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Sample records for gene amplification confers

  1. EPSPS gene amplification conferring resistance to glyphosate in windmill grass (Chloris truncata) in Australia.

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    Ngo, The D; Malone, Jenna M; Boutsalis, Peter; Gill, Gurjeet; Preston, Christopher

    2017-03-20

    Five glyphosate-resistant populations of Chloris truncata originally collected from New South Wales were compared with one susceptible (S) population from South Australia to confirm glyphosate resistance and elucidate possible mechanisms of resistance. Based on the amounts of glyphosate required to kill 50% of treated plants (LD50 ), glyphosate resistance (GR) was confirmed in five populations of C. truncata (A536, A528, T27, A534 and A535.1). GR plants were 2.4-8.7-fold more resistant and accumulated less shikimate after glyphosate treatment than S plants. There was no difference in glyphosate absorption and translocation between GR and S plants. The EPSPS gene did not contain any point mutation that had previously been associated with resistance to glyphosate. The resistant plants (A528 and A536) contained up to 32-48 more copies of the EPSPS gene than the susceptible plants. This study has identified EPSPS gene amplification contributing to glyphosate resistance in C. truncata. In addition, a Glu-91-Ala mutation within EPSPS was identified that may contribute to glyphosate resistance in this species. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  2. Gene amplification in carcinogenesis

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    Lucimari Bizari

    2006-01-01

    Full Text Available Gene amplification increases the number of genes in a genome and can give rise to karyotype abnormalities called double minutes (DM and homogeneously staining regions (HSR, both of which have been widely observed in human tumors but are also known to play a major role during embryonic development due to the fact that they are responsible for the programmed increase of gene expression. The etiology of gene amplification during carcinogenesis is not yet completely understood but can be considered a result of genetic instability. Gene amplification leads to an increase in protein expression and provides a selective advantage during cell growth. Oncogenes such as CCND1, c-MET, c-MYC, ERBB2, EGFR and MDM2 are amplified in human tumors and can be associated with increased expression of their respective proteins or not. In general, gene amplification is associated with more aggressive tumors, metastases, resistance to chemotherapy and a decrease in the period during which the patient stays free of the disease. This review discusses the major role of gene amplification in the progression of carcinomas, formation of genetic markers and as possible therapeutic targets for the development of drugs for the treatment of some types of tumors.

  3. Identification of genetic elements associated with EPSPS gene amplification

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    Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene evolved to confer resistance to glyphosate, the world's most important herbicide, in the wee...

  4. Identification of genetic elements associated with EPSPs gene amplification.

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    Todd A Gaines

    Full Text Available Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS gene evolved in the weed species Amaranthus palmeri to confer resistance to glyphosate, the world's most important herbicide. However, the gene amplification mechanism is unknown. We sequenced the EPSPS gene and genomic regions flanking EPSPS loci in A. palmeri, and searched for mobile genetic elements or repetitive sequences. The EPSPS gene was 10,229 bp, containing 8 exons and 7 introns. The gene amplification likely proceeded through a DNA-mediated mechanism, as introns exist in the amplified gene copies and the entire amplified sequence is at least 30 kb in length. Our data support the presence of two EPSPS loci in susceptible (S A. palmeri, and that only one of these was amplified in glyphosate-resistant (R A. palmeri. The EPSPS gene amplification event likely occurred recently, as no sequence polymorphisms were found within introns of amplified EPSPS copies from R individuals. Sequences with homology to miniature inverted-repeat transposable elements (MITEs were identified next to EPSPS gene copies only in R individuals. Additionally, a putative Activator (Ac transposase and a repetitive sequence region were associated with amplified EPSPS genes. The mechanism controlling this DNA-mediated amplification remains unknown. Further investigation is necessary to determine if the gene amplification may have proceeded via DNA transposon-mediated replication, and/or unequal recombination between different genomic regions resulting in replication of the EPSPS gene.

  5. Heat induces gene amplification in cancer cells

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    Yan, Bin, E-mail: yanbin@mercyhealth.com [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  6. RNA amplification for successful gene profiling analysis

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    Wang Ena

    2005-07-01

    Full Text Available Abstract The study of clinical samples is often limited by the amount of material available to study. While proteins cannot be multiplied in their natural form, DNA and RNA can be amplified from small specimens and used for high-throughput analyses. Therefore, genetic studies offer the best opportunity to screen for novel insights of human pathology when little material is available. Precise estimates of DNA copy numbers in a given specimen are necessary. However, most studies investigate static variables such as the genetic background of patients or mutations within pathological specimens without a need to assess proportionality of expression among different genes throughout the genome. Comparative genomic hybridization of DNA samples represents a crude exception to this rule since genomic amplification or deletion is compared among different specimens directly. For gene expression analysis, however, it is critical to accurately estimate the proportional expression of distinct RNA transcripts since such proportions directly govern cell function by modulating protein expression. Furthermore, comparative estimates of relative RNA expression at different time points portray the response of cells to environmental stimuli, indirectly informing about broader biological events affecting a particular tissue in physiological or pathological conditions. This cognitive reaction of cells is similar to the detection of electroencephalographic patterns which inform about the status of the brain in response to external stimuli. As our need to understand human pathophysiology at the global level increases, the development and refinement of technologies for high fidelity messenger RNA amplification have become the focus of increasing interest during the past decade. The need to increase the abundance of RNA has been met not only for gene specific amplification, but, most importantly for global transcriptome wide, unbiased amplification. Now gene

  7. Amplification and characterization of eukaryotic structural genes.

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    Maniatis, T; Efstratiadis, A; Sim, G K; Kafatos, F

    1978-05-01

    An approach to the study of eukaryotic structural genes which are differentially expressed during development is described. This approach involves the isolation and amplification of mRNA sequences by in vitro conversion of mRNA to double-stranded cDNA followed by molecular cloning in bacterial plasmids. This procedure provides highly specific hybridization probes that can be used to identify genes and their contiguous DNA sequences in genomic DNA, and to detect specific RNA transcripts during development. The nature of the method allows the isolation of individual mRNA sequences from a complex population of molecules at different stages of development.

  8. Mutualism breakdown by amplification of Wolbachia genes.

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    Chrostek, Ewa; Teixeira, Luis

    2015-02-01

    Most insect species are associated with vertically transmitted endosymbionts. Because of the mode of transmission, the fitness of these symbionts is dependent on the fitness of the hosts. Therefore, these endosymbionts need to control their proliferation in order to minimize their cost for the host. The genetic bases and mechanisms of this regulation remain largely undetermined. The maternally inherited bacteria of the genus Wolbachia are the most common endosymbionts of insects, providing some of them with fitness benefits. In Drosophila melanogaster, Wolbachia wMelPop is a unique virulent variant that proliferates massively in the hosts and shortens their lifespan. The genetic bases of wMelPop virulence are unknown, and their identification would allow a better understanding of how Wolbachia levels are regulated. Here we show that amplification of a region containing eight Wolbachia genes, called Octomom, is responsible for wMelPop virulence. Using Drosophila lines selected for carrying Wolbachia with different Octomom copy numbers, we demonstrate that the number of Octomom copies determines Wolbachia titers and the strength of the lethal phenotype. Octomom amplification is unstable, and reversion of copy number to one reverts all the phenotypes. Our results provide a link between genotype and phenotype in Wolbachia and identify a genomic region regulating Wolbachia proliferation. We also prove that these bacteria can evolve rapidly. Rapid evolution by changes in gene copy number may be common in endosymbionts with a high number of mobile elements and other repeated regions. Understanding wMelPop pathogenicity and variability also allows researchers to better control and predict the outcome of releasing mosquitoes transinfected with this variant to block human vector-borne diseases. Our results show that transition from a mutualist to a pathogen may occur because of a single genomic change in the endosymbiont. This implies that there must be constant selection on

  9. Mechanism of Gene Amplification via Yeast Autonomously Replicating Sequences

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    Shelly Sehgal

    2015-01-01

    Full Text Available The present investigation was aimed at understanding the molecular mechanism of gene amplification. Interplay of fragile sites in promoting gene amplification was also elucidated. The amplification promoting sequences were chosen from the Saccharomyces cerevisiae ARS, 5S rRNA regions of Plantago ovata and P. lagopus, proposed sites of replication pausing at Ste20 gene locus of S. cerevisiae, and the bend DNA sequences within fragile site FRA11A in humans. The gene amplification assays showed that plasmid bearing APS from yeast and human beings led to enhanced protein concentration as compared to the wild type. Both the in silico and in vitro analyses were pointed out at the strong bending potential of these APS. In addition, high mitotic stability and presence of TTTT repeats and SAR amongst these sequences encourage gene amplification. Phylogenetic analysis of S. cerevisiae ARS was also conducted. The combinatorial power of different aspects of APS analyzed in the present investigation was harnessed to reach a consensus about the factors which stimulate gene expression, in presence of these sequences. It was concluded that the mechanism of gene amplification was that AT rich tracts present in fragile sites of yeast serve as binding sites for MAR/SAR and DNA unwinding elements. The DNA protein interactions necessary for ORC activation are facilitated by DNA bending. These specific bindings at ORC promote repeated rounds of DNA replication leading to gene amplification.

