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Length Polymorphism Analysis (length + polymorphism_analysis)
Kinds of Length Polymorphism Analysis Selected AbstractsEvolutionary Relationships of the Dutch Elm Disease Fungus Ophiostoma novo-ulmi to Other Ophiostoma Species Investigated by Restriction Fragment Length Polymorphism Analysis of the rDNA RegionJOURNAL OF PHYTOPATHOLOGY, Issue 9-10 2000N. D. Pipe Restriction fragment length polymorphisms (RFLPs) in the ribosomal RNA gene (rDNA) region were used to assess relationships between the Dutch elm disease fungi Ophiostoma novo-ulmi and Ophiostoma ulmi, the recently described Himalayan Dutch elm disease pathogen, Ophiostoma himal-ulmi, the morphologically similar sapstain fungi, Ophiostoma piceae and Ophiostoma quercus, and several Ophiostoma species from hardwood trees, including Ophiostoma stenoceras and Ophiostoma proliferum. A distance matrix and cluster analysis indicated that the rDNA region of O. himal-ulmi is more closely related to those of O. novo-ulmi and O. ulmi than to those of O. piceae and O. quercus and is more distantly related to O. stenoceras and the other Ophiostoma species, which formed a separate clade. The rDNA region of O. quercus was found to be at least as closely related to that of O. novo-ulmi and O. ulmi as it is to that of O. piceae. The implications of these results for the evolution of the Dutch elm disease fungi are discussed. [source] Characterization of Phytoplasmas Associated With Almond Diseases in IranJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2009L. Zirak Abstract In recent years, almond witches'-broom disease has been prevalent in almond growing areas in the centre and south of Iran. Furthermore, almond trees showing different symptoms of phytoplasma diseases such as little leaf, leaf rolling, dieback of branches, rosette and yellowing were observed in the central regions of Iran. DNA isolated from symptomatic almond trees was used to amplify 16S rDNA and 16S-23S rDNA intergenic spacer (IS) fragments by nested polymerase chain reaction (PCR) using phytoplasma universal primer pairs (P1/P7, R16F2/R2, PA2F/R and NPA2F/R). Phytoplasmas were detected in symptomatic almonds in two major almond-growing regions, Isfahan and ChaharMahal-O-Bakhtiari. Restriction fragment length polymorphism analyses of nested PCR products using endonuclease enzymes HpaII and TaqI revealed that phytoplasmas associated with infected almonds are genetically different. Sequence analyses of amplified fragments of 16S rDNA and IS region indicated that the almond phytoplasmas in Iran are closely related to ,Candidatus (Ca.) Phytoplasma aurantifolia', ,Ca. Phytoplasma phoenicium', ,Ca. Phytoplasma solani' and ,Ca. Phytoplasma trifolii'. The phytoplasmas related to ,Ca. Phytoplasma aurantifolia' were more prevalent than other phytoplasmas in the central regions of Iran. [source] Occurrence, Symptom Expression and Characterization of Phytoplasma Associated with Pear Decline Disease in Catalonia (Spain)JOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2003M. Garcia-Chapa Abstract A total area of 1500 ha of commercial plots was surveyed to study the extent of pear decline disease and its relative importance in northeastern Spain. A preliminary evaluation indicated that around 7% of the plots had symptoms of the disease. At the same time, pear decline incidence was evaluated in 45 plots, by visual inspection of 500 trees in each plot. In September, the incidence of trees with symptoms ranged from 8 to 59% depending on the cultivar selected. The presence of pear decline (PD) phytoplasma in these plots was confirmed by polymerase chain reaction (PCR) amplification of phytoplasma DNA with universal or group-specific primers. Restriction fragment length polymorphism analyses also showed the presence of a unique phytoplasma strain. The symptom expression of PD disease in different cultivars was evaluated throughout the year. The relationship between the presence of symptoms and detection of PD by PCR in these cultivars was also studied. Results showed that the nested-PCR, using specific primers to detect the DNA from PD phytoplasma, is the most accurate method to identify the total percentage of affected trees. [source] Distinct C-terminus of the B subunit of factor XIII in a population-associated major phenotype: the first case of complete allele-specific alternative splicing products in the coagulation and fibrinolytic systemsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2009H. IWATA Summary.,Objectives: The purpose of this study was to elucidate the molecular bases of the heterogeneity of the B subunit of coagulation factor XIII (FXIII-B), classified by isoelectric focusing into its three population-associated major phenotypes. Methods and Results: By genetic sequencing and polymerase chain reaction (PCR),restriction fragment length polymorphism analyses, a C-to-G change was identified in intron K for the Asian-associated major phenotype FXIII-B*3. A transcript containing the novel exon XII, was detected by reverse transcription PCR using hepatocyte cell lines with this allele. The exclusive existence of a novel C-terminal peptide in a homozygote of FXIII-B*3 was also detected by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The FXIII-B*3 isoform had a C-terminus 15 residues longer than the other isoforms, containing two additional basic amino acids and one extra acidic amino acid. Accordingly, the C-to-G nucleotide substitution created an efficient splice acceptor AG dinucleotide, which resulted in allele-specific alternative splicing in intron K. When compared with FXIII-B*1, the third major phenotype, FXIII-B*2, had an A-to-G change in exon III, converting His95 to Arg, and a rare phenotype, FXIII-B*4, had an A-to-T change in exon VII, converting Glu368 to Val. Conclusions: We found an extremely rare event of complete allele-specific alternative splicing for FXIII-B. The FXIII-B*3 isoform had a distinct C-terminal