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Restriction Fragment Length Polymorphism (restriction + fragment_length_polymorphism)
Kinds of Restriction Fragment Length Polymorphism Terms modified by Restriction Fragment Length Polymorphism Selected AbstractsAssociation of ABCB1 genetic variants 3435C>T and 2677G>T to ABCB1 mRNA and protein expression in brain tissue from refractory epilepsy patientsEPILEPSIA, Issue 9 2008Igor Mosyagin Summary Purpose: There is evidence from studies in rodents that P-glycoprotein (P-gp) overexpression is implicated in the causation of refractory epilepsy. Genetic variants in the human ABCB1 (MDR1) gene were shown to affect the expression levels of the transporter in various tissues and to be associated with refractory epilepsy. However, the effect of the genetic variants on the P-gp level in epileptogenic brain tissue is poorly investigated. In the present study, we examined the impact of putatively functional polymorphisms 3435C>T and 2677G>T in the ABCB1 gene on the ABCB1 mRNA expression and P-gp content in human brain tissue from epileptogenic foci of the patients with refractory epilepsy. Methods: Fresh brain tissue specimens were obtained from therapy-refractory epilepsy patients during neurosurgery of the epileptogenic focus. We determined the ABCB1 mRNA expression in 23 samples using 5, exonuclease-based real-time polymerase chain reaction (PCR) as well as the P-gp content in 32 samples determined by immunohistochemistry, genotyping was performed by PCR/restriction fragment length polymorphism (RFLP). Results: There was lack of association of 3435C>T and 2677G>T as well as diplotype configurations on ABCB1 mRNA expression and P-gp content in epileptogenic brain tissues. Conclusions: We cannot exclude an association of ABCB1 variants on P-gp function, but our results suggest that brain ABCB1 mRNA and protein expression is not substantially influenced by major ABCB1 genetic variants thus explaining in part results from case-control studies obtaining lack of association of ABCB1 polymorphisms to the risk of refractory epilepsy. [source] Functional polymorphism of the myeloperoxidase gene (G-463A) in depressive patientsACTA NEUROPSYCHIATRICA, Issue 5 2010Piotr Ga, ecki Ga,ecki P, Florkowski A, Bobi,ska K, ,migielski J, Bie,kiewicz M, Szemraj J. Functional polymorphism of the myeloperoxidase gene (G-463A) in depressive patients. Objective: Myeloperoxidase (MPO) is an enzyme involved in the production of hypochloric acid as well as other reactive oxygen species. This enzyme plays a significant role in inflammatory processes. In view of the observed associations between depression and such inflammatory processes, as well as of the reports that confirm the presence of oxidative stress in depression, this study was designed to assess the correlation, if any, between the single nucleotide polymorphism G-463A of the MPO gene and the risk of recurrent depressive disorders (DD). Methods: The study was carried out in a group of 149 patients with recurrent DD and 149 healthy control subjects. Genotyping was performed by PCR/restriction fragment length polymorphism. Results: A comparison between healthy controls and depressive patients showed a statistically significant difference in genotype distribution and allele frequency in the studied groups. Genotype distribution and allele frequency did not correlate with clinical variables of the patients. Conclusion: The obtained results of the study allow us to draw a cautious conclusion about the role of the analysed G-463A MPO polymorphism in recurrent DD development, which, however, requires eventual confirmation in further studies. [source] Occupational tuberculosis following extremely short exposureTHE CLINICAL RESPIRATORY JOURNAL, Issue 1 2009Zaza Kamper-Jørgensen Abstract Introduction:, Transmission of Mycobacterium tuberculosis (MT) in most cases requires extended exposure. Objectives:, To document that MT transmission may occur even after very short exposure. Material and Methods:, All first-time culture-confirmed tuberculosis (TB) cases in Denmark have since 1992 been subjected to genotyping, using the IS6110 -Restriction Fragment Length Polymorphism (RFLP) technique. A young nurse with no risk factors developed pulmonary TB: the DNA pattern of her MT strain was compared to The Danish TB Subtyping Database, comprising >6000 DNA patterns from TB patients nationwide. Results:, Only one single MT DNA pattern matched the DNA profile of the isolate from the nurse. The pattern originated from a patient shortly admitted to the department where she worked at the time. MT transmission had occurred in spite of very short exposure. Conclusion:, By adding modern molecular epidemiological methods to traditional epidemiological surveys, a more detailed picture of MT-transmission pathways can be obtained, showing that MT transmission can occur even after extremely short exposure. This stresses the necessity for adequate respiratory protection among hospital staff taking care of patients with pulmonary symptoms suspected for TB. Please cite this paper as: Kamper-Jørgensen Z, Lillebaek T and Andersen ÅB. Occupational tuberculosis following extremely short exposure. The Clinical Respiratory Journal 2009; 3: 55,57. [source] A novel mutation in the ATP2C1 gene is associated with Hailey,Hailey disease in a Chinese familyINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 1 2009Zhou Jiang Liu MD Background, A three-generation Chinese family with Hailey,Hailey disease (HHD) was identified and characterized. The proband developed HHD with severe recurrent blisters and crusted erosions involving the body folds. Skin biopsy studies showed epidermal hyperkeratosis and defects in cell-to-cell adhesion. Three other members in the family were also affected with HHD and had the same clinical manifestations. The purpose of this study was to identify the pathogenic gene or mutation in the family. Methods, All exons and exon,intron boundaries of ATP2C1 were polymerase chain reaction (PCR) amplified and sequenced with DNA samples from the proband. Restriction fragment length polymorphism (RFLP) analysis for the intron 23,exon 24 boundary of ATP2C1 was performed in all family members and in 100 normal control subjects. Results, A novel 2-bp deletion (c.2251delGT) was detected in exon 24 of the ATP2C1 gene. The mutation