Denaturing Gradient Gel Electrophoresis Patterns Research Articles

Enumeration and detection of anaerobic ferrous iron-oxidizing, nitrate-reducing bacteria from diverse European sediments.

Anaerobic, nitrate-dependent microbial oxidation of ferrous iron was recently recognized as a new type of metabolism. In order to study the occurrence of three novel groups of ferrous iron-oxidizing, nitrate-reducing bacteria (represented by strains BrG1, BrG2, and BrG3), 16S rRNA-targeted oligonucleotide probes were developed. In pure-culture experiments, these probes were shown to be suitable for fluorescent in situ hybridization, as well as for hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns. However, neither enumeration by in situ hybridization nor detection by the DGGE-hybridization approach was feasible with sediment samples. Therefore, the DGGE-hybridization approach was combined with microbiological methods. Freshwater sediment samples from different European locations were used for enrichment cultures and most-probable-number (MPN) determinations. Bacteria with the ability to oxidize ferrous iron under nitrate-reducing conditions were detected in all of the sediment samples investigated. At least one of the previously described types of bacteria was detected in each enrichment culture. MPN studies showed that sediments contained from 1 x 10(5) to 5 x 10(8) ferrous iron-oxidizing, nitrate-reducing bacteria per g (dry weight) of sediment, which accounted for at most 0.8% of the nitrate-reducing bacteria growing with acetate. Type BrG1, BrG2, and BrG3 bacteria accounted for an even smaller fraction (0.2% or less) of the ferrous iron-oxidizing, nitrate-reducing community. The DGGE patterns of MPN cultures suggested that more organisms than those isolated thus far are able to oxidize ferrous iron with nitrate. A comparison showed that among the anoxygenic phototrophic bacteria, organisms that have the ability to oxidize ferrous iron also account for only a minor fraction of the population.

Biofilm dynamics studied with microsensors and molecular techniques

Here we present preliminary data on the development of a biofilm from a wastewater treatment plant studied with microsensors and molecular techniques. The development during biofilm growth of oxygen, sulfide and pH profiles was measured with microsensors. Anoxic zones developed within one week and further increased during the following weeks. However, sulfide production was delayed and was first detected in a six-week-old biofilm. With denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments the sequence of the bacterial community was followed showing an increasing complexity of the biofilm community during development. In a mature biofilm the influence of nitrate on sulfide production was studied by measuring oxygen, sulfide, pH, nitrite and nitrate profiles with microsensors. Sulfide production was detected deeper in the biofilm and in lower concentrations, when nitrate was added to the medium. The DGGE pattern of the mature biofilm showed both differences and similarities with the DGGE pattern of the 12-week-old biofilm. In particular the RNA pattern changed when nitrate was added to the medium, indicating a change in activity of certain strains.

Biofilm dynamics studied with microsensors and molecular techniques

Here we present preliminary data on the development of a biofilm from a wastewater treatment plant studied with microsensors and molecular techniques. The development during biofilm growth of oxygen, sulfide and pH profiles was measured with microsensors. Anoxic zones developed within one week and further increased during the following weeks. However, sulfide production was delayed and was first detected in a six-week-old biofilm. With denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 165 rRNA gene fragments the sequence of the bacterial community was followed showing an increasing complexity of the biofilm community during development. In a mature biofilm the influence of nitrate on sulfide production was studied by measuring oxygen, sulfide, pH, nitrite and nitrate profiles with microsensors. Sulfide production was detected deeper in the biofilm and in lower concentrations, when nitrate was added to the medium. The DGGE pattern of the mature biofilm showed both differences and similarities with the DGGE pattern of the 12-week-old biofilm. In particular the RNA pattern changed when nitrate was added to the medium, indicating a change in activity of certain strains.

Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis of specifically amplified 18s rDNA.