peptide, while the FXIII-B*2 and FXIII-B*4 isoforms had His95 to Arg and Glu368 to Val substitutions, respectively, which led to differential isoelectric points of these isoforms. Such variations in the amino acid sequence of FXIII-B may have profound effects on its structure,function relationship, plasma FXIII levels, and disease susceptibility. [source] Faecal microbiota profile of Crohn's disease determined by terminal restriction fragment length polymorphism analysisALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 1 2009A. ANDOH Summary Background, Terminal restriction fragment length polymorphism (T-RFLP) analyses are powerful tools to assess the diversity of complex microbiota. T-RFLPs permit rapid comparisons of microbiota from many samples. Aim, To perform T-RFLP analyses of faecal microbiota in Crohn's disease (CD) patients to investigate potential alterations in faecal microbial communities and furthermore to analyse the effects of elemental diet on faecal microbiota profiles. Methods, Thirty-four patients with CD and 30 healthy individuals were enrolled in the study. DNA was extracted from stool samples and 16S rRNA genes were amplified by PCR. PCR products were digested with BslI restriction enzymes and T-RF lengths were determined. Results, Faecal microbial communities were classified into seven clusters. Almost all healthy individuals (28/30) were included in cluster I, II and III, but the majority of CD patients (25/34) could be divided into another four clusters (cluster IV,VII). Prediction of bacteria based on the BslI-digested T-RFLP database showed a significant decrease in Clostridium cluster IV, Clostridium cluster XI and subcluster XIVa in CD patients. In contrast, Bacteroides significantly increased in CD patients. Significant increases in Enterobacteriales were also observed in CD patients. Furthermore, elemental diets modulated faecal bacterial communities in CD patients. Conclusions, Terminal restriction fragment length polymorphism analyses showed that the diversity of faecal microbiota in patients with CD differed from that of healthy individuals. Furthermore, elemental diets modulated faecal microbiota composition, and this effect may be involved in mechanisms of clinical effects of elemental diet. [source] Genetic drift outweighs balancing selection in shaping post-bottleneck major histocompatibility complex variation in New Zealand robins (Petroicidae)MOLECULAR ECOLOGY, Issue 12 2004HILARY C. MILLER Abstract The Chatham Island black robin, Petroica traversi, is a highly inbred, endangered passerine with extremely low levels of variation at hypervariable neutral DNA markers. In this study we investigated variation in major histocompatibility complex (MHC) class II genes in both the black robin and its nonendangered relative, the South Island robin Petroica australis australis. Previous studies have shown that Petroica have at least four expressed class II B MHC genes. In this study, the sequences of introns flanking exon 2 of these loci were characterized to design primers for peptide-binding region (PBR) sequence analysis. Intron sequences were comprised of varying numbers of repeated units, with highly conserved regions immediately flanking exon 2. Polymerase chain reaction primers designed to this region amplified three or four sequences per black robin individual, and eight to 14 sequences per South Island robin individual. MHC genes are fitness-related genes thought to be under balancing selection, so they may be more likely to retain variation in bottlenecked populations. To test this, we compared MHC variation in the black robin with artificially bottlenecked populations of South Island robin, and with their respective source populations, using restriction fragment length polymorphism analyses and DNA sequencing of the PBR. Our results indicate that the black robin is monomorphic at class II B MHC loci, while both source and bottlenecked populations of South Island robin have retained moderate levels of variation. Comparison of MHC variation with minisatellite DNA variation indicates that genetic drift outweighs balancing selection in determining MHC diversity in the bottlenecked populations. However, balancing selection appears to influence MHC diversity over evolutionary timescales, and the effects of gene conversion are evident. [source] CYP2D6 gene deletion allele in patients with neuroleptic malignant syndrome: Preliminary reportPSYCHIATRY AND CLINICAL NEUROSCIENCES, Issue 4 2005DAIJI KATO md Abstract, Neuroleptic malignant syndrome (NMS) is a potentially fatal adverse reaction to psychopharmacologic treatment. Reported herein are two NMS patients with schizophrenia who were found to possess a CYP2D6 gene deletion allele (CYP2D6*5). The deletion results in decreased CYP2D6 activity, possibly leading to drug accumulation. Both patients with NMS had been treated with neuroleptics, including CYP2D6 substrates. Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analyses and long PCR were performed to detect CYP2D6 genotype. One patient was found to possess *5/*10; the other had a *1/*5 genotype. The present preliminary report suggests that pharmacokinetic factors cannot be excluded and the CYP2D6 polymorphism is possibly associated with the etiology of NMS. [source] Use of microsatellite DNA and amplified fragment length polymorphism for Cherry salmon (Oncorhynchus masou) complex identificationAQUACULTURE RESEARCH, Issue 9 2010Te-Hua Hsu Abstract Formosa landlocked salmon (Oncorhynchus masou formosanus), an endemic, critically endangered subspecies of Cherry salmon (Oncorhynchus masou) complex, is only found in Taiwan. Because the eyed eggs and ungutted carcasses of Pacific salmons (genus Oncorhynchus) are imported for aquaculture and food to Taiwan from overseas every year, the requirement for preventing illegal trade or accidental commercial imports to avoid unwanted fish from contaminating the gene pool of Formosa landlocked salmon and infect them with