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DNA Sequencing (dna + sequencing)
Kinds of DNA Sequencing Terms modified by DNA Sequencing Selected AbstractsGEITLERINEMA SPECIES (OSCILLATORIALES, CYANOBACTERIA) REVEALED BY CELLULAR MORPHOLOGY, ULTRASTRUCTURE, AND DNA SEQUENCING,JOURNAL OF PHYCOLOGY, Issue 3 2009Maria Do Carmo Bittencourt-Oliveira Geitlerinema amphibium (C. Agardh ex Gomont) Anagn. and G. unigranulatum (Rama N. Singh) Komárek et M. T. P. Azevedo are morphologically close species with characteristics frequently overlapping. Ten strains of Geitlerinema (six of G. amphibium and four of G. unigranulatum) were analyzed by DNA sequencing and transmission electronic and optical microscopy. Among the investigated strains, the two species were not separated with respect to cellular dimensions, and cellular width was the most varying characteristic. The number and localization of granules, as well as other ultrastructural characteristics, did not provide a means to discriminate between the two species. The two species were not separated either by geography or environment. These results were further corroborated by the analysis of the cpcB- cpcA intergenic spacer (PC-IGS) sequences. Given the fact that morphology is very uniform, plus the coexistence of these populations in the same habitat, it would be nearly impossible to distinguish between them in nature. On the other hand, two of the analyzed strains were distinct from all others based on the PC-IGS sequences, in spite of their morphological similarity. PC-IGS sequences indicate that these two strains could be a different species of Geitlerinema. Using morphology, cell ultrastructure, and PC-IGS sequences, it is not possible to distinguish G. amphibium and G. unigranulatum. Therefore, they should be treated as one species, G. unigranulatum as a synonym of G. amphibium. [source] HLA-B27 typing: Evaluation of an allele-specific PCR melting assay and two flow cytometric antigen assaysCYTOMETRY, Issue 1 2005Michael T. Seipp Abstract Background Human leukocyte antigen B27 (HLA-B27) is a major histocompatibility complex class 1 molecule that is strongly associated with the disease ankylosing spondylitis. Testing for HLA-B27 is of diagnostic value because 90% of patients with ankylosing spondylitis have the B27 antigen. Two commonly used HLA-B27 flow cytometric assays are commercially available. Methods An allele-specific polymerase chain reaction (PCR) melting assay for HLA-B27 was compared with two available antigen assays on 371 clinical samples. The accuracy of the assays was measured by receiver operating characteristic analysis using the PCR method and sequencing as the reference standard. Results When PCR results were compared with those of the antigen assays, complete concordance was observed except for five discrepant results that were resolved by sequence analysis. Using DNA sequencing as the gold standard, the sensitivity and specificity of PCR were 99.6 and 100.0, those of the best single antigen assay were 98.2 and 97.6, and those of a reflex combination of both antigen assays were 98.8 and 97.6. Conclusions The allele-specific PCR melting assay for HLA-B27 genotyping is easy to perform and has better sensitivity and specificity than antigen assays. The performance of the two flow cytometric antigen assays depends on the antibody used and the positive cutoff values assigned. © 2004 Wiley-Liss, Inc. [source] Universal multiplex PCR and CE for quantification of SMN1/SMN2 genes in spinal muscular atrophyELECTROPHORESIS, Issue 7 2009Chun-Chi Wang Abstract We established a universal multiplex PCR and CE to calculate the copy number of survival motor neuron (SMN1 and SMN2) genes for clinical screening of spinal muscular atrophy (SMA). In this study, one universal fluorescent primer was designed and applied for multiplex PCR of SMN1, SMN2 and two internal standards (CYBB and KRIT1). These amplicons were separated by conformation sensitive CE. Mixture of hydroxyethyl cellulose and hydroxypropyl cellulose were used in this CE system. Our method provided the potential to separate two 390-bp PCR products that differ in a single nucleotide. Differentiation and quantification of SMN1 and SMN2 are essential for clinical screening of SMA patients and carriers. The DNA samples included 22 SMA patients, 45 parents of SMA patients (obligatory carriers) and 217 controls. For evaluating accuracy, those 284 samples were blind-analyzed by this method and denaturing high pressure liquid chromatography (DHPLC). Eight of the total samples showed different results. Among them, two samples were diagnosed as having only SMN2 gene by DHPLC, however, they contained both SMN1 and SMN2 by our method. They were further confirmed by DNA sequencing. Our method showed good agreement with the DNA sequencing. The multiplex ligation-dependent probe amplification (MLPA) was used for confirming the other five samples, and showed the same results with our CE method. For only one sample, our CE showed different results with MLPA and DNA sequencing. One out of 284 samples (0.35%) belonged to mismatching. Our method provided a better accurate method and convenient method for clinical genotyping of SMA disease. [source] Cover Picture: Electrophoresis 23'2008ELECTROPHORESIS, Issue 23 2008Article first published online: 3 DEC 200 Issue 23 is a paper symposium on "DNA sequencing and genotyping". It consists of 22 contributions covering a wide spectrum of investigations in the area of DNA sequencing by capillary electrophoresis and the latest technologies for DNA sequencing and genotyping on microfluidic devices. Ten contributions deal with microchip-based electrophoresis analyses of DNA, an indication that miniaturized devices may surge to the fore for medical sequencing applications on individual DNA samples, as well as for forensic applications. [source] Quantification of SMN1 and SMN2 genes by capillary electrophoresis for diagnosis of spinal muscular atrophyELECTROPHORESIS, Issue 13 2008Chun-Chi Wang Abstract We present the first CE method for the separation and quantification of SMN1 and SMN2 genes. Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder deleted or mutated in SMN1 gene and retained at least one copy of SMN2 gene. However, these two genes are highly homologous, differentiation and quantification of SMN1 and SMN2 are therefore required in diagnosis to identify SMA patients and carriers. We developed a fluorescence-labeled conformation-sensitive CE method to quantitatively analyze PCR products covering the variable position in the SMN1/SMN2 genes using a copolymer solution composed of hydroxyethylcellulose and hydroxypropylcellulose. The DNA samples included 24 SMA patients, 52 parents of SMA patients (obligatory carriers), and 255 controls. Those 331 samples were blind analyzed to evaluate the method, and the results compared with those obtained using denaturing HPLC (DHPLC). Validation of accuracy was performed by comparing the results with those of DHPLC. Nine of total samples showed different results. Diagnosis of one fetus DNA among them was related to abortion or not, which was further confirmed by gel electrophoresis and DNA sequencing. Our method showed good coincidence with them, and proved the misdiagnosis of DHPLC. This simple and reliable CE method is a powerful tool for clinical genotyping of large populations to detect carriers and SMA patients. [source] Cover Picture: Electrophoresis 11/2008ELECTROPHORESIS, Issue 11 2008Article first published online: 11 JUN 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. Issue 11 also offers a series of 9 papers on bioanalysis related to proteomics, protein analysis, genes, DNA sequencing, SNP, genotyping and SSCP. [source] Using DNA sequencing electrophoresis compression artifacts as reporters of stable mRNA structures affecting gene expressionELECTROPHORESIS, Issue 21 2007Divya Kapoor Abstract The formation of secondary structure in oligonucleotide DNA is known to lead to "compression" artifacts in electropherograms produced through DNA sequencing. Separately, the formation of secondary structure in mRNA is known to suppress translation; in particular, when such structures form in a region covered by the ribosome either during, or shortly after, initiation of translation. Here, we demonstrate how a DNA sequencing compression artifact provides important clues to the location(s) of translation-suppressing secondary structural elements in mRNA. Our study involves an engineered version of a gene sourced from Rhodothermus marinus encoding an enzyme called Cel12A. We introduced this gene into Escherichia coli with the intention of overexpressing it, but found that it expressed extremely poorly. Intriguingly, the gene displayed a remarkable compression artifact during DNA sequencing electrophoresis. Selected "designer" silent mutations destroyed the artifact. They also simultaneously greatly enhanced the expression of the cel12A gene, presumably by destroying stable mRNA structures that otherwise suppress translation. We propose that this method of finding problem mRNA sequences is superior to software-based analyses, especially if combined with low-temperature CE. [source] Novel design of multicapillary arrays for high-throughput DNA sequencingELECTROPHORESIS, Issue 14 2006Andriy Tsupryk Dr. Abstract A novel approach to design and optimize linear multicapillary arrays,(LMCAs) for high-throughput DNA sequencing is proposed. A significant increase in the number of capillary lanes is obtained due to the use of composite insertions alternately placed between working capillaries of the array and a specific combination of refractive indices of the DNA separation matrix, capillary glass, the insertions and a medium which surrounds the capillary array. Theoretical and experimental studies showed that in conjunction with a dual-side laser illumination scheme, the proposed LMCA design allows a simultaneous uniform irradiation of as many as 550,working capillaries. [source] The biotin-streptavidin interaction can be reversibly broken using water at elevated temperaturesELECTROPHORESIS, Issue 3 2005Anders Holmberg Abstract The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, Kd, in the order of 4×10,14 M. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 70°C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluidics and nanotechnology. [source] A microfabricated hybrid device for DNA sequencingELECTROPHORESIS, Issue 21 2003Shaorong LiuArticle first published online: 6 NOV 200 Abstract We have created a hybrid device of a microfabricated round-channel twin-T injector incorporated with a separation capillary in order to extend the straight separation distance for high speed and long readlength DNA sequencing. Semicircular grooves on glass wafers are obtained using a photomask with a narrow line-width and a standard isotropic photolithographic etching process. Round channels are made when two etched wafers are face-to-face aligned and bonded. A two-mask fabrication process has been developed to make channels of two different diameters. The twin-T injector is formed by the smaller channels whose diameter matches the bore of the separation capillary, and the "usual" separation channel, now called the connection channel, is formed by the larger ones whose diameter matches the outer diameter of the separation capillary. The separation capillary is inserted through the connection channel all the way to the twin-T injector to allow the capillary bore flush with the twin-T injector channels. The total dead-volume of the connection is estimated to be , 5 pL. To demonstrate the efficiency of this hybrid device, we have performed four-color DNA sequencing on it. Using a 200 ,m twin-T injector coupled with a separation capillary of 20 cm effective separation distance, we have obtained readlengths of 800 plus bases at an accuracy of 98.5% in 56 min, compared to about 650 bases in 100 min on a conventional 40 cm long capillary sequencing machine under similar conditions. At an increased separation field strength and using a diluted sieving matrix, the