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PubMed2004

Development of an exonuclease protection mediated PCR bioassay for sensitive detection of Ah receptor agonists.

The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. Here we developed a novel method to detect the presence of AhR ligands using Exonuclease Protection Mediated PCR bioassay (EPM-PCR). This assay measures the ability of a chemical to activate AhR DNA binding in vitro. In the presence of AhR ligand, an expected length PCR product was observed on electrophoresis, but no signal was detected in the absence of ligand. Real-time quantitative PCR was performed to quantify DNA bound to ligand:AhR complex. We obtained a standard curve with TCDD concentration to bound DNA copies in the range of 0.01 pM-10 nM of TCDD. Minimal detection limit of the assay was below 0.01 pM TCDD, and the whole detection time was less than 5 h. In comparison to the chemical-activated luciferase gene expression (CALUX) bioassay, EPM-PCR bioassay is more sensitive and easier to perform. These results suggest that this assay is useful for detection and quantification of TCDD and related AhR ligands in a cell-free system without the use of radioactivity.

Animals↗
PubMed2004

The gp64 locus of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus contains a 3' repair exonuclease homologue and lacks v-cath and ChiA genes.

Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) is one of the most successful biological insecticides. In this study, we cloned and sequenced a 12.5 kbp BamHI-D restriction endonuclease fragment of the AgMNPV isolate 2D genome that includes the gp64 gene. We compared this highly conserved region with that of other baculoviruses. AgMNPV contained two genes, p22.2 and v-trex, in common with Choristoneura fumiferana MNPV (CfMNPV) that were not present in other baculoviruses. The v-trex gene has homology to a eukaryotic 3' repair exonuclease and appears to have been acquired from an invertebrate host. The v-trex gene product has the potential to be involved in virus recombination or UV-light tolerance. Multigene phylogenetic analysis suggested that AgMNPV is most closely related to Orgyia pseudotsugata MNPV (OpMNPV). AgMNPV differed from other group I NPVs in that ChiA and v-cath gene homologues were missing from the region downstream of the gp64 gene. Proteinase assays and genetic probes suggest the v-cath gene is absent from AgMNPV.

3' Untranslated Regions↗
PubMed1996

Sequence-specific priming and exonuclease-released fluorescence detection of HLA-DQB1 alleles.

Molecular typing of HLA DQB1 alleles, employing sequence-specific primers (SSP) for PCR amplification, was used to test a novel method that eliminates the requirement for subsequent gel electrophoresis or additional hybridization steps by directly detecting positive reactions. We have evaluated the performance of this fluorescence-based oligonucleotide probe assay to assign the most common DQB1 alleles on DNA from 14 homozygous cell lines and in a blind study of 50 diabetic patient samples that had been previously typed at the DQB1 locus using SSOP and conventional SSP-based approaches. We used a panel of 14 DQB1 SSP primer pairs, internal control primers, and a combination of 4 fluorescent oligonucleotide probes to detect 14 alleles or groups of alleles and controls. We can reliably detect single-base allelic differences, observe 100% concordance with the results obtained using both of the standard methods, and are able to further subtype several alleles that are not easily distinguished using SSOP (e.g. DQB1 *0401/0402 and DQB1 *0302/ 0303). Sequence-specific priming and exonuclease-released fluorescence (SSPERF) detection is technically simple and can be performed in less than 2 hours, including DNA extraction, PCR amplification, data analysis and allele identification. This method is particularly useful for the analysis of large numbers of samples, for which high throughput is critical and for which gel-based approaches are difficult to perform. This technique may also be useful for small-scale class I and class II molecular typing in clinically oriented laboratories.

Alleles↗
PubMed1963

PROCESS OF INFECTION WITH PHI-X174: EFFECT OF EXONUCLEASES ON THE REPLICATIVE FORM.

When a preparation containing the DNA of the bacteriophage øX174 in its replicative form together with DNA from Escherichia coli is treated with exonucleases, the replicative form retains its characteristic physical properties, while the bacterial DNA is degraded. The evidence suggests that the mature replicative form exists as a closed ring.

Coliphages↗
PubMed1976

Genetic mapping of xthA, the structural gene for exonuclease III in Escherichia coli K-12.

The genes xthA, pncA, and pabB were ordered relative to others by two- and three-factor transductional crosses with bacteriophage P1. The genes studied span 2 min (2%) of the genetic map of Escherichia coli K-12 in the clockwise sequence pheS-pfkB-xthA-pncA-gap-pabB-fadD. Eleven independently derived xth mutations were examined; all were known to affect exonuclease III and its associated endonuclease II activity, and all were mapped in the xthA region. pncA mutations were found to confer resistance to 6-aminonicotinamide, whereas some pheS mutations are known to specify resistance to p-fluorphenylalanine. xth mutations were readily transferred into other strains by selecting for these co-transducible drug resistance markers.