was present in the three other affected family members and in two asymptomatic young carriers, but not in the other normal family members or the 100 normal controls. The mutation resulted in a frameshift change and led to the formation of a premature termination codon (PTC) four amino acid residues downstream from the sixth transmembrane domain. Conclusions, Our results indicate that the novel c.2251delGT (p.V751fs) mutation in the ATP2C1 gene is responsible for HHD in this Chinese family. This study expands the spectrum of ATP2C1 mutations associated with HHD. [source] No association of SUMO4 M55V with autoimmune diabetes in Asian-Indian patientsINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 2 2007S. K. Sedimbi Summary Autoimmune diabetes [type 1 diabetes mellitus (T1DM), latent autoimmune diabetes in adults (LADA) and part of malnutrition-related diabetes] has been shown to have genetic predisposition. Studies in IDDM 5 have lead to the discovery of a novel polymorphism 163 A,G, of SUMO4 (small ubiquitin-related modifier) gene, associated with risk to T1DM in Asians, but not in Caucasians. We studied patients with T1DM (n = 134), patients with LADA (n = 101), patients with malnutrition-modulated diabetes mellitus (n = 66) and patients with fibrocalculous pancreatic diabetes (n = 43) and healthy controls subjects (n = 114) from Cuttack, India. Polymerase chain reaction,sequence-specific primer (PCR-SSP) was used to amplify the 163 A,G sequences. Restriction fragment length polymorphism (RFLP) was performed using restriction enzyme Taq I (PCR-RFLP). Differences in the allelic frequencies of the A and the G alleles were tested statistically using Fisher's exact test or chi-squared test wherever appropriate. P -values were considered significant when equal to or less than 0.05. No significant association was detected between SUMO4 M55V and T1DM susceptibility in Asian-Indians. Comparison of the A and G alleles with HLA DR3-DR4 did not result in any significant P -values. No significant association was found between SUMO4 M55V and LADA or malnutrition-related diabetes mellitus (MRDM). Our results show that Asian-Indians with T1DM are different from other Asian populations. Asian-Indians show more similarity to Caucasians with respect to the association of SUMO4 M55V variant in T1DM. Association studies on Asian-Indian patients with LADA and MRDM showed no significant difference in the presence of the A and the G alleles when compared to healthy controls. [source] Phenotypic, serological and genetic characterization of Flavobacterium psychrophilum strains isolated from salmonids in ChileJOURNAL OF FISH DISEASES, Issue 4 2009S Valdebenito Abstract Characterization of 20 Flavobacterium psychrophilum strains isolated from farmed Atlantic salmon and rainbow trout in Chile was done using phenotypic, antigenic and genetic techniques. Experimental infections were also performed to assess the virulence of two representative isolates and of the type strain. Biochemical and physiological analyses showed that Chilean F. psychrophilum strains, regardless of the host species, constitute a phenotypically very homogeneous group matching with previous descriptions of this pathogen. However, serological assays indicated the existence of antigenic heterogeneity with four patterns of serological reactions. The first group contained most (14 of 20) of the F. psychrophilum isolates showing cross-reaction with the antisera obtained against Atlantic salmon and rainbow trout isolates. Group 2 corresponded to four other rainbow trout isolates (1658, 1731, 1762 and 29009) that did not agglutinate with anti-1150 serum. Two minor serological groups were identified for the remaining isolates (Groups 3 and 4). Marked homogeneity was also revealed by genetic studies including 16S rRNA alleles, random amplified polymorphic DNA and REP-PCR showing that a major genetic group of F. psychrophilum may be dominant in disease outbreaks in farms. Restriction fragment length polymorphism of PCR analysis showed that gyrase genotypes B-S or B-R were found in Chilean isolates from rainbow trout and Atlantic salmon, whereas genotype A was not found. Virulence assays using Atlantic salmon indicated no relationship between the degree of pathogenicity and the host origin of the F. psychrophilum strains. [source] Viruses Associated with Cassava Mosaic Disease in Senegal and Guinea ConakryJOURNAL OF PHYTOPATHOLOGY, Issue 2 2004G. Okao-Okuja Abstract A survey in Senegal and Guinea Conakry established the presence and incidence of cassava mosaic virus disease (CMD) in both countries. CMD occurred in all the fields surveyed, although its incidence was higher in Senegal (83%) than in Guinea (64%). Populations of the whitefly vector, Bemisia tabaci, were low in both countries averaging 1.7 adults per shoot in Guinea and 3.2 in Senegal. Most infections were attributed to the use of infected cuttings, 86 and 83% in Senegal and Guinea, respectively, and there was no evidence of rapid current-season, whitefly-borne infection at any of the sampled locations. Disease severity was generally low in the two countries and averaged 2.5 in Guinea and 2.3 in Senegal. No plants with unusually severe CMD symptoms characteristic of the CMD pandemic in East and Central Africa were observed. Restriction fragment length polymorphism (RFLP)-based diagnostics revealed that African cassava mosaic virus (ACMV) is exclusively associated with CMD in both the countries. Neither East African cassava mosaic virus (EACMV), nor the recombinant Uganda variant (EACMV-UG2) was detected in any sample. These survey data indicate that CMD could be effectively controlled in both countries by phytosanitation, involving the use of CMD-free planting material and the removal of diseased plants. [source] A non-syndromic hearing loss caused by very low levels of the mtDNA A3243G mutationACTA NEUROLOGICA SCANDINAVICA, Issue 1 2004M. Mancuso We described a patient with progressive non-syndromic hearing loss (NSHL) harboring the A3243G mutation in the mitochondrial DNA (mtDNA). Muscle biopsy showed scattered ragged-red, cytochrome c oxidase negative fibers, whereas the biochemical analysis of the mitochondrial respiratory chain complexes was normal. Restriction fragment length polymorphism (RFLP) analysis showed A3243G mtDNA transition, present at very low in