggest that viral-mediated delivery of E4orf6, combined with localized external beam radiation, could be a useful approach for the treatment of radioresistant solid tumors such as glioblastomas.

Adenoviridae↗
PubMed1990

Production of human papillomavirus type 16 virions in a keratinocyte cell line.

Human papillomavirus type 16 (HPV-16) is strongly associated with carcinoma of the cervix, but the complete life cycle of the virus cannot be studied because no experimental system is available in which HPV-16 progeny are produced, and there is currently no source of HPV-16 virus particles. Most cell lines that harbor HPV-16 DNA contain the viral genome as integrated or concatenated DNA in which open reading frames are disrupted or deleted, but a human cervical keratinocyte cell line has been described which maintains HPV-16 DNA in monomeric episomal form (M.A. Stanley, H.M. Brown, M.W. Appleby, and A.C. Minson, Int. J. Cancer 43:672-676, 1989). This cell line was induced to form a stratified differentiating epithelium by grafting onto nude mice. Long-term grafts displayed the histological features of a low-grade cervical dysplasia, and terminally differentiated cells contained amplified levels of HPV-16 DNA, virus capsid antigen, and virus particles. This experimental system appears to permit the completion of the HPV-16 life cycle in virus-containing keratinocytes.

Cell Line↗
PubMed2000

Shear-induced assembly of lambda-phage DNA.

Recombinant DNA technology, which is based on the assembly of DNA fragments, forms the backbone of biological and biomedical research. Here we demonstrate that a uniform shear flow can induce and control the assembly of lambda-phage DNA molecules: increasing shear rates form integral DNA multimers of increasing molecular weight. Spontaneous assembly and grouping of end-blunted lambda-phage DNA molecules are negligible. It is suggested that shear-induced DNA assembly is caused by increasing the probability of contact between molecules and by stretching the molecules, which exposes the cohesive ends of the otherwise undeformed lambda-phage DNA molecules. We apply this principle to enhance the kinetics and extent of DNA concatenation in the presence of ligase. This novel approach to controlled DNA assembly could form the basis for improved approaches to gene-chip and recombinant DNA technologies and provide new insight into the rheology of associating polymers.

Bacteriophage lambda↗
PubMed1991

Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).

Animals↗
PubMed1996

An efficient method for generation and subcloning of tandemly repeated DNA sequences with defined length, orientation and spacing.

Tandemly repeated DNA sequences generated from single synthetic oligonucleotide monomers are useful for many purposes. With conventional ligation procedures low yields and random orientation of oligomers makes cloning of defined repeated sequences difficult. We solved these problems using 2 bp overhangs to direct orientation and random incorporation of linkers containing restriction sites during ligation. Ligation products are amplified by PCR using the linker oligonucleotides as primers. Restriction digestion of the PCR products generate multimer distributions whose length is controlled by the monomer/linker ratio. The concatenated DNA fragments of defined length, orientation and spacing can be directly used for subcloning or other applications without further treatment.

Base Sequence↗
PubMed2004

Diverse roles for E4orf3 at late times of infection revealed in an E1B 55-kilodalton protein mutant background.

Species C human adenovirus mutants that fail to express open reading frame 3 of early region 4 (E4orf3) are phenotypically indistinguishable from the wild-type virus when evaluated in cells cultured in vitro. However, E4orf3 gene function has been productively studied in the context of additional viral mutations. This study identifies diverse roles for the E4orf3 protein that are evident in the absence of early region 1B 55-kDa protein (E1B-55K) function. In an E1B-55K-deficient background, the E4orf3 protein promotes viral replication by increasing both the burst size and the probability that an infected cell will produce virus. Early viral gene expression is not impaired in E1B-55K/E4orf3 double mutant virus-infected cells. Cells infected with the double mutant virus accumulated concatemers of viral DNA. However, the E1B-55K/E4orf3 double mutant virus did not replicate any better in MO59J cells, in which viral DNA concatemers did not accumulate, than in MO59K cells, in which viral DNA concatemers were produced, suggesting that viral DNA concatenation is not the primary growth defect of the E1B-55K/E4orf3 double mutant virus. Accumulation of viral mRNA in the nucleus and cytoplasm of E1B-55K/E4orf3 double mutant virus-infected cells was severely reduced compared to that on wild-type virus-infected cells. Thus, in an E1B-55K mutant background, the E4orf3 protein promotes the accumulation of late viral RNA and enhances late gene expression. Finally, within the context of an E1B-55K mutant virus, the E4orf3 protein acts to suppress host cell translation and preserve the viability of cells at moderately late times of infection.