  10. Laser amplifier based on Raman amplification in plasma (Conference Presentation)

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    Vieux, Gregory; Cipiccia, Silvia; Lemos, Nuno R. C.; Ciocarlan, Cristian; Grant, Peter A.; Grant, David W.; Ersfeld, Bernhard; Hur, MinSup; Lepipas, Panagiotis; Manahan, Grace; Reboredo Gil, David; Subiel, Anna; Welsh, Gregor H.; Wiggins, S. Mark; Yoffe, Samuel R.; Farmer, John P.; Aniculaesei, Constantin; Brunetti, Enrico; Yang, Xue; Heathcote, Robert; Nersisyan, Gagik; Lewis, Ciaran L. S.; Pukhov, Alexander; Dias, João. Mendanha; Jaroszynski, Dino A.

    2017-05-01

    The increasing demand for high laser powers is placing huge demands on current laser technology. This is now reaching a limit, and to realise the existing new areas of research promised at high intensities, new cost-effective and technically feasible ways of scaling up the laser power will be required. Plasma-based laser amplifiers may represent the required breakthrough to reach powers of tens of petawatt to exawatt, because of the fundamental advantage that amplification and compression can be realised simultaneously in a plasma medium, which is also robust and resistant to damage, unlike conventional amplifying media. Raman amplification is a promising method, where a long pump pulse transfers energy to a lower frequency, short duration counter-propagating seed pulse through resonant excitation of a plasma wave that creates a transient plasma echelon that backscatters the pump into the probe. Here we present the results of an experimental campaign conducted at the Central Laser Facility. Pump pulses with energies up to 100 J have been used to amplify sub-nanojoule seed pulses to near-joule level. An unprecedented gain of eight orders of magnitude, with a gain coefficient of 180 cm-1 has been measured, which exceeds high-power solid-state amplifying media by orders of magnitude. High gain leads to strong competing amplification from noise, which reaches similar levels to the amplified seed. The observation of 640 Jsr-1 directly backscattered from noise, implies potential overall efficiencies greater than 10%.

  11. A mechanism of gene amplification driven by small DNA fragments.

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    Kuntal Mukherjee

    Full Text Available DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s. Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation

  12. Somatic recombination, gene amplification and cancer.

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    Ramel, C; Cederberg, H; Magnusson, J; Vogel, E; Natarajan, A T; Mullender, L H; Nivard, J M; Parry, J M; Leyson, A; Comendador, M A; Sierra, L M; Ferreiro, J A; Consuegra, S

    1996-06-12

    The principle objective of this research programme, to analyse chemical induction of somatic recombination and related endpoints, i.e., mobilization of transposing elements and gene amplification, has been approached by means of several assay systems. These have included Drosophila, Saccharomyces and mammalian cell cultures. 6.1. Screening assays for mitotic recombination. A large number of chemicals have been investigated in the three Drosophila assay systems employed--the multiple wing hair/flare wing spot system developed by Graf et al., 1984, the white-ivory system developed by Green et al., 1986 and the white/white+ eye spot assay developed by Vogel (Vogel and Nivard, 1993). Particularly the screening of 181 chemicals, covering a wide array of chemical classes, by the last mentioned assay has shown that measurement of somatic recombination in Drosophila constitutes a sensitive and efficient short-term test which shows a remarkably good correlation with the agent score of 83 short-term tests analysed by ICPEMC (Mendelsohn et al., 1992; Table 2) as well as the assay performance in international collaborative programmes measuring carcinogen/non-carcinogens (de Serres and Ashby, 1981; Ashby et al., 1985, 1988). Also the wing spot assay has gained wide international recognition as a similarly sensitive test. These two assay systems in Drosophila measure both intrachromosomal events and interchromosomal recombination. The white-ivory system on the other hand is based on the loss of a tandem duplication in the white locus, the mechanism of which is less known, but probably involves intrachromosomal recombination. The difference in the mechanism between this assay and the former two was indicated by the lack of response to methotrexate in the white-ivory assay, while this compound was strongly recombinogenic in both the wing spot and white/white+ assays. The use of different strains of Drosophila with the white/white+ assay demonstrated the importance of the

  13. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases

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    Pravas Ranjan Sahoo

    2016-05-01

    Full Text Available India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

  14. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases.

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    Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

    2016-05-01

    India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

  15. On the mechanism of gene amplification induced under stress in Escherichia coli.

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    2006-04-01

    Full Text Available Gene amplification is a collection of processes whereby a DNA segment is reiterated to multiple copies per genome. It is important in carcinogenesis and resistance to chemotherapeutic agents, and can underlie adaptive evolution via increased expression of an amplified gene, evolution of new gene functions, and genome evolution. Though first described in the model organism Escherichia coli in the early 1960s, only scant information on the mechanism(s of amplification in this system has been obtained, and many models for mechanism(s were possible. More recently, some gene amplifications in E. coli were shown to be stress-inducible and to confer a selective advantage to cells under stress (adaptive amplifications, potentially accelerating evolution specifically when cells are poorly adapted to their environment. We focus on stress-induced amplification in E. coli and report several findings that indicate a novel molecular mechanism, and we suggest that most amplifications might be stress-induced, not spontaneous. First, as often hypothesized, but not shown previously, certain proteins used for DNA double-strand-break repair and homologous recombination are required for amplification. Second, in contrast with previous models in which homologous recombination between repeated sequences caused duplications that lead to amplification, the amplified DNAs are present in situ as tandem, direct repeats of 7-32 kilobases bordered by only 4 to 15 base pairs of G-rich homology, indicating an initial non-homologous recombination event. Sequences at the rearrangement junctions suggest nonhomologous recombination mechanisms that occur via template switching during DNA replication, but unlike previously described template switching events, these must occur over long distances. Third, we provide evidence that 3'-single-strand DNA ends are intermediates in the process, supporting a template-switching mechanism. Fourth, we provide evidence that lagging

  16. Amplification of kinetic oscillations in gene expression

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    Zhdanov, V. P.

    2008-10-01

    Because of the feedbacks between the DNA transcription and mRNA translation, the gene expression in cells may exhibit bistability and oscillations. The deterministic and stochastic calculations presented illustrate how the bistable kinetics of expression of one gene in a cell can be influenced by the kinetic oscillations in the expression of another gene. Due to stability of the states of the bistable kinetics of gene 1 and the relatively small difference between the maximum and minimum protein amounts during the oscillations of gene 2, the induced oscillations of gene 1 are found to typically be related either to the low-or high-reactive state of this gene. The quality of the induced oscillations may be appreciably better than that of the inducing oscillations. This means that gene 1 can serve as an amplifier of the kinetic oscillations of gene 2.

  17. Tandem amplification of a chromosomal segment harboring 5-enolpyruvylshikimate-3-phosphate synthase locus confers glyphosate resistance in Kochia scoparia.

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    Jugulam, Mithila; Niehues, Kindsey; Godar, Amar S; Koo, Dal-Hoe; Danilova, Tatiana; Friebe, Bernd; Sehgal, Sunish; Varanasi, Vijay K; Wiersma, Andrew; Westra, Philip; Stahlman, Phillip W; Gill, Bikram S

    2014-11-01

    Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.