patient's muscle (3%) and in urinary sediments (1%), and not detectable in blood and buccal mucosa. The patient was submitted to a bilateral cochlear implantation with post-operative excellent hearing and communicative outcomes. Our findings indicate that A3243G mutation may be responsible both for SHL and NSHL, may be depending on the levels of mutated mtDNA. Patients with hearing loss due to mtDNA mutations should be considered as good candidates for cochlear implantation. [source] Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probesJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2000G. Frech A total of 65 epidemiologically unrelated tetracycline-resistant isolates of the six Salmonella enterica subsp. enterica (Salm.) serovars Dublin, Choleraesuis, Typhimurium, Enteritidis, Hadar and Saintpaul were investigated for the presence of tetracycline resistance genes. For this, specific gene probes of the tetracycline resistance genes (tet) of the hybridization classes A, B, C, D, E and G were constructed by cloning PCR-amplified internal segments of the respective tet structural genes. These gene probes were sequenced and used in hybridization experiments with plasmid DNA or endonuclease digested whole cell DNA as targets. Only tet(A) genes were detected on plasmids in all Salm. Dublin isolates as well as in single isolates of Salm. Choleraesuis and Salm. Typhimurium. Genes of the hybridization classes B, C, D and G, but also in some cases those of class A, were located in the chromosomal DNA of the corresponding Salmonella isolates. Restriction fragment length polymorphisms (RFLPs) of tet gene carrying fragments were detected in chromosomally tetracycline-resistant isolates. These RFLPs might represent valuable additional tools for the identification and characterization of tetracycline-resistant Salmonella isolates. [source] Apolipoprotein AI-CIII-AIV gene cluster polymorphisms in relation to cholesterol gallstoneJOURNAL OF DIGESTIVE DISEASES, Issue 1 2007You Gui YAO OBJECTIVE: To investigate the frequency of variants at Xmn I, Msp I sites of apolipoprotein (Apo), A I-CIII-AIV gene cluster, and its relation to cholesterol gallstones in Chinese patients. METHODS: Restriction fragment length polymorphisms (RFLP) at Xmn I, Msp I sites of ApoAI-CIII-AIV gene cluster were studied using a polymerase chain reaction (PCR) in 161 patients with cholesterol gallstones and 94 healthy subjects from a Chinese population in Sichuan Province. RESULTS: In both the cholesterol gallstone group and the healthy control group, X1 and M1 alleles were the major alleles and homozygous X1X1 and M1M1 genotypes were the most frequent. However, the frequency of X2 allele mutation in female patients of the cholesterol gallstones group was significantly higher than that in women in the healthy control group (P < 0.05), but no difference was found in the frequency of M2 alleles mutation (P > 0.05). CONCLUSION: The data showed that Xmn I RFLP of ApoAI-CIII-AIV gene cluster is associated to some extent with cholesterol gallstones in female Chinese patients. [source] Molecular characterization of the fish-pathogenic fungus Aphanomyces invadansJOURNAL OF FISH DISEASES, Issue 5 2003J H Lilley Abstract Aphanomyces invadans (Saprolegniaceae) is a peronosporomycete fungus associated with the serious fish disease, epizootic ulcerative syndrome (EUS), also known as mycotic granulomatosis. In this study, interspecific relationships were examined between A. invadans isolates and other aquatic animal pathogenic Saprolegniaceae, and saprophytic Saprolegniaceae from EUS-affected areas. Restriction fragment length polymorphisms and sequences of ribosomal DNA confirmed that A. invadans is distinct from all other species studied. A sequence from the internal transcribed spacer region ITS1, unique to A. invadans, was used to design primers for a PCR-based diagnostic test. Intraspecific relationships were also examined by random amplification of polymorphic DNA using 20 isolates of A. invadans from six countries. The isolates showed a high degree of genetic homogeneity using 14 random ten-mer primers. This provides evidence that the fungus has spread across Asia in one relatively rapid episode, which is consistent with reports of outbreaks of EUS. Physiological distinctions between A. invadans and other Aphanomyces species based on a data set of 16 growth parameters showed remarkable taxonomic congruence with the molecular phylogeny. [source] Evolutionary 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] A molecular diagnostic for identifying central African forest artiodactyls from faecal pelletsANIMAL CONSERVATION, Issue 1 2010S. Ntie Abstract Small to medium-sized central African forest artiodactyls constitute a diverse yet heavily hunted group composed primarily of species within the genera Cephalophus, Neotragus, Tragelaphus and Hyemoschus. Of these genera, Cephalophus is the richest with as many as seven sympatric species known to occur in central African forests. However, differentiating species from their faeces or from tissue where the whole carcass is unavailable is very difficult. In order to develop a robust molecular diagnostic for species identification, a database of mitochondrial cytochrome b (553 bp) and control region (,675 bp) sequences was compiled from all forest Cephalophus species and other similarly sized, sympatric Tragelaphus, Neotragus and Hyemoschus species. Reference phylogenies from each marker were then used to recover the identity of sequences obtained from unknown faecal samples collected in the field. Results were then compared to determine which region best recovered species identity with the highest statistical support. Restriction fragment length polymorphisms (RFLPs) were also assessed as an alternative method for rapid species identification. Of the methods examined, tree-based analyses built on a geographically comprehensive database of control region sequences was the best means of reliably recovering species identity from central African duikers. However, three sister taxa appear indistinguishable (Cephalophus callipygus, Cephalophus ogilbyi and Cephalophus weynsi) and not all species were monophyletic. This lack of monophyly may be due to incomplete lineage sorting commonly observed in recently derived taxa, hybridization or the presence of nuclear translocated