Adenovirus E1B Proteins↗
PubMed2005

Lineage-specific variations of congruent evolution among DNA sequences from three genomes, and relaxed selective constraints on rbcL in Cryptomonas (Cryptophyceae).

BACKGROUND: Plastid-bearing cryptophytes like Cryptomonas contain four genomes in a cell, the nucleus, the nucleomorph, the plastid genome and the mitochondrial genome. Comparative phylogenetic analyses encompassing DNA sequences from three different genomes were performed on nineteen photosynthetic and four colorless Cryptomonas strains. Twenty-three rbcL genes and fourteen nuclear SSU rDNA sequences were newly sequenced to examine the impact of photosynthesis loss on codon usage in the rbcL genes, and to compare the rbcL gene phylogeny in terms of tree topology and evolutionary rates with phylogenies inferred from nuclear ribosomal DNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA), and nucleomorph SSU rDNA. RESULTS: Largely congruent branching patterns and accelerated evolutionary rates were found in nucleomorph SSU rDNA and rbcL genes in a clade that consisted of photosynthetic and colorless species suggesting a coevolution of the two genomes. The extremely accelerated rates in the rbcL phylogeny correlated with a shift from selection to mutation drift in codon usage of two-fold degenerate NNY codons comprising the amino acids asparagine, aspartate, histidine, phenylalanine, and tyrosine. Cysteine was the sole exception. The shift in codon usage seemed to follow a gradient from early diverging photosynthetic to late diverging photosynthetic or heterotrophic taxa along the branches. In the early branching taxa, codon preferences were changed in one to two amino acids, whereas in the late diverging taxa, including the colorless strains, between four and five amino acids showed changes in codon usage. CONCLUSION: Nucleomorph and plastid gene phylogenies indicate that loss of photosynthesis in the colorless Cryptomonas strains examined in this study possibly was the result of accelerated evolutionary rates that started already in photosynthetic ancestors. Shifts in codon usage are usually considered to be caused by changes in functional constraints and in gene expression levels. Thus, the increasing influence of mutation drift on codon usage along the clade may indicate gradually relaxed constraints and reduced expression levels on the rbcL gene, finally correlating with a loss of photosynthesis in the colorless Cryptomonas paramaecium strains.

Asparagine↗
PubMed2001

Molecular characterisation of aureocin A70, a multi-peptide bacteriocin isolated from Staphylococcus aureus.

Staphylococcus aureus A70 produces a heat-stable bacteriocin designated aureocin A70. Aureocin A70 is encoded within a mobilisable 8 kb plasmid, pRJ6, and is active against Listeria monocytogenes. Experiments of transposition mutagenesis and gene cloning had shown that aureocin A70 production and immunity were associated with the HindIII-A and B fragments of pRJ6. Therefore, a 6332 bp region of the plasmid, encompassing both these fragments, was sequenced using a concatenation DNA sequencing procedure. DNA sequence and genetic analyses revealed the presence of three transcriptional units that appear to be involved in bacteriocin activity. The first transcriptional unit contains a single gene, aurT, which encodes a protein that resembles an ATP-dependent transporter, similar to those involved in lantibiotic export. AurT is required for aureocin A70 production and it appears to be essential for mobilisation of pRJ6. The second putative operon contains two open reading frames (ORFs); the first gene, orfA, is predicted to encode a protein similar to small repressor proteins found in some Archaea, whose function remains to be elucidated. The second gene, orfB, codes for an 138 amino acid residue protein which shares a number of characteristics (high pI and hydrophobicity profile) with proteins associated with immunity, needed for self-protection against bacteriocin. Four other genes are present in the third operon, aurABCD. aurABCD encode four related peptides that are small (30-31 amino acid residues), strongly cationic (pI of 9.85 to 10.04) and highly hydrophobic. Theses peptides also have a high content of small amino acid residues like glycine and alanine, and no cysteine residue. Tn917-lac insertional mutations, which affected aureocin A70 activity, reside within operon aurABCD. Analysis of purified bacteriocin preparations by mass spectrometry demonstrated that all four peptides encoded by aurABCD operon are produced, expressed and excreted without post-translational modifications. Thus, aureocin A70 is a multi-peptide non-lantibiotic bacteriocin, which is transported without processing.

Amino Acid Sequence↗
PubMed1987

Changes in the catalytic properties of DNA ligases during early sea urchin development.

