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At least 631 records · Page 35Linked to original sources
PubMed1977

Characterization of DNA used to assay sera for anti-DNA antibodies; determination of the specificities of anti-DNA antibodies in SLE and non-SLE rheumatic disease states.

Commercial 14C-labeled KB cell DNA, widely used to assay sera for anti-DNA antibodies, was chromatographed on benzoylated-naphthoylated-DEAE-cellulose (BNDC) and on hydroxyapatite (HAP). On BNDC, only 25% of the 14C label eluted with 1 M NaC1 (KB fraction I) characteristic of ds-DNA. Fifty-five percent of the label eluted with 50% formamide-1 M NaC1 (KB fraction II) characteristic of ss or denatured DNA. On HAP, however, none of the 14C label eluted with 0.2 M phosphate buffer as anticipated for ss-DNA, but, rather, all of the 14C label eluted with 0.4 M phosphate, characteristic of ds-DNA. after pretreatment with S1 endonuclease of Aspergillus oryzae, which selectively digests ss regions, however, 42% of the 14C label was lost from the 0.4 M phosphate peak. These results indicated that more than half of this 14C-KB-cell DNA preparation was ds-DNA with ss regions which was undetectable by HAP chromatography. 3H-ds-DNA and circular 3H-ss-DNA prepared from T7 and phiX174 bacteriophage, respectively, were found to be chromatographically pure on both BNDC and HAP. None of 10 non-SLE sera (rheumatoid arthritis 3, mixed connective tissue disease 4, scleroderma 1, ulcerative colitis 1, and pulmonary fibrosis with chronic active hepatitis 1), previously believed to contain anti-ds-DNA antibodies on the basis of KB cell DNA testing and detectable antibodies against KB fraction 1 or T7 DNA: all of 10 KB cell DNA positive SLE sera had antibodies against both. Additionally, none of the 10 non-SLE sera had antibodies against KB cell DNA when retested with DNA that had been pretreated with S1 endonuclease. Seven of these 10, however, as well as all 10 SLE sera, had antibodies against phiX174 DNA, KB fraction II DNA and alkali-denatured T7 DNA. The data support the conclusions that 1) false positive tests for anti-ds-DNA antibodies can result from contamination of ds-DNA with ds-DNA having ss regions, and 2) non-SLE sera do not contain antibodies specific for ds-DNA at levels comparable to those found in SLE sera but rather contain high levels of antibodies reacting with ss regions or mixed DNA.

Antibodies↗
PubMed2003

DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone in V79 cells expressing human cytochrome P450 enzymes.

Diesel exhaust is known to induce tumours in animals. Of the compounds found in diesel exhaust 3-nitrobenzanthrone (3-NBA) is particularly a powerful mutagen. Recently we showed that 3-NBA is genotoxic in vivo in rats by forming specific DNA adducts derived from nitroreduction. In this study a panel of genetically engineered V79 Chinese hamster cell lines expressing various human cytochrome P450 (CYP) enzymes (CYP1A1, CYP3A4) and/or human NADPH:CYP oxidoreductase (CYPOR) was used to identify CYP enzymes involved in the metabolic activation of 3-NBA. We analyzed the formation of specific DNA adducts by 32P-postlabelling after exposing cells to 1 microM 3-NBA. A similar pattern with a total of four distinct 3-NBA-DNA adducts was found in all cells, identical to those detected previously in DNA from rats treated with 3-NBA in vivo. Total adduct levels ranged from 75 to 132 using nuclease P1 and from 103 to 220 adducts per 10(8) nucleotides, using butanol enrichment. Comparison of DNA binding between different V79MZ derived cells revealed that human CYPOR and CYP3A4 were involved in the metabolic activation of 3-NBA. Furthermore, dose-dependent high adduct levels were detected after exposure to 0.01, 0.1 or 1 microM 3-NBA in the subclone V79NH which exhibits high activities of nitroreductase and N,O-acetyltransferase. Our results suggest that nitroreduction is the major pathway in the human bioactivation of 3-NBA. Moreover, acetylation of the initially formed N-hydroxy arylamine intermediates may contribute to the high genotoxic potential of 3-NBA.

Animals↗
PubMed1998

A nondenaturing purification scheme for the DNA-binding domain of poly(ADP-ribose) polymerase, a structure-specific DNA-binding protein.