6-Aminonicotinamide↗
PubMed1979

rorA mutation of Escherichia coli K-12 affects the recB subunit of exonuclease V.

The ATP-dependent nuclease, exonuclease V, of Escherichia coli plays an important role in repair and recombination. The enzyme is composed of two subunits, one of which is the product of the recB and recC genes. In this communication it is shown by mapping and complementation experiments that the rorA mutation, which results in radiation sensitivity but not the loss of recombination ability, is an allele of the recB gene.

Chromosome Mapping↗
PubMed1995

The old exonuclease of bacteriophage P2.

The Old protein of bacteriophage P2 is responsible for interference with the growth of phage lambda and for killing of recBC mutant Escherichia coli. We have purified Old fused to the maltose-binding protein to 95% purity and characterized its enzymatic properties. The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides.

Adenosine Triphosphatases↗
PubMed2002

5' exonuclease assay for detection of serogroup Y Neisseria meningitidis.

The incidence of serogroup Y meningococcal disease has increased recently in the United States. Here, we describe the development of a 5' exonuclease assay for the detection of serogroup Y Neisseria meningitidis and demonstrate the usefulness of this assay for resolving serogroup identification of strains that are resistant to conventional serogrouping and for the nonculture identification of serogroup Y meningococcal disease.

Blood↗
PubMed1977

In vivo effects of recBC DNase, exonuclease I, and DNA polymerases of Escherichia coli on the infectivity of native and single-stranded DNA of bacteriophage T7.

The effect of several enzymes of the DNA metabolism of Escherichia coli on the biological activity of native and single-stranded T7 DNA was studied by transfection of lysozyme-EDTA spheroplasts prepared from various E. coli mutants. It is shown that the presence of the recBC DNase in the recipient cells decreases the infectivity of native and denatured DNA by about 100- and 10-fold, respectively. Lack of exonuclease I did not stimulate transfection by single-stranded DNA. Separated light (l) and heavy (r) strands of T7 DNA are fully infective, with a linear dependence on DNA concentrations, whereas heat-denatured DNA shows a two-hit kinetics. Single-stranded DNA was observed to depend on a functional DNA polymerase III for infectivity in polAB cells, whereas transfection with native T7 DNA was independent of the host DNA polymerases. The results are discussed with respect to the mode of T7 DNA replication.

Coliphages↗
PubMed1978

Replication of bacteriophage M13. XIV. Differential inhibition of the replication of M13 and M13 miniphage in a mutant of Escherichia coli defective in the 5' leads to 3' exonuclease associated with DNA polymerase I.

Previous studies have shown that M13 single-strand synthesis is inhibited at nonpermissive temperature in Escherichia coli polAexl, a temperature-sensitive mutant defective in the 5' leads to 3' exonuclease activity of polymerase I (T.-C. Chen and D. S. Ray, J. Mol. Biol. 106:589-604, 1976). Under these conditions the formation of covalently closed replicative form (RF) molecules is greatly reduced, and miniature forms of RF accumulate. We show here that the accumulation of mini-RFs is the consequence of a differential inhibition of the replication of unit-length phage and preexisting miniphage rather than a de novo production of miniphage. Mini-RFs do not accumulate even after as many as nine cycles of growth in the mutant host infected only with unit-length phage. Mixed infections of the mutant host with plaque-purified unit-length phage and a single cloned miniphage show that discontinuities in the mini-RFs are joined with higher efficiency than are those contained in unit-length RFs. After a shift to nonpermissive temperature during single-strand synthesis in cells infected with plaque-purified phage alone, M13 RFs are found largely as RFII molecules (RF form having one or more single-strand discontinuities) containing only a single discontinuity in the viral strand. The inability of the accumulated unit-length RFII molecules to actively replicate may reflect the presence of either a bound protein or RNA primer on the 5' terminus of the viral strand and provides further support for the existence of distinct initiation and termination events in the synthesis of the viral strand.

Coliphages↗
PubMed1987

B-cell nuclear proteins binding in vitro to the human immunoglobulin kappa enhancer: localization by exonuclease protection.

