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Molecular Identification (molecular + identification)

Distribution by Scientific Domains

Life Sciences	64%
Medical Sciences	17%
Chemistry	12%

Distribution within Life Sciences

Entomology	18%
Applied Microbiology	15%

Selected Abstracts

Multiple Displacement Amplification of Isolated DNA from Human Gallstones: Molecular Identification of Helicobacter DNA by Means of 16S rDNA-Based Pyrosequencing Analysis

HELICOBACTER, Issue 6 2005
Isabelle Nilsson
ABSTRACT Background., Molecular typing of Helicobacter spp. in clinical biopsy specimens has become increasingly important. By means of nested polymerase chain reaction (PCR) amplification and Southern blot analysis of the PCR amplicons, we have shown that Helicobacter spp. DNA is present in human gallstones. In this study we have investigated the possibility of using multiple displacement amplification (MDA) of isolated gallstone DNA and pyrosequencing analysis for the molecular identification of Helicobacter spp. Materials and Methods., DNA isolated from the nucleus of 33 human gallstones and one control strain were used in a MDA assay. Subsequently, pyrosequencing analysis was performed either directly on MDA-DNA using primers flanking the Helicobacter spp. 16S rDNA variable V3 region or on PCR amplicons derived from broad-range primers flanking the 16S rDNA variable V3, V4, and V9 regions. Results., Pyrosequencing analysis of 16S rDNA derived from MDA-DNA revealed that Helicobacter spp.-like DNA was present in 25 of 33 (approximately 76%) gallstones. Using an H. pylori- specific Southern blot analysis, Helicobacter spp.-like DNA was present in 20 of 33 [approximately 61%] of the gallstones. Using MDA-DNA directly in pyrosequencing analysis, Helicobacter spp.-like DNA was present in 13 of 33 [approximately 39%] gallstones. Conclusions., We conclude that multiple displacement amplification combined with pyrosequencing enables a rapid and accurate molecular typing of Helicobacter spp. from small and precious biopsy specimens. [source]

Molecular Identification of Phytophthora spp.

JOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2009
Affecting some Economically Important Crops in Eastern India through ITS-RFLP, Sequencing of the ITS Region
Abstract Molecular identification of the Phytophthora spp. affecting betelvine (Piper betel), brinjal (Solanum melongena), guava (Psidium guajava), roselle (Hibiscus subdariffa), black pepper (Piper nigrum), sesame (Sesamum indicum), taro (Colocasia esculenta), chilli (Capsicum annuum), pointed gourd (Trichosanthes dioica), papaya (Carica papaya) was performed through rDNA ITS-RFLP and also additionally by sequencing the Internal Transcriber spacer (ITS) ITS1 and ITS2 regions. Phytophthora nicotianae, Phytophthora capsici, Phytophthora colocasiae, Phytophthora melonis and Phytophthora palmivora isolates from these 10 different crops were accessioned and the ITS sequences were deposited in Genbank. ITS sequences for Phytophthora isolates from most of these crops are being reported here for the first time. In this study, a review of all earlier Indian reports based on morphology from the above crops and their molecular corroboration has been attempted. This study revealed that not only is P. nicotianae the most prevalent species but also there is the presence of both P. nicotianae and P. capsici, but not P. palmivora on betelvine; as well as possible first reports of P. nicotianae on pepper, P. capsici on chilli and P. palmivora on papaya from this vegetable growing Eastern region of the country. Mating type assays and RAPD markers were used to assess the genotypic diversity of the population. This detection of diversity is a first and critical step for helping to devise and adopt strategies for control and quarantine of these pathogens in this region. [source]

Isolation of a Thermotolerant Paravahlkampfia sp. from Lizard Intestine: Biology and Molecular Identification

THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 5 2003
FREDERICK L. SCHUSTER
ABSTRACT. An amoeba was isolated from the intestines of several moribund pink-tongued skinks (lizards), Hemisphaeriodon ger-rardi Unusual features of this isolate were its ability to grow at temperatures of , 37 °C, and its inability to use Escherichia coli as a food source or to grow axenically on a variety of enriched culture media suitable for other soil amoeba isolates. Growth was abundant, however, on tissue culture cells, with amoebae clearing cell monolayers in ,48 h at 37 °C. Trophozoites had a vahlkampfiid-like morphology, moving by means of an anterior eruptive pseudopod. Cysts, round to slightly ovoid and lacking exit pores, were formed in culture. Tests for enflagellation of trophic amoebae were negative. Indirect immunofluorescence staining was negative for Naegleria fowleri and Willaertia sp. The isolate was sensitive to azithromycin, but not to amphotericin B, pentamidine isethionate, fluconazole, 5-fluorocytosine, and sulfadiazine. Phylogenetic analysis based on the PCR-amplified small subunit ribosomal DNA, identified the organism as Paravahlkampfia ustiana, an amoeba not previously isolated from either poikilothermic or homeothermic hosts. No evidence of pathology was seen in stained sections of lizard intestine, suggesting that the ameba was part of the normal fauna of the lizard gut. Its diet in the lizard intestine is unknown and the organism may have unusual growth requirements. Thus, P. ustiana joins other soil amoebae that have been isolated from mammals, amphibia, fish, and reptiles, which have the potential of becoming opportunistic pathogens. [source]