Poly(ADP-ribose) polymerase (PARP) is thought to be involved in DNA repair given its ability to recognize and bind to DNA strand breaks. During apoptosis, PARP is proteolytically cleaved into two stable fragments, the N-terminal 25-kDa DNA-binding domain (DBD) and the 85-kDa fragment containing the automodification and catalytic domains. To understand the DNA-binding properties of PARP, we expressed a recombinant hexahistidine tagged protein (His-DBD) in Escherichia coli. We modified expression to facilitate protein folding by including zinc and reducing the induction temperature. Properly folded, the DNA-binding domain of PARP binds to single- and double-stranded DNA in a structure-specific manner. To eliminate contamination with bacterial DNA that occurred during the purification process, a purification procedure was developed to produce DNA-free protein. In addition, to remove the hexahistidine tag from the recombinant protein, thrombin cleavage was carried out while the recombinant protein was bound to a DNA column. This procedure stabilized the recombinant protein and resulted in nearly 100% cleavage with no appreciable loss to unwanted proteolytic degradation. This nondenaturing purification scheme results in high-quality, native PARP-DBD for use in structural and biochemical studies.

Amino Acid Sequence↗
PubMed2004

The Etruscans: a population-genetic study.

The origins of the Etruscans, a non-Indo-European population of preclassical Italy, are unclear. There is broad agreement that their culture developed locally, but the Etruscans' evolutionary and migrational relationships are largely unknown. In this study, we determined mitochondrial DNA sequences in multiple clones derived from bone samples of 80 Etruscans who lived between the 7th and the 3rd centuries b.c. In the first phase of the study, we eliminated all specimens for which any of nine tests for validation of ancient DNA data raised the suspicion that either degradation or contamination by modern DNA might have occurred. On the basis of data from the remaining 30 individuals, the Etruscans appeared as genetically variable as modern populations. No significant heterogeneity emerged among archaeological sites or time periods, suggesting that different Etruscan communities shared not only a culture but also a mitochondrial gene pool. Genetic distances and sequence comparisons show closer evolutionary relationships with the eastern Mediterranean shores for the Etruscans than for modern Italian populations. All mitochondrial lineages observed among the Etruscans appear typically European or West Asian, but only a few haplotypes were found to have an exact match in a modern mitochondrial database, raising new questions about the Etruscans' fate after their assimilation into the Roman state.

Bone and Bones↗
PubMed1999

Analysis of ancient DNA from a prehistoric Amerindian cemetery.

The Norris Farms No. 36 cemetery in central Illinois has been the subject of considerable archaeological and genetic research. Both mitochondrial DNA (mtDNA) and nuclear DNA have been examined in this 700-year-old population. DNA preservation at the site was good, with about 70% of the samples producing mtDNA results and approximately 15% yielding nuclear DNA data. All four of the major Amerindian mtDNA haplogroups were found, in addition to a fifth haplogroup. Sequences of the first hypervariable region of the mtDNA control region revealed a high level of diversity in the Norris Farms population and confirmed that the fifth haplogroup associates with Mongolian sequences and hence is probably authentic. Other than a possible reduction in the number of rare mtDNA lineages in many populations, it does not appear as if European contact significantly altered patterns of Amerindian mtDNA variation, despite the large decrease in population size that occurred. For nuclear DNA analysis, a novel method for DNA-based sex identification that uses nucleotide differences between the X and Y copies of the amelogenin gene was developed and applied successfully in approximately 20 individuals. Despite the well-known problems of poor DNA preservation and the ever-present possibility of contamination with modern DNA, genetic analysis of the Norris Farms No. 36 population demonstrates that ancient DNA can be a fruitful source of new insights into prehistoric populations.

Amelogenin↗
PubMed1985

Analysis of transfer of tumor-inducing plasmids from Agrobacterium tumefaciens to Petunia protoplasts.

Petunia protoplasts were infected with the virulent Agrobacterium tumefaciens strain A348 or the avirulent strain A136 (lacking a Ti plasmid). The infection process was stopped at various time intervals up to 24 h after inoculation, and the DNA from the plant cells was isolated. Southern blot analysis indicated that the DNA isolated from infected Petunia cells was not detectably contaminated by bacterial DNA from lysed Agrobacterium cells. Analysis of the DNA from the virulent infections suggested that the transferred DNA (T-DNA) may be transferred to the plant cell rapidly (within 2 to 6 h) after the bacteria bind to the cell wall and that the T-DNA may exist in a rearranged state which is stable over the time period investigated. Dot blot analysis indicated that regions far outside the T-DNA may be transferred to the plant cell. Most of the DNA transferred to the plant cell during the initial hours of infection is rapidly degraded.

Base Sequence↗
PubMed1999

Efficient DNA fingerprinting method for the identification of cross-culture contamination of cell lines.

In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.

Base Sequence↗
PubMed2006

Dna damage and external lesions in brown bullheads (Ameiurus Nebulosus) from contaminated habitats.