diseases is critical for the conservation of Formosa landlocked salmon. Traditional morphology-based species identification is impossible for salmon eggs and larvae that lack clearly defined morphological features. In the present study, the genetic differences among four subspecies (Oncorhynchus masou ishikawae, O. masou subsp., Oncorhynchus masou masou and O. masou formosanus) of Cherry salmon complex were determined from microsatellite DNA and amplified fragment length polymorphism analyses. We successfully generated a genetic marker to aid traditional taxonomy and investigate the integrity of the current taxonomic status among members of Cherry salmon complex. Use of molecular markers, in combination with traditional morphological identification, is a promising tool for identifying four closely related subspecies of Cherry salmon complex. [source] Human Papillomavirus and Overexpression of P16INK4a in Nonmelanoma Skin CancerDERMATOLOGIC SURGERY, Issue 3 2004Ingo Nindl PhD Background. P16INK4a overexpression has been identified as a specific biomarker in high-risk human papillomavirus (HPV),infected cervical (pre)cancer lesions. Objective. To evaluate the overexpression of this cyclin-dependent kinase inhibitor in skin tumors depending on HPV infections, we analyzed normal skin, benign skin disease, and skin cancer specimens. Methods. Biopsies of 23 patients with normal histology (3), psoriasis (2), verrucae vulgaris (2), actinic keratoses (5), squamous cell carcinoma (SCC) in situ (3), Bowen's carcinoma (1), and SCC (7) were analyzed. Specimens of 23 patients were immunostained using the monoclonal antibody E6H4 specific for p16INK4a. HPV status was assessed by a polymerase chain reaction (PCR) system to detect all currently known HPV types. MY (MY09/MY11 and MYN9/MYN10)-, CP (CP65/CP70 and CP66/CP69)-nested PCR, and three single PCR methods CN1, CN3, and CN4 were used in a first step, and HPV typing was performed by restriction fragment length polymorphism analysis. Only ,-globin,positive patients were included in this study. Results. HPV DNA was detected in all actinic keratoses, SCC in situ, Bowen's carcinoma, and SCC, in 50% (one of two) of verrucae vulgaris, in 66% (two of three) of normal skin, and in none of two psoriasis. P16INK4a expression was not detected in normal skin, psoriasis, and verrucae vulgares. Overexpression of p16INK4a was detected in a subset of dysplastic cells (10% to 80%) of all skin (pre)cancer lesions such as actinic keratoses, SCC in situ, Bowen's carcinoma, and SCC infected with HPV independent of sun exposure. Conclusion. P16INK4a appears to be overexpressed in a portion of dysplastic cells from actinic keratoses and SCC. Further studies to examine the association of HPV infection and the overexpression of p16INK4a are warranted. [source] The p73 polymorphisms are not associated with susceptibility to esophageal squamous cell carcinoma in a high incidence region of ChinaDISEASES OF THE ESOPHAGUS, Issue 4 2007H. Ge SUMMARY., P73, a p53 homolog, has some p53-like activities and plays an important role in modulating cell cycle, apoptosis and DNA repair. The two linked polymorphisms in the non-coding region of exon2 of p73 gene, named G4C14-A4T14, may alter translation efficiency of the gene. The transcription of p73 gene is initiated by three promoters, termed P1-P3. There is a single nucleotide substitution (,386G/A) in the P3 promoter region with unknown function. To test the hypothesis that the genetic variations in the exon2 and P3 promoter play a role in the etiology of esophageal squamous cell carcinoma (ESCC), we conducted a population-based case-control study in 348 ESCC patients and 583 healthy controls from a high incidence region of Hebei province, China. The p73 polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP). The results showed that the family history of upper gastrointestinal cancer (UGIC) significantly increased the risk of developing ESCC (the age, sex and smoking status adjusted OR = 2.02, 95% CI = 1.54,2.67). The overall distribution of the p73 genotype, allelotype and haplotype in cancer patients and controls were not significantly different (all P -values are above 0.05). Stratification analysis by smoking status and family history of UGIC also did not show the significant influence of the polymorphisms on the risk of ESCC development. The results suggested that the p73 exon2 G4C14-A4T14 and P3 promoter ,386G/A polymorphisms might not be used as potential markers to predicate the risk of ESCC development in northern China. [source] Cover Picture: Electrophoresis 19'2008ELECTROPHORESIS, Issue 19 2008Article first published online: 28 OCT 200 This issue has a dual emphasis: "APCE 2007" and "Fundamentals and Methodologies" with the aim of providing the readers of the Journal with the latest developments in the field. APCE 2007, which was held in Singapore, December 17 , 19, 2007, is the premier forum in the Asia-Pacific countries for communicating advances in capillary- and chip-based electroseparation techniques and their applications to genomics, proteomics, and chemical and biochemical analysis. The emphasis part on APCE 2007 is a "mini proceeding" that groups 7 representative research articles, which deal with microchip electrophoresis, restriction fragment length polymorphism analysis by CE, gradient MEKC, stacking and sweeping in CE, in-line pre-concentration in CE, and food and drug analysis by CE. In addition, issue 19 has a "Fast Track" article on the principles for different modes of multiple-injection CZE and provides equations that facilitate the transfer from single-injection CZE to one or more suitable modes of multiple injection CZE. [source] Bacteria associated with the rapid tissue necrosis of stony coralsENVIRONMENTAL MICROBIOLOGY, Issue 7 2007G. M. Luna Summary The rapid tissue necrosis (RTN) is a common disease of both wild and captive stony corals, which causes a fast tissue degradation (peeling) and death of the colony. Here we report the results of an investigation carried out on the stony coral Pocillopora damicornis, affected by an RTN-like disease. Total abundance of prokaryotes in tissue samples, determined by epifluorescence microscopy, was significantly higher in diseased than in healthy corals, as well as bacterial counts on MB2216 agar plates. Further experiments performed by fluorescent in situ hybridization using a 16S rDNA Vibrio -specific probe showed that vibrios were significantly more abundant in diseased than in healthy corals. Accordingly, bacterial counts on TCBS agar plates were higher in diseased than in healthy tissues. 