separation time has been reduced to 20 min with a readlength of 700 bases at 98.5% base-calling accuracy. [source] Polyphyletic photosynthetic reaction centre genes in oligotrophic marine GammaproteobacteriaENVIRONMENTAL MICROBIOLOGY, Issue 6 2007Jang-Cheon Cho Summary Ecological studies indicate that aerobic anoxygenic phototrophic bacteria (AAP) that use bacteriochlorophyll to support phototrophic electron transport are widely distributed in the oceans. All cultivated marine AAP are alpha-3 and alpha-4 Proteobacteria, but metagenomic evidence indicates that uncultured AAP Gammaproteobacteria are important members of ocean surface microbial communities. Here we report the description of obligately oligotrophic, marine Gammaproteobacteria that have genes for aerobic anoxygenic photosynthesis. Three strains belonging to the OM60 clade were isolated in autoclaved seawater media. Polymerase chain reaction assays for the pufM gene show that these strains contain photosynthetic reaction centre genes. DNA sequencing and phylogenetic analysis indicate that the pufM genes are polyphyletic, suggesting multiple instances of lateral gene transfer. Peptide sequences from six photosynthesis genes (pufL, pufM, pufC, pufB, pufA and puhA) were detected by proteomic analyses of strain HTCC2080 cells grown aerobically in seawater. They closely match predicted peptides from an environmental seawater bacterial artificial chromosome clone of gammaproteobacterial origin, thus identifying the OM60 clade as a significant source of gammaproteobacterial AAP genes in marine systems. The cell yield and rate of growth of HTCC2080 in autoclaved, aerobic seawater increased in the light. These findings identify the OM60 clade as a source of Gammaproteobacteria AAP genes in coastal oceans, and demonstrate that aerobic, anoxygenic photosynthetic metabolism can enhance the productivity of marine oligotrophic bacteria that also grow heterotrophically in darkness. [source] Evolution of a chlorobenzene degradative pathway among bacteria in a contaminated groundwater mediated by a genomic island in RalstoniaENVIRONMENTAL MICROBIOLOGY, Issue 3 2003Tina Andrea Müller Summary The genetic structure of two Ralstonia spp., strain JS705 and strain JS745, isolated from the same groundwater aquifer, was characterized with respect to the degradation capacities for toluene and chlorobenzene degradation. Cosmid library construction, cloning, DNA sequencing and mating experiments indicated that the genes for chlorobenzene degradation in strain JS705 were a mosaic of the clc genes, previously described for Pseudomonas sp. strain B13, and a 5 kb fragment identical to strain JS745. The 5 kb fragment identical to both JS705 and JS745 was flanked in JS705 by one complete and one incomplete insertion (IS) element. This suggested involvement of the IS element in mobilizing the genes from JS745 to JS705, although insertional activity of the IS element in its present configuration could not be demonstrated. The complete genetic structure for chlorobenzene degradation in strain JS705 resided on a genomic island very similar to the clc element (Ravatn, R., Studer, S., Springael, D., Zehnder, A.J., van der Meer, J.R. 1998. Chromosomal integration, tandem amplification, and deamplification in Pseudomonas putida F1 of a 105-kilobase genetic element containing the chlorocatechol degradative genes from Pseudomonas sp. strain B13. J Bacteriol 180: 4360,4369). The unique reconstruction of formation of a metabolic pathway through the activity of IS elements and a genomic island in the chlorobenzene-degrading strain JS705 demonstrated how pathway evolution can occur under natural conditions in a few ,steps'. [source] Cadmium-regulated gene fusions in Pseudomonas fluorescensENVIRONMENTAL MICROBIOLOGY, Issue 4 2000Silvia Rossbach To study the mechanisms soil bacteria use to cope with elevated concentrations of heavy metals in the environment, a mutagenesis with the lacZ -based reporter gene transposon Tn5 -B20 was performed. Random gene fusions in the genome of the common soil bacterium Pseudomonas fluorescens strain ATCC 13525 were used to create a bank of 5000 P. fluorescens mutants. This mutant bank was screened for differential gene expression in the presence of the toxic metal cadmium. Fourteen mutants were identified that responded with increased or reduced gene expression to the presence of cadmium. The mutants were characterized with respect to their metal-dependent gene expression and their metal tolerance. Half the identified mutants reacted with differential gene expression specifically to the metal cadmium, whereas some of the other mutants also responded to elevated concentrations of copper and zinc ions. One of the mutants, strain C8, also showed increased gene expression in the presence of the solvent ethanol, but otherwise no overlap between cadmium-induced gene expression and general stress response was detected. Molecular analysis of the corresponding genetic loci was performed using arbitrary polymerase chain reaction (PCR), DNA sequencing and comparison of the deduced protein products with sequences deposited in genetic databases. Some of the genetic loci targeted by the transposon did not show any similarities to any known genes; thus, they may represent ,novel' loci. The hypothesis that genes that are differentially expressed in the presence of heavy metals play a role in metal tolerance was verified for one of the mutants. This mutant, strain C11, was hypersensitive to cadmium and zinc ions. In mutant C11, the transposon had inserted into a genetic region displaying similarity to genes encoding the sensor/regulator protein pairs of two-component systems that regulate gene expression in metal-resistant bacteria, including czcRS of Ralstonia eutropha, czrRS of Pseudomonas aeruginosa and copRS of Pseudomonas syringae. Although the P. fluorescens strain used in this study had not been isolated from a metal-rich environment, it nevertheless contained at least one genetic region enabling it to cope with elevated concentrations