The Comet assay was used to compare levels of DNA damage in brown bullheads (Ameiurus nebulosus) collected from three known contaminated locations, the Cuyahoga River (OH, U.S.A.), Ashtabula River (OH, U.S.A.; both tributaries to Lake Erie, USA), and Ashumet Pond (Cape Cod, MA, U.S.A.), with brown bullheads collected from three paired reference sites, Old Woman Creek (OH, U.S.A.), Conneaut River (OH, U.S.A.; both tributaries to Lake Erie), and Great Herring Pond (mainland MA, U.S.A.), respectively. Blood was sampled from each fish, and the Comet assay was conducted on erythrocytes. The assay results demonstrate that fish from the three contaminated sites each suffered higher DNA damage compared with fish from their respective reference sites. The results also show that the genetic damage was associated with the occurrence of external lesions and deformities in fish. The Comet assay is sufficiently sensitive to detect exposure of natural fish populations to environmental levels of genotoxic contaminants.

Animals↗
PubMed2002

"In-gel" purified ditags direct synthesis of highly efficient SAGE Libraries.

BACKGROUND: SAGE (serial analysis of gene expression) is a recently developed technique for systematic analysis of eukaryotic transcriptomes. The most critical step in the SAGE method is large scale amplification of ditags which are then are concatemerized for the construction of representative SAGE libraries. Here, we report a protocol for purifying these ditags via an 'in situ' PAGE purification method. This generates ditags free of linker contaminations, making library construction simpler and more efficient. RESULTS: Ditags used to generate SAGE libraries were demarcated 'in situ' on preparative polyacrylamide gels using XC and BPB dyes, which precisely straddle the ditag band when a 16% PAGE gel (19:1 acrylamide:bis, 5% cross linker) is used to resolve the DNA bands. Here, the ditag DNA was directly excised from gel without visualization via EtBr or fluorescent dye staining, resulting in highly purified ditag DNA free of contaminating linkers. These ditags could be rapidly self ligated even at 4 degrees C to generate concatemers in a controlled manner, which in turn enabled us to generate highly efficient SAGE libraries. This reduced the labor and time necessary, as well as the cost. CONCLUSIONS: This approach greatly simplified the ditag purification procedure for constructing SAGE libraries. Since the traditional post-run staining with EtBr or fluorescent dyes routinely results in cross contamination of a DNA band of interest by other DNA in the gel, the dry gel DNA excision method described here may also be amenable to other molecular biology techniques in which DNA purity is critically important.

Journal Article↗
PubMed2001

Species-specific PCR for identification of common contaminant mollicutes in cell culture.

Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5' end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5' end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.

Cell Line↗
PubMed2002

Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis.

Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.

Bacteria↗
PubMed2000

Effects of growth medium selection on plasmid DNA production and initial processing steps.

Cultures of recombinant Escherichia coli containing the plasmid pSVbeta were grown in three medium formulations to assess their effects on the characteristics of supercoiled plasmid DNA production for plasmid-based gene therapy. A semi-defined medium containing casamino acids (SDCAS) was found to support higher cell densities and higher plasmid stability than a similar medium containing soya amino acids (SDSOY) or Luria-Bertani medium (LB). Differences were observed in the cell harvest characteristics, plasmid DNA primary recovery, plasmid DNA yield and quality between cells grown on LB and on SDCAS medium. Cells grown on SDCAS medium were more difficult to resuspend after harvest than those grown in LB medium and were less susceptible to alkaline lysis. The plasmid DNA content from SDCAS was predominantly supercoiled and was less contaminated by chromosomal DNA than plasmid DNA extracts derived from cells grown on LB medium. It was hypothesised that the different carbon:nitrogen ratio (C:N) of the medium may have been responsible for changing the cell wall polysaccharide composition resulting in the change in cell harvest and lysis characteristics. Results indicated that changing the C:N ratio of SDCAS medium between 1.21:1 and 12.08:1 resulted in no alteration in cell wall polysaccharide composition or in cell susceptibility to chemical lysis or physical breakage. Plasmid DNA yields increased ten-fold with ten-fold increase in the C:N ratio of SDCAS medium.

Biotechnology↗
PubMed2001

DNA chips: a new tool for genetic analysis and diagnostics.

DNA chips are miniaturized microsystems based on the ability of DNA to spontaneously find and bind its complementary sequence in a highly specific and reversible manner, known as hybridization. Labeled DNA molecules in a sample are analyzed by DNA probes tethered at distinct sites on a solid support. The composition of the DNA sample is then deduced by analyzing the signal generated by labels present at each probe site. Applications are widespread: fundamental research, cancer or microbiology diagnostics, genotyping, gene expression, pharmacogenomics, and environmental control. Medical application consists, for example, in the identification and detection of mutations in genes responsible for cancers, or DNA chip analysis of individual polymorphisms which may provide a guide towards the most efficient treatment. In the environmental and agro-industrial fields, DNA chips show great promise in rapidly testing microorganism content, contamination or pathogenicity. DNA chip dimensions offer hybridization sites in the 50-200 micron range, producing arrays ranging from 100 to 1,000,000 different probes per cm2.

DNA Probes↗
PubMed2002

Molecular analysis of ancient microbial infections.