16S rDNA sequencing identified as Vibrio colonies from diseased tissues only. Cultivated vibrios were dominated by a single ribotype, which displayed 99% of similarity with Vibrio harveyi strain LB4. Bacterial ribotype richness, assessed by terminal-restriction fragment length polymorphism analysis of the 16S rDNA, was significantly higher in diseased than in healthy corals. Using an in silico software, we estimated that a single terminal restriction fragment, putatively assigned to a Vibrio sp., accounted for >,15% and < 5% of the total bacterial assemblage, in diseased and healthy corals respectively. These results let us hypothesize that the RTN in stony corals can be an infectious disease associated to the presence of Vibrio harveyi. However, further studies are needed to validate the microbial origin of this pathology. [source] Methanogenesis and methanogenic pathways in a peat from subarctic permafrostENVIRONMENTAL MICROBIOLOGY, Issue 4 2007Martina Metje Summary Few studies have dealt so far with methanogenic pathways and populations in subarctic and arctic soils. We studied the effects of temperature on rates and pathways of CH4 production and on the relative abundance and structure of the archaeal community in a mildly acidic peat from a permafrost region in Siberia (67°N). We monitored the production of CH4 and CO2 over time and measured the consumption of Fe(II), ethanol and volatile fatty acids. All experiments were performed with and without specific inhibitors [2-bromoethanesulfonate (BES) for methanogenesis and CH3F for acetoclastic methanogenesis]. The optimum temperature for methanogenesis was between 26°C and 28°C [4.3 ,mol CH4 (g dry weight),1 day,1], but the activity was high even at 4°C [0.75 ,mol CH4 (g dry weight),1 day,1], constituting 17% of that at 27°C. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Acetoclastic methanogenesis accounted for about 70% of the total methanogenesis. Most 16S rRNA gene sequences clustered with Methanosarcinales, correlating with the prevalence of acetoclastic methanogenesis. In addition, sequences clustering with Methanobacteriales were recovered. Fe reduction occurred in parallel to methanogenesis. At lower and higher temperatures Fe reduction was not affected by BES. Because butyrate was consumed during methanogenesis and accumulated when methanogenesis was inhibited (BES and CH3F), it is proposed to serve as methanogenic precursor, providing acetate and H2 by syntrophic oxidation. In addition, ethanol and caproate occurred as intermediates. Because of thermodynamic constraints, homoacetogenesis could not compete with hydrogenotrophic methanogenesis. [source] The 3020insC mutation of the NOD2/CARD15 gene in patients with periodontal diseaseEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 4 2004Matthias Folwaczny The 3020insC mutation of the NOD2/CARD15 gene leads to impaired activation of nuclear factor-kappa B (NF- ,B) in vitro. As the destruction of periodontal tissue is mediated via activation of NF- ,B, with subsequent transcription of proinflammatory cytokines, the c-insertion mutation of the NOD2/CARD15 gene might contribute to the proposed genetic background of periodontitis. The present study analysed the frequency of this mutation in 80 patients with chronic periodontal disease and 122 healthy controls. The 3020insC mutation was identified by employing the polymerase chain reaction followed by restriction fragment length polymorphism analysis. The prevalence of the 3020insC mutation of the NOD2/CARD15 protein in patients with periodontitis was 1.9% (three of 160) and that for the control group was 2.0% (five of 244) (P = 0.942). Hence, unlike in Crohn's disease, the 3020insC mutation of the NOD2/CARD15 gene does not seem to influence the pathophysiology of periodontitis. [source] Influence of arbuscular mycorrhizal mycelial exudates on soil bacterial growth and community structureFEMS MICROBIOLOGY ECOLOGY, Issue 2 2007Jonas F. Toljander Abstract Plant root systems colonized by arbuscular mycorrhizal (AM) fungi have previously been shown to influence soil bacterial populations; however, the direct influence of the AM extraradical mycelium itself on bacterial growth and community composition is not well understood. In this study, we investigated the effects of exudates produced by AM extraradical mycelia on the growth and development of an extracted soil bacterial community in vitro. The chemical composition of the mycelial exudates was analysed using proton nuclear magnetic resonance spectrometry. Following the addition of exudates to a bacterial community extracted from soil, bacterial growth and vitality were determined using a bacterial vitality stain and fluorescence microscopy. Changes in community composition were also analysed at various times over the course of 3 days by terminal restriction fragment length polymorphism analysis, in combination with cloning and sequencing of 16S rRNA genes. Mycelial exudates increased bacterial growth and vitality and changed bacterial community composition. Several Gammaproteobacteria, including a taxon within the Enterobacteriaceae, increased in frequency of occurrence in response to AM mycelial exudates. This study is the first attempt to identify carbohydrates from the extraradical mycelium of an AM fungus, and demonstrates the direct effects of mycelial exudates