Proteins capable of interacting with the enhancer of the immunoglobulin kappa gene in vitro have been detected in extracts of nuclei from human B cells and from human, mouse, and rabbit spleens. The experiments, based on an exonuclease protection technique, demonstrate nuclear protein factors binding to a 30- to 35-base-pair domain containing both the simian virus 40 enhancer core element (TTTCCA) and the octamer CAGGTGGC that was previously identified as the consensus sequence for protein-binding sites in the murine immunoglobulin heavy-chain enhancer. This 30- to 35-base-pair domain in the human kappa enhancer is homologous to a site of protein binding detected in the murine kappa enhancer by other investigators using a gel retardation assay. Our results complement in vivo dimethyl sulfate footprinting studies of the human immunoglobulin kappa enhancer which demonstrated B cell-specific changes in guanine reactivity immediately 5' to the consensus octamer. Together, these findings suggest that DNA-binding proteins in B-cell nuclei interact with the 5' portion of the human kappa-gene enhancer. Such proteins could play a role in the B cell-specific transcription of the human immunoglobulin kappa gene.

B-Lymphocytes↗
PubMed2004

Messenger RNA decay in mammalian cells: the exonuclease perspective.

The majority of messenger RNA (mRNA) decay in mammalian cells appears to be the work of a series of RNA exoribonucleases. A set of multiple poly(A)-specific deadenylases has been identified, some, if not most, of which are likely to play a role in the key first step of mRNA turnover--the regulated shortening of the poly(A) tail. After deadenylation, the transcript likely gets degraded by either a 5'-to-3' or a 3'-to-5' exonucleolytic pathway. Interestingly, multiple exonucleases have been identified for both of these pathways that appear to form multicomponent complexes with diverse roles in cellular biology. Therefore these enzymes appear not only to be important components of the mRNA turnover machinery, but also may function in a networked fashion in the post-transcriptional control of gene expression.

Animals↗
PubMed1978

Characterization of the Bacillus subtilis bacteriophage PBS2-induced DNA polymerase and its associated exonuclease activity.

The DNA polymerase induced by Bacillus subtilis bacteriophage PBS2 has a Stokes radius of 7.2 in buffers of high ioninc strength, suggesting a molecular weight in the range 145,000 to 195,000. The polypeptide bands observed on gel electrophoresis in dodecyl sulfate have apparent molecular weights of 78,000 and 69,000 (and possibly another 27,000) in equimolar amounts. In buffers of low ionic strength, the enzyme appears to form large aggregates and even precipitates, with about 90% loss of activity. A nuclease activity co-purifies with the PBS2 DNA polymerase and shows similar responses to changes in pH, MgCl2, N-ethylmaleimide, temperature, and dextran sulfate levels. The nuclease produces deoxyribonucleoside 5'monophosphates from denatured DNA containing thymine or uracil. No endonuclease activity is detectable on supercoiled DNA. The inhibition of nuclease activity by added deoxyribonucleoside triphosphates, the DNA-dependent turnover of triphosphates, to free monophosphates during DNA polymerization, the inhibition of nuclease activity by 3'-phosphates on the DNA template-primer, and the pattern of digestion of 5'-[32P]phosphate-labeled DNA all indicate that the PBS2 DNA polymerase-associated hydrolytic activity is a 3' leads to 5'-exonuclease.

Bacillus subtilis↗
PubMed2003

[Effect of 3' exonuclease activity of polymerase on extension of phosphorothioate-modified primers].

OBJECTIVE: To determine whether 3'phosphorothioate-modified-2 terminal mismatched primers can turn off DNA polymerization mediated by Exo(+) polymerase. METHODS: Two-directional primer extension was performed using polymerase with and without 3' exonuclease activity. The effects of unmodified primers and 3' phosphorothioate-modified primers on primer extension were evaluated. RESULTS: Exo(-) polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3' phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported "off-switch" of Exo(+) polymerase. CONCLUSION: These data suggested that the "off-switch" can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.

DNA Primers↗
PubMed1992

An exonuclease requiring an intact helical stem for specificity produces the 3' end of Acanthamoeba castellanii 5 S RNA.

A nuclear extract from Acanthamoeba castellanii which contains all of the components necessary for specific transcription of a 5 S RNA gene was separated into fractions required for specific transcription initiation and an additional fraction needed in the reconstituted system to produce the 3' end characteristic of mature 5 S RNA. The latter fraction contained a novel processing activity characterized by an exonuclease specific for highly structured RNAs, including 5 S RNA. An intact helical stem formed between the 5' and 3' ends of the 5 S RNA precursor determines the 3' nucleotide. In addition, the presence of ATP is required for specific processing. However, the possibility has not been ruled out that ATP inhibits a nonspecific ribonuclease in the extract since processing proceeds into the helical stem in its absence.

Acanthamoeba↗