  18. Amplification biases: possible differences among deviating gene expressions

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    Piumi Francois

    2008-01-01

    Full Text Available Abstract Background Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT and polymerase chain reaction (PCR, the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored. Results Screening a bovine cDNA array dedicated to embryonic stages with embryonic (n = 3 and somatic tissues (n = 2, we proceeded to moderate amplifications starting from 1 μg of total RNA (global PCR or IVT one round. Whatever the tissue, 16% of the probes were involved in deviating gene expressions due to amplification defaults. These distortions were likely due to the molecular features of the affected sequences (position within a gene, GC content, hairpin number but also to the relative abundance of these transcripts within the tissues. These deviating genes mainly encoded housekeeping genes from physiological or cellular processes (70% and constituted 2 subsets which did not overlap (molecular features, signal intensities, gene ID. However, the differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions and relevant (biologically validated. In addition, the relative expression levels of those genes were biologically similar between amplified and unamplified samples. Conclusion Conversely to the most recent reports which challenged the use of intense amplification procedures on minute amounts of RNA, we chose moderate PCR and IVT amplifications for our gene profiling study. Conclusively, it appeared that systematic biases arose even with moderate amplification procedures, independently of (i the sample used: brain, ovary or embryos, (ii

  19. Hormonal Involvement in Breast Cancer Gene Amplification

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    2010-10-01

    and s ubsequently amp lified at the Yale University sequenc ing facility for Illumina sequencing. However, it required a lot of effort to obtain this...and Polyak K. (2008). Genome-wide functi onal synergy between amp lified and mutated genes in human breast cancer. Cancer Res. 68: 9532-9540...east cancer patient samples. Other co-amp lified genes, within the HER2 amplicon and/or at other regions, could serve as additional novel target s for

  20. Whole genome amplification of DNA for genotyping pharmacogenetics candidate genes.

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    Santosh ePhilips

    2012-03-01

    Full Text Available Whole genome amplification (WGA technologies can be used to amplify genomic DNA when only small amounts of DNA are available. The Multiple Displacement Amplification Phi polymerase based amplification has been shown to accurately amplify DNA for a variety of genotyping assays; however, it has not been tested for genotyping many of the clinically relevant genes important for pharmacogenetic studies, such as the cytochrome P450 genes, that are typically difficult to genotype due to multiple pseudogenes, copy number variations, and high similarity to other related genes. We evaluated whole genome amplified samples for Taqman™ genotyping of SNPs in a variety of pharmacogenetic genes. In 24 DNA samples from the Coriell human diversity panel, the call rates and concordance between amplified (~200-fold amplification and unamplified samples was 100% for two SNPs in CYP2D6 and one in ESR1. In samples from a breast cancer clinical trial (Trial 1, we compared the genotyping results in samples before and after WGA for four SNPs in CYP2D6, one SNP in CYP2C19, one SNP in CYP19A1, two SNPs in ESR1, and two SNPs in ESR2. The concordance rates were all >97%. Finally, we compared the allele frequencies of 143 SNPs determined in Trial 1 (whole genome amplified DNA to the allele frequencies determined in unamplified DNA samples from a separate trial (Trial 2 that enrolled a similar population. The call rates and allele frequencies between the two trials were 98% and 99.7%, respectively. We conclude that the whole genome amplified DNA is suitable for Taqman™ genotyping for a wide variety of pharmacogenetically relevant SNPs.

  1. Genome-wide gene amplification during differentiation of neural progenitor cells in vitro.

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    Fischer, Ulrike; Keller, Andreas; Voss, Meike; Backes, Christina; Welter, Cornelius; Meese, Eckart

    2012-01-01

    DNA sequence amplification is a phenomenon that occurs predictably at defined stages during normal development in some organisms. Developmental gene amplification was first described in amphibians during gametogenesis and has not yet been described in humans. To date gene amplification in humans is a hallmark of many tumors. We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells. Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells. We identified several amplified genes in neural progenitor cells that are known to be amplified in malignant tumors. There is also a striking overlap of amplified chromosomal regions between differentiating neural progenitor cells and malignant tumor cells derived from astrocytes. Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

  2. Copy number change: evolving views on gene amplification.

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    Elliott, Kathryn T; Cuff, Laura E; Neidle, Ellen L

    2013-07-01

    The rapid pace of genomic sequence analysis is increasing the awareness of intrinsically dynamic genetic landscapes. Gene duplication and amplification (GDA) contribute to adaptation and evolution by allowing DNA regions to expand and contract in an accordion-like fashion. This process affects diverse aspects of bacterial infection, including antibiotic resistance and host-pathogen interactions. In this review, microbial GDA is discussed, primarily using recent bacterial examples that demonstrate medical and evolutionary consequences. Interplay between GDA and horizontal gene transfer further impact evolutionary trajectories. Complementing the discovery of gene duplication in clinical and environmental settings, experimental evolution provides a powerful method to document genetic change over time. New methods for GDA detection highlight both its importance and its potential application for genetic engineering, synthetic biology and biotechnology.

  3. Gene amplification during differentiation of mammalian neural stem cells in vitro and in vivo.

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    Fischer, Ulrike; Backes, Christina; Raslan, Abdulrahman; Keller, Andreas; Meier, Carola; Meese, Eckart

    2015-03-30

    In development of amphibians and flies, gene amplification is one of mechanisms to increase gene expression. In mammalian cells, gene amplification seems to be restricted to tumorigenesis and acquiring of drug-resistance in cancer cells. Here, we report a complex gene amplification pattern in mouse neural progenitor cells during differentiation with approximately 10% of the genome involved. Half of the amplified mouse chromosome regions overlap with amplified regions previously reported in human neural progenitor cells, indicating conserved mechanisms during differentiation. Using fluorescence in situ hybridization, we verified the amplification in single cells of primary mouse mesencephalon E14 (embryonic stage) neurosphere cells during differentiation. In vivo we confirmed gene amplifications of the TRP53 gene in cryosections from mouse embryos at stage E11.5. Gene amplification is not only a cancer-related mechanism but is also conserved in evolution, occurring during differentiation of mammalian neural stem cells.

  4. Gene amplification of the Hps locus in Glycine max

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    Kuflu Kuflom

    2006-03-01

    Full Text Available Abstract Background Hydrophobic protein from soybean (HPS is an 8 kD cysteine-rich polypeptide that causes asthma in persons allergic to soybean dust. HPS is synthesized in the pod endocarp and deposited on the seed surface during development. Past evidence suggests that the protein may mediate the adherence or dehiscence of endocarp tissues during maturation and affect the lustre, or glossiness of the seed surface. Results A comparison of soybean germplasm by genomic DNA blot hybridization shows that the copy number and structure of the Hps locus is polymorphic among soybean cultivars and related species. Changes in Hps gene copy number were also detected by comparative genomic DNA hybridization using cDNA microarrays. The Hps copy number polymorphisms co-segregated with seed lustre phenotype and HPS surface protein in a cross between dull- and shiny-seeded soybeans. In soybean cultivar Harosoy 63, a minimum of 27 ± 5 copies of the Hps gene were estimated to be present in each haploid genome. The isolation and analysis of genomic clones indicates that the core Hps locus is comprised of a tandem array of reiterated units, with each 8.6 kb unit containing a single HPS open reading frame. Conclusion This study shows that polymorphisms at the Hps locus arise from changes in the gene copy number via gene amplification. We present a model whereby Hps copy number modulates protein expression levels and seed lustre, and we suggest that gene amplification may result from selection pressures imposed on crop plants.

  5. Amplification of LDH gene from Indian strains of Plasmodium vivax

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    Ritu Berwal, N. Gopalan, Kshitij Chandel, Shri Prakash ,K. Sekhar

    2006-09-01

    Full Text Available Background & objectives: Plasmodium vivax is geographically widespread and responsible for >50% of malaria cases in India. Increased drug resistance of the parasite highlights the immediaterequirement of early and accurate diagnosis as well as new therapeutics. In view of this, the presentstudy was undertaken to amplify P. vivax (Indian strains lactate dehydrogenase gene (PvLDHwhich has been identified as a good target for antimalarials as well as diagnostics.Methods: P. vivax infected clinical blood samples were collected from southern part of India andwere tested with established diagnostic parameters (ICT, Giemsa staining. Total DNA was extractedfrom blood samples and subjected to PCR using two sets of primers, one for the amplification of fullPvLDH gene (951bp and the other for a partial PvLDH gene fragment (422bp, covering a variableantigenic region (140aa as compared to other plasmodial species.Results & conclusion: PCRs for both the full and partial gene targets were optimised and found to beconsistent when tested on several P. vivax positive clinical samples. In addition, full gene PCR wasfound to specifically detect only P. vivax DNA and could be used as a specific molecular diagnostictool. These amplified products can be cloned and expressed as a recombinant protein that might beuseful for the development and screening of antimalarials as well as for diagnostic purposes.