Molecular identification and localization of cellular titin, a novel titin isoform in the fibroblast stress fiber

CYTOSKELETON, Issue 6 2007
Peter J. Cavnar
Abstract We previously discovered a large titin-like protein,c-titin,in chicken epithelial brush border and human blood platelet extracts that binds ,-actinin and organizes arrays of myosin II bipolar filaments in vitro. RT-PCR analysis of total RNA from human megakaryoblastic (CHRF-288-11) and mouse fibroblast (3T3) nonmuscle cells reveal sequences identical to known titin gene exon sequences that encode parts of the Z-line, I-band, PEVK domain, A-band, and M-line regions of striated muscle titins. In the nonmuscle cells, these sequences are differentially spliced in patterns not reported for any striated muscle titin isoform. Rabbit polyclonal antibodies raised against expressed protein fragments encoded by the Z-repeat and kinase domain regions react with the c-titin band in Western blot analysis of platelet extracts and immunoprecipitate c-titin in whole platelet extracts. Immunofluorescent localization demonstrates that the majority of the c-titin colocalizes with ,-actinin and actin in 3T3 and Indian Muntjac deer skin fibroblast stress fibers. Our results suggest that differential expression of titin gene exons in nonmuscle cells yields multiple novel isoforms of the protein c-titin that are associated with the actin stress fiber structures. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]

Molecular identification and expression study of differentially regulated genes in the Pacific oyster Crassostrea gigas in response to pesticide exposure

FEBS JOURNAL, Issue 2 2005
Arnaud Tanguy
The effects of pesticide contamination on the metabolism of marine molluscs are poorly documented. We investigated the response of a marine bivalve, the Pacific oyster, Crassostrea gigas, using a suppression subtractive hybridization method to identify up- and down-regulated genes after a 30-day exposure period to herbicides (a cocktail of atrazine, diuron and isoproturon, and to the single herbicide glyphosate). A total of 137 unique differentially expressed gene sequences was identified, as well as their associated physiological process. The expression of 18 of these genes was analyzed by RT-PCR under laboratory experimental conditions. The metabolic functions they are associated with include xenobiotic detoxification, energy production, immune system response and transcription. This study provides a preliminary basis for studying the response of marine bivalves to long-term herbicide exposure in terms of regulated gene expression and characterizes new potential genetic markers of herbicide contamination. [source]

Molecular identification and phylogeny of East African Simulium damnosum s.l. and their relationship with West African species of the complex (Diptera: Simuliidae)

INSECT MOLECULAR BIOLOGY, Issue 1 2000
A. Krüger
Abstract The phylogenetic relationships of East and West African members of the Simulium damnosum complex were studied by sequence analyses of the mitochondrial 16s ribosomal RNA (rRNA) gene. Results suggest that: (i) the S. damnosum complex is divided into an East and a West African clade, and (ii) S. pandanophilum and the cytoform ,Kiwira' form an East African subbranch distinct from the ,Sanje' group. In contrast to former assumptions from cytogenetic analyses, our molecular data do not support a direct relationship between the East African S. kilibanum and the West African S. squamosum. Length differences of the rDNA internal transcribed spacer 1 (ITS-1) turned out to be useful to distinguish between cytoforms. [source]

Molecular identification and population dynamics of two species of Pemphigus (Homoptera: Pemphidae) on cabbage

INSECT SCIENCE, Issue 2 2009
Naiqi Chen
Abstract The poplar petiole gall aphid, Pemphigus populitransversus Riley, has been one of the major pests on cruciferous vegetable in the Rio Grande Valley (LRGV) of Texas since the late 1940s. It normally migrates from poplar trees to cruciferous vegetables in the fall, and migrates back to the trees in early spring of the coming year. Some root-feeding aphids were found on cruciferous vegetables in late spring and early summer in 1998 and the following years. Those aphids have been identified as Pemphigus obesinymphae Moran. This discovery completely changed the current knowledge about the root-feeding aphids on cruciferous vegetables in the LRGV. Due to their small size, morphological and feeding similarities between P. populitransversus and P. obesinymphae, their identification and distinction are difficult. In this study, random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to distinguish these two species over a period of time when the two species occurred together, or separately, in cabbage fields. The two species occurred on cabbage at different times of the year, and overlapped from October to June. From May to October, both species migrated to their primary hosts. The apterous aphids found on cabbage in winter contained mainly P. obesinymphae, whereas in early spring more apterous P. populitransversus were recovered. The root-feeding aphids would feed on cabbage plants as long as this host was available even during the hot, dry summer in the LRGV, although their populations were generally low. Both RAPD and AFLP techniques were efficient in discriminating the two species that showed obviously genetic variability. These molecular techniques confirmed the existence of the two aphid species in apterous samples collected from the soil in cabbage fields in the LRGV, and the results performed by RAPD were confirmed by AFLP. Furthermore, the results suggest that RAPD technique was a better choice despite its reproducibility problem, as it was less time-consuming and required less technology, labor and expense than AFLP. [source]

Molecular Identification of Phytophthora spp.

