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PubMed1994

An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis.

A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonuclease PalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi including Gossypium hirsucum (cotton), Pseudomonas, Bacillus, Streptomyces, and Colletotrichum. Up to 100 micrograms DNA/g (wet weight) of soil and 400 micrograms DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonuclease PalI, contained 12-20 DNA fragments, appearing as sample "fingerprints." Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.

Base Sequence↗
PubMed2006

A novel transposon for functional expression of DNA libraries.

Environmental DNA libraries are important sources for novel biocatalyst genes but activity screening for relevant enzymes is often inefficient. Therefore, we have constructed the transposon MuExpress which randomly integrates in vitro into existing bacterial artificial chromosome (BAC) or cosmid libraries and permits the inducible expression of its flanking regions in both directions. Furthermore, this transposon allows the bidirectional sequencing of the respective clones starting from unique primer binding sites.

Chromosomes, Artificial, Bacterial↗
PubMed2006

Capture probe conjugated to paramagnetic nanoparticles for purification of Alexandrium species (Dinophyceae) DNA from environmental samples.

AIMS: To develop a rapid, cost-effective and selective Alexandrium DNA extraction procedure from environmental samples in order to provide good-quality template for the downstream PCR-based detection assay. METHODS AND RESULTS: In this study, we tested a DNA extraction method based on silica-coated, superparamagnetic nanoparticles conjugated to a DNA-capture sequence (probe) complementary to a specific region of 5.8S rDNA of the genus Alexandrium. Cultured Alexandrium catenella cells were used as the harmful algal bloom species for the DNA extraction. Then, a PCR assay was performed with primers specific for the genus Alexandrium to assess the specificity and sensitivity of the nucleic acid extraction method. This method was applied to both cultured and field samples, reaching in both cases a detection limit of one A. catenella cell. CONCLUSIONS: The results suggest that the use of probe-conjugated paramagnetic nanoparticles could be effective for the specific purification of microalgal DNA in cultured or environmental samples, ensuring sensitivity and specificity of the subsequent PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The DNA extraction method optimized in this study represents a progress towards the rapid and efficient direct detection of Alexandrium cells in seawater monitoring. In fact, this method requires no other equipment than a magnet and a hybridization oven and, in principle, can be adapted to different toxic microalgal species and can be automated, allowing the processing of a high number of samples.

Animals↗
PubMed2004

[Risk assessment of environmental carcinogenesis using WBC DNA adducts].

Environmental carcinogens are enzymatically activated to form intermediates that can react with cellular DNA and form DNA adducts. Several kind of carcinogens bind to several sites of DNA. The measurement of WBC DNA adducts is a useful indicator for environmental carcinogen exposure monitoring. The 32P-Postlabeling method is a most popular and very sensitive method for DNA adduct analysis. We can analyze 1 adduct/10(8) nucleotides. In this review, I show some data of DNA adduct analysis for PAH exposure. These results demonstrated that inter-individual variation was very large. There were some confounding factors, such as metabolism or repair variations. I also showed some limitations of DNA adduct analysis. The method of adduct analysis is very complicated with several steps. We need to improve the accuracy. Do the data from WBC explain the target organ, such as the lung or liver? Almost all previous studies have been cross-sectional. We need a large-sized cohort study to evaluate whether adducts are a predictor of cancer. DNA adducts should be an important factor in gene-environment interaction.

Carcinogens, Environmental↗
PubMed1985

The dependence of the surface electrostatic potential of B-DNA on environmental factors.

The electrostatic potential of B-DNA is calculated on its surface envelope for two homopolymeric base pair sequences using models representing the effects of both counterion binding and of aqueous solution. The influence of these two factors on the resulting potentials is established and the significance of calculations which omit such effects is discussed.

Base Composition↗
PubMed2003

Environmental exposure, DNA methylation, and gene regulation: lessons from diethylstilbesterol-induced cancers.