Marram grass (Ammophila arenaria L.), a sand-stabilizing plant species in coastal dune areas, is affected by a specific pathosystem thought to include both plant-pathogenic fungi and nematodes. To study the fungal component of this pathosystem, we developed a method for the cultivation-independent detection and characterization of fungi infecting plant roots based on denaturing gradient gel electrophoresis (DGGE) of specifically amplified DNA fragments coding for 18S rRNA (rDNA). A nested PCR strategy was employed to amplify a 569-bp region of the 18S rRNA gene, with the addition of a 36-bp GC clamp, from fungal isolates, from roots of test plants infected in the laboratory, and from field samples of marram grass roots from both healthy and degenerating stands from coastal dunes in The Netherlands. PCR products from fungal isolates were subjected to DGGE to examine the variation seen both between different fungal taxa and within a single species. DGGE of the 18S rDNA fragments could resolve species differences from fungi used in this study yet was unable to discriminate between strains of a single species. The 18S rRNA genes from 20 isolates of fungal species previously recovered from A. arenaria roots were cloned and partially sequenced to aid in the interpretation of DGGE data. DGGE patterns recovered from laboratory plants showed that this technique could reliably identify known plant-infecting fungi. Amplification products from field A. arenaria roots also were analyzed by DGGE, and the major bands were excised, reamplified, sequenced, and subjected to phylogenetic analysis. Some recovered 18S rDNA sequences allowed for phylogenetic placement to the genus level, whereas other sequences were not closely related to known fungal 18S rDNA sequences. The molecular data presented here reveal fungal diversity not detected in previous culture-based surveys.

Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments

Denaturing gradient gel electrophoresis (DGGE) is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial populations. DGGE was evaluated for the identification of ammonia oxidizers of the beta subdivision of the Proteobacteria based on the mobility of PCR-amplified 16S rDNA fragments and for the analysis of mixtures of PCR products from this group generated by selective PCR of DNA extracted from coastal sand dunes. Degenerate PCR primers, CTO189f-GC and CTO654r, incorporating a 5' GC clamp, were designed to amplify a 465-bp 16S rDNA region spanning the V-2 and V-3 variable domains. The primers were tested against a representative selection of clones and cultures encompassing the currently recognized beta-subdivision ammonia oxidizer 16S rDNA sequence diversity. Analysis of these products by DGGE revealed that while many of the sequences could be separated, some which were known to be different migrated similarly in the denaturant system used. The CTO primer pair was used to amplify 16S rDNA sequences from DNA extracted from soil sampled from Dutch coastal dune locations of differing in pH and distance from the beach. The derived DGGE patterns were reproducible across multiple DNA isolations and PCRs. Ammonia oxidizer-like sequences from different phylogenetic groupings isolated from gene libraries made from the same sand dune DNA samples but prepared with different primers gave DGGE bands which comigrated with most of the bands detected from the sand dune samples. Bands from the DGGE gels of environmental samples were excised, reamplified, and directly sequenced, revealing strong similarity or identity of the recovered products to the corresponding regions of library clones. Six of the seven sequenced clusters of beta-subdivision ammonia oxidizers were detected in the dune systems, and differences in community structure between some sample sites were demonstrated. The most seaward dune site contained sequences showing affinity with sequence clusters previously isolated only from marine environments and was the only site where sequences relate to Nitrosomonas genes could be detected. Nitrosospira-like sequences were present in all sites, and there was some evidence of differences between Nitrosospira populations in acid and alkaline dune soils. Such differences in community structure may affect physiological differences within beta-subdivision ammonia oxidizers, with consequent effects on nitrification rates in response to key environmental factors.

Prenatal Diagnosis of β-Thalassemia Among Indians using Denaturing Gradient Gel Electrophoresis

We have offered first trimester prenatal diagnosis to 55 couples at risk for β-thalassemia, originating from various parts of India, using polymerase chain reaction and denaturing gradient gel electrophoresis. Apart from the six common mutations, codon 30 (CAG→CAA), Cap site +1 (A→C), as well as three uncharacterized mutations were seen among the parents. in the majority of cases, the diagnosis was possible by scanning only one fragment (B) where most of the Indian mutations are situated. in 18 out of 55 cases, framework analysis could also have been used to offer prenatal diagnosis without characterizing the β-thalassemia mutations. in the two cases where the mutations were uncharacterized, prenatal diagnosis was done only on the basis of the anomalous denaturing gradient gel electrophoresis patterns seen in the parents and in previously affected children. This is the first attempt of prenatal analysis using denaturing gradient gel electrophoresis in the extremely diverse Indian population where the profile of mutations has not yet been fully elucidated.