of heavy metals. [source] Phylogenetic relationship and antifouling activity of bacterial epiphytes from the marine alga Ulva lactucaENVIRONMENTAL MICROBIOLOGY, Issue 3 2000Brief report It is widely accepted that bacterial epiphytes can inhibit the colonization of surfaces by common fouling organisms. However, little information is available regarding the diversity and properties of these antifouling bacteria. This study assessed the antifouling traits of five epiphytes of the common green alga, Ulva lactuca. All isolates were capable of preventing the settlement of invertebrate larvae and germination of algal spores. Three of the isolates also inhibited the growth of a variety of bacteria and fungi. Their phylogenetic positions were determined by 16S ribosomal subunit DNA sequencing. All isolates showed a close affiliation with the genus Pseudoalteromonas and, in particular, with the species P. tunicata. Strains of this bacterial species also display a variety of antifouling activities, suggesting that antifouling ability may be an important trait for members of this genus to be highly successful colonizers of animate surfaces and for such species to protect their host against fouling. [source] Co-inheritance of Hb Hershey [,70(E14) Ala,Gly] and Hb La Pommeraie [,133(H11)Val,Met] in a Sicilian subjectEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2010Antonino Giambona Abstract Objectives:,This report represents the first observation in Sicily of two rare , -globin gene variants, Hb Hershey [,70(E14) Ala,Gly] and Hb La Pommeraie [,133(H11)Val,Met], found in a 35-year-old male patient from Messina, in the north-east of Sicily during population screening for hemoglobinopathies. Methods: The occurrence of the Hb variants was assessed by cation exchange chromatography while complete blood counts were obtained using automatic cell counters. Red cell lysates were analyzed by electrophoresis at alkaline and acid pH. Stability of hemoglobin was checked by the isopropanol precipitation test and by the heat tests while inclusion bodies and reticulocyte count were determined by incubation of blood samples with brilliant cresyl blue. Molecular analysis was performed by DNA sequencing of ,- and , -globin genes. Results: We observed an abnormally high performance liquid chromatography elution with a slight reduction in mean corpuscular volume and mean corpuscular haemoglobin parameters and mutations at codon 70 GCC,GGC (Hb Hershey) and at codon 133 GTG,ATG (Hb La Pommeraie) in , -globin gene. Conclusion: Family analysis of three generations demonstrated the presence of these two mutations in trans. So it was possible to describe the phenotypes of these variants in a heterozygous state and in double heterozygous state. [source] Defective DNA methylation and CD70 overexpression in CD4+ T cells in MRL/lpr lupus-prone miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2007Abstract We have determined that abnormal DNA methylation in T cells coincides with the development of autoimmunity, using a mouse model that exhibits an age-dependent lupus-like disease (MRL/lpr mice). Splenic CD4+ T cells were isolated from these mice at 5 and 16,wk of age (before and after autoimmunity is established) and the expression of DNA methyltransferase,1 (Dnmt1) and the methylation-sensitive gene Tnfsf7 (CD70) was measured. Bisulfite DNA sequencing was used to monitor the methylation status of the Tnfsf7 gene. We found that Dnmt1 steady-state mRNA levels were significantly lower in 16-wk-old MRL/lpr mice, which had established autoimmunity, compared to the 5-wk-old MRL/lpr mice. Furthermore, the expression of CD70 was higher in MRL/lpr mice at 16,wk. CD70 was overexpressed in MRL/lpr mice compared to age- and sex-matched MRL+/+ controls. Bisulfite DNA sequencing of the Tnfsf7 gene in MRL/lpr mice revealed that at 16,wk, CG pairs were hypomethylated compared to 5-wk-old mice, and that Tnfsf7 from MRL/lpr mice was hypomethylated at 16,wk relative to age-matched MRL+/+ controls. Our data indicate that decreased expression of Dnmt1 and the corresponding T cell DNA hypomethylation correlate with the development of age-dependent autoimmunity in MRL/lpr mice. [source] Human alcoholism studies of genes identified through mouse quantitative trait locus analysisADDICTION BIOLOGY, Issue 4 2002Marissa A. Ehringer Coding region DNA sequence variants have been recently identified in several QTL candidate genes in a mouse model of differential sensitivity to alcohol [inbred long-sleep (ILS) and inbred short-sleep (ISS)]. This work has been extended into a human population characterized for their initial level of response to alcohol (LR). The coding region of one of the most promising of these candidate genes, zinc finger 133 (Znf133), has been sequenced completely in 50 individuals who participated in alcohol challenges at approximately age 20 and have been followed subsequently for the last 15 years. PCR products were obtained for the protein coding region of ZNF133 using human genomic DNA and directly sequenced using automated sequencers. Novel single nucleotide polymorphisms (SNPs) were detected by analyzing the sequence data using a suite of bioinformatics programs including Consed, Phred, Phrap and Polyphred. Five human SNPs were detected, two that correspond to amino acid changes in the protein, two that are silent DNA changes and one located in an intron. In this small sample, no significant association between any of the SNPs and alcohol diagnosis was detected. A follow-up of these SNPs in a larger sample should allow a more definitive conclusion to be reached. Significantly, the data presented here demonstrate the feasibility of directly testing genes in human alcoholic populations that had been identified first by comparative DNA sequencing of candidate genes located within mouse alcohol-related QTLs, even without detailed knowledge of the gene's function. [source] A clinical and genetic study of 56 Saudi Wilson disease patients: identification of Saudi-specific mutationsEUROPEAN JOURNAL OF