  6. No fitness cost of glyphosate resistance endowed by massive EPSPS gene amplification in Amaranthus palmeri.

    Science.gov (United States)

    Vila-Aiub, Martin M; Goh, Sou S; Gaines, Todd A; Han, Heping; Busi, Roberto; Yu, Qin; Powles, Stephen B

    2014-04-01

    Amplification of the EPSPS gene has been previously identified as the glyphosate resistance mechanism in many populations of Amaranthus palmeri, a major weed pest in US agriculture. Here, we evaluate the effects of EPSPS gene amplification on both the level of glyphosate resistance and fitness cost of resistance. A. palmeri individuals resistant to glyphosate by expressing a wide range of EPSPS gene copy numbers were evaluated under competitive conditions in the presence or absence of glyphosate. Survival rates to glyphosate and fitness traits of plants under intra-specific competition were assessed. Plants with higher amplification of the EPSPS gene (53-fold) showed high levels of glyphosate resistance, whereas less amplification of the EPSPS gene (21-fold) endowed a lower level of glyphosate resistance. Without glyphosate but under competitive conditions, plants exhibiting up to 76-fold EPSPS gene amplification exhibited similar height, and biomass allocation to vegetative and reproductive organs, compared to glyphosate susceptible A. palmeri plants with no amplification of the EPSPS gene. Both the additive effects of EPSPS gene amplification on the level of glyphosate resistance and the lack of associated fitness costs are key factors contributing to EPSPS gene amplification as a widespread and important glyphosate resistance mechanism likely to become much more evident in weed plant species.

  7. Gene Amplification of the Multidrug Resistance 1 Gene of Plasmodium vivax Isolates from Thailand, Laos, and Myanmar▿

    Science.gov (United States)

    Imwong, Mallika; Pukrittayakamee, Sasithon; Pongtavornpinyo, Wirichada; Nakeesathit, Supatchara; Nair, Shalini; Newton, Paul; Nosten, Francois; Anderson, Timothy J. C.; Dondorp, Arjen; Day, Nicholas P. J.; White, Nicholas J.

    2008-01-01

    Plasmodium vivax mdr1 gene amplification, quantified by real-time PCR, was significantly more common on the western Thailand border (6 of 66 samples), where mefloquine pressure has been intense, than elsewhere in southeast Asia (3 of 149; P = 0.02). Five coding mutations in pvmdr1, independent of gene amplification, were also found. PMID:18443118

  8. The Study on Gene Amplification of EGFR in Bronchioloalveolar Carcinoma and Conventional Adenocarcinoma of the Lung

    Directory of Open Access Journals (Sweden)

    Xin SONG

    2009-08-01

    Full Text Available Background and objective Patients with adenocarcinoma of the lung have disproportionately response to the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI. The aim of this study is to analyze the difference of EGFR gene amplification in bronchioloalveolar carcinoma (BAC, adenocarcinma mixed subtype and conventional adenocarcinoma of the lung and provide some information to clinical therapies. Methods Lung cancer cases were collected and reviewed from the archives of the Department of Pathology, Chinese PLA General Hospital during the time period from 2004 to 2006. The definite diagnosis of BAC based on 2004 WHO classification of lung tumors was made by two pathologists. Fluorescence in situ hybridization (FISH was performed to detect EGFR gene amplification in pure BAC, adenocarcinma mixed subtype and conventional adenocarcinoma. Results Conventional adenocarcinoma had higher EGFR amplification compared with pure BAC and adenocarcinma mixed subtype (χ2=11.632, P<0.05. EGFR gene amplification was found in 45.45% of conventional adenocarcinoma, 14.81% in pure BACs, and 22.58% in adenocarcinma mixed subtype. EGFR gene amplification was observed as scattered signals in most cases. Conclusion EGFR gene amplification was seen more frequently in the invasive components than in BAC. EGFR gene amplification might be associated with the development of adenocarcinoma of the lung.

  9. KRAS and MAPK1 Gene Amplification in Type II Ovarian Carcinomas

    Directory of Open Access Journals (Sweden)

    Noriyuki Ishikawa

    2013-07-01

    Full Text Available In this study, we examined the clinical significance of KRAS and MAPK1 amplification and assessed whether these amplified genes were potential therapeutic targets in type II ovarian carcinoma. Using fluorescence in situ hybridization, immunohistochemistry, and retrospectively collected clinical data, KRAS and MAPK1 amplifications were identified in 9 (13.2% and 5 (7.4% of 68 type II ovarian carcinoma tissue samples, respectively. Interestingly, co-amplification of KRAS and MAPK1 seemed to be absent in the type II ovarian carcinomas tested, except one case. Active phospho-ERK1/2 was identified in 26 (38.2% out of 68 type II ovarian carcinomas and did not correlate with KRAS or MAPK1 amplification. There was no significant relationship between KRAS amplification and overall or progression-free survival in patients with type II ovarian carcinoma. However, patients with MAPK1 amplification had significantly poorer progression-free survival than patients without MAPK1 amplification. Moreover, type II ovarian carcinoma cells with concomitant KRAS amplification and mutation exhibited dramatic growth reduction following treatment with the MEK inhibitor PD0325901. These findings indicate that KRAS/MAPK1 amplification is critical for the growth of a subset of type II ovarian carcinomas. Additionally, RAS/RAF/MEK/ERK pathway-targeted therapy may benefit selected patients with type II ovarian carcinoma harboring KRAS/MAPK1 amplifications.

  10. SPERM RNA AMPLIFICATION FOR GENE EXPRESSION PROFILING BY DNA MICROARRAY TECHNOLOGY

    Science.gov (United States)

    Sperm RNA Amplification for Gene Expression Profiling by DNA Microarray TechnologyHongzu Ren, Kary E. Thompson, Judith E. Schmid and David J. Dix, Reproductive Toxicology Division, NHEERL, Office of Research and Development, US Environmental Protection Agency, Research Triang...

  11. Study on the protein expression and amplification of HER2 gene in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Sunan Wang; Yingying Li; Zhengshun Xu; Wenzhao Zhao; Tian Yun; Wuling Zhu; Yangkun Wang

    2014-01-01

    Objective:The aim of the study was to investigate the human epidermal growth factor receptor 2 (HER2) gene amplification and protein expression and interpretation points in the stomach mixed carcinomas. Methods:Immunohisto-chemistry (IHC) and fluorescence in situ hybridization (FISH) technique were used to detect HER2 gene amplification and ex-pression of HER2 protein in 442 cases of gastric mixed carcinoma. Results:The expression rate of HER2 protein was 41.2%(182/442):the HER2 protein expression IHC 3+extensive type in 18 cases, partial type in 21 cases, focal type in 8 cases, accounting for 10.6%(47/442);the HER2 protein expression IHC 2+extensive type in 23 cases, partial type in 28 cases, focal type in 11 cases, accounting for 14.0%(62/442);the HER2 protein expression IHC 1+extensive type in 27 cases, partial type in 31 cases, focal type in 15 cases, accounting for 16.5%(73/442). HER2 gene amplification rate of 442 cases was 16.1%(71/442). In 182 cases of HER2 protein positive expression, the HER2 gene cluster amplification rate was 14.8%(27/182), large granular amplification rate 11.0%(20/182), punctate amplification rate 6.0%(11/182) and high polysomy 7.1%(13/182). In 71 cases of HER2 gene amplification, there was 42 cases of HER2 protein expression IHC 3+, 22 cases of HER2 protein expression IHC 2+, and 7 cases of IHC 1+. Conclusion:HER2 detection of gastric mixed carcinoma has great heterogeneity, HER2 protein positive expression is divided into extensive type, partial type and focal type, and HER2 gene positive amplifica-tion is divided into cluster amplification, large granular amplification, punctate amplification and high polysomy. These typing of HER2 protein expression and HER2 gene amplification provide reference index to quantify for targeted therapeutic ef ect of anticancer drugs.