copies of mitochondrial DNA. The high level of intra-specific variation and lack of robust species-specific diagnostic sites made an RFLP-based approach to duiker species identification difficult to implement. The tree-based control region diagnostic presented here has many important applications including fine-scale mapping of species distributions, identification of confiscated tissue and environmental impact assessments. [source] SPR1 gene near HLA-C is unlikely to be a psoriasis susceptibility geneEXPERIMENTAL DERMATOLOGY, Issue 3 2003Y. T. Chang Abstract:, Although genetics analyses have identified the HLA-Cw6 allele to be the major risk allele for psoriasis vulgaris (PV) in many racial groups, it has been proposed that other putative genes near the HLA-C locus are involved in PV susceptibility and that the association of Cw6 is a result of linkage disequilibrium. The SPR1 gene, a predicted gene located 128 kb telomeric to the HLA-C locus, is considered to be one potential candidate gene of PV. Until now, no association study of the SPR1 gene has been conducted on psoriasis patients. We investigated the SPR1 gene for disease association by direct sequencing of the SPR1 gene in 116 Chinese patients with PV and 116 normal subjects. Genotyping for HLA-Cw6 was also carried out using polymerase chain reaction/restriction fragment length polymorphism. Significant increase of the HLA-Cw6 allele was found in psoriasis patients (32.8% vs. 13.8%, P = 0.001). We found that the SPR1 gene is a highly polymorphic gene containing 13 single nucleotide polymorphisms (SNPs), two of which have not been previously reported, and four SNPs cause amino acid change. No significantly different allelic distribution of 13 SPR1 SNPs could be found between the patients with PV and controls after correction for multiple testing. If the frequencies of SPR1 SNPs were compared between the early onset psoriatics and control subjects, early onset patients were more likely to have G allele at position 988 (60% vs. 35.3%, P = 0.001). However, the significance disappeared upon stratification for the Cw6 status. Haplotype-based association analysis showed two susceptibility haplotypes (types 8 and 19) in early onset psoriasis patients. Nonetheless, the significance also disappeared after stratification of the Cw6 status. Our results suggest that HLA-Cw6 remains the major risk allele in Chinese psoriatics, and that the SPR1 gene might not play an important role in the causation of PV. [source] Phylogenetic Reanalysis of the Saudi Gazelle and Its Implications for ConservationCONSERVATION BIOLOGY, Issue 4 2001Robert L. Hammond The Saudi gazelle ( Gazella saudiya) was endemic to the Arabian peninsula but is now considered extinct in the wild and is potentially a candidate for captive breeding and reintroduction. Using 375 base pairs of mitochondrial DNA (mtDNA) cytochrome b gene derived from museum samples collected from the wild prior to the presumed extinction of this species, we show that G. saudiya is the sister taxon of the African dorcas gazelle ( G. dorcas). Reciprocal monophyly of G. saudiya mtDNA haplotypes with G. dorcas, coupled with morphological distinctiveness, suggests that it is an evolutionarily significant unit. These data indicate that captive populations identified previously as potential sources of G. saudiya for captive breeding appear incorrectly designated and are irrelevant to the conservation of G. saudiya. The polymerase chain reaction,restriction fragment length polymorphism ( PCR-RFLP) analysis of several private collections of living gazelles in Saudi Arabia provides no evidence for the survival of G. saudiya. We recommend that field surveys be undertaken to establish whether G. saudiya is indeed extinct in the wild and that other private collections within the Arabian peninsula be screened genetically. We urge caution when captive animals of unknown provenance are used to investigate the phylogenetics of cryptic species groups. Resumen: La identificación de poblaciones taxonómicamente apropiadas de especies en peligro para programas de reproducción en cautiverio y de reintroducción es fundamental para su éxito. La Gacela Saudi (Gazella saudiya) fue endémica a la península de Arabia pero ahora está considerada como extinta en su medio y es un candidato potencial para reproducción en cautiverio y reintroducción. Utilizando 375 pares de bases de ADN mitocondrial (ADNmt) del gene citocromo b derivados de muestras de museos colectadas en el medio silvestre antes de la extinción de la especie, mostramos que G. saudiya es el taxón hermano de la gacela dorcas africana (G. dorcas). La monofilia recíproca de haplotipos de ADNmt de G. saudiya con G. dorcas, aunado a diferencias morfológicas, sugiere que es una unidad evolutiva significativa. Estos datos indican que las poblaciones cautivas identificadas previamente como fuente potencial de G. saudiya para reproducción en cautiverio están incorrectamente identificadas y son irrelevantes para la conservación de G. saudiya. El análisis PCR-RFLP de varias colecciones privadas de gacelas vivas en Arabia Saudita no proporcionan evidencia para la supervivencia de G. saudiya. Recomendamos que se realicen muestreos en el campo para establecer si en efecto G. saudiya está extinta en su hábitat y que se examinen genéticamente las otras colecciones privadas en la península Arábiga. Recomendamos precaución cuando animales cautivos de origen desconocido son utilizados para investigar la filogenia de grupos de especies crípticas. [source] Lack of association between serum adiponectin levels and the Pro12Ala polymorphism in Asian IndiansDIABETIC MEDICINE, Issue 4 2007V. Radha Abstract Aims, The aim of the study was to investigate the association of serum adiponectin levels with the Pro12Ala polymorphism of the peroxisome proliferator activated receptor-, (PPARG) gene in Asian Indians. Methods, We selected 400 diabetic subjects, 200 with the Pro12Pro genotype (100 male and 100 female) and 200 with the Pro12Ala genotype (100 male and 100 female) and 400 age- and sex-matched normal glucose tolerance subjects with similar genotype profiles from the Chennai Urban Rural Epidemiology Study. Fasting serum adiponection levels were measured using radioimmunoassay. The Pro12Ala polymorphism was genotyped by PCR,restriction fragment length polymorphism using