Isolation of Pseudomonas spp. from Diseased Capsicum chinense (Habanero Pepper) Plants in Yucatan, Mexico

JOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2007
F. Moguel-Salazar
Abstract Capsicum chinense (habanero pepper) grown in Yucatan, Mexico, is frequently diseased by plant bacterial pathogens, but the bacterial agents remain unidentified. Bacteria associated with diseased C. chinense were isolated and characterized. Two isolates, ChA11 and ChA14, induced hypersensitive response in C. chinense plantlets and caused rot in C. chinense fruit and potato slices. Molecular identification showed both to be Pseudomonas spp. This is the first report identifying Pseudomonas spp. associated with C. chinense grown in Yucatan, and may represent a first step towards developing control measures against this insidious pathogen. [source]

Molecular identification of five commercial flatfish species by PCR,RFLP analysis of a 12S rRNA gene fragment

JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 8 2003
Angel S Comesaña
Abstract Refrigerated or frozen fillets of commercial flatfish species are sometimes mislabelled, and identification of those products is needed to avoid fraudulent substitution. Molecular identification of five commercial flatfish species (order Pleuronectiformes), ie Lepidorhombus whiffiagonis (megrim), Platichthys flesus (flounder), Reinhardtius hippoglossoides (Greenland halibut), Scophthalmus maximus (turbot) and Solea vulgaris (= S solea) (sole), has been carried out on the basis of the amplification of an approximately 433 bp segment from the mitochondrial 12S rRNA gene using the polymerase chain reaction (PCR) and universal primers. Direct DNA sequencing from two PCR products for each flatfish species was carried out, and sequences were used to select six restriction enzymes. PCR products of 15 individuals of each species were cut with each enzyme, resulting in species-specific restriction fragment length polymorphism (RFLP). The five flatfish species could be identified by application of the restriction enzyme AluI as well as by using different combinations of a pair of enzymes, ie DdeI and either AciI or MwoI. No intraspecific genetic polymorphism was found for any of the six enzymes. Results confirmed the usefulness of this technique to distinguish and genetically characterise refrigerated or frozen pieces of these five flatfish species. Copyright © 2003 Society of Chemical Industry [source]

Molecular identification and characterization of rifampicin-resistant Mycobacterium tuberculosis isolates by line probe assay: an approach for rapid diagnosis of multidrug-resistant tuberculosis

LETTERS IN APPLIED MICROBIOLOGY, Issue 3 2008
C. Bicmen
Abstract Aim:, Early identification and characterization of rifampicin-resistant (Rr) Mycobacterium tuberculosis isolates recovered from the samples of tuberculosis (TB) patients in the Aegean (West Anatolian) Region was intended. Methods and Results:, Sixty isolates [47 (78·3%) multidrug-resistant (MDR)], which were identified as M. tuberculosis complex and phenotypically resistant to rifampicin by both BACTEC mycobacteria growth indicator tube (MGIT) 960 and 460 systems were analysed by a commercial line probe assay (INNO-LiPA Rif TB). The concordance of LiPA with the in vitro susceptibility test was found as 98·3%. Among the isolates, S531L (R5 pattern; 46·7%) and L511P/R, S512T, Q513L/K (,S1 pattern; 11·7%) were the most frequent mutation patterns. As compared with the BACTEC systems and conventional techniques for cultivation, identification and in vitro susceptibility testing, INNO-LiPA Rif TB after cultivation in BACTEC MGIT 960 system provided an average of 20 days early diagnosis of RrM. tuberculosis isolates. Conclusions:, Rapid molecular identification and characterization of RrM. tuberculosis isolates after BACTEC MGIT 960 cultivation would be useful for faster diagnosis, infection control and planning of accurate treatment in MDR-TB patients. Significance and Impact of the Study:, Patients with MDR-TB need a specified treatment and efficient follow-up strategies. Rapid and practical methodologies to diagnose and follow these patients should be applied in routine use. [source]

Molecular identification of some forensically important blowflies of southern Africa and Australia

MEDICAL AND VETERINARY ENTOMOLOGY, Issue 4 2003
M. L. Harvey
Abstract., One major aspect of research in forensic entomology is the investigation of molecular techniques for the accurate identification of insects. Studies to date have addressed the corpse fauna of many geographical regions, but generally neglected the southern African calliphorid species. In this study, forensically significant calliphorids from South Africa, Swaziland, Botswana and Zimbabwe and Australia were sequenced over an 1167 base pair region of the COI gene. Phylogenetic analysis was performed to examine the ability of the region to resolve species identities and taxonomic relationships between species. Analyses by neighbour-joining, maximum parsimony and maximum likelihood methods all showed the potential of this region to provide the necessary species-level identifications for application to post-mortem interval (PMI) estimation; however, higher level taxonomic relationships did vary according to method of analysis. Intraspecific variation was also considered in relation to determining suitable maximum levels of variation to be expected during analysis. Individuals of some species in the study represented populations from both South Africa and the east coast of Australia, yet maximum intraspecific variation over this gene region was calculated at 0.8%, with minimum interspecific variation at 3%, indicating distinct ranges of variation to be expected at intra- and interspecific levels. This region therefore appears to provide southern African forensic entomologists with a new technique for providing accurate identification for application to estimation of PMI. [source]