  12. Her2 expression and gene amplification is rarely detectable in patients with oral squamous cell carcinomas.

    Science.gov (United States)

    Hanken, Henning; Gaudin, Robert; Gröbe, Alexander; Fraederich, Meike; Eichhorn, Wolfgang; Smeets, Ralf; Simon, Ronald; Sauter, Guido; Grupp, Katharina; Izbicki, Jacob R; Sehner, Susanne; Heiland, Max; Blessmann, Marco

    2014-04-01

    Her2 (ErbB2) transforms cells when overexpressed and is an important therapeutic target in breast cancer. Contrary to breast cancer, studies on Her2 overexpression and gene amplification in squamous cell carcinomas of the head and neck region described largely different results. This study was undertaken to learn more on the prevalence and clinical significance of HER2 amplification and overexpression in squamous cell carcinomas of the head and neck. Her2 expression and gene amplification was analyzed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) on two tissue microarrays composed of 427 squamous cell carcinomas of the head and neck region and 222 oral squamous cell carcinomas. Results were compared with clinicopathological features. Her2 expression and gene amplification was rarely detectable in squamous cell carcinomas of the head and neck region and unrelated to tumor phenotype or survival of the patients with oral squamous carcinoma. Our results demonstrate that Her2 protein and gene amplification was only detectable in a small subset of squamous cell carcinomas of the head and neck region as well as oral squamous cell carcinomas. However, it can be speculated that those few patients with Her2 overexpressing and gene amplificated tumors may possibly benefit from an anti-Her2 therapy. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Analysis of HER2 gene amplification using Differential PCR in breast cancer patients of Isfahan Province

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    Zohreh Hojati

    2014-10-01

    Full Text Available Background: Amplification of HER2 is seen in 20-30% of breast cancer cases. Measurement of HER2 gene amplification appears to be of vital importance in planning the treatment schedule for patients with breast carcinoma. The aim of our study was to evaluate HER2 amplification status in malignant and benign breast tumors by differential PCR (dPCR. Materials and Methods: The genomic DNA was extracted using the phenol/chloroform extraction procedure from 76 different breast tissues. Differential PCR was performed using the DNA samples isolated from fresh and paraffin- embedded breast cancer tissues. The relative copy number ratio of target gene (HER2 to control gene ( INF-γ was measured. dPCR products were then separated by electrophoresis using 2% agarose gel. The intensity of HER2 and INFγ bands were determined for each sample by ImageJ software. Results: According to the ratio between the band intensity of HER2 to INFγ in tumour and also normal samples, 7% and 26% rates of HER2 amplification were observed in benign and malignant samples respectively. The ratio showed a 2-5 fold increase in HER2 gene copy number for tissues with HER2 amplification whereas, a one-fold increase was found in other samples. Conclusion: Differential PCR provides a relatively rapid and inexpensive technique to assess the HER2 gene amplification, especially alongside immunohistochemistry as a routine assessing method .

  14. Flux variability scanning based on enforced objective flux for identifying gene amplification targets

    Directory of Open Access Journals (Sweden)

    Park Jong

    2012-08-01

    Full Text Available Abstract Background In order to reduce time and efforts to develop microbial strains with better capability of producing desired bioproducts, genome-scale metabolic simulations have proven useful in identifying gene knockout and amplification targets. Constraints-based flux analysis has successfully been employed for such simulation, but is limited in its ability to properly describe the complex nature of biological systems. Gene knockout simulations are relatively straightforward to implement, simply by constraining the flux values of the target reaction to zero, but the identification of reliable gene amplification targets is rather difficult. Here, we report a new algorithm which incorporates physiological data into a model to improve the model’s prediction capabilities and to capitalize on the relationships between genes and metabolic fluxes. Results We developed an algorithm, flux variability scanning based on enforced objective flux (FVSEOF with grouping reaction (GR constraints, in an effort to identify gene amplification targets by considering reactions that co-carry flux values based on physiological omics data via “GR constraints”. This method scans changes in the variabilities of metabolic fluxes in response to an artificially enforced objective flux of product formation. The gene amplification targets predicted using this method were validated by comparing the predicted effects with the previous experimental results obtained for the production of shikimic acid and putrescine in Escherichia coli. Moreover, new gene amplification targets for further enhancing putrescine production were validated through experiments involving the overexpression of each identified targeted gene under condition-controlled batch cultivation. Conclusions FVSEOF with GR constraints allows identification of gene amplification targets for metabolic engineering of microbial strains in order to enhance the production of desired bioproducts. The algorithm

  15. Carboxylesterase gene amplifications associated with insecticide resistance in Aedes albopictus: Geographical distribution and evolutionary origin.

    Directory of Open Access Journals (Sweden)

    Linda Grigoraki

    2017-04-01

    Full Text Available Aedes albopictus is one of the most invasive human disease vectors. Its control has been largely based on insecticides, such as the larvicide temephos. Temephos resistance has been associated with the up-regulation, through gene amplification, of two carboxylesterase (CCE genes closely linked on the genome, capable of sequestering and metabolizing temephos oxon, the activated form of temephos.Here, we investigated the occurrence, geographical distribution and origin of the CCE amplicon in Ae. albopictus populations from several geographical regions worldwide. The haplotypic diversity at the CCEae3a locus revealed high polymorphism, while phylogenetic analysis showed an absence of correlation between haplotype similarity and geographic origin. Two types of esterase amplifications were found, in two locations only (Athens and Florida: one, previously described, results in the amplification of both CCEae3a and CCEae6a; the second is being described for the first time and results in the amplification of CCEae3a only. The two amplification events are independent, as confirmed by sequence analysis. All individuals from Athens and Florida carrying the CCEae3a-CCEae6a co-amplicon share a common haplotype, indicating a single amplification event, which spread between the two countries.The importance of passive transportation of disease vectors, including individuals carrying resistance mechanisms, is discussed in the light of efficient and sustainable vector control strategies.

  16. Inducible amplification of gene copy number and heterologous protein production in the yeast Kluyveromyces lactis.

    Science.gov (United States)

    Morlino, G B; Tizzani, L; Fleer, R; Frontali, L; Bianchi, M M

    1999-11-01

    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1beta, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.

  17. Amplification of epidermal growth factor receptor gene in renal cell carcinoma

    DEFF Research Database (Denmark)

    Harper, Peter; El-Hariry, Iman; Powles, Thomas;

    2010-01-01

    Expression of epidermal growth factor receptor (EGFR) may be of prognostic value in renal cell cancer (RCC). Gene amplification of EGFR was investigated in a cohort of 315 patients with advanced RCC from a previously reported randomised study. Using fluorescent in situ hybridisation, only 2...

  18. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

    NARCIS (Netherlands)

    Ziesemer, K.A.; Mann, A.E.; Sankaranarayanan, K.; Schroeder, H.; Ozga, A.T.; Brandt, B.W.; Zaura, E.; Waters-Rist, A.; Hoogland, M.; Salazar-García, D.C.; Aldenderfer, M.; Speller, C.; Hendy, J.; Weston, D.A.; MacDonald, S.J.; Thomas, G.H.; Collins, M.J.; Lewis, C.M.; Hofman, C.; Warinner, C.

    2015-01-01

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gen

  19. Complex Oncogenic Translocations with Gene Amplification are Initiated by Specific DNA Breaks in Lymphocytes

    OpenAIRE

    2009-01-01

    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. While chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double strand break repair in suppression of oncogenic genome instability, the genomic elements requir...

  20. Expression of FGFR3 Protein and Gene Amplification in Urinary Bladder Lesions in Relation to Schistosomiasis

    Directory of Open Access Journals (Sweden)

    Olfat Hammam

    2017-04-01

    CONCLUSIONS: FGFR3 overexpression in malignant cases was significantly higher than in chronic cystitis. FGFR3 gene amplification was reported mainly in low grade and NNMBIC tumours. FGFR3 may be further studied as a subject for target therapy of bladder cancer.

  1. Amplification and overexpression of the EGF receptor gene in primary human glioblastomas.

    Science.gov (United States)

    Libermann, T A; Nusbaum, H R; Razon, N; Kris, R; Lax, I; Soreq, H; Whittle, N; Waterfield, M D; Ullrich, A; Schlessinger, J

    1985-01-01

    The expression of epidermal growth factor (EGF) receptor in brain tumours of glial origin was studied at the protein, mRNA and genomic levels. Four out of 10 glioblastomas that overexpress EGF receptor also have gene amplification. The amplified genes appear to be rearranged, generating an aberrant mRNA in at least one of these tumours. Such receptor defects may be relevant to tumorigenesis of human glioblastomas.

  2. Super resolution imaging of HER2 gene amplification

    Science.gov (United States)

    Okada, Masaya; Kubo, Takuya; Masumoto, Kanako; Iwanaga, Shigeki

    2016-02-01

    HER2 positive breast cancer is currently examined by counting HER2 genes using fluorescence in situ hybridization (FISH)-stained breast carcinoma samples. In this research, two-dimensional super resolution fluorescence microscopy based on stochastic optical reconstruction microscopy (STORM), with a spatial resolution of approximately 20 nm in the lateral direction, was used to more precisely distinguish and count HER2 genes in a FISH-stained tissue section. Furthermore, by introducing double-helix point spread function (DH-PSF), an optical phase modulation technique, to super resolution microscopy, three-dimensional images were obtained of HER2 in a breast carcinoma sample approximately 4 μm thick.