Two distinct DNA ligases are expressed during early sea urchin embryogenesis. A light form (50 kDa) is found in unfertilized eggs (oocyte form) and a heavier enzyme (110 kDa) is observed at the two-cell stage (embryonic form). The chronology of the change reveals that the embryonic form is detected 90 min after fertilization. After the two proteins were purified, their catalytic properties were studied using different substrates. The oocyte ligase acts only on deoxypolymers while the embryonic form also ligates heteropolymers. The two enzymes were found to undergo both nick and cohesive-end ligation. With different kinds of restriction sites it was observed that the embryonic enzyme could also ligate blunt-ended DNA. These catalytic properties account for sealing of exogenous DNA and concatenation following DNA injection into eggs. The role of the oocyte form of the enzyme is unclear; one speculation is a role in repair of DNA breaks which might accumulate during long-term sperm and oocyte storage in the gonad.

Animals↗
PubMed1996

Two distinct apolipoprotein B alleles in mice generated by a single 'in-out' targeting.

'In-out' gene targeting using a hypoxanthine phosphoribosyltransferase (HPRT) minigene was applied to generate two new alleles in the gene (Apob) coding for apolipoprotein B (apo B) in murine embryonic stem (ES) cells. Homologous integration of the targeting vector during the 'in step' disrupted the Apob gene leading to an allele encoding apo B81, having a 19% carboxyl-terminal truncation. All six targeted cells obtained had more than one insert at the locus, and the chromosomal target sequence in four of them was changed during the recombination. These results suggest that concatenation of the targeting vector prior to insertion was needed to generate sufficient gene product to yield the HPRT+ phenotype, and that recombination between the concatenated DNA and endogenous DNA was a gene replacement more frequently than a simple insertion. The 'out step' recombination event which occurs between sequences duplicated in the 'in step', was planned to replace the sequences encoding the putative LDL receptor-binding domains of apo B100 with sequences encoding human beta-globin peptides (designated apo B100-beta). 6-Thioguanine (6-TG) resistant colonies were obtained from all the 'in-step' cell lines tested at frequencies of 10(-5) to 10(-4), but the frequency of physical loss of the HPRT sequences accompanied by retention of the modified Apob sequence was variable, indicating that mechanisms other than a simple excision are responsible for the generation of 6-TG resistance. Mice from the 'in-step' produce apo B81 and display characteristics of familial hypobetalipoproteinemia; some homozygotes develop hydrocephaly or exencephaly. Mice from the 'out-step' produce apo B100-beta and secrete lipoprotein particles containing the modified protein; their phenotypic changes are subtle, suggesting the lack of the putative LDL receptor-binding domains is not sufficient to increase the steady-state level of apo B100-beta particles above that of apo B100 particles in control mice.

Alleles↗
PubMed2005

Morphogenesis and cytopathic effects of the Diachasmimorpha longicaudata entomopoxvirus in host haemocytes.

The Diachasmimorpha longicaudata entomopoxvirus (DlEPV), the first reported symbiotic entomopoxvirus, occurs in the venom apparatus of D. longicaudata female wasps and is introduced into Anastrepha suspensa larvae during parasitism. The DlEPV 250-300 kb double stranded DNA genome encodes putative proteins having 30 to >60% amino acid identity with poxvirus homologs such as DNA helicase, DNA-dependent RNA polymerase, and the poxvirus-specific rifampicin resistance protein. Although the molecular characterization of DlEPV is progressing, little is known about its morphogenesis in and effects on host haemocytes. This paper describes (1) haemocytes of third instar A. suspensa, (2) DlEPV infection and morphogenesis, and (3) DlEPV-induced changes in haemocytes. A. suspensa third instars have 3-4 haemocyte morphotypes. Dot blots of DNA from infected haemocytes hybridized with a digoxigenin-labeled DlEPV genomic probe as early as 4 h post-parasitism (hpp) and the intensity of the signal increased with time through 40 hpp. Immunofluorescence microscopy localized DlEPV proteins in cytoplasmic (but not nuclear) sites of infected haemocytes, within 24-36 hpp. Electron microscopy confirmed the presence of viral envelopes, immature spheroids with centric nucleoids, budding virus, and extracellular enveloped virus in three haemocyte types, 24-84 hpp and later. Infected haemocytes exhibited blebbing, DNA concatenation, and inability to encapsulate sephadex beads in vitro. These data indicate that DlEPV disrupts the normal function of host haemocytes, thereby insuring the successful development of D. longicaudata offspring and as such should be regarded as a symbiont of the wasp.