NEUROLOGY, Issue 2 2004M. Al Jumah Wilson disease (WD) is a hereditary disorder, with recessive transmission and genetic heterogeneity. Several mutations of ATP7B, the gene underlying WD, were reported in many ethnic groups. In this study, mutation screening in ATP7B of 56 Saudi Arabian WD patients was undertaken. The clinical data of all patients were recorded. The entire ATP7B coding sequence, including intron,exon boundaries were screened for mutation by the polymerase chain reaction (PCR)-based mutation detection technique and DNA sequencing. Thirty-nine patients were symptomatic at presentation and 17 subjects were pre-symptomatic siblings of affected patients. Fourteen patients had neurological, 11 patients had mixed (hepatic and neurological), and 14 patients had hepatic presentations. Family history suggestive of WD was present in 72% of cases and 68% had consanguineous parents. Genetic analysis showed disease-causing mutations in three exons (exons 8, 19 and 21) of the ATP7B gene in 28 patients (50%). Mutations in exons 21 (18 cases) and 19 (one case) were unique for Saudis. This large series of Saudi patients with WD has shown wide variability in the genomic substrate of WD. There is no correlation between genotype and clinical presentation. [source] PERSPECTIVE: GENE DIVERGENCE, POPULATION DIVERGENCE, AND THE VARIANCE IN COALESCENCE TIME IN PHYLOGEOGRAPHIC STUDIESEVOLUTION, Issue 6 2000ScottV. Abstract Molecular methods as applied to the biogeography of single species (phylogeography) or multiple codistributed species (comparative phylogeography) have been productively and extensively used to elucidate common historical features in the diversification of the Earth's biota. However, only recently have methods for estimating population divergence times or their confidence limits while taking into account the critical effects of genetic polymorphism in ancestral species become available, and earlier methods for doing so are underutilized. We review models that address the crucial distinction between the gene divergence, the parameter that is typically recovered in molecular phylogeographic studies, and the population divergence, which is in most cases the parameter of interest and will almost always postdate the gene divergence. Assuming that population sizes of ancestral species are distributed similarly to those of extant species, we show that phylogeographic studies in vertebrates suggest that divergence of alleles in ancestral species can comprise from less than 10% to over 50% of the total divergence between sister species, suggesting that the problem of ancestral polymorphism in dating population divergence can be substantial. The variance in the number of substitutions (among loci for a given species or among species for a given gene) resulting from the stochastic nature of DNA change is generally smaller than the variance due to substitutions along allelic lines whose coalescence times vary due to genetic drift in the ancestral population. Whereas the former variance can be reduced by further DNA sequencing at a single locus, the latter cannot. Contrary to phylogeographic intuition, dating population divergence times when allelic lines have achieved reciprocal monophyly is in some ways more challenging than when allelic lines have not achieved monophyly, because in the former case critical data on ancestral population size provided by residual ancestral polymorphism is lost. In the former case differences in coalescence time between species pairs can in principle be explained entirely by differences in ancestral population size without resorting to explanations involving differences in divergence time. Furthermore, the confidence limits on population divergence times are severely underestimated when those for number of substitutions per site in the DNA sequences examined are used as a proxy. This uncertainty highlights the importance of multilocus data in estimating population divergence times; multilocus data can in principle distinguish differences in coalescence time (T) resulting from differences in population divergence time and differences in T due to differences in ancestral population sizes and will reduce the confidence limits on the estimates. We analyze the contribution of ancestral population size (,) to T and the effect of uncertainty in , on estimates of population divergence (,) for single loci under reciprocal monophyly using a simple Bayesian extension of Takahata and Satta's and Yang's recent coalescent methods. The confidence limits on , decrease when the range over which ancestral population size , is assumed to be distributed decreases and when increases; they generally exclude zero when /(4Ne) > 1. We also apply a maximum-likelihood method to several single and multilocus data sets. With multilocus data, the criterion for excluding = 0 is roughly that l/(4Ne)> 1, where l is the number of loci. Our analyses corroborate recent suggestions that increasing the number of loci is critical to decreasing the uncertainty in estimates of population divergence time. [source] Prevalence of the MspI and Ile462Val SNPs of Cytochrome P-450 1A1 in Hidradenitis SuppurativaEXPERIMENTAL DERMATOLOGY, Issue 6 2010Ansgar Lukowsky Please cite this paper as: Prevalence of the MspI and Ile462Val SNPs of Cytochrome P-450 1A1 in Hidradenitis Suppurativa. Experimental Dermatology 2010; 19: 541,542. Abstract:, Hidradenitis Suppurativa (HS) is a chronic inflammatory skin disease that affects the hair follicles in the axillary, perianal and inguinal area. Its cause and pathogenesis are unknown, but cigarette smoking increases the risk of developing HS conceivably by accumulating toxic metabolites in sweat. The xenobiotic compounds from tobacco are metabolized by the cytochromes P-450. The cytochrome P-450 1A1 (CYP1A1), one of the most active isoenzymes, harbours several polymorphisms. Two of them, MspI and Ile462Val single nucleotide polymorphism (SNP), are associated with enhanced activity and inducibility. Performing direct DNA sequencing, we investigated the