on a soil bacterial community. [source] High vertical and low horizontal diversity of Prochlorococcus ecotypes in the Mediterranean Sea in summerFEMS MICROBIOLOGY ECOLOGY, Issue 2 2007Laurence Garczarek Abstract Natural populations of the marine cyanobacterium Prochlorococcus exist as two main ecotypes, inhabiting different layers of the ocean's photic zone. These so-called high light- (HL-) and low light (LL-) adapted ecotypes are both physiologically and genetically distinct. HL strains can be separated into two major clades (HLI and HLII), whereas LL strains are more diverse. Here, we used several molecular techniques to study the genetic diversity of natural Prochlorococcus populations during the Prosope cruise in the Mediterranean Sea in the summer of 1999. Using a dot blot hybridization technique, we found that HLI was the dominant HL group and was confined to the upper mixed layer. In contrast, LL ecotypes were only found below the thermocline. Secondly, a restriction fragment length polymorphism analysis of PCR-amplified pcb genes (encoding the major light-harvesting proteins of Prochlorococcus) suggested that there were at least four genetically different ecotypes, occupying distinct but overlapping light niches in the photic zone. At comparable depths, similar banding patterns were observed throughout the sampled area, suggesting a horizontal homogenization of ecotypes. Nevertheless, environmental pcb gene sequences retrieved from different depths at two stations proved all different at the nucleotide level, suggesting a large genetic microdiversity within those ecotypes. [source] Cosmopolitan distribution of phlD -containing dicotyledonous crop-associated biocontrol pseudomonads of worldwide originFEMS MICROBIOLOGY ECOLOGY, Issue 2 2001Chunxia Wang Abstract In biocontrol fluorescent pseudomonads, phlD encodes a polyketide synthase required for the synthesis of the antifungal compound 2,4-diacetylphloroglucinol (Phl). Here, PCR-restriction fragment length polymorphism analysis was used to compare phlD alleles in 77 dicot-associated pseudomonads originating from various countries worldwide and 10 counterparts from a monocotyledonous host (wheat). The 16 restriction patterns obtained were mostly unrelated to geographic location or dicot host. Cluster analysis distinguished eight phlD clusters at a similarity level of 0.63. One cluster grouped 18 pseudomonads that produced also the antifungal polyketide pyoluteorin but could not assimilate D -galactose, D -galactonate lactone, D -sorbitol, L -arabinose, D -saccharate or D -xylose. These 18 pseudomonads, along with the eight pseudomonads from a second phlD cluster, were the only isolates that failed to deaminase 1-aminocyclopropane-1-carboxylate (ACC), a rare root growth promotion trait. Overall, assessment of phlD polymorphism, ACC deaminase activity and catabolic profiles pointed to a cosmopolitan distribution of Phl-producing biocontrol fluorescent pseudomonads of worldwide origin associated with dicotyledonous crop plants. [source] Structure and diversity of Gram-negative sulfate-reducing bacteria on rice rootsFEMS MICROBIOLOGY ECOLOGY, Issue 2-3 2001Daniel Scheid Abstract Specific PCR assays were used to amplify the 16S rRNA genes of the Desulfobacteriaceae and the Desulfovibrionaceae from extracted environmental DNA from rice roots. 16S rDNA-based community patterns of the Desulfobacteriaceae were generated via terminal restriction fragment length polymorphism analysis from rice roots and compared with bulk soil. The molecular fingerprints showed no significant difference between rice roots and bulk soil, but changes during the vegetation period. 16S rDNA clone libraries and sequencing showed that the predominant terminal restriction fragments represented distinct phylogenetic groups. The 16S rDNA clone sequences of the Desulfobacteriaceae fell in the phylogenetic radiation of Desulfonema and Desulfosarcina or grouped within the Desulforhabdus,Syntrophobacter assemblage. Three of the latter sequences were closely affiliated with the MPN isolate EZ-2C2 from rice roots. All Desulfovibrionaceae 16S rDNA clone sequences, with one exception, were affiliated with the MPN isolate F1-7b from rice roots. The clustering of the clone sequences and the close phylogenetic affiliation with isolates from MPN enrichments from the same habitat in two cases indicated that these sequence clusters may represent predominant Gram-negative sulfate reducers on rice roots. Quantification of the bacterial abundances was accomplished by rRNA dot blot hybridization. In total the Gram-negative sulfate reducers accounted for approximately 2,3% of the total rRNA content. The relative rRNA abundance of the Desulfobacteriaceae was, at 1.4%, higher than that of the Desulfovibrionaceae (0.5%). [source] Molecular epidemiology of clinical and environmental isolates of the Cryptococcus neoformans species complex reveals a high genetic diversity and the presence of the molecular type VGII mating type a in ColombiaFEMS YEAST RESEARCH, Issue 4 2006Patricia Escandón Abstract The aim of this study was to investigate the epidemiological relationships of clinical and environmental isolates of the Cryptococcus neoformans species complex in Colombia. The current study reflects data from 1987 to 2004. In Colombia serotypes A and B are most frequently recovered from patients and the environment. Of the 178 clinical isolates studied, 91.1% were of serotype A, 8.4% serotype B and 0.5% serotype C. Of the 247 environmental isolates, 44.2% were of serotype A, 42.6% serotype B and 13.2% serotype C. No serotype D isolates were isolated. Serotype AD has not been recovered in Colombia. PCR fingerprinting with the primers M13, (GACA)4 and (GTG)5 and URA5 gene restriction fragment length polymorphism analysis grouped the majority of clinical serotype A and environmental serotype B isolates into the molecular types VNI (98.1%) and VGII (100%), respectively. Mating type , was determined in 99.3% of