Lack of mutations in K- ras codons 12 and 13 in human atherosclerotic lesions

In the framework of a project investigating the possible involvement of cancer biomarkers in human atherogenesis, we evaluated the occurrence of K- ras mutations in the DNA extracted from smooth muscle cells of abdominal aorta atherosclerotic lesions. The molecular analysis of the DNA from 32 surgical specimens, using PCR-based denaturing gradient gel electrophoresis (DGGE), did not reveal any variant in K- ras codons 12 and 13, which are the most frequently involved codons among the ras genes mutated in various types of human tumors. Analysis of the DNA extracted from four cell lines carrying known K- ras mutational alleles showed typically positive DGGE patterns. Thus, on the whole, the conclusions of this study and of previous studies using the same biological material are consistent with the occurrence of DNA adducts in human atherosclerotic lesions but in the absence of p53 involvement or of K- ras mutations in codons 12 and 13. The search for candidate genes which may possibly be involved in the atherogenetic process warrants further studies.

Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments.

The sulfate-reducing bacterial populations of a stratified marine water column, Mariager Fjord, Denmark, were investigated by molecular and culture-dependent approaches in parallel. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA and DNA encoding rRNA (rDNA) isolated from the water column indicated specific bacterial populations in different water column layers and revealed a highly differentiated pattern of rRNA- and rDNA-derived PCR amplificates, probably reflecting active and resting bacterial populations. Hybridization of DGGE patterns with rRNA probes indicated the increased presence and activity (by at least 1 order of magnitude) of sulfate-reducing bacteria within and below the chemocline. Parallel to this molecular approach, an approach involving most-probable-number (MPN) counts was used, and it found a similar distribution of cultivable sulfate-reducing bacteria in the water column of Mariager Fjord, Approximately 25 cells and 250 cells per ml above and below the chemocline, respectively, were found. Desulfovibrio- and Desulfobulbus-related strains occurred in the oxic zone. DGGE bands from MPN cultures were sequenced and compared with those obtained from nucleic acids extracted from water column samples. The MPN isolates were phylogenetically affiliated with sulfate-reducing delta subdivision proteobacteria (members of the genera Desulfovibrio, Desulfobulbus, and Desulfobacter), whereas the molecular isolates constituted an independent lineage of the delta subdivision proteobacteria. DGGE of PCR-amplified nucleic acids with general eubacterial PCR primers conceptually revealed the general bacterial population, whereas the use of culture media allowed cultivable sulfate-reducing bacteria to be selected. A parallel study of Mariager Fjord biogeochemistry, bacterial activity, and bacterial counts complementing this investigation has been presented elsewhere (N.B. Ramsing, H. Fossing, T. G. Ferdelman, F. Andersen, and B. Thamdrup, Appl. Environ.

Phenotypic presentation of the FH-Cincinnati type 5 low density lipoprotein receptor mutation.

Familial hypercholesterolaemia (FH) is an autosomal dominant hereditary disease of lipid metabolism that in most families is caused by mutations in the low density lipoprotein receptor (LDLR) gene. Though more than 150 mutations are known, the clinical picture associated with most of these is not known. Genetic FH diagnosis may soon become routine in the setting of genetic counselling, and therefore thorough information on the phenotype-genotype relationship of different mutations is now important. In this study, index patients from each of 14 Danish FH families were screened for mutations in exon 2 of the LDLR gene using a denaturing gradient gel electrophoresis (DGGE)-based mutation screening assay. A deviating DGGE pattern identified two index patients, where subsequent sequencing revealed heterozygosity for the FH Cincinnati type 5 Trp23-to-Stop LDLR mutation. Data from three generations of the families allowed the first clinical and biochemical description of this mutation. Evidence that genetic analysis adds independent diagnostic information compared to traditional clinical/biochemical FH diagnosis was documented by demonstrating the presence of the FH Cincinnati mutation in a family member with a completely normal lipid profile. By comparison to non-FH family members, it was documented that carrier status for the FH Cincinnati mutation is associated with a significant risk of cardiovascular disease. Thus, genetic analysis may improve diagnostic precision and help to define more precisely which of the members of FH families are in need of preventive interventions and may aid in establishing phenotype-genotype relationships allowing more refined genetic counselling in FH.