  3. Concurrent AURKA and MYCN Gene Amplifications Are Harbingers of Lethal TreatmentRelated Neuroendocrine Prostate Cancer

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    Juan Miguel Mosquera

    2013-01-01

    Full Text Available Neuroendocrine prostate cancer (NEPC, also referred to as anaplastic prostate cancer, is a lethal tumor that most commonly arises in late stages of prostate adenocarcinoma (PCA with predilection to metastasize to visceral organs. In the current study, we explore for evidence that Aurora kinase A (AURKA and N-myc (MYCN gene abnormalities are harbingers of treatment-related NEPC (t-NEPC. We studied primary prostate tissue from 15 hormone naïve PCAs, 51 castration-resistant prostate cancers, and 15 metastatic tumors from 72 patients at different stages of disease progression to t-NEPC, some with multiple specimens. Histologic evaluation, immunohistochemistry, and fluorescence in situ hybridization were performed and correlated with clinical variables. AURKA amplification was identified in overall 65% of PCAs (hormone naïve and treated from patients that developed t-NEPC and in 86% of metastases. Concurrent amplification of MYCN was present in 70% of primary PCAs, 69% of treated PCAs, and 83% of metastases. In contrast, in an unselected PCA cohort, AURKA and MYCN amplifications were identified in only 5% of 169 cases. When metastatic t-NEPC was compared to primary PCA from the same patients, there was 100% concordance of ERG rearrangement, 100% concordance of AURKA amplification, and 60% concordance of MYCN amplification. In tumors with mixed features, there was also 100% concordance of ERG rearrangement and 94% concordance of AURKA and MYCN co-amplification between areas of NEPC and adenocarcinoma. AURKA and MYCN amplifications may be prognostic and predictive biomarkers, as they are harbingers of tumors at risk of progressing to t-NEPC after hormonal therapy.

  4. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

    Science.gov (United States)

    Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

    2015-11-13

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

  5. Single Cell Analysis of Dystrophin and SRY Gene by Using Whole Genome Amplification

    Institute of Scientific and Technical Information of China (English)

    徐晨明; 金帆; 黄荷凤; 陶冶; 叶英辉

    2001-01-01

    Objective To develop a reliable and sensitive method for detection of sex and multiloci of Duchenne muscular dystrophy (DMD) gene in single cell Materials & methods Whole genome of single cell were amplified by using 15-base random primers (primer extension preamplification, PEP), then a small aliquot of PEP product were analyzed by using locus-specific nest PCR amplification. The procedure was evaluated by detection dystrophin exons 8, 17, 19, 44, 45, 48 and human testis-determining gene (SRY)in single lymphocytes from known sources and single blastomeres from the couples with no family history of DMD.Results The amplification efficiency rate of six dystrophin exons from single lymphocytes and single blastomeres were 97. 2% (175/180) and 100% (60/60) respectively.Results of SRY showed that 100% (15/15) amplification in single male-derived lymphocytes and 0% (0/15) amplification in single female-derived lymphocytes. Conclusion The technique of single cell PEP-nest PCR for dystrophin exons 8, 17,19, 44, 45, 48 and SRY is highly specifc. PEP-nest PCR is suitable for Preimplantation genetic diagnosis (PGD) of DMD at single cell level.

  6. Cyclin E gene (CCNE) amplification and hCDC4 mutations in endometrial carcinoma.

    Science.gov (United States)

    Cassia, Raúl; Moreno-Bueno, Gema; Rodríguez-Perales, Sandra; Hardisson, David; Cigudosa, Juan C; Palacios, José

    2003-12-01

    Cyclin E overexpression occurs in a subset of endometrial carcinomas (ECs), but the molecular mechanisms underlying this alteration remain to be established. The present study has analysed amplification of the cyclin E gene (CCNE) and mutation in hCDC4, the gene coding for the F-box protein, which tags phosphorylated cyclin E for proteosomal degradation, to ascertain whether these alterations might be responsible for cyclin E overexpression in ECs. Cyclin E and p53 expression was studied by immunohistochemistry in eight atypical endometrial hyperplasias (AEHs), 51 endometrioid endometrial carcinomas (EECs), and 22 non-endometrioid endometrial carcinomas (NEECs). CCNE amplification was analysed by fluorescence in situ hybridization (FISH). Mutations in exons 2-11 of the hCDC4 gene were screened by PCR-SSCP-sequencing. Finally, the polymorphic marker D4S1610 was used to assess loss of heterozygosity (LOH) in the hCDC4 gene. Cyclin E overexpression was found in 26/81 (32%) cases and was associated with the histological type of the lesion, since it was not found in any AEHs but was present in 27% of EECs and 54.5% of NEECs (p=0.035). Cyclin E overexpression was associated with histological grade (p=0.011) and p53 immunostaining in EECs (p=0.033). CCNE amplification was found in 6 of 37 (16%) ECs examined. There was a significant association between CCNE amplification and the histological type of the lesion, since five (83%) of the six cases with amplification were NEECs (p=0.008). One EEC harboured an hCDC4 mutation: a CGA to CAA (Arg/Gln) change at codon 479. In addition, D4S1610 LOH was found in 7 of 23 (30%) informative cases analysed, but no correlation with cyclin E overexpression was found. However, the tumour with hCDC4 mutation also showed LOH. This is the first study demonstrating that cyclin E overexpression is associated with gene amplification in ECs, these alterations being more frequent in NEECs. Although hCDC4 exhibits a low mutation frequency in ECs

  7. Upregulated, 7q21-22 amplicon candidate gene SHFM1 confers oncogenic advantage by suppressing p53 function in gastric cancer.

    Science.gov (United States)

    Tamilzhalagan, Sembulingam; Muthuswami, Muthulakshmi; Periasamy, Jayaprakash; Lee, Ming Hui; Rha, Sun Young; Tan, Patrick; Ganesan, Kumaresan

    2015-06-01

    Chromosomal aberrations are hallmarks of cancers and the locus of frequent genomic amplifications often harbors key cancer driver genes. Many genomic amplicons remain larger with hundreds of genes and the key drivers remain to be identified by an amplification-wide systematic analysis. The 7q21.12-q22.3 genomic amplification is frequent in gastric cancers which occur in ~10% of the patients and multiple cell lines. This 7q21.12-q22.3 amplicon has not yet been completely analyzed towards identifying the driver genes and their functional contribution in oncogenesis. The amplitude and prevalence indicate the important role conferred by this amplicon in gastric cancers. Among the 159 genes of this amplicon, 12 genes are found over-expressed in primary gastric tumors and cell lines. Many of the over-expressed genes show negative association with p53 transcriptional activity. RNAi based functional screening of the genes reveal, SHFM1 as key gastric cancer driver gene. SHFM1 confers cell cycle progression and resistance to p53 stabilizing drugs in gastric cancer cells. SHFM1 also activates Src, MAPK/ERK and PI3K/Akt signaling pathways. This is the first integrative genomic investigation of 7q21.12-q22.3 amplicon revealing the potential oncogenic candidacy of 12 genes. The oncogenic contribution of SHFM1, mediated by the p53 suppressive feature has been demonstrated in gastric cancer cells.

  8. The Association between EGFR Gene Amplification and the Prognosis in Non-small Cell Lung Cancer: A meta-analysis

    Directory of Open Access Journals (Sweden)

    Jie WANG

    2009-12-01

    Full Text Available Background and objective Many studies concluded that epidermal growth factor receptor (EGFR gene amplification might be related with the prognosis in non-small cell lung cancer. However, the results were not consistent. To identify whether EGFR gene amplification could affect the prognosis, a meta-analysis was conducted based on the published works. Methods According to inclusion and exclusion criteria, articles were selected from medical electronic databases searching, including PubMed, Cochrane library and CNKI. The qualities of included articles were scored based on the European Lung Cancer Working Party scale, and the hazard ratio (HR and the 95% confidence interval (CI were also collected. Meta-analysis was completed using software Review Manager 4.2. Results Forest plot from 12 studies in which 1 221 included patients were all treated with EGFR-TKI showed that the prognosis of patients harboring EGFR gene amplification were superior to negative ones (HR=0.82, 95%CI: 0.68-0.99, P=0.04, and for Caucasian patients who received EGFR-TKI, the survivals were longer in EGFR gene amplification positive cases than those without amplification (HR=0.42, 95%CI: 0.31-0.57, P < 0.001. Meta results from 8 studies in which 658 included patients did not receive EGFR-TKI indicated that the casualty hazard of amplification positive patients was comparable with negative ones. However, for Asia patients who failed to receive EGFR-TKI, the prognosis of patients with EGFR gene amplification were inferior to negative ones (HR=1.88, 95%CI: 1.21-2.93, P=0.005. Conclusion EGFR gene amplification may be a positive predictive factor in terms of survival for Caucasian patients receiving EGFR-TKI rather than Asia patients. However, the prognosis was inferior in Asia patients with EGFR amplification to those without the amplification, if they failed to receive EGFR-TKI.