Molecular identification of prey in predator diets

MOLECULAR ECOLOGY, Issue 4 2002
W. O. C. Symondson
Abstract In many situations prey choice by predators in the field cannot be established or quantified using direct observation. The remains of some prey may be visually identified in the guts and faeces of predators but not all predators ingest such hard remains and even those that do consume them may also ingest soft-bodies prey that leave no recognizable remnants. The result is, at best, a biased picture of prey choice. A range of molecular techniques and applications are reviewed that allow prey remains to be identified, often to the species and even stage level. These techniques, all of which are still in use, include enzyme electrophoresis, a range of immunological approaches using polyclonal and monoclonal antibodies to detect protein epitopes, and recently developed polymerase chain reaction (PCR)-based methods for detecting prey DNA. Analyses may be postmortem, on invertebrate and vertebrate predators collected from the field, or noninvasive assays of the remains in regurgitated bird pellets or vertebrate faeces. It was concluded that although monoclonal antibodies are currently the most effective method in use today, PCR-based techniques have proved to be highly effective and versatile in recent laboratory trials and are likely to rapidly displace all other approaches. [source]

Molecular identification of the extinct mountain goat, Oreamnos harringtoni (Bovidae)

BOREAS, Issue 1 2010
PAULA F. CAMPOS
Campos, P. F., Willerslev, E., Mead, J. I., Hofreiter, M. & Gilbert, M. T. P. 2009: Molecular identification of the extinct mountain goat, Oreamnos harringtoni (Bovidae). Boreas, 10.1111/j.1502-3885.2009.00111.x. ISSN 0300-9483. Harrington's mountain goat (Oreamnos harringtoni), an extinct North American herbivore, is one of the least known mammals of the Pleistocene. Fossil specimens are predominantly known from dry cave localities throughout the arid American west , the Grand Canyon, Colorado Plateau, Nevada and Mexico. Morphological analysis of the recovered fossils suggests a close phylogenetic relationship between Harrington's mountain goat and the extant mountain goats from the American northwest (Oreamnos americanus). However, the degree of genetic similarity between the two species, and their overall placement within the Caprinae, is not clear. In this study, we recovered and sequenced the first DNA fragments from O. harringtoni in order to investigate these relationships. Genetic analysis further supports the morphological hypothesis that O. harringtoni and O. americanus are two distinct species. [source]

Minor histocompatibility antigens as targets for immunotherapy using allogeneic immune reactions

CANCER SCIENCE, Issue 8 2007
Yoshiki Akatsuka
Minor histocompatibility antigens (mHag) were originally identified as antigens causing graft rejection or graft-versus-host disease in human leukocyte antigen (HLA)-matched allogeneic transplantation. Molecular identification has revealed most to be major histocompatibility complex (MHC)-bound short peptide fragments encoded by genes which are polymorphic due to single nucleotide polymorphisms (SNP). Genotypic disparity of SNP between transplantation donors and recipients gives rise to mHag as non-self antigens for both the donor and the recipient. Subsequently, mHag have been explored as immunotherapeutic antigens for use against recurring hematological malignancies after allogeneic hematopoietic cell transplantation (HCT), because mHag expressed only on hematopoietic cells are considered to augment graft-versus-leukemia/lymphoma (GVL) effects without increasing the risk of life-threatening graft-versus-host disease (GVHD). Accumulating evidence suggests that T-cell responses to mHag aberrantly expressed on solid tumor cells are also involved in the eradication of sensitive tumors such as renal cell carcinomas following HCT. Over the past decade, the number of putative GVL-directed mHag has increased to a level that covers more than 30% of the Japanese patient population, so that clinical trials may now be executed in the setting of either vaccination or adoptive immunotherapy. As it is expected that immune responses to alloantigens are more powerful than to tumor antigens mostly derived from overexpressed self-proteins, mHag-based immunotherapy may lead to a new treatment modality for high-risk malignancies following allogeneic HCT. (Cancer Sci 2007; 98: 1139,1146) [source]