DNA methylation is an epigenetic mechanism that regulates chromosomal stability and gene expression. Abnormal DNA methylation patterns have been observed in many types of human tumors, including those of the breast, prostate, colon, thyroid, stomach, uterus, and cervix. We and others have shown that exposure to a wide variety of xenobiotics during critical periods of mammalian development can persistently alter the pattern of DNA methylation, resulting in potentially adverse biological effects such as aberrant gene expression. Thus, this epigenetic mechanism may underlie the observed increased risk in adulthood of several chronic diseases, including cancer, in response to xenobiotic exposures early in life. We present here the lessons learned from studies on the effects of perinatal diethylstilbesterol (DES) exposure on the methylation pattern of the promoters of several estrogen-responsive genes associated with the development of reproductive organs. Perinatal DES exposure, which induces epithelial tumors of the uterus in mice and is associated with several reproductive tract abnormalities and increased vaginal and cervical cancer risk in women, provides a clear example of how estrogenic xenobiotic exposure during a critical period of development can abnormally demethylate DNA sequences during organ development and possibly increase cancer risk later in life. In addition, nutritional factors and stress may also alter DNA methylation during early life and modulate the risk of cancer and other chronic diseases in adulthood. We suggest that DNA methylation status may be influenced by environmental exposures in early life, leading to increased risk of cancer in adulthood.

Animals↗
PubMed2006

Comparison of methods of extracting Salmonella enterica serovar Enteritidis DNA from environmental substrates and quantification of organisms by using a general internal procedural control.

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 x 10(3) to 1.8 x 10(3) CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.

Animals↗
PubMed1988

B lymphocyte activation in systemic lupus erythematosus: spontaneous production of IgG antibodies to DNA and environmental antigens in cultures of blood mononuclear cells.

IgG antibodies to DNA, influenza virus haemagglutinin (HA), adenovirus hexon (HX) and mannan from Candida albicans (MN) have been determined in supernatants from 2-day unstimulated cultures of peripheral blood mononuclear cells from SLE patients and controls. Mean values were much higher in the SLE group, with from 20% (MN) to 85% (DNA) of patients giving values above the normal range. Although a significant correlation was observed between anti-DNA and anti-HA production, anti-HX and anti-MN showed no such correlations. The specificity of the ELISA assays was demonstrated by inhibition tests. It is concluded that a selective form of polyclonal activation in SLE results in the production of antibodies to foreign as well as to self antigens.

Antibodies, Antinuclear↗
PubMed1999

A rapid procedure for the isolation of plasmid DNA from environmental bacteria.

The INSTA-MINI-PREP method, a rapid protocol for plasmid DNA extraction, was originally developed to prepare plasmid DNA from 1 to 3 ml miniprep Escherichia coli cultures. Direct extraction of plasmid DNA is achieved by a two-phase solution which is separated by centrifugation in the presence of the INSTA-PREP gel barrier material. This method has been successfully tested on various environmental Salmonella strains, although it was not suitable for Pseudomonas aeruginosa and enterococci strains. The INSTA-MINI-PREP method is a new alternative procedure to screen plasmid contents of Salmonella and E. coli strains rapidly and easily.

Bacteriological Techniques↗
PubMed1999

Comet assay application in environmental monitoring: DNA damage in human leukocytes and plant cells in comparison with bacterial and yeast tests.