  9. ANALYSIS OF C-HA-RAS GENE AMPLIFICATION AND MUTATION IN LARYNGEAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    刘世喜; 林代诚; 洪邦泰; 黄光琦

    1995-01-01

    In order to study the ahered molecular events during laryngeal carcinogenesis and elucidate the role of Ha-ras oncogene amplification and mutation, we have examined their profile by polymerase chain reaction (PCR) and selective oligonucleoride hybridization. We analyzed the mutational status of codon 12 of Ha-ras in 22 laryngeal carcinomas and 10 normal tissues, and found that 7 of 22 laryngeal carcinomas con-tained a Ha-ras mutation at codon 12. The frequency of mutation was 32%. None of the normal tissues re-vealed mutation. Moreover, no amplification was found in cancers when compared to the normal. Our findings indicated that the aefivmed Ha-ras gene existed in laryngeal carcinoma, and activation of the Ha-ras gene by mutation at codon 12 might play a key role in laryngeal carcinogenesis.

  10. Biologically relevant effects of mRNA amplification on gene expression profiles

    Directory of Open Access Journals (Sweden)

    Smits Jos FM

    2006-04-01

    Full Text Available Abstract Background Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Results Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other. Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P Conclusion This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left

  11. Microarray analysis after RNA amplification can detect pronounced differences in gene expression using limma

    Directory of Open Access Journals (Sweden)

    Orengo Christine

    2006-10-01

    Full Text Available Abstract Background RNA amplification is necessary for profiling gene expression from small tissue samples. Previous studies have shown that the T7 based amplification techniques are reproducible but may distort the true abundance of targets. However, the consequences of such distortions on the ability to detect biological variation in expression have not been explored sufficiently to define the true extent of usability and limitations of such amplification techniques. Results We show that expression ratios are occasionally distorted by amplification using the Affymetrix small sample protocol version 2 due to a disproportional shift in intensity across biological samples. This occurs when a shift in one sample cannot be reflected in the other sample because the intensity would lie outside the dynamic range of the scanner. Interestingly, such distortions most commonly result in smaller ratios with the consequence of reducing the statistical significance of the ratios. This becomes more critical for less pronounced ratios where the evidence for differential expression is not strong. Indeed, statistical analysis by limma suggests that up to 87% of the genes with the largest and therefore most significant ratios (p -20 in the unamplified group have a p-value below 10e-20 in the amplified group. On the other hand, only 69% of the more moderate ratios (10e-20 -10 in the unamplified group have a p-value below 10e-10 in the amplified group. Our analysis also suggests that, overall, limma shows better overlap of genes found to be significant in the amplified and unamplified groups than the Z-scores statistics. Conclusion We conclude that microarray analysis of amplified samples performs best at detecting differences in gene expression, when these are large and when limma statistics are used.

  12. Patterns of HER2 Gene Amplification and Response to Anti-HER2 Therapies.

    Directory of Open Access Journals (Sweden)

    Rocio Vicario

    Full Text Available A chromosomal region that includes the gene encoding HER2, a receptor tyrosine kinase (RTK, is amplified in 20% of breast cancers. Although these tumors tend to respond to drugs directed against HER2, they frequently become resistant and resume their malignant progression. Gene amplification in double minutes (DMs, which are extrachromosomal entities whose number can be dynamically regulated, has been suggested to facilitate the acquisition of resistance to therapies targeting RTKs. Here we show that ~30% of HER2-positive tumors show amplification in DMs. However, these tumors respond to trastuzumab in a similar fashion than those with amplification of the HER2 gene within chromosomes. Furthermore, in different models of resistance to anti-HER2 therapies, the number of DMs containing HER2 is maintained, even when the acquisition of resistance is concomitant with loss of HER2 protein expression. Thus, both clinical and preclinical data show that, despite expectations, loss of HER2 protein expression due to loss of DMs containing HER2 is not a likely mechanism of resistance to anti-HER2 therapies.

  13. Epidermal growth factor receptor gene amplification and protein expression in glioblastoma multiforme: prognostic significance and relationship to other prognostic factors.

    Science.gov (United States)

    Layfield, Lester J; Willmore, Carlynn; Tripp, Sheryl; Jones, Claudia; Jensen, Randy L

    2006-03-01

    Epidermal growth factor receptor (EGFR) overexpression occurs in a significant percentage of cases of glioblastoma multiforme (GBM), and amplification has been found in approximately 40% of these neoplasms. Controversy exists as to the prognostic significance of EGFR gene amplification: some reports have indicated that amplification is associated with a poor prognosis, while other authors have reported no relationship between gene amplification and prognosis. Some reports have found a poor prognosis to be associated with amplification of the EGFR gene in patients of all ages with GBM, while other authors have found EGFR amplification to be an independent predictor of prolonged survival in patients with GBM who are older than 60 years of age. The authors studied a series of 34 specimens (32 patients) with histologically proven GBM by immunohistochemistry for the presence of EGFR overexpression and by fluorescence in situ hybridization (FISH) for gene amplification of the EGFR gene. Results of these studies and data on patient age, sex, functional status, therapy, and survival were correlated to determine which variables were predictive of survival. p53 expression was also determined by immunohistochemistry and correlated with the other variables and survival.

  14. Amplification of thermostable lipase genes fragment from thermogenic phase of domestic waste composting process

    Science.gov (United States)

    Nurhasanah, Nurbaiti, Santi; Madayanti, Fida; Akhmaloka

    2015-09-01

    Lipases are lipolytic enzymes, catalyze the hydrolysis of fatty acid ester bonds of triglycerides to produce free fatty acids and glycerol. The enzyme is widely used in various fields of biotechnological industry. Hence, lipases with unique properties (e.g.thermostable lipase) are still being explored by variation methods. One of the strategy is by using metagenomic approach to amplify the gene directly from environmental sample. This research was focused on amplification of lipase gene fragment directly from the thermogenic phase of domestic waste composting in aerated trenches. We used domestic waste compost from waste treatment at SABUGA, ITB for the sample. Total chromosomal DNA were directly extracted from several stages at thermogenic phase of compost. The DNA was then directly used as a template for amplification of thermostable lipase gene fragments using a set of internal primers namely Flip-1a and Rlip-1a that has been affixed with a GC clamp in reverse primer. The results showed that the primers amplified the gene from four stages of thermogenic phase with the size of lipase gene fragment of approximately 570 base pairs (bp). These results were further used for Denaturing Gradient Gel Electrophoresis (DGGE) analysis to determine diversity of thermostable lipase gene fragments.

  15. Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    B. Radhika

    2016-01-01

    Full Text Available Aim: The loop mediated isothermal amplification (LAMP was standardized for rapid detection of Clostridium perfringens. Materials and Methods: A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene. Results: Out of 120 samples screened 112 (93.3% samples were positive by both LAMP and polymerase chain reaction (PCR for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis. Conclusion: Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases.

  16. CDK4 amplification is an alternative mechanism to p16 gene homozygous deletion in glioma cell lines

    National Research Council Canada - National Science Library

    He, J; Allen, J R; Collins, V P; Allalunis-Turner, M J; Godbout, R; Day, 3rd, R S; James, C D

    1994-01-01

    ... those established from malignant gliomas. Here we have examined 32 glioma cell lines for amplification-associated overexpression of the CDK4 gene as an alternative mechanism for abrogating the growth-regulatory effects of p16...

  17. Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips.

    Science.gov (United States)

    Kosuri, Sriram; Eroshenko, Nikolai; Leproust, Emily M; Super, Michael; Way, Jeffrey; Li, Jin Billy; Church, George M

    2010-12-01

    Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ∼35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

  18. Dissection of the beta-globin replication-initiation region reveals specific requirements for replicator elements during gene amplification.

    Directory of Open Access Journals (Sweden)

    Naoya Okada

    Full Text Available Gene amplification plays a pivotal role in malignant transformation of human cells. A plasmid with both a mammalian replication-initiation region (IR/origin/replicator and a nuclear matrix-attachment region (MAR is spontaneously amplified in transfected cells by a mechanism that involves amplification at the extrachromosomal site, followed by amplification at the chromosomal arm, ultimately generating a long homogeneously staining region (HSR. Several observations suggest that replication initiation from IR sequences might mediate amplification. To test this idea, we previously dissected c-myc and DHFR IRs to identify the minimum sequence required to support amplification. In this study, we applied an improved analysis that discriminates between two amplification steps to the ß-globin RepP IR, which contains separate elements already known to be essential for initiation on the chromosome arm. The IR sequence was required at least for the extrachromosomal amplification step. In addition to the vector-encoded MAR, amplification also required an AT-rich region and a MAR-like element, consistent with the results regarding replicator activity on the chromosome. However, amplification did not require the AG-rich tract necessary for replicator activity, but instead required a novel sequence containing another AG-rich tract. The differential sequence requirement might be a consequence of extrachromosomal replication.