frequencies of these SNP in 51 patients with HS, 45 of these were smokers. We found similar overall SNP rates in our patients in comparison with previous data for Caucasian or German controls. Obviously, there is no relation between the occurrence of these SNPs and the risk of developing HS. [source] Isolation, characterization, sequencing and crystal structure of charybdin, a type 1 ribosome-inactivating protein from Charybdis maritima agg.FEBS JOURNAL, Issue 12 2006Eleftherios Touloupakis A novel, type 1 ribosome-inactivating protein designated charybdin was isolated from bulbs of Charybdis maritima agg. The protein, consisting of a single polypeptide chain with a molecular mass of 29 kDa, inhibited translation in rabbit reticulocytes with an IC50 of 27.2 nm. Plant genomic DNA extracted from the bulb was amplified by PCR between primers based on the N-terminal and C-terminal sequence of the protein from dissolved crystals. The complete mature protein sequence was derived by partial DNA sequencing and terminal protein sequencing, and was confirmed by high-resolution crystal structure analysis. The protein contains Val at position 79 instead of the conserved Tyr residue of the ribosome-inactivating proteins known to date. To our knowledge, this is the first observation of a natural substitution of a catalytic residue at the active site of a natural ribosome-inactivating protein. This substitution in the active site may be responsible for the relatively low in vitro translation inhibitory effect compared with other ribosome-inactivating proteins. Single crystals were grown in the cold room from PEG6000 solutions. Diffraction data collected to 1.6 Ĺ resolution were used to determine the protein structure by the molecular replacement method. The fold of the protein comprises two structural domains: an ,,+ , N-terminal domain (residues 4,190) and a mainly ,-helical C-terminal domain (residues 191,257). The active site is located in the interface between the two domains and comprises residues Val79, Tyr117, Glu167 and Arg170. [source] Characterization of sequence variations in human histone H1.2 and H1.4 subtypesFEBS JOURNAL, Issue 14 2005Bettina Sarg In humans, eight types of histone H1 exist (H1.1,H1.5, H1°, H1t and H1oo), all consisting of a highly conserved globular domain and less conserved N- and C-terminal tails. Although the precise functions of these isoforms are not yet understood, and H1 subtypes have been found to be dispensable for mammalian development, it is now clear that specific functions may be assigned to certain individual H1 subtypes. Moreover, microsequence variations within the isoforms, such as polymorphisms or mutations, may have biological significance because of the high degree of sequence conservation of these proteins. This study used a hydrophilic interaction liquid chromatographic method to detect sequence variants within the subtypes. Two deviations from wild-type H1 sequences were found. In K562 erythroleukemic cells, alanine at position 17 in H1.2 was replaced by valine, and, in Raji B lymphoblastoid cells, lysine at position 173 in H1.4 was replaced by arginine. We confirmed these findings by DNA sequencing of the corresponding gene segments. In K562 cells, a homozygous GCC,GTC shift was found at codon 18, giving rise to H1.2 Ala17Val because the initial methionine is removed in H1 histones. Raji cells showed a heterozygous AAA,AGA codon change at position 174 in H1.4, corresponding to the Lys173Arg substitution. The allele frequency of these sequence variants in a normal Swedish population was found to be 6.8% for the H1.2 GCC,GTC shift, indicating that this is a relatively frequent polymorphism. The AAA,AGA codon change in H1.4 was detected only in Raji cells and was not present in a normal population or in six other cell lines derived from individuals suffering from Burkitt's lymphoma. The significance of these sequence variants is unclear, but increasing evidence indicates that minor sequence variations in linker histones may change their binding characteristics, influence chromatin remodeling, and specifically affect important cellular functions. [source] Superantigen gene profile diversity among clinical group A streptococcal isolatesFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2008Linda Maripuu Abstract This study examines the diversity of superantigen gene profiles between and within emm -genotypes of 92 clinical group A streptococcal isolates (30 STSS, 24 sepsis, 25 erysipelas, and 12 tonsillitis) collected in Sweden between 1986 and 2001. The emm- genotype and the distribution of smeZ, speG, speJ, speA, speC, speH, speI, speK/L, speL/M, speM, and ssa genes, and the smeZ allelic variant were determined using PCR and DNA sequencing. Forty-five emm1 isolates revealed 10 superantigen gene profiles. One profile dominated and was identified in 22 isolates collected over 14 years. The results indicate that a selective advantage maintained this genotype in circulation. The superantigen content among the emm1 isolates ranged from three to seven, with smeZ-1, speG, and speA present in all but one profile. The 47 isolates of 27 other emm -genotypes exhibited 29 superantigen gene profiles. Thus, the distribution of superantigen genes was highly variable within isolates regardless of emm -genotype. Two novel emm1 subtypes and 14 novel smeZ allelic variants were identified. The 22 smeZ alleles were generally linked to the emm -genotype. The results of the investigation show that superantigen gene profiling is useful for tracking spread of clones in the community. [source] Organization of butyrate synthetic genes in human colonic bacteria: phylogenetic conservation and horizontal gene transferFEMS MICROBIOLOGY LETTERS, Issue 2 2007Petra Louis Abstract Butyrate producers constitute an important bacterial group in the human large intestine. Butyryl-CoA is formed from two molecules of acetyl-CoA in a process resembling ,-oxidation in reverse. Three different arrangements of the six genes coding for this pathway have been found in low