Animals↗
PubMed2003

Cell line-specific accumulation of the baculovirus non-hr origin of DNA replication in infected insect cells.

Successive viral passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in the S. exigua cell line Se301 leads to the rapid accumulation of the non-hr origin of DNA replication (ori) as large concatemers. Passage of SeMNPV in two other S. exigua cell lines, SeUCR1 and SeIZD2109, did not show the accumulation of such concatemers. When introduced into SeUCR1 and SeIZD2109 cells, the non-hr ori concatemers generated in Se301 cells were maintained but did not increase. This suggests that the non-hr ori confers a strong selective advantage in Se301 cells, but not or to a lesser extent in the other cell lines. The cell line-specific accumulation of non-hr ori concatemers might be due to a higher intrinsic recombination frequency in Se301 cells and may reflect tissue related differences involving some host cell factor(s). Since non-hr ori concatemers in Se301 cells were more abundant in intracellular than in extracellular viral DNA preparations, episomal replication and the requirement of a minimal DNA size for packaging into nucleocapsids is hypothesized.

Animals↗
PubMed2004

Most chloroplast DNA of maize seedlings in linear molecules with defined ends and branched forms.

We used pulsed-field gel electrophoresis, restriction fragment mapping, and fluorescence microscopy of individual DNA molecules to analyze the structure of chloroplast DNA (cpDNA) from shoots of ten to 14 day old maize seedlings. We find that most of the cpDNA is in linear and complex branched forms, with only 3-4% as circles. We find the ends of linear genomic monomers and head-to-tail (h-t) concatemers within inverted repeat sequences (IRs) near probable origins of replication, not at random sites as expected from broken circles. Our results predict two major and three minor populations of linear molecules, each with different ends and putative origins of replication. Our mapping data predict equimolar populations of h-t linear concatemeric molecules differing only in the relative orientation (inversion) of the single copy regions. We show how recombination during replication can produce h-t linear concatemers containing an inversion of single copy sequences that has for 20 years been attributed to recombinational flipping between IRs in a circular chromosome. We propose that replication is initiated predominantly on linear, not circular, DNA, producing multi-genomic branched chromosomes and that most replication involves strand invasion of internal regions by the ends of linear molecules, rather than the generally accepted D-loop-to-theta mechanism. We speculate that if the minor amount of cpDNA in circular form is useful to the plant, its contribution to chloroplast function does not depend on the circularity of these cpDNA molecules.

Chloroplasts↗
PubMed2004

Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation.

Catalysis of DNA recombination by Tn3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites (res). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent 'accessory' binding sites II and III. 'Hyperactive' resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli. Some single mutants can resolve a res x site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I x site I plasmid. Site I x site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2-3' interface that abolish activity of wild-type resolvase. Activating mutations may allow regulatory mechanisms of the wild-type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.

Binding Sites↗
PubMed2003

Mhr1p-dependent concatemeric mitochondrial DNA formation for generating yeast mitochondrial homoplasmic cells.

Mitochondria carry many copies of mitochondrial DNA (mtDNA), but mt-alleles quickly segregate during mitotic growth through unknown mechanisms. Consequently, all mtDNA copies are often genetically homogeneous within each individual ("homoplasmic"). Our previous study suggested that tandem multimers ("concatemers") formed mainly by the Mhr1p (a yeast nuclear gene-encoded mtDNA-recombination protein)-dependent pathway are required for mtDNA partitioning into buds with concomitant monomerization. The transmission of a few randomly selected clones (as concatemers) of mtDNA into buds is a possible mechanism to establish homoplasmy. The current study provides evidence for this hypothesis as follows: the overexpression of MHR1 accelerates mt-allele-segregation in growing heteroplasmic zygotes, and mhr1-1 (recombination-deficient) causes its delay. The mt-allele-segregation rate correlates with the abundance of concatemers, which depends on Mhr1p. In G1-arrested cells, concatemeric mtDNA was labeled by [14C]thymidine at a much higher density than monomers, indicating concatemers as the immediate products of mtDNA replication, most likely in a rolling circle mode. After releasing the G1 arrest in the absence of [14C]thymidine, the monomers as the major species in growing buds of dividing cells bear a similar density of 14C as the concatemers in the mother cells, indicating that the concatemers in mother cells are the precursors of the monomers in buds.

DNA, Concatenated↗