Urban airborne particulate is a complex mixture of air pollutants, many of which have not been identified. However, short-term mutagenesis tests together with chemicophysical parameter analysis are able to better assess air quality and genotoxic load. The findings of continuous monitoring (January 1991-August 1998) of urban air genotoxicity of a Po Valley town (Italy) on Salmonella typhimurium and Saccharomyces cerevisiae are reported. During this period, various measures (catalytic devices, unleaded fuels, annual vehicle overhaul, etc.) to improve air-dispersed pollutant control were enforced. However, a continuous presence of genotoxic compounds is shown and more qualitative than quantitative changes are evident. We also demonstrate the ability of the Comet assay to detect DNA-damaging agents in airborne particulate samples. We applied the test to human leukocytes and, with major improvements, to plant cells (Allium cepa roots and epigean tissues of Impatiens balsamina). The first findings on human leukocytes confirm the sensitivity of this assay, its peculiarity and its applicability in assessing genotoxicity in environmental samples. The capability of plants to show the response of multicellular organisms to environmental pollutants largely counterbalances a probable lowering in sensitivity. Moreover, application of the Comet test to epigean tissues could be useful in estimating the bioavailability of and genotoxic damage by air pollutants, including volatile compounds (ozone, benzene, nitrogen oxides, etc.) to higher plants.

Cells, Cultured↗
PubMed2005

Cell-free cloning using phi29 DNA polymerase.

We describe conditions for rolling-circle amplification (RCA) of individual DNA molecules 5-7 kb in size by >10(9)-fold, using phi29 DNA polymerase. The principal difficulty with amplification of small amounts of template by RCA using phi29 DNA polymerase is "background" DNA synthesis that usually occurs when template is omitted, or at low template concentrations. Reducing the reaction volume while keeping the amount of template fixed increases the template concentration, resulting in a suppression of background synthesis. Cell-free cloning of single circular molecules by using phi29 DNA polymerase was achieved by carrying out the amplification reactions in very small volumes, typically 600 nl. This procedure allows cell-free cloning of individual synthetic DNA molecules that cannot be cloned in Escherichia coli, for example synthetic phage genomes carrying lethal mutations. It also allows cell-free cloning of genomic DNA isolated from bacteria. This DNA can be sequenced directly from the phi29 DNA polymerase reaction without further amplification. In contrast to PCR amplification, RCA using phi29 DNA polymerase does not produce mutant jackpots, and the high processivity of the enzyme eliminates stuttering at homopolymer tracts. Cell-free cloning has many potential applications to both natural and synthetic DNA. These include environmental DNA samples that have proven difficult to clone and synthetic genes encoding toxic products. The method may also speed genome sequencing by eliminating the need for biological cloning.

Bacillus Phages↗
PubMedDate not supplied

Morphology, phylogeny and biology of Gliocephalis hyalina, a biotrophic contact mycoparasite of Fusarium species.

Gliocephalis hyalina, a rarely seen microfungus with a morphology similar to the hyphomycete genus Aspergillus but with slimy conidia was found in a mixed microbial culture from soybean roots. This species has been reported sporadically since 1899, each time in association with other fungi or bacteria. Gliocephalis hyalina has not been maintained in monoxenic culture and requires other fungi to grow. Light and scanning electron microcope studies indicate that it is a biotrophic contact parasite of Fusarium species. The fungus may penetrate the cells but has no apparent deleterious effect on the growth or plant pathogenicity of its host. Phylogenetic analyses of partial nuclear small subunit rDNA sequences place G. hyalina near the Laboulbeniales, an order of obligate insect parasitic microfungi, and the related mycelial genus Pyxidiophora. Gliocephalis hyalina is mycoparasitic along with many Pyxidiophora species. These discoveries suggest that some "unculturable" microorganisms or "cryptic DNA" recovered from environmental DNA samples might represent obligate biotrophs that could be cultured and studied with simple techniques.

Ascomycota↗
PubMed2005

Suicide polymerase endonuclease restriction, a novel technique for enhancing PCR amplification of minor DNA templates.