mol% G+C-content Gram-positive human colonic bacteria using DNA sequencing and degenerate PCR. Gene arrangements were strongly conserved within phylogenetic groups defined by 16S rRNA gene sequence relationships. In the case of one of the genes, encoding ,-hydroxybutyryl-CoA dehydrogenase, however, sequence relationships were strongly suggestive of horizontal gene transfer between lineages. The newly identified gene for butyryl-CoA CoA-transferase, which performs the final step in butyrate formation in most known human colonic bacteria, was not closely linked to these central pathway genes. [source] DNA Sensing Using Nanocrystalline Surface-Enhanced Al2O3 Nanopore SensorsADVANCED FUNCTIONAL MATERIALS, Issue 8 2010Bala Murali Venkatesan Abstract A new solid-state, Al2O3 nanopore sensor with enhanced surface properties for the real-time detection and analysis of individual DNA molecules is reported. Nanopore formation using electron-beam-based decomposition transforms the local nanostructure and morphology of the pore from an amorphous, stoichiometric structure (O to Al ratio of 1.5) to a heterophase crystalline network, deficient in O (O to Al ratio of ,0.6). Direct metallization of the pore region is observed during irradiation, thereby permitting the potential fabrication of nanoscale metallic contacts in the pore region with application to nanopore-based DNA sequencing. Dose-dependent phase transformations to purely , and/or ,-phase nanocrystallites are also observed during pore formation, allowing for surface-charge engineering at the nanopore/fluid interface. DNA transport studies reveal an order-of-magnitude reduction in translocation velocities relative to alternate solid-state architectures, accredited to high surface-charge density and the nucleation of charged nanocrystalline domains. The unique surface properties of Al2O3 nanopore sensors make them ideal for the detection and analysis of single-stranded DNA, double-stranded DNA, RNA secondary structures, and small proteins. These nanoscale sensors may also serve as useful tools in studying the mechanisms driving biological processes including DNA,protein interactions and enzyme activity at the single-molecule level. [source] Relevance of translocation type in myxoid liposarcoma and identification of a novel EWSR1-DDIT3 fusionGENES, CHROMOSOMES AND CANCER, Issue 11 2007B. Bode-Lesniewska The clinical course of myxoid/round cell liposarcoma (MRCL) is characterized by frequent local recurrences and metastases at unusual sites. MRCLs carry specific translocations, t(12;16) or rarely t(12;22), linking the FUS or the EWSR1 gene with the DDIT3 gene, respectively. Nine FUS/DDIT3 and three EWSR1/DDIT3 variants of fusion transcripts have been described thus far. In search of prognostic markers for MRCL, we analyzed the translocation types of 31 patients and related them to the event free and overall survival. Using break-apart FISH and RT-PCR combined with DNA sequencing, we detected FUS/DDIT3 fusions in 30 sarcomas, while an EWSR1/DDIT3 translocation was identified in one tumor. FUS/DDIT3 type II (exons 5-2) was most commonly detected (20 cases), followed by type I (7-2) (7 cases) and type III (8-2) (3 cases). A single tumor carrying a t(12;22) translocation expressed a hitherto unknown EWSR1-DDIT3 fusion transcript (13-3) linking the complete RNA-binding domain of EWSR1 with a short piece of the 5,-UTR and the entire open reading frame of the DDIT3 gene. Interestingly, five of six patients with type I (7-2) FUS/DDIT3 fusions displayed local recurrences and/or metastatic spread within the first 3 years, generally requiring chemotherapeutical treatment (median disease-free survival 17 months). In contrast, 9 of 13 patients with type II FUS/DDIT3 translocations remained at 3 years disease-free (median disease-free survival 75 months). Since the total number of patients is still limited, further studies are required to verify a putative association of type I FUS/DDIT3 -fusion transcripts with a prognosis of MRCL. © 2007 Wiley-Liss, Inc. [source] Use of combined molecular biomarkers for prediction of clinical outcomes in locally advanced tonsillar cancers treated with chemoradiotherapy aloneHEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 1 2009Yih-Lin Chung MD Abstract Background. Environmental exposures to tobacco, alcohol, human papillomavirus (HPV) and/or Epstein-Barr virus (EBV), all of which can perturb multiple cell cycle proteins or tumor suppressors, have been implicated in the pathogenesis of different subsets of head and neck cancers. The aim of this study was to investigate to which extent the virus infection by itself, and/or the altered cell cycle proteins, contributes to prognosis in locally advanced tonsillar squamous cell carcinomas (TSCCs) treated with concurrent chemoradiotherapy (CCRT) alone. Methods. Serial tumor tissue arrays from archival samples were tested for the presence of HPV genome integration or EBV episome by means of DNA sequencing, real-time polymerase chain reaction (PCR), and in situ hybridization. Alterations of cell cycle proteins (p53, pRb, and p21) were evaluated by immunohistochemical staining. The association of viral presence with altered cell cycle proteins was correlated to clinical outcomes. Results. Of the 46 patients with the same T2N2bM0 stage IVA among consecutive patients with TSCC, 23 (50%) had integrated HPV DNA and only 1 (2%) had EBV episome. The HPV types detected were almost all HPV-16. A reduced expression pattern of p53, pRb, and p21 was noted in HPV-positive tumors, and the incremental number of alterations in the 3 proteins was significantly associated with HPV-negative tumors. The presence or absence of HPV together with the number of altered expression of the 3 cell cycle markers resulted in further identification of 4 biologically and clinically distinct subgroups with different outcomes after CCRT. Conclusions. Use of combined biomarkers of oncogenic HPV and tumor suppressors of p53, pRb, and p21 in advanced TSCC provides prognostic molecular classification