serotype A isolates, but 96.6% of serotype B isolates were of mating type a. Similar profiles between clinical and environmental isolates suggest that the patients may have acquired the infection from the environment. The data presented form part of the Colombian contribution to the ongoing global survey of the C. neoformans species complex. [source] Evidence for multiple interspecific hybridization in Saccharomyces sensu stricto speciesFEMS YEAST RESEARCH, Issue 4 2002Miguel de Barros Lopes Abstract Fluorescent amplified fragment length polymorphism analysis demonstrates a high level of gene exchange between Saccharomyces sensu stricto species, with some strains having undergone multiple interspecific hybridization events with subsequent changes in genome complexity. Two lager strains were shown to be hybrids between Saccharomyces cerevisiae and the alloploid species Saccharomyces pastorianus. The genome structure of CBS 380T, the type strain of Saccharomyces bayanus, is also consistent with S. pastorianus gene transfer. The results indicate that the cider yeast, CID1, possesses nuclear DNA from three separate species. Mating experiments show that there are no barriers to interspecific conjugation of haploid cells. Furthermore, the allopolyploid strains were able to undergo further hybridizations with other Saccharomyces sensu stricto yeasts. These results demonstrate that introgression between the Saccharomyces sensu stricto species is likely. [source] Advances in molecular ecology: tracking trophic links through predator,prey food-websFUNCTIONAL ECOLOGY, Issue 5 2005S. K. SHEPPARD Summary 1It is not always possible to track trophic interactions between predators and prey by direct observation. This is especially true when observing small or elusive animals with cryptic food-web ecology. Gut and/or faecal analysis can sometimes allow prey remains to be identified visually but is only possible when a component of the diet is resistant to digestion. In some cases there are no solid remains, and when there are it can lead to bias in interpretation of prey choice. 2Numerous invasive and non-invasive methods have been developed to characterize predator,prey interactions but two principal areas dominate ,molecular' research. These are reviewed under the headings of monoclonal antibodies and DNA-based techniques. 3Early ,molecular' studies of predator,prey food webs were dominated by the development of monoclonal antibodies. These methods continue to be used for mass-screening of field-collected arthropods for insect-specific proteins. 4The application of species-specific primer design, polymerase chain reaction (PCR), restriction fragment length polymorphism analysis (RFLP), DNA cloning and sequencing, comparative sequence analysis (e.g. BLAST; basic local alignment search tool), high-resolution gel electrophoresis, Temperature/denaturing gradient gel electrophoresis (TGGE/DGGE) and automated fragment analysis with fluorescent probes is reviewed. The development of molecular techniques for use in predator,prey studies is primarily limited by their cost and the development of new procedures and equipment that complement them. [source] Role of the NFKB1 ,94ins/delATTG promoter polymorphism in IBD and potential interactions with polymorphisms in the CARD15/NOD2, IKBL, and IL-1RN genesINFLAMMATORY BOWEL DISEASES, Issue 7 2006Jürgen Glas MD Abstract Background: Recently, an association of the NFKB1 polymorphism ,94ins/delATTG with ulcerative colitis (UC) has been reported. This 4-bp insertion/deletion polymorphism is localized in the promoter region of the NFKB1 gene and appears to be functionally relevant. The aim of the present study was to confirm the association of the ,94ins/delATTG (W/D) NFKB1 promoter polymorphism with UC in a population of German origin and to test for a potential association with Crohn's disease (CD). Furthermore, potential interactions of the ,94ins/delATTG polymorphism with the IKBL and the IL-1RN genes should be determined. Materials and Methods: The study population comprised 630 patients with CD, 365 patients with UC, and 974 healthy controls. Genotyping was performed using polymerase chain reaction and restriction fragment length polymorphism analysis. For statistical evaluation, the chi-square test and the Fisher exact test were used. Results: No significant association of the W/D NFKB1 polymorphism with CD or UC was detected. In addition, no significant interactions between the ,94ins/delATTG NFKB1 polymorphism and polymorphisms within the IKBL and the IL-1RN genes, respectively, were found in CD or UC. Also, no significant interactions of the NFKB1 polymorphism with mutations of the CARD15/NOD2 gene and with clinical phenotypes were detected in CD. Moreover, no associations of the NFKB1 polymorphism were found in UC depending on disease localization. Conclusions: The present study could not confirm the reported association of the ,94ins/delATTG NFKB1 polymorphism with UC and also found no evidence for a role of this polymorphism in CD. The results do not give evidence for a role of this NFKB1 polymorphism in the pathogenesis of UC and CD. [source] Association of polymorphisms in the interleukin-18 gene in patients with Crohn's disease depending on the CARD15/NOD2 genotypeINFLAMMATORY BOWEL DISEASES, Issue 12 2005Jürgen Glas MD Abstract Background: An increased expression of interleukin-18 (IL-18), a proinflammatory cytokine inducing interferon-,, has been found in Crohn's disease (CD). In the IL-18 gene, several partly functional relevant polymorphisms are known. This study sought to investigate associations of IL-18 polymorphisms in inflammatory bowel disease and CD according to CARD15/NOD2 mutation status and clinical phenotypes. Methods: The IL-18 polymorphisms ,607, ,137, and the third position of codon 35 (c35/3) were genotyped in 210 patients with CD, 140 patients with ulcerative colitis, and 265 healthy controls using polymerase chain reaction and restriction fragment