BstUI. Results, All clinical and biochemical parameters were similar in the subjects with the Pro12Pro and Pro12Ala genotypes. There was no significant difference in serum adiponectin values between subjects with the Pro12Pro and Pro12Ala genotypes (males 5.4 vs. 5.8 µg/ml, P = 0.546; females 6.9 vs. 7.2 µg/ml, P = 0.748). Adiponectin values did not differ among these two genotypes even when categorized based on their diabetes status (normal glucose tolerance Pro12Pro 7.9 vs. Pro12Ala 7.7 µg/ml, P = 0.994; diabetes Pro12Pro 4.7 vs. Pro12Ala 5.4 µg/ml, P = 0.622). Conclusion, The Pro12Ala polymorphism of the PPARG gene is not associated with serum adiponectin levels in Asian Indians. [source] Islet autoimmunity and genetic mutations in Chinese subjects initially thought to have Type 1B diabetesDIABETIC MEDICINE, Issue 1 2006D. Zhang Abstract Aims To explore the contribution of islet autoimmunity and genetic mutations in Chinese patients initially thought to have Type 1B diabetes. Methods A group of 33 Chinese patients with newly diagnosed Type 1B diabetes, were identified by the absence of autoantibodies to glutamic acid decarboxylase (GAD), IA-2, insulin, thyroid globulin or thyroid peroxidase, or high-risk HLA-DQ haplotypes. The cohort was further characterized by measurement of autoantibodies to carboxypeptidase H (CPH) and SOX13 using radioligand assays, and testing for genetic mutations associated with MODY3/MODY6 and mitochondrial diabetes. Mutations of HNF-1, (MODY3) and neuroD1/,2 (MODY6) genes were screened using the single-strand conformation polymorphism (SSCP) technique and sequencing. Mitochondrial DNA mutations were analysed with polymerase chain reaction,restriction fragment length polymorphism (PCR-RFLP). Results Within the cohort, we found one patient with a novel mutation, R321H (CGC,CAC) in exon 5 of the HNF-1, gene, one with ND1 mt3316 G,A mutation in mitochondrial DNA, five with Ala45Thr polymorphisms in the neuroD1/,2 gene, and two patients with autoantibodies to SOX13. Conclusions Some of the Chinese patients originally thought to have Type 1B diabetes do have other evidence of islet autoimmunity and genetic mutations involved in the underlying aetiology. This suggests that more rigorous screening for these conditions is needed before classifying subjects as having Type 1B diabetes. [source] Peroxisome proliferator-activated receptor-, co-activator-1, (PGC-1,) gene polymorphisms and their relationship to Type 2 diabetes in Asian IndiansDIABETIC MEDICINE, Issue 11 2005K. S. Vimaleswaran Abstract Aims The objective of the present investigation was to examine the relationship of three polymorphisms, Thr394Thr, Gly482Ser and +A2962G, of the peroxisome proliferator activated receptor-, co-activator-1 alpha (PGC-1,) gene with Type 2 diabetes in Asian Indians. Methods The study group comprised 515 Type 2 diabetic and 882 normal glucose tolerant subjects chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The three polymorphisms were genotyped using polymerase chain reaction,restriction fragment length polymorphism (PCR,RFLP). Haplotype frequencies were estimated using an expectation,maximization (EM) algorithm. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. Results The three polymorphisms studied were not in linkage disequilibrium. With respect to the Thr394Thr polymorphism, 20% of the Type 2 diabetic patients (103/515) had the GA genotype compared with 12% of the normal glucose tolerance (NGT) subjects (108/882) (P = 0.0004). The frequency of the A allele was also higher in Type 2 diabetic subjects (0.11) compared with NGT subjects (0.07) (P = 0.002). Regression analysis revealed the odds ratio for Type 2 diabetes for the susceptible genotype (XA) to be 1.683 (95% confidence intervals: 1.264,2.241, P = 0.0004). Age adjusted glycated haemoglobin (P = 0.003), serum cholesterol (P = 0.001) and low-density lipoprotein (LDL) cholesterol (P = 0.001) levels and systolic blood pressure (P = 0.001) were higher in the NGT subjects with the XA genotype compared with GG genotype. There were no differences in genotype or allelic distribution between the Type 2 diabetic and NGT subjects with respect to the Gly482Ser and +A2962G polymorphisms. Conclusions The A allele of Thr394Thr (G , A) polymorphism of the PGC-1 gene is associated with Type 2 diabetes in Asian Indian subjects and the XA genotype confers 1.6 times higher risk for Type 2 diabetes compared with the GG genotype in this population. [source] In situ measurement of methane fluxes and analysis of transcribed particulate methane monooxygenase in desert soilsENVIRONMENTAL MICROBIOLOGY, Issue 10 2009Roey Angel Summary Aerated soils are a biological sink for atmospheric methane. However, the activity of desert soils and the presence of methanotrophs in these soils have hardly been studied. We studied on-site atmospheric methane consumption rates as well as the diversity and expression of the pmoA gene, coding for a subunit of the particulate methane monooxygenase, in arid and hyperarid soils in the Negev Desert, Israel. Methane uptake was only detected in undisturbed soils in the arid region (,90 mm year,1) and vertical methane profiles in soil showed the active layer to be at 0,20 cm depth. No methane uptake was detected in the hyperarid soils (,20 mm year,1) as well as in disturbed soils in the arid region (i.e. agricultural field and a mini-catchment). Molecular analysis of the methanotrophic community using terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene detected methanotrophs in the active soils, whereas the inactive ones were dominated by sequences of the homologous gene amoA, coding for a subunit of the ammonia monooxygenase. Even in the active soils, methanotrophs (as well as in situ activity) could not be detected in the soil crust, which is the biologically most important layer in desert soils. All pmoA sequences belonged to yet uncultured strains. Transcript analysis showed dominance of sequences clustering within the JR3, formerly identified in Californian grassland soils. Our results show that although active methanotrophs are prevalent in arid soils they seem to be absent or inactive in hyperarid and disturbed arid soils. Furthermore, we postulate that methanotrophs of the yet uncultured JR3 cluster are the dominant atmospheric methane oxidizers in this ecosystem. [source] The role of