superior to the TNM stage system and identifies low-risk patients for organ preservation by CCRT alone and high-risk patients who might benefit from planned tonsillectomy and neck dissection before or after CCRT. © 2008 Wiley Periodicals, Inc. Head Neck, 2009 [source] Pseudomigraine With Lymphocytic Pleocytosis: A Calcium Channelopathy?HEADACHE, Issue 8 2003Clinical Description of 10 Cases, Genetic Analysis of the Familial Hemiplegic Migraine Gene CACNA1A Objective.,To report the clinical findings of 10 patients diagnosed with pseudomigraine with lymphocytic pleocytosis and the results of mutational analysis of the CACNA1A gene in 8 of these patients. Background.,Pseudomigraine with lymphocytic pleocytosis, also referred to as headache with neurologic deficits and cerebrospinal fluid lymphocytosis (HaNDL), is characterized by episodic transient neurologic dysfunction associated with moderate to severe headache and cerebrospinal fluid lymphocytic pleocytosis. Episodes are recurrent and the condition is self-limiting. The etiology of this sporadic condition remains unknown, but the episodic nature and its ability to be triggered by angiography is somewhat reminiscent of the phenotypic features of familial hemiplegic migraine, a condition caused by mutations in the CACNA1A gene. Design/Methods.,Utilizing retrospective chart review, we describe the clinical features of pseudomigraine with lymphocytic pleocytosis in 10 patients. Whole blood was taken from 8 patients (2 were lost to follow-up) and used for DNA testing. The CACNA1A gene was screened for mutations using heteroduplex analysis and direct DNA sequencing. Results.,Clinical features of pseudomigraine with lymphocytic pleocytosis included transient episodes of weakness, sensory and visual symptoms, aphasia, and confusion lasting minutes up to 4 hours. Sensory symptoms, typically affecting the face and arm, were the most common presentation. Localization of symptoms did not conform to vascular territories. Headache was typically throbbing and most often bilateral. Genetic analysis did not identify any mutations in the CACNA1A gene. Conclusions.,Similarities between familial hemiplegic migraine and pseudomigraine with lymphocytic pleocytosis include recurrent headache with reversible neurologic deficit, cerebrospinal fluid lymphocytic pleocytosis, and triggers such as angiography. Even so, heteroduplex analysis and DNA sequencing failed to identify any sporadic mutations or shared polymorphisms in the exons or the intron/exon boundaries of the CACNA1A gene. These results do not support a role of the CACNA1A gene in the etiology of pseudomigraine with lymphocytic pleocytosis. [source] Distinctiveness of the cagA Genotype in Children and Adults with Peptic Symptoms in South ChinaHELICOBACTER, Issue 4 2009Juan Li Abstract Background:,Helicobacter pylori infection is different between children and adults, not only in infection rate but also in virulence genotypes. However, the 3, region of CagA, important in stomach carcinogenesis, still remains unclear in children. The present study aims to compare the frequency of cagA and the distribution of its subtypes between children and adults in South China. Materials and Methods:, One hundred and twenty-eight children and 99 adults with peptic symptoms were enrolled in our research. Histology, rapid urease test, and real-time polymerase chain reaction (PCR) assay were used to diagnose H. pylori infection. vacA s1 was detected by real-time PCR, and EPIYA motifs in the 3, region of CagA by conventional PCR and DNA sequencing. Results:,H. pylori infection was diagnosed in 53 children and 62 adults. vacA s1 was identified in 90.6% and 91.9% of infected children and adults, respectively. Furthermore, cagA was identified in 73.6% and 82.3% of infected children and adults, respectively. No patient with multiple cagA subtypes was observed. A higher prevalence of more virulent cagA genotype was found in children compared to adults (p < .05). Thirty-eight of 39 (97.4%) cagA -positive children were found to have EPIYA-ABD and only one (2.6%) with EPIYA-ABC. In adults, four types of EPIYA motifs , ABC (29.4%), ABD (64.7%), ABAB (2%), and AAD (3.9%) , were identified, and the ABD type was found more commonly in severe diseases, such as atrophic gastritis (53.3%) and gastric cancer (71.4%). Conclusion:,cagA genotypes in children and in adults are different, and EPIYA-ABD may have potential clinical implication in the development of gastric cancer in South China. [source] Epigenetic and genetic alterations of PTEN in hepatocellular carcinomaHEPATOLOGY RESEARCH, Issue 5 2007Li Wang Aim:, To investigate the roles of epigenetic and genetic alterations of the phosphatase and tensin homologue on chromosome 10 gene (PTEN) in carcinogenesis and the development of hepatocellular carcinomas (HCC). Methods:, A total of 56 cases of HCC tissues and six liver cell lines were studied for the expression of PTEN by immunohistochemistry and Western blot analysis. The PTEN gene mutations in exon5 and exon8 were detected by a combination of single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. Methylation-specific PCR (MSP) was used to identify PTEN promoter methylation. Results:, Of the 56 cases of HCC, 24 (42.9%) expressed the PTEN protein. All surrounding liver tissues of the hepatoma (32 cases) were positive for PTEN. Of the six cell lines, three liver cancer cell lines showed a low expression of PTEN. Five mutations of 56 HCC samples were detected. All of them were located at intron4. No mutation was found in exon5 and exon8. After MSP analysis, we found nine cases of PTEN promoter methylation in 56 specimens (16.1%). However, no CpG island of PTEN was found to be methylated in all six liver cell lines. Conclusion:, The level of PTEN protein was altered in part of the HCC. The downregulation of PTEN expression may not be mainly associated with the PTEN mutations, but partly due to PTEN promoter methylation and other epigenetic regulation. [source] | ||||||||||||||||||||||||||||||||||||||||||||||