The detection of ancient microbial DNA offers a new approach for the study of infectious diseases, their occurrence, frequency and host-pathogen interaction in historic times and populations. Moreover, data obtained from skeletal and mummified tissue may represent an important completion of contemporary phylogenetic analyses of pathogens. In the last few years, a variety of bacterial, protozoal and viral infections have been detected in ancient tissue samples by amplification and characterization of specific DNA fragments. This holds particularly true for the identification of the Mycobacterium tuberculosis complex, which seems to be more robust than other microbes due to its waxy, hydrophobic and lipid-rich cell wall. These observations provided useful information about the occurrence, but also the frequency of tuberculosis in former populations. Moreover, these studies suggest new evolutionary models and indicate the route of transmission between human and animals. Until now, other pathogens, such as Mycobacterium leprae, Yersinia pestis, Plasmodium falciparum and others, have occasionally been identified - mostly in single case studies or small sample sizes - as well, although much less information is available on these pathogens in ancient settings. The main reason therefore seems to be the degradation and modification of ancient DNA by progressive oxidative damage. Furthermore, the constant risk of contamination by recent DNA forces to take time and cost effective measures and renders the analysis of ancient microbes difficult. Nevertheless, the study of microbial ancient DNA significantly contributes to the understanding of transmission and spread of infectious diseases, and potentially to the evolution and phylogenetic pathways of pathogens.

Animals↗
PubMed2005

DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone after intratracheal instillation in rats.

3-Nitrobenzanthrone (3-NBA) is an environmental pollutant and suspected human carcinogen found in emissions from diesel and gasoline engines and on the surface of ambient air particulate matter; human exposure to 3-NBA is likely to occur primarily via the respiratory tract. In our study female Sprague Dawley rats were treated by intratracheal instillation with a single dose of 0.2 or 2 mg/kg body weight of 3-NBA. Using the butanol enrichment version of the (32)P-postlabeling method, DNA adduct formation by 3-NBA 48 hr after intratracheal administration in different organs (lung, pancreas, kidney, urinary bladder, heart, small intestine and liver) and in blood was investigated. The same adduct pattern consisting of up to 5 DNA adduct spots was detected by thin layer chromatography in all tissues and blood and at both doses. Highest total adduct levels were found in lung and pancreas (350 +/- 139 and 620 +/- 370 adducts per 10(8) nucleotides for the high dose and 39 +/- 18 and 55 +/- 34 adducts per 10(8) nucleotides for the low dose, respectively) followed by kidney, urinary bladder, heart, small intestine and liver. Adduct levels were dose-dependent in all organs (approximately 10-fold difference between doses). It was demonstrated by high performance liquid chromatography (HPLC) that all 5 3-NBA-derived DNA adducts formed in rats after intratracheal instillation are identical to those formed by other routes of application and are, as previously shown, formed from reductive metabolites bound to purine bases. Although total adduct levels in the blood were much lower (41 +/- 27 and 9.5 +/- 1.9 adducts per 10(8) nucleotides for the high and low dose, respectively) than those found in the lung, they were related to dose and to the levels found in lung. These results show that uptake of 3-NBA by the lung induces high levels of specific DNA adducts in several organs of the rat and an identical adduct pattern in DNA from blood. Therefore, 3-NBA-DNA adducts present in the blood are useful biomarkers for exposure to 3-NBA and may help to assess the effective biological dose in humans exposed to it.

Animals↗
PubMed2002

Contamination of environmental surfaces by genital human papillomaviruses.

OBJECTIVE: To investigate contamination of environmental surfaces with human papillomaviruses (HPV) DNA in two genitourinary medicine (GUM) clinics and in an on-site staff leisure and fitness centre. METHODS: Samples were collected from the treatment rooms and patients' toilets of two GUM clinics situated at two hospital sites and from the toilets of the staff leisure and fitness centre on one of the sites. Samples were tested for the presence of HPV DNA by nested polymerase chain reaction (PCR), and HPV amplicons were typed by reverse line hybridisation using HPV type specific oligonucleotide probes complementary to 35 HPV types. All samples were also tested for beta globin DNA by PCR in order to assess their quality. RESULTS: HPV DNA was found to be present at more than 50% of the sites sampled in one of the GUM clinics, but was absent in the second, and also from the staff leisure and fitness centre. All HPV DNA detected was found to be cell associated. The most commonly found HPV types were 6, 11, and 16, respectively. HPV infected cells were found to be localised mainly to surfaces used predominantly by medical staff. CONCLUSIONS: This study has identified contamination of the environment of a GUM clinic. Possible sources for the contamination of the clinic toilets were from genital sites via hands to the environment. Within the treatment rooms the most likely route of HPV DNA contamination of the environment was via the doctor's gloved hands.

Ambulatory Care Facilities↗