Variovorax and other Comamonadaceae in sulfur transformations by microbial wheat rhizosphere communities exposed to different sulfur fertilization regimesENVIRONMENTAL MICROBIOLOGY, Issue 6 2008Achim Schmalenberger Summary Sulfonates are a key component of the sulfur present in agricultural soils. Their mobilization as part of the soil sulfur cycle is mediated by rhizobacteria, and involves the oxidoreductase AsfA. In this study, the effect of fertilization regime on rhizosphere bacterial asfA distribution was examined at the Broadbalk long-term wheat experiment, Rothamsted, UK, which was established in 1843, and has included a sulfur-free treatment since 2001. Direct isolation of desulfonating rhizobacteria from the wheat rhizospheres led to the identification of several Variovorax and Polaromonas strains, all of which contained the asfA gene. Rhizosphere DNA was isolated from wheat rhizospheres in plots fertilized with inorganic fertilizer with and without sulfur, with farmyard manure or from unfertilized plots. Genetic profiling of 16S rRNA gene fragments [denaturing gradient gel electrophoresis (DGGE)] from the wheat rhizospheres revealed that the level of inorganic sulfate in the inorganic fertilizer was correlated with changes in the general bacterial community structure and the betaproteobacterial community structure in particular. Community analysis at the functional gene level (asfA) showed that 40% of clones in asfAB clone libraries were affiliated to the genus Variovorax. Analysis of asfAB -based terminal restriction fragment length polymorphism (T-RFLP) fingerprints showed considerable differences between sulfate-free treatments and those where sulfate was applied. The results suggest the occurrence of desulfonating bacterial communities that are specific to the fertilization regime chosen and that arylsulfonates play an important role in rhizobacterial sulfur nutrition. [source] Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acidsENVIRONMENTAL MICROBIOLOGY, Issue 2 2008Minita Shrestha Summary Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH4 into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH4 flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using 13C-labelled CH4 directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to 13CH4 for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms. [source] A methane-driven microbial food web in a wetland rice soilENVIRONMENTAL MICROBIOLOGY, Issue 12 2007Jun Murase Summary Methane oxidation is a key process controlling methane emission from anoxic habitats into the atmosphere. Methanotrophs, responsible for aerobic methane oxidation, do not only oxidize but also assimilate methane. Once assimilated, methane carbon may be utilized by other organisms. Here we report on a microbial food web in a rice field soil driven by methane. A thin layer of water-saturated rice field soil was incubated under opposing gradients of oxygen and 13C-labelled methane. Bacterial and eukaryotic communities incorporating methane carbon were analysed by RNA-stable isotope probing (SIP). Terminal restriction fragment length polymorphism (T-RFLP) and cloning showed that methanotrophs were the most prominent group of bacteria incorporating methane carbon. In addition, a few Myxobacteria -related sequences were obtained from the ,heavy' rRNA fraction. Denaturing gradient gel electrophoresis (DGGE) targeting 18S rRNA detected various groups of protists in the ,heavy' rRNA fraction including naked amoeba (Lobosea and Heterolobosea), ciliates (Colpodea) and flagellates (Cercozoa). Incubation of soil under different methane concentrations in air resulted in the development of distinct protozoan communities. These results suggest that methane carbon is incorporated into non-methanotrophic pro- and microeukaryotes probably via grazing, and that methane oxidation is a shaping force of the microeukaryotic community depending on methane availability. [source] Microbial succession of nitrate-reducing bacteria in the rhizosphere of Poa alpina across a glacier foreland in the Central AlpsENVIRONMENTAL MICROBIOLOGY, Issue 9 2006K. Deiglmayr Summary Changes in community structure and activity of the dissimilatory nitrate-reducing community were investigated across a glacier foreland in the Central Alps to gain insight into the successional pattern of this functional group and the driving environmental factors. Bulk soil and rhizosphere soil of Poa alpina was sampled in five replicates in August during the flowering stage and in September after the first snowfalls along a gradient from 25 to 129 years after deglaciation and at a reference site outside the glacier foreland (> 2000 years deglaciated). In a laboratory-based assay, nitrate reductase activity was determined colorimetrically after 24 h of anaerobic incubation. In selected rhizosphere soil samples, the community structure of nitrate-reducing microorganisms was analysed by restriction fragment length polymorphism (RFLP) analysis using degenerate primers for the narG gene encoding the active site of the membrane-bound nitrate reductase. Clone libraries of the early (25 years) and late (129 years) succession were constructed and representative clones sequenced. The activity of the nitrate-reducing community increased significantly with age mainly due to higher carbon and nitrate availability in the late succession. The community structure, however, only showed a small shift over the 100 years of soil formation with pH explaining a major part (19%) of the observed variance. Clone library analysis of the early and late succession pointed to a trend of declining diversity with progressing age. Presumably, the pressure of competition on the nitrate reducers was relatively low in the early successional stage due to minor densities of microorganisms compared with the late stage; hence, a higher diversity could persist in this sparse environment. These results suggest that the nitrate reductase activity is regulated by environmental factors other than those shaping the genetic structure of the nitrate-reducing community. [source] Vertical profiles of methanogenesis and methanogens in two contrasting acidic peatlands in central New York State, USAENVIRONMENTAL MICROBIOLOGY, Issue 8 2006Hinsby Cadillo-Quiroz Summary Northern acidic peatlands are important sources of atmospheric methane, yet