Molecular identification of coliform bacteria from colicky breastfed infants

ACTA PAEDIATRICA, Issue 10 2009
F Savino
Abstract Objective:, To determine the presence of intestinal coliform bacteria in colicky vs healthy infants. Study design:, We isolated coliform strains from faeces and performed quantitative bacterial cultures in 41 colicky and 39 healthy breastfed infants, identified using PCR with species-specific primers, strain-specific Automated Ribotyping and the API-50E kit for Enterobacteriaceae to identify the most frequent strains. Results:, Coliform strains were more abundant in colicky infants (median 6.04 log10 CFU/g faeces, range 2.00,8.76) vs controls (median 4.47 log10 CFU/g faeces, range 1.00,8.08) (p = 0.026). Escherichia coli, Klebsiella pneumoniae, K. oxytoca, Enterobacter cloacae, E. aerogenes and Enterococcus faecalis were the predominant species in colicky and healthy infants. The counts of each bacterial species differed between the two groups, and the difference was significant (p = 0.002) for E. coli: median 6.30 log10 CFU/g faeces (range 3.00,8.74) in colicky infants, and median 4.70 log10 CFU/g faeces (range 2.00,5.85) in controls. Conclusions:, This is the first study to evaluate the colonization patterns of gas-forming coliforms in colicky infants and healthy controls identified by molecular methods. Coliform bacteria, particularly Escherichia coli, were found to be more abundant in colicky infants. Our data could help to shed light on the cause of infantile colic. [source]

Surveys reveal the occurrence of phytoplasmas in plants at different geographical locations in Peru

ANNALS OF APPLIED BIOLOGY, Issue 1 2009
J. Hodgetts
Abstract Two independent surveys were performed in Peru during February and November 2007 to detect the presence of phytoplasmas within any crops showing symptoms resembling those caused by phytoplasmas. Molecular identifications and characterisations were based on phytoplasma 16S and 23S rRNA genes using nested PCR and terminal restriction fragment length polymorphism (T-RFLP). The surveys indicated that phytoplasmas were present in most of the locations sampled in Peru in both cultivated crops, including carrots, maize, native potatoes, improved potato, tomato, oats, papaya and coconut, and in other plants such as dandelion and the ornamental Madagascar periwinkle (Catharanthus roseus). Phylogenetic analysis of the sequences confirmed that while most of the isolates belong to the 16SrI aster yellows group, which is ubiquitous throughout other parts of South America, one isolate from potato belongs to the 16SrII peanut witches' broom group, and one isolate from tomato and one from dandelion belong to the 16SrIII X-disease group. The use of T-RFLP was validated for the evaluation of phytoplasma-affected field samples and provided no evidence for mixed infection of individual plants with more than one phytoplasma isolate. These data represent the first molecular confirmation of the presence of phytoplasmas in a broad range of crops in Peru. [source]

BIODIVERSITY RESEARCH: Genetic diversity in two introduced biofouling amphipods (Ampithoe valida & Jassa marmorata) along the Pacific North American coast: investigation into molecular identification and cryptic diversity

DIVERSITY AND DISTRIBUTIONS, Issue 5 2010
Erik M. Pilgrim
Abstract Aim, We investigated patterns of genetic diversity among invasive populations of Ampithoe valida and Jassa marmorata from the Pacific North American coast to assess the accuracy of morphological identification and determine whether or not cryptic diversity and multiple introductions contribute to the contemporary distribution of these species in the region. Location, Native range: Atlantic North American coast; Invaded range: Pacific North American coast. Methods, We assessed indices of genetic diversity based on DNA sequence data from the mitochondrial cytochrome c oxidase subunit I (COI) gene, determined the distribution of COI haplotypes among populations in both the invasive and putative native ranges of A. valida and J. marmorata and reconstructed phylogenetic relationships among COI haplotypes using both maximum parsimony and Bayesian approaches. Results, Phylogenetic inference indicates that inaccurate species-level identifications by morphological criteria are common among Jassa specimens. In addition, our data reveal the presence of three well supported but previously unrecognized clades of A. valida among specimens in the north-eastern Pacific. Different species of Jassa and different genetic lineages of Ampithoe exhibit striking disparity in geographic distribution across the region as well as substantial differences in genetic diversity indices. Main conclusions, Molecular genetic methods greatly improve the accuracy and resolution of identifications for invasive benthic marine amphipods at the species level and below. Our data suggest that multiple cryptic introductions of Ampithoe have occurred in the north-eastern Pacific and highlight uncertainty regarding the origin and invasion histories of both Jassa and Ampithoe species. Additional morphological and genetic analyses are necessary to clarify the taxonomy and native biogeography of both amphipod genera. [source]

Approaches to prokaryotic biodiversity: a population genetics perspective

ENVIRONMENTAL MICROBIOLOGY, Issue 11 2002
Francisco Rodríguez-Valera
Summary The study of prokaryotic diversity has blossomed during the last 10,15 years as a result of the introduction of molecular identification, mostly based on direct 16S rRNA gene polymerase chain reaction (PCR) amplification and sequencing from natural samples. A large amount of information exists about the diversity of this specific gene. However, data from the field of bacterial population genetics and genomics make questionable the value of information regarding just one gene. Even if we accept 16S rRNA genes as useful for species identification, intraspecific variation in bacteria is so high that species catalogues are often of little value. The gene pools represented by an operational species are yet impossible to predict. On the other hand, adaptive features in prokaryotes are often coded in gene clusters (genomic islands) that can be cloned directly from the environment, sequenced and even expressed in a surrogate host. Thus, the study of the environmental genome or metagenome appears as an alternative that could eventually lead to a more realistic understanding of prokaryotic biodiversity, provide biotechnology with new tools and maybe even contribute to develop a model of prokaryotic evolution. [source]