Mutations in the low density lipoprotein receptor gene of familial hypercholesterolemic patients detected by denaturing gradient gel electrophoresis and direct sequencing.

Familial hypercholesterolemia (FH) results from mutations in the low density lipoprotein receptor (LDLR) gene. We applied denaturing gradient gel electrophoresis (DGGE) to screen for sequence variations in the coding and splice site consensus sequences of the LDLR gene. For amplification of each exon by the polymerase chain reaction (PCR), optimal pairs of primers were designed by the MELT 87 computer algorithm. To increase the sensitivity, an artificial GC-clamp was included in either the 5'- or the 3'-end of each fragment. DGGE screening of 32 apparently unrelated heterozygous FH patients revealed 16 unique different aberrant DGGE patterns in 27 patients, while in a group of 32 normal subjects none of these DGGE patterns could be observed, suggesting that the aberrant patterns represent disease-causing mutations. Interestingly, 16 out of 27 patients showed an aberrant DGGE pattern in the part of the gene encoding the ligand binding domain (exons 2-6). Direct solid-phase sequencing of the corresponding exon-specific PCR products revealed the nature of the mutations: three nonsense, four splicing, two frameshift, one silent, and six missense mutations. Six of the mutations have been previously reported, while ten are novel mutations. These results indicate that DGGE provides a reliable method for the detection of the presence of point mutations in the LDLR gene of FH patients, thereby facilitating the introduction of rapid DNA diagnosis for this common and genetically heterogeneous disorder.

Genetic diagnosis with the denaturing gradient gel electrophoresis technique improves diagnostic precision in familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal dominant inherited disorder of lipid metabolism caused by mutations in the LDL receptor gene. FH is characterized clinically by elevated LDL cholesterol level and premature coronary disease. Diagnosing FH on clinical grounds may be difficult, and previous genetic methods are too cumbersome for routine use except in the few populations with FH-founder mutations. A simple mutation screening technique based on denaturing gradient gel electrophoresis (DGGE) has been highly useful in detecting mutations in other genes, and in the present study we evaluated the diagnostic potential of this method for the diagnosis of FH. Conditions for screening exon 3 of the LDL receptor gene using the DGGE technique were established and 14 Danish FH families were examined. An index patient from 1 family had an abnormal DGGE pattern; consequently, an examination of exon 3 of the LDL receptor gene in 21 members of this patient's family was done. The DGGE pattern was seen only in patients with a definite clinical diagnosis of FH. Subsequent sequencing of exon 3 of the LDL receptor gene in these individuals revealed the presence of the French-Canadian type 4 Trp66-Gly mutation. However, in 4 of 11 cases in which a definite clinical diagnosis of FH had been made, the inheritance of the French-Canadian type 4 mutation could be rejected on the basis of genetic analysis. Introduction of a simple genetic analysis based on DGGE may improve the precision of diagnosis in FH families.

Mutation screening of the codon 3500 region of the apolipoprotein B gene by denaturing gradient-gel electrophoresis

Familial defective apolipoprotein B (FDB) is a clinical condition resembling familial hypercholesterolemia. The underlying genetic defects are mutations in the apolipoprotein B-100 (apo B-100) gene. Two mutations (Arg3500 --> Gln and Arg3531 --> Cys) are known to date. We designed a denaturing gradient-gel electrophoresis (DGGE) technique to detect sequence variations in codons 3456-3553 of the apo B-100 gene. In 46 heterozygous FDB patients with the predominant codon 3500 mutation, a uniform four-band DGGE pattern was seen, whereas 57 non-FDB patients showed the uniform single-band pattern expected in normal homozygotes. The recently described codon 3531 mutation and a previously unpublished Arg --> Pro mutation in codon 3480 showed unique DGGE patterns, allowing unambiguous differentiation of the three mutations. The DGGE method thus both detects known FDB mutations and screens for other mutations in codons 3456-3553 of the low-density lipoprotein receptor binding region of apo B-100; it can be used as a first-line screening method for FDB.