the methanogens in them are poorly characterized. We examined methanogenic activities and methanogen populations at different depths in two peatlands, McLean bog (MB) and Chicago bog (CB). Both have acidic (pH 3.5,4.5) peat soils, but the pH of the deeper layers of CB is near-neutral, reflecting its previous existence as a neutral-pH fen. Acetotrophic and hydrogenotrophic methanogenesis could be stimulated in upper samples from both bogs, and phylotypes of methanogens using H2/CO2 (Methanomicrobiales) or acetate (Methanosarcinales) were identified in 16S rRNA gene clone libraries and by terminal restriction fragment length polymorphism (T-RFLP) analyses using a novel primer/restriction enzyme set that we developed. Particularly dominant in the upper layers was a clade in the Methanomicrobiales, called E2 here and the R10 or fen group elsewhere, estimated by quantitative polymerase chain reaction to be present at ,108 cells per gram of dry peat. Methanogenic activity was considerably lower in deeper samples from both bogs. The methanogen populations detected by T-RFLP in deeper portions of MB were mainly E2 and the uncultured euryarchaeal rice cluster (RC)-II group, whereas populations in the less acidic CB deep layers were considerably different, and included a Methanomicrobiales clade we call E1-E1,, as well as RC-I, RC-II, marine benthic group D, and a new cluster that we call the subaqueous cluster. E2 was barely detectable in the deeper samples from CB, further evidence for the associations of most organisms in this group with acidic habitats. [source] DNA extraction procedure: a critical issue for bacterial diversity assessment in marine sedimentsENVIRONMENTAL MICROBIOLOGY, Issue 2 2006Gian Marco Luna Summary In order to evaluate whether different DNA extraction procedures can affect estimates of benthic bacterial diversity, based on 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) fingerprinting technique, we compared two in situ lysis procedures (a SDS-based protocol and a commercial kit for DNA recovery) and one cell-extraction protocol on a variety of marine sediments. Despite the two in situ lysis procedures resulted in significantly different DNA yields (highest with the SDS in situ lysis), estimates of bacterial diversity provided a not significantly different ribotype richness, as well as similar values of the Shannon-Wiener (H,) and Margalef (d) indices of biodiversity and of evenness (Pielou index, J). Conversely, the cell-extraction procedure for DNA extraction resulted always in a significantly lower ribotype richness and diversity. The analysis of similarities (anosim) among the T-RFLP electropherograms allowed concluding that ribotypes composition did not change significantly using different protocols. However, the analysis of ,-diversity (turnover diversity) revealed that a large number of ribotypes was observed exclusively with one of the three protocols utilized. When unshared ribotypes from in situ lysis and cell extraction were pooled together, total ribotype richness resulted much higher (up to 80%). Our results indicate that estimates of ribotype diversity based on a single protocol of DNA extraction can significantly underestimate the total number of bacterial ribotypes present in the benthic domain. We recommend that future studies will not only integrate different DNA extraction procedures, but also will explore the possibility of integrating two or more different genetic markers in order to increase our ability to detect the actual bacterial diversity in environmental samples. [source] Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary systemENVIRONMENTAL MICROBIOLOGY, Issue 9 2005Anne E. Bernhard Summary Ammonia-oxidizing bacteria (AOB) play an important role in nitrogen cycling in estuaries, but little is known about AOB diversity, distribution and activity in relation to the chemical and physical changes encountered in estuary systems. Although estuarine salinity gradients are well recognized to influence microbial community structure, few studies have examined the influence of varying salinity on the diversity and stability of AOB populations. To investigate these relationships, we collected sediment samples from low-, mid- and high-salinity sites in Plum Island Sound estuary, MA, during spring and late summer over 3 years. Ammonia-oxidizing bacteria distribution and diversity were assessed by terminal restriction fragment length polymorphism (TRFLP) analysis of the ammonia monooxygenase (amoA) gene, and fragments were identified by screening amoA clone libraries constructed from each site. Most striking was the stability and low diversity of the AOB community at the high-salinity site, showing little variability over 3 years. Ammonia-oxidizing bacteria at the high-salinity site were not closely related to any cultured AOB, but were most similar to Nitrosospira spp. Ammonia-oxidizing bacteria at the mid- and low-salinity sites were distributed among Nitrosospira- like sequences and sequences related to Nitrosomonas ureae/oligotropha and Nitrosomonas sp. Nm143. Our study suggests that salinity is a strong environmental control on AOB diversity and distribution in this estuary. [source] Niche heterogeneity determines bacterial community structure in the termite gut (Reticulitermes santonensis)ENVIRONMENTAL MICROBIOLOGY, Issue 7 2005Hong Yang Summary Differences in microenvironment and interactions of microorganisms within and across habitat boundaries should influence structure and diversity of the microbial communities within an ecosystem. We tested this hypothesis using the well characterized gut tract of the European subterranean termite Reticulitermes santonensis as a model. By cloning and sequencing analysis and molecular fingerprinting (terminal restriction fragment length polymorphism), we characterized the bacterial microbiota in the major intestinal habitats , the midgut, the wall of the hindgut paunch, the hindgut fluid and the intestinal protozoa. The bacterial community was very diverse (> 200 ribotypes) and comprised representatives of several phyla, including Firmicutes (mainly clostridia, streptococci and Mycoplasmatales -related clones), Bacteroidetes, Spirochaetes and a number of Proteobacteria, all of which were unevenly distributed among the four habitats. The largest group of clones fell into the so-called