Multiple Displacement Amplification of Isolated DNA from Human Gallstones: Molecular Identification of Helicobacter DNA by Means of 16S rDNA-Based Pyrosequencing Analysis

Molecular modifiers of T cell antigen receptor triggering threshold: the mechanism of CD28 costimulatory receptor

IMMUNOLOGICAL REVIEWS, Issue 1 2003
Oreste Acuto
Summary:, CD28 was thought to represent a prototypic membrane receptor responsible for delivering the classically defined ,second signal' needed to avoid T cell paralysis when recognizing antigen presented by appropriate antigen presenting cells (APCs). Almost two decades after its molecular identification, the mechanism by which this ,second receptor' facilitates clonal expansion and differentiation upon antigen encounter is still not fully elucidated. There may be at least two reasons for this partially gray picture: the use of nonphysiological experimental conditions to study it and the fact that the action of CD28 may be partly masked by the presence of additional T cell surface receptors that also provide some costimulatory signals, although not equivalent to the one delivered through CD28. Thus, instead of aging, the study of CD28 is still a topical subject. What is appearing through work of recent years is that far from being purely qualitative, the CD28 signal provides a key quantitative contribution to potently boost the T cell antigen receptor (TCR) signal. In other words, CD28 is in part a signaling ,sosia' of the TCR. Also, it is clear now that CD28 operates via multiple molecular effects. Still, what we do not understand is the ,qualitative' part of this signal, perhaps due to lack of identification of unique signaling components and/or pathways activated by CD28 only. Here we review a series of recent findings pointing towards novel avenues to better understand the molecular basis of CD28 function. [source]

Ultrastructural and molecular identification of a Wolbachia endosymbiont in a spider, Nephila clavata

INSECT MOLECULAR BIOLOGY, Issue 5 2000
Hyun Woo Oh
Abstract Wolbachia -like bacteria were observed in the egg cells of golden orb-weaving spider, Nephila clavata, by means of transmission electron microscopy. The bacteria exhibited the typical morphology of Wolbachia, including three enveloping membranes. Based on the amplification and sequencing of partial 16S rDNA and ftsZ gene, the bacteria were identified as Wolbachia, intracellular, transovarially inherited ,-proteobacteria in invertebrates. Phylogenetic analysis based on 16S rDNA and ftsZ gene sequences invariably indicated that the intracellular bacteria from N. clavata belonged to group A Wolbachia, which were found only from insects. Clustering of Wolbachia from N. clavata with group A Wolbachia indicates that the bacteria were probably transferred horizontally between insects and the spider. [source]

PCR-BASED TECHNIQUE FOR IDENTIFICATION AND DETECTION OF TRICHOGRAMMA SPP. (HYMENOPTERA: TRICHOGRAMMATIDAE) WITH SPECIFIC PRIMERS

INSECT SCIENCE, Issue 3 2002
LI Zheng-xi
Abstract The rDNA-ITS2 regions of T. dendrolimi Matsumura and T. ostriniae Pang et Chen (Hymenoptera: Trichogrammatidae) were cloned and sequenced. The homologous sequences available in GenBank were retrieved and analyzed, and then specific primers were designed for molecular identification and detection of T. dendrolimi. Repeated screening showed that PCR amplification by the diagnostic primers enabled the differentiation of not only bulk samples and single adult (male or female), but also eggs and juveniles, which was not possible by conventional methods. The advantage of this system over morphology-based systems is that non-specialists are able to identify individuals or trace specimens efficiently. The derived molecular detection technique was then used to identify 12 specimens collected from different localities on the Chinese mainland; the results showed that this protocol could be applied to molecular monitoring of Trichogramma species in the field. Finally, 1132s of 6 geographical populations of T. dendrolimi (TdCHA, TDJL, TdXZ, TdKH, TdCZ and TdYBL) were cloned and sequenced. The multialignment analysis of intraspecific ITS2 sequences showed that the diagnostic primers have their own theoretical bases. [source]

Application of recA and rpoB sequence analysis on phylogeny and molecular identification of Geobacillus species

JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2009
F.Y. Weng
Abstract Aims:, Some Geobacillus species have highly similar 16S rRNA gene sequences, making 16S rDNA sequence analysis-based identification problematic. To overcome this limitation, recA and rpoB sequence analysis was evaluated as an alternative for distinguishing Geobacillus species. Methods and Results:, The phylogram of 16S rRNA gene sequences inferred from the neighbour-joining method showed that nine clusters of Geobacillus species were characterized with bootstrap values >90%. The recA and rpoB sequences of 10 reference strains in clusters V, VIb and VIc were amplified and sequenced using consensus primers. Alignment of recA sequences in clusters V, VIb and VIc revealed three types of recA genes, consistent with the putative amino acid sequences and in vivo recA splicing analysis. The phylogram constructed from rpoB sequences showed more divergence than that constructed from 16S rRNA gene sequences. Conclusions:,recA and rpoB sequence analysis differentiated closely-related Geobacillus species and provided direct evidence for reclassifying some species dubiously categorized as Geobacilli. Additionally, this study revealed three types of recA genes in the different Geobacillus species. Significance and Impact of the Study:, This study highlights the advantage of recA and rpoB sequence analysis to supplement 16S rRNA gene sequence analysis for efficient and convenient determination of Geobacillus species. [source]