length polymorphism analysis. Results: Frequencies of alleles and genotypes of the 3 polymorphisms and of the respective haplotypes and diplotypes displayed no significant differences between the whole groups of patients with CD and ulcerative colitis, respectively, compared with the controls. After stratification of patients with CD for CARD15/NOD2 status, significant associations of genotypes ,137 CC (P = 0.018) and c35/3 CC (P = 0.010) and of the diplotype 2-2 (P = 0.018) were found in cases carrying CARD15/NOD2 mutations. Associations of genotypes ,137 GG (P = 0.015) and c35/3 AA (P = 0.030) with colonic disease only in cases positive for CARD15/NOD2 mutations and of the genotype ,607 AA (P = 0.007) with fistulas in cases negative for CARD15/NOD2 mutations were observed. Conclusions: In this study, significant differences of several genotypes and diplotypes within the IL-18 gene in CD depending on CARD15/NOD2 status have been found. In context with an increased expression of IL-18 in CD, it remains to be shown whether the expression of IL-18 is influenced by CARD15/NOD2 mutation status. [source] Ribosomal DNA spacer genotypes of the Anopheles bancroftii group (Diptera: Culicidae) from Australia and Papua New GuineaINSECT MOLECULAR BIOLOGY, Issue 5 2001N. W. Beebe Abstract Mosquitoes of the Anopheles bancroftii group collected from Northern Australia and Papua New Guinea (PNG) were investigated for sequence variation within the ribosomal DNA ITS2. Wing fringe morphology originally used to identify members of this group was compared to genotypes identified by restriction fragment length polymorphism analysis (RFLP) and heteroduplex analysis (HDA) of the rDNA ITS2. Members of this group separated into four RFLP genotypes (A, B, C and D) with some genotypes displaying wing fringe polymorphisms. Heteroduplex analysis of the ITS2 within and between populations identified genotype A as containing two geographically separate ITS2 sequences: A1 from the Northern Territory of Australia and A2 from Queensland and the Western Province of PNG. Genotypes B and C and genotypes C and D were found sympatric and appeared to be evolving independently suggesting the possibility of cryptic species. Genotype C contained two ITS2 sequence types within the genome. [source] Protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene R620W variant and sporadic idiopathic hypoparathyroidism in Asian IndiansINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 4 2006D. Ray Summary Recently, a gain of function variant C1858T of the lymphoid-specific protein tyrosine phosphatase non-receptor (LYP, PTPN22) gene has been reported to be associated with several autoimmune disorders including Graves' disease, type 1 diabetes, rheumatoid arthritis and vitiligo. The present study was carried out in 80 patients with sporadic idiopathic hypoparathyroidism (SIH) [43 males and 37 females, mean ± SD age and duration of symptoms 32.5 ± 14.1 years and 6.7 ± 7.2 years (range 1 day to 35 years), respectively] and 193 healthy controls (male : female ratio 91:102, mean ± SD age, 43.1 ± 11.6 years) to assess association of 1858T allele with the disease. Polymerase chain reaction,restriction fragment length polymorphism analysis was performed to genotype C1858T variant. The frequency of occurrence of 1858T allele was 4/160 (2.5%) in SIH and 5/386 (1.3%) in the control alleles (odds ratio 1.95, 95% CI 0.51,7.37). Thus, the present study reveals that 1858T allele is rare (1.3%) in Asian Indians. The trend of higher prevalence of 1858T allele in patients with SIH needs to be studied further in other population with higher rate of the allele to support the autoimmune basis of the disease. [source] Amplified fragment length polymorphism analysis to identify the genetic structure of the Gymnocypris przewalskii (Kessler, 1876) population from the Qinghai Basin, ChinaJOURNAL OF APPLIED ICHTHYOLOGY, Issue 3 2005D. Chen Summary Amplified fragment length polymorphism (AFLP) was used to analyse the genetic structure of 45 individuals of Gymnocypris przewalskii (Kessler, 1876), an endangered and state-protected rare fish species, from three areas [the Heima (HM), Buha (BH) and Shaliu rivers (SL), all draining into Qinghai Lake]. A total of 563 polymorphic loci were detected. The HM, BH and SL populations have 435, 433 and 391 loci, respectively (Zhu and Wu, 1975), which account for 77.26%, 76.91% and 69.45% of the total number of polymorphic loci of each population, respectively. The Nei indices of genetic diversities (H) of the three populations were calculated to be 0.2869 (HM), 0.2884 (BH) and 0.2663 (SL), respectively. Their Shannon informative indices are 0.4244, 0.4251 and 0.3915, respectively. Research results show that the mean genetic distance between HM and BH is the smallest (0.0511), between BH and SL is the second shortest (0.0608), and between HM and SL is the largest (0.0713), with the mean genetic distance among the three populations being over 0.05. Data mentioned above indicate that the three populations have a certain genetic differentiation. The total genetic diversity (Ht = 0.3045) and the mean value of genetic diversity within the population (Hs = 0.2786) indicate that the variations have mainly come from within the population. [source] Assessment of anaerobic wastewater treatment failure using terminal restriction fragment length polymorphism analysisJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2005C. Scully Abstract Aims:, The suitability of genetic fingerprinting to study the microbiological basis of anaerobic bioreactor failure is investigated. Methods and Results:, Two laboratory-scale anaerobic expanded granular sludge bed bioreactors, R1 and R2, were used for the mesophilic (37°C) treatment of high-strength [10 g chemical oxygen demand (COD) l,1] synthetic industrial-like wastewater over a 100-day trial period. A successful start up was achieved by both bioreactors with COD removal over 90%. Both reactors were operated