Detection of a single base deletion in codon 424 of the low density lipoprotein receptor gene in a Danish family with familial hypercholesterolemia

We performed a screening of exon 9 of the low density lipoprotein receptor (LDLR) gene in 14 Danish families with familial hypercholesterolemia (FH) using the denaturing gradient gel electrophoresis (DGGE) technique. In one of the probands from these families an abnormal band pattern in the gradient gel was detected. Subsequent DGGE analysis of the family of this index patient revealed that the DGGE pattern cosegregated with the disease in this family. Sequencing of the exon showed a deletion of a C in codon 424 of the LDLR gene resulting in a frame shift with the introduction of a stop codon 5 codons further downstream. The mutation is referred to as FH-Odense. The predicted truncated receptor protein consists of the 428 amino terminal amino acids. Consequently, the cytosolic and membrane spanning parts of the mature LDL receptor, which normally secure the receptor in the plasma membrane, are missing. The FH-Odense mutation results in severe premature coronary atherosclerosis as shown by the clinical expression in 5 generations of the affected family.

Defects in type IIA von Willebrand disease: a cysteine 509 to arginine substitution in the mature von Willebrand factor disrupts a disulphide loop involved in the interaction with platelet glycoprotein Ib‐IX

Type IIA von Willebrand disease (vWD) is characterized by the loss of high and intermediate weight multimers of von Willebrand factor (vWF) from plasma. The 3' end of exon 28 in the vWF gene from four type IIA vWD patients was amplified by the polymerase chain reaction, cloned and sequenced. Sequencing identified two potential missense mutations resulting in the amino acid substitutions Arg 834-->Gln and Glu 875-->Lys in the mature vWF subunit within an area of vWF where mutations in type IIA vWD have been reported. Neither of these amino acid substitutions was found in over 100 normal alleles tested by allele specific oligonucleotide hybridization. A polymorphism (Val 802-->Leu) was identified in another patient. Other areas of exon 28 were analysed by denaturing gradient gel electrophoresis (DGGE) and DNA from one patient demonstrated an irregular DGGE pattern on the 5' end of the exon. Sequencing demonstrated an amino acid substitution of an arginine for cysteine at position 509 adjacent to an area of vWF where defects associated with type IIB vWD have been found. This substitution was not found in 100 normal chromosomes tested by restriction enzyme digestion. The Cys 509-->Arg substitution eliminates an intramolecular disulphide bridge formed by Cys 509 and Cys 695 which is important to maintain the configuration of vWF functional domains that interact with platelet glycoprotein Ib-IX.

Impact of Sublethal Oil and Emulsion Exposure on the Reproductive Success of Leach's Storm-Petrels: Short and Long-Term Effects

To investigate the effects of different forest stands in subtropical China on the communities of soil ammonia-oxidizing microorganisms, we characterized the abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB), and the community structure of AOA in soils under stands of broad-leaved (BF) , Chinese fir (CF) , Pinus massoniana (PF) and moso bamboo (MB) forests using real-time quantitative PCR and denaturing gradient gel electrophoresis (DGGE). The results showed that the AOA gene copy numbers (1.62 x 10(6)-1.88 x 10(7) per gram of dry soil) were significantly higher than those of AOB genes (2.41 x 10(5)-4.36 x 10(5) per gram of dry soil). Significantly higher soil AOA abundance was detected in the MB than that in the CF (P < 0.05), and the latter was significantly higher than that in the BF and PF soils (P < 0.05). There were no significant differences in the soil AOB abundance among the four forest stands. As indicated by DGGE pattern, soil AOA species varied among the four forest stands. There was a difference in the soil AOA communities between the CF and MB stands. The AOA demonstrated a competitive advantage over the AOB in the soils under these major subtropical forests. Soil pH, concentrations of soil available potassium and organic carbon as well as the forest type were the main factors that influence the variation of AOA community structure and diversity.