Grapevine yellows in Northern Italy: molecular identification of Flavescence dorée phytoplasma strains and of Bois Noir phytoplasmas

JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2007
S. Botti
Abstract Aims:, Verify the presence and the molecular identity of phytoplasmas in Northern and Central Italy vineyards where yellows diseases are widespread. Methods and Results:, Phytoplasma presence and identity were determined by PCR/RFLP analyses on 16S ribosomal gene testing 1424 symptomatic samples. The 65% of samples resulted phytoplasma infected; in particular 256 samples were found positive to phytoplasmas belonging to group 16SrV (mainly Flavescence dorée associated), and the remaining 37% was infected by phytoplasmas belonging to ribosomal subgroup 16SrXII-A (Stolbur or Bois Noir associated). 16SrV ribosomal group representative strains were further typed for variability in SecY and rpS3 genes. The results showed the presence of phytoplasmas belonging to 16SrV-C, 16SrV-D and to a lesser extent, 16SrV-A subgroup. Conclusions:, Possible relationships between genetic polymorphisms of phytoplasma strains belonging to subgroup 16SrV-C and their geographic distribution and/or epidemic situations were detected. Significance and Impact of the Study:, Bois Noir and Flavescence dorée phytoplasmas are present in significant percentages in the areas under investigation. Molecular tools allowed to identify phytoplasma-infected plants and the genes employed as polymorphism markers resulted useful in distinguishing and monitoring the spreading of the diseases associated with diverse phytoplasmas belonging to 16SrV subgroup in vineyards. [source]

Diversity, dynamics and reproduction in a community of small mammals in Upper Guinea, with emphasis on pygmy mice ecology

AFRICAN JOURNAL OF ECOLOGY, Issue 3 2010
Elisabeth Fichet-Calvet
Abstract As part of a large survey on reservoirs of Lassa fever in Guinea, three villages were investigated in high endemic zone, close to Sierra Leone border. Biodiversity of the small mammal community is presented in this study through a standardized trapping in houses, cultivations and forest. Identification of the small mammals was based on morphology and by molecular technique for sibling species. Of the 1123 specimens collected in 2003,2005, we identified seventeen species (thirteen Muridae, four Soricidae), leading to high diversity (Shannon index = 1.6,1.8) and high equitability (evenness index = 0.7,0.8) in cultivations and forest. In houses conversely, the rodent community was dominated by Mastomys natalensis (95,98%), leading to low diversity and equitability. Dynamics and reproduction were investigated in two species of pygmy mice, Mus mattheyi and Mus minutoides, two species of Praomys, P. daltoni and P. rostratus, and in Mastomys erythroleucus. The pygmy mice were abundant in cultivations in early rainy season, and reproduced from rainy to dry season. Praomys daltoni was also found more abundant in cultivations and seemed to reproduce between rainy and dry season, whereas P. rostratus preferred forest and cultivations in late rainy season, and reproduced throughout the year. Finally, M. erythroleucus was more abundant in forest in dry season, and seemed to reproduce from late rainy to dry season. This species had a low occurrence (6.5%) in the Faranah's zone, and probably lived at its southern limit in Guinea. The presence of other Murinae, such as M. natalensis, Praomys spp as possible competitors in the same habitats, is discussed. For the first time, this study relates population biology of pygmy mice with molecular identification. Résumé Dans le cadre d'une vaste étude des réservoirs de la fièvre de Lassa en Guinée, trois villages ont étéétudiés dans une zone de forte endémie près de la frontière de la Sierra Leone. La biodiversité de la communauté de petits mammifères est présentée dans cette étude grâce à un piégeage standardisé dans les maisons, les cultures et les forêts. L'identification des petits mammifères est réalisée sur la base de leur morphologie et de techniques moléculaires dans le cas d'espèces jumelles. Parmi les 1123 spécimens récoltés de 2003 à 2005, nous avons identifié dix-sept espèces (treize Muridae, quatre Soricidae), indiquant une grande diversité (Indice de Shannon = 1,6 à 1,8) et une grande équitabilité (indice d'équitabilité = 0,7 à 0,8) dans les cultures et les forêts. Dans les maisons en revanche, la communauté de rongeurs était dominée par Mastomys natalensis (95 à 98%), conduisant à une faible diversité et équitabilité. La dynamique et la reproduction de deux espèces de souris naines, Mus mattheyi et M. minutoides, deux espèces de Praomys, P. daltoni et P. rostratus, et de Mastomys erythroleucus ont étéétudiées également. Les souris naines étaient abondantes dans les cultures au début de la saison des pluies, se reproduisant de la saison des pluies à la saison sèche. Praomys daltoni fut aussi trouvé en plus grand nombre dans les cultures et semblait se reproduire entre la saison des pluies et la saison sèche alors que P. rostratus préférait les forêts et les cultures à la fin de la saison des pluies et se reproduisait toute l'année. Enfin, M. erythroleucusétait plus abondant en forêt en saison sèche et semblait se reproduire de la fin de la saison des pluies à la saison sèche. Cette espèce était peu présente (6,5%) dans la zone de Faranah et était probablement en Guinée à la limite sud de son aire de répartition. La présence d'autres Murinae, comme M. natalensis et Praomys spp. est discutée en tant que compétiteurs possibles dans les même habitats. Pour la première fois, cette étude relate la biologie des populations de souris naines grâce à une identification moléculaire. [source]