under identical parameters; however, increased organic loading during the trial induced a reduction in the COD removal of R1, while R2 maintained satisfactory performance (COD removal >90%) throughout the experiment. Specific methanogenic activity measurements of biomass from both reactors indicated that the main route of methane production was hydrogenotrophic methanogenesis. Terminal restriction fragment length polymorphism (TRFLP) analysis was applied to the characterization of microbial community dynamics within the system during the trial. The principal differences between the two consortia analysed included an increased abundance of Thiovulum - and Methanococcus -like organisms and uncultured Crenarchaeota in R1. Conclusions:, The results indicated that there was a microbiological basis for the deviation, in terms of operational performance, of R1 and R2. Significance and Impact of the Study:, High-throughput fingerprinting techniques, such as TRFLP, have been demonstrated as practically relevant for biomonitoring of anaerobic reactor communities. [source] Clustering of Saccharomyces boulardii strains within the species S. cerevisiae using molecular typing techniquesJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2002G. Mitterdorfer Aims: This study was undertaken to characterize and differentiate therapeutically relevant Saccharomyces yeasts. Among the isolates were so-called Saccharomyces boulardii strains, which are considered as probiotic agents, but whose taxonomic assignment is controversial. Moreover, the discriminative power of the applied molecular typing techniques should be evaluated. Methods and Results: Genotyping was performed using species-specific polymerase chain reaction (PCR), randomly amplified polymorphic DNA-PCR, restriction fragment length polymorphism analysis of rDNA spacer regions and pulsed-field gel electrophoresis. Species-specific PCR assigned all of the product isolates to the species S. cerevisiae. By combining the other techniques, all isolates could be discriminated. Moreover, it could be demonstrated that probiotic S. boulardii strains form a separate cluster located within the species. Conclusions: With the exception of species-specific PCR, all of the applied methodologies were suitable for subspecies typing and indicated a close relationship between the probiotic strains. Significance and Impact of the Study: The methods applied in this study are considered powerful tools for quality control of therapeutically relevant yeasts. It is of crucial importance, especially regarding S. boulardii yeasts, to verify the identity of the correct strain, since the beneficial properties are considered to be strain-specific. [source] Characterization of yellow-pigmented and motile enterococci isolated from intestines of the garden snail Helix aspersaJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2002Aims: Enterococci associated with garden snails (Helix aspersa) were studied in order to obtain reliable species identification and characterization. Methods and Results: Twelve yellow-pigmented and motile enterococci, isolated from the intestines of garden snails, were phenotypically close to Enterococcus casseliflavus, but they showed certain unusual biochemical characteristics. tRNA intergenic length polymorphism analysis (tDNA-PCR) divided all strains studied into two groups, in full agreement with biochemical test results. 16S rDNA sequencing, DNA base composition analysis and DNA-DNA hybridization results showed unambiguously that the enterococci studied belonged to the species Ent. casseliflavus. The representative strains of described ecovars were deposited in the Czech Collection of Microorganisms (CCM) as Ent. casseliflavus CCM 4868, 4869, 4870 and 4871. Conclusions:Enterococcus casseliflavus associated with garden snails can be subdivided into groups. Significance and Impact of the Study:Enterococcus casseliflavus differs from other enterococcal species in that it is typically associated with plants, soil, water and invertebrate animals. The different groups that can be found in these widely occurring bacteria are possibly source-specific ecovars, as exemplified by the Ent. casseliflavus inhabiting the intestines of snails. [source] Cytochrome P450 CYP2C19 genotypes in Nigerian sickle-cell disease patients and normal controlsJOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 4 2010C. P. Babalola PhD Summary Background and objective:, Subjects with different CYP2C19 genotypes may metabolize proguanil, a pro-drug used for malaria prophylaxis differently and the frequency of the different alleles may be different in patients with sickle-cell disease (SCD) and normal controls. The objective of this study was to evaluate CYP2C19 *1, *2 and *3 allele and genotype frequencies in Nigerian normal controls and SCD patients, and to further compare variant CYP2C19 frequencies in Nigerians with other African populations. Methods:, Genotyping was carried out with PCR and restriction fragment length polymorphism analysis. Results and discussion:, CYP2C19 *1 (84·3 vs. 84·9%) or *2 allele frequency (15·7 vs. 15·1%) was not significantly different between patients with SCD and normal subjects. No *3 allele was detected in the cohort. The SCD group exhibited a statistically significantly lower frequency of *1/*1 genotype (69·6%) compared with normal controls (74·4%). Frequency of *2/*2 was significantly lower in SCD (0·9%) compared with normal controls (4·7%). Frequencies of *1/*2 (29·6 vs. 20·9%) were no different in SCD and normal controls. Conclusion:, Prevalence of CYP2C19 polymorphisms was defined for the first time in Nigerian normal and SCD populations. Nigerian SCD patients exhibited significantly lower CYP2C19 *1/*1 and *2/*2 frequencies than normal controls. No differences were detected in CYP2C19 allele or genotype frequencies in normal subjects between this study and previous reports in other African populations. [source] | |||||||||||||||||||||||||||||||