PCR-based molecular analyses can be hindered by the presence of unwanted or dominant DNA templates that reduce or eliminate detection of alternate templates. We describe here a reaction in which such templates can be exclusively digested by endonuclease restriction, leaving all other DNAs unmodified. After such a modification, the digested template is no longer available for PCR amplification, while nontarget DNAs remain intact and can be amplified. We demonstrate the application of this method and use denaturing gradient gel electrophoresis to ascertain the removal of target DNA templates and the subsequent enhanced amplification of nondigested DNAs. Specifically, plastid 16S rRNA genes were exclusively digested from environmental DNA extracted from plant roots. In addition, pure culture and environmental DNA extracts were spiked with various amounts of genomic DNA extracted from Streptomyces spp., and selective restriction of the Streptomyces 16S rRNA genes via the suicide polymerase endonuclease restriction PCR method was employed to remove the amended DNA.

Arachis↗
PubMed2005

Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora.

Salinispora is the first obligate marine genus within the order Actinomycetales and a productive source of biologically active secondary metabolites. Despite a worldwide, tropical or subtropical distribution in marine sediments, only two Salinispora species have thus far been cultivated, suggesting limited species-level diversity. To further explore Salinispora diversity and distributions, the phylogenetic diversity of more than 350 strains isolated from sediments collected around the Bahamas was examined, including strains cultured using new enrichment methods. A culture-independent method, using a Salinispora-specific seminested PCR technique, was used to detect Salinispora from environmental DNA and estimate diversity. Overall, the 16S rRNA gene sequence diversity of cultured strains agreed well with that detected in the environmental clone libraries. Despite extensive effort, no new species level diversity was detected, and 97% of the 105 strains examined by restriction fragment length polymorphism belonged to one phylotype (S. arenicola). New intraspecific diversity was detected in the libraries, including an abundant new phylotype that has yet to be cultured, and a new depth record of 1,100 m was established for the genus. PCR-introduced error, primarily from Taq polymerase, significantly increased clone library sequence diversity and, if not masked from the analyses, would have led to an overestimation of total diversity. An environmental DNA extraction method specific for vegetative cells provided evidence for active actinomycete growth in marine sediments while indicating that a majority of sediment samples contained predominantly Salinispora spores at concentrations that could not be detected in environmental clone libraries. Challenges involved with the direct sequence-based detection of spore-forming microorganisms in environmental samples are discussed.

Bacteriological Techniques↗
PubMed1995

Extraction and purification of microbial DNA from petroleum-contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis.

We investigated the use of multiplex polymerase chain reaction (PCR) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWW0), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWW0, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 10(0) to 10(6) cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 microL) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradative cells, at a lower detection limit of approximately 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.

Alcaligenes↗
PubMed2003

Amplified functional DNA restriction analysis to determine catechol 2,3-dioxygenase gene diversity in soil bacteria.

To determine phylogenetic diversity of a functional gene from strain collections or environmental DNA amplifications, new and fast methods are required. Catechol 2,3-dioxygenase (C23O) subfamily I.2.A genes, known to be of crucial importance for aromatic degradation, were used as a model to adapt the amplified ribosomal DNA restriction analysis to functional genes. Sequence data of C23O genes from 13 reference strains, representing the main branches of the C23O family I.2.A phylogeny, were used for simulation of theoretical restriction patterns. Among other restriction enzymes, Sau3A1 theoretically produce characteristic profiles from each subfamily I.2.A member and their similarities reassembled the main divergent branches of C23O gene phylogeny. This enzyme was used to perform an amplified functional DNA restriction analysis (AFDRA) on C23O genes of reference strains and 19 isolates. Cluster analyses of the restriction fragment profiles obtained from isolates showed patterns with distinct similarities to the reference strain profiles, allowing to distinguish four different groups. Sequences of PCR fragments from isolates were in close agreement with the phylogenetic correlations predicted with the AFDRA approach. AFDRA thus provided a quick assessment of C23O diversity in a strain collection and insights of its gene phylogeny affiliation among known family members. It cannot only be easily applied to a vast number of isolates but also to define the predominant polymorphism of a functional gene present in environmental DNA extracts. This approach may be useful to differentiate functional genes also for many other gene families.

Base Sequence↗