Histone deacetylases as transducers and targets of nuclear signaling

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2008
Donald R. Walkinshaw
Abstract Histone deacetylase (HDAC) activity was first discovered about 40 years ago, but it was not until the molecular identification of the first HDACs in 1996 that this family of enzymes gained prominence. In addition to histones, HDACs reverse lysine acetylation of various non-histone proteins located in the nucleus and the cytoplasm. Here, we examine the nuclear roles of these enzymes, with a specific focus on their active crosstalk with different chromatin regulators. J. Cell. Biochem. 104: 1541,1552, 2008. © 2008 Wiley-Liss, Inc. [source]

Genetic Variability Analysis and Molecular Detection of Fusarium oxysporum f.sp. eustomae Isolated from Eustoma grandiflorum in Northern Italy

JOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2010
Yuan Li
Abstract A total of 35 isolates of Fusarium oxysporum f.sp. eustomae obtained from diseased Eustoma grandiflorum plants in northern Italy, showing typical Fusarium wilt symptoms, were analysed for their genetic variability and molecular identification. Genetic diversity of the isolates was studied by using random amplified polymorphic DNA (RAPD). This analysis clustered the isolates into three groups at a genetic similarity of 69%. Sequence analysis of RAPD fragments led to the design of a pair of specific primers that amplify a 505-bp SCAR (sequence characterized amplified region) marker (SCAR505) which was used to rapidly detect F. oxysporum f.sp. eustomae on Eustoma grandiflorum plants. In a temperature-controlled chamber, detection of the pathogen by PCR was 100% successful in root and stem samples of infected but still symptomless plants. The diagnostic procedure could be completed in 1 day and allowed rapid and reliable detection of the pathogen in asymptomatic plants in the early stages of disease development. [source]

Occurrence and Distribution of Microdochium and Fusarium Species Isolated from Durum Wheat in Northern Tunisia and Detection of Mycotoxins in Naturally Infested Grain

JOURNAL OF PHYTOPATHOLOGY, Issue 9 2009
Lobna Gargouri Kammoun
Abstract An outbreak of Fusarium Head Blight of durum wheat occurred in 2004 being localized in sub-humid and higher semi-arid region of Northern Tunisia. A mycological survey carried out throughout these regions, revealed that 78% of the prospected fields were infested. Results of the morphological and molecular identification, showed that the most common species isolated from diseased wheat spikes was Microdochium nivale var. nivale (63.5%), followed by Fusarium culmorum (26%), F. pseudograminearum (9%) and F. avenaceum (1.5%). To evaluate mycotoxin content of naturally infected grain, the amounts of trichothecene mycotoxin deoxynivalenol (DON) in harvested grain from 45 fields were quantified by RIDASCREEN DON Enzyme Immunoassay Kit (ELISA). This study showed that the infection levels in freshly harvested grain were very low and the maximum deoxynivalenol (DON) level of the positive samples was 53 ppb. This is the first report on the natural occurrence of DON in naturally infected wheat grain sampled from Northern Tunisia. [source]

Fluorescence and Raman spectra on painting materials: reconstruction of spectra with mathematical methods

JOURNAL OF RAMAN SPECTROSCOPY, Issue 10 2006
Iacopo Osticioli
Abstract SERDS (shift excitation difference spectroscopy) and SSRS (subtracted shifted Raman spectroscopy) methods were applied for fluorescence-background rejection in the Raman spectra of colored materials. These techniques are based on the assumption that the fluorescence contribution can be completely eliminated by subtracting two Raman spectra acquired at two shifted laser excitation frequencies. For the SERDS method a micro-Raman experimental apparatus coupled with a tunable diode laser (central emission at 684 nm) was set up. SSRS measurements were made on a commercial micro-Raman instrument; in this case the shifted spectrum was obtained by moving the spectrometer grating. Raman spectra were then reconstructed by applying the difference deconvolution method that automatically converts the difference signals in Raman peaks through a deconvolution operation. These techniques were tested on two reference colors (ultramarine and 6,6,-dibromoindigotine) and two colored samples of unknown composition (a Pompeian pink powder and a blue paint from a XVII century painting). Fluorescence-background subtraction and the following operation of spectra reconstruction took place successfully with no errors in Raman peaks, width and wavenumber position. In addition, even weak spectral details were revealed favoring the comparison with reference data for a molecular identification. Copyright © 2006 John Wiley & Sons, Ltd. [source]