Termite group 1 (TG-1) phylum, which has no cultivated representatives. The majority of the TG-1 clones were associated with the protozoa and formed two phylogenetically distinct clusters, which consisted exclusively of clones previously retrieved from the gut of this and other Reticulitermes species. Also the other clones represented lineages of microorganisms that were exclusively recovered from the intestinal tract of termites. The termite specificity of these lineages was underscored by the finding that the closest relatives of the bacterial clones obtained from R. santonensis were usually derived also from the most closely related termites. Overall, differences in diversity between the different gut habitats and the uneven distribution of individual phylotypes support conclusively that niche heterogeneity is a strong determinant of the structure and spatial organization of the microbial community in the termite gut. [source] Novel microbial diversity adherent to plant biomass in the herbivore gastrointestinal tract, as revealed by ribosomal intergenic spacer analysis and rrs gene sequencingENVIRONMENTAL MICROBIOLOGY, Issue 4 2005Ross Larue Summary It is well recognized that a dynamic biofilm develops upon plant biomass in the herbivore gastrointestinal tract, but this component of the microbiome has not previously been specifically sampled, or directly compared with the biodiversity present in the planktonic fraction of digesta. In this study, the digesta collected from four sheep fed two different diets was separated into three fractions: the planktonic phase, and the microbial populations either weakly or tightly adherent to plant biomass. The community DNA prepared from each fraction was then subjected to both ribosomal intergenic spacer analysis (RISA) and denaturing gradient gel electrophoresis (DGGE). Both types of analysis showed that dietary factors influence community structure, and that the adherent fractions produced more complex profiles. The RIS-clone libraries prepared from the planktonic and adherent populations were then subjected to restriction fragment length polymorphism (RFLP) and DNA sequence analyses, which resulted in a far greater degree of discrimination among the fractions. Although many of the sequenced clones from the adherent populations were assigned to various clusters within the low G+C Gram-positive bacteria, the clone libraries from animals consuming an all-grass diet were largely comprised of novel lineages of Clostridium, while in animals consuming the starch-containing diet, Selenomonas and Ruminococcus spp. were the dominant low G+C Gram-positive bacteria. Additionally, the libraries from hay-fed animals also contained clones most similar to asaccharolytic Clostridia, and other Gram-positive bacteria that specialize in the transformation of plant phenolic compounds and the formation of cinnamic, phenylacetic and phenylpropionic acids. These results reveal, for the first time, the phylogeny of adherent subpopulations that specialize in the transformation of plant lignins and other secondary compounds, which potentiate polysaccharide hydrolysis by other members of the biofilm. [source] Diversity of functional genes of methanogens, methanotrophs and sulfate reducers in deep-sea hydrothermal environmentsENVIRONMENTAL MICROBIOLOGY, Issue 1 2005Olivier Nercessian Summary To contribute to the identification of methanogens, methanotrophs and sulfate-reducing bacteria (SRB) in microbial communities from the 13°N (East Pacific Rise) and Rainbow (Mid-Atlantic Ridge) hydrothermal vent fields, we investigated the diversity of mcrA, pmoA and dsrAB genes sequences. Clone libraries were obtained using DNA isolated from fragments of diffuse vents, sediment and in situ samplers. The clones were categorized by restriction fragment length polymorphism, and representatives of each group were sequenced. Sequences were related to that of hyperthermophilic (order Methanopyrales and family Methanocaldococcaceae), thermophilic and mesophilic (family Methanococcaceae) methanogens, thermophilic (proposed genus ,Methylothermus') and mesophilic type I methanotrophs, and hyperthermophilic (order Archaeoglobales), thermophilic (order Thermodesulfobacteriales) and mesophilic (family Desulfobulbaceae) SRB. Several of the obtained sequences were distantly related to the genes of cultivated organisms, providing evidence of the existence of novel lineages in the three functional groups. This study provides for the first time an insight into the diversity of several functional genes of deep-sea hydrothermal system microorganisms. [source] Microsite-dependent changes in methanogenic populations in a boreal oligotrophic fenENVIRONMENTAL MICROBIOLOGY, Issue 11 2003Pierre E. Galand Summary Wetlands, including peatlands, are the main source of natural methane emission. Well-defined fen microsites have different methane emissions rates, but it is not known whether the methane-producing Archaea communities vary at these sites. Possible horizontal variations of communities, in a natural oligotrophic fen, were analysed by characterizing the methanogens from two well-defined microsites: Eriophorum lawn and Hummock. Community structures were studied at two different layers of the fen, showing, respectively, high and low methane production. The structure of methanogen populations was determined using molecular techniques targeting the 16SrRNA gene and combined denaturing gradient gel electrophoresis (DGGE) and restriction fragment length polymorphism (RFLP) analysis. Results subjected to non-metric multidimensional scaling (MDS), diversity indices calculation and phylogenetic analysis revealed that upper layer communities changed with site while deeper layer communities remained the same. Phylogenetic analyses revealed six different clusters of sequences grouping with only two known orders of methanogens. Upper layers of Hummock were dominated by sequences clustering with members of Methanomicrobiales and sequences dominating the upper part of the Eriophorum lawn were related to members of the order Methanosarcinales. Novel methanogenic sequences were found at both sites at both depths. Vegetation characterizing the microsites probably influences the microbial communities in the layers of the fen where methane is produced. [source] | |||||||||||||||||||||||||||||||||||||||||||