  19. Genome-wide copy number profiling to detect gene amplifications in neural progenitor cells

    Directory of Open Access Journals (Sweden)

    U. Fischer

    2014-12-01

    Full Text Available DNA sequence amplification occurs at defined stages during normal development in amphibians and flies and seems to be restricted in humans to drug-resistant and tumor cells only. We used array-CGH to discover copy number changes including gene amplifications and deletions during differentiation of human neural progenitor cells. Here, we describe cell culture features, DNA extraction, and comparative genomic hybridization (CGH analysis tailored towards the identification of genomic copy number changes. Further detailed analysis of amplified chromosome regions associated with this experiment, was published by Fischer and colleagues in PLOS One in 2012 (Fischer et al., 2012. We provide detailed information on deleted chromosome regions during differentiation and give an overview on copy number changes during differentiation induction for two representative chromosome regions.

  20. Duplication and amplification of antibiotic resistance genes enable increased resistance in isolates of multidrug-resistant Salmonella Typhimurium

    Science.gov (United States)

    During normal bacterial DNA replication, gene duplication and amplification (GDA) events occur randomly at a low frequency in the genome throughout a population. In the absence of selection, GDA events that increase the number of copies of a bacterial gene (or a set of genes) are lost. Antibiotic ...

  1. Amplification of complete gag gene sequences from geographically distinct equine infectious anemia virus isolates.

    Science.gov (United States)

    Boldbaatar, Bazartseren; Bazartseren, Tsevel; Koba, Ryota; Murakami, Hironobu; Oguma, Keisuke; Murakami, Kenji; Sentsui, Hiroshi

    2013-04-01

    In the current study, primers described previously and modified versions of these primers were evaluated for amplification of full-length gag genes from different equine infectious anemia virus (EIAV) strains from several countries, including the USA, Germany and Japan. Each strain was inoculated into a primary horse leukocyte culture, and the full-length gag gene was amplified by reverse transcription polymerase chain reaction. Each amplified gag gene was cloned into a plasmid vector for sequencing, and the detectable copy numbers of target DNA were determined. Use of a mixture of two forward primers and one reverse primer in the polymerase chain reaction enabled the amplification of all EIAV strains used in this study. However, further study is required to confirm these primers as universal for all EIAV strains. The nucleotide sequence of gag is considered highly conserved, as evidenced by the use of gag-encoded capsid proteins as a common antigen for the detection of EIAV in serological tests. However, significant sequence variation in the gag genes of different EIAV strains was found in the current study.

  2. Isolation, amplification and characterization of foodborne pathogen disease bacteria gene for rapid kit test development

    Science.gov (United States)

    Nurjayadi, M.; Santoso, I.; Kartika, I. R.; Kurniadewi, F.; Saamia, V.; Sofihan, W.; Nurkhasanah, D.

    2017-07-01

    There is a lot of public concern over food safety. Food-safety cases recently, including many food poisoning cases in both the developed and developing countries, considered to be the national security threats which involved police investigation. Quick and accurate detection methods are needed to handle the food poisoning cases with a big number of sufferers at the same time. Therefore, the research is aimed to develop a specific, sensitive, and rapid result molecular detection tool for foodborne pathogen bacteria. We, thus, propose genomic level approach with Polymerase Chain Reaction. The research has successfully produced a specific primer to perform amplification to fim-C S. typhi, E. coli, and pef Salmonella typhimurium genes. The electrophoresis result shows that amplification products are 95 base pairs, 121 base pairs, and 139 base pairs; and all three genes are in accordance with the size of the in silico to third genes bacteria. In conclusion, the research has been successfully designed a specific detection tool to three foodborne pathogen bacteria genes. Further stages test and the uses of Real-time PCR in the detection are still in the trial process for better detection method.

  3. DNMT3B gene amplification predicts resistance to DNA demethylating drugs.

    Science.gov (United States)

    Simó-Riudalbas, Laia; Melo, Sónia A; Esteller, Manel

    2011-07-01

    Disruption of the DNA methylation landscape is one of the most common features of human tumors. However, genetic alterations of DNA methyltransferases (DNMTs) have not been described in carcinogenesis. Herein, we show that pancreatic and breast cancer cells undergo gene amplification of the DNA methyltransferase 3B (DNMT3B). The presence of extra copies of the DNMT3B gene is linked to higher levels of the corresponding mRNA and protein. Most importantly, the elevated gene dosage of DNMT3B is associated with increased resistance to the growth-inhibitory effect mediated by DNA demethylating agents. In particular, cancer cells harboring DNMT3B gene amplification are less sensitive to the decrease in cell viability caused by 5-azacytidine (Vidaza), 5-aza-2-deoxycytidine (Decitabine), and SGI-1027. Overall, the data confirm DNMT3B as a bona fide oncogene in human cancer and support the incorporation of the DNMT3B copy number assay into current clinical trials assessing the efficacy of DNA demethylating drugs in solid tumors.

  4. Prognostic value of FGFR gene amplification in patients with different types of cancer: a systematic review and meta-analysis.

    Directory of Open Access Journals (Sweden)

    Jinjia Chang

    Full Text Available BACKGROUND: Fibroblast growth factor receptor (FGFR gene amplification has been reported in different types of cancer. We performed an up-to-date meta-analysis to further characterize the prognostic value of FGFR gene amplification in patients with cancer. METHODS: A search of several databases, including MEDLINE (PubMed, EMBASE, Web of Science, and China National Knowledge Infrastructure, was conducted to identify studies examining the association between FGFR gene amplification and cancer. A total of 24 studies met the inclusion criteria, and overall incidence rates, hazard risk (HR, overall survival, disease-free survival, and 95% confidence intervals (CIs were calculated employing fixed- or random-effects models depending on the heterogeneity of the included studies. RESULTS: In the meta-analysis of 24 studies, the prevalence of FGFR gene amplification was FGFR1: 0.11 (95% CI: 0.08-0.13 and FGFR2: 0.04 (95% CI: 0.02-0.06. Overall survival was significantly worse among patients with FGFR gene amplification: FGFR1 [HR 1.57 (95% CI: 1.23-1.99; p = 0.0002] and FGFR2 [HR 2.27 (95% CI: 1.73-3.00; p<0.00001]. CONCLUSIONS: Current evidence supports the conclusion that the outcomes of patients with FGFR gene amplified cancers is worse than for those with non-FGFR gene amplified cancers.

  5. MDM2 gene amplification: a new independent factor of adverse prognosis in non-small cell lung cancer (NSCLC).

    Science.gov (United States)

    Dworakowska, Dorota; Jassem, Ewa; Jassem, Jacek; Peters, Brigitte; Dziadziuszko, Rafał; Zylicz, Maciej; Jakóbkiewicz-Banecka, Joanna; Kobierska-Gulida, Grazyna; Szymanowska, Amelia; Skokowski, Jan; Roessner, Albert; Schneider-Stock, Regine

    2004-03-01

    The prognostic impact of MDM2 amplification in non-small cell lung cancer (NSCLC) remains unknown. In this study, we investigated the occurrence of MDM2 amplification in surgically treated NSCLC patients. Molecular data were correlated with clinicopathological factors and evaluated for their prognostic value. The study group included 116 NSCLC patients who underwent pulmonary resection between 1996 and 1999. MDM2 amplification was assessed by real-time PCR using hybridization probe format on a LightCycler (Roche). The calculated ratio was a MDM2 value normalized to the amplification of the housekeeping gene phenylalaninhydroxylase (PAH). Survival curves were drawn according to the Kaplan-Meier method and compared with the use of the log-rank test. Multivariate analysis was based on Cox regression analysis. MDM2 amplification was found in 24 patients (21%). There was no relationship between MDM2 amplification and clinicopathological factors, such as sex, age and stage of disease, pT, pN, histology and tumor differentiation. Median disease-free survival (DFS) in patients with and without MDM2 amplification was 3 and 31 months, and 5-year DFS 24 and 33%, respectively (log-rank, P = 0.02). Likewise, median overall survival (OS) in patients with and without MDM2 amplification was 9 and 33 months, respectively, and 5-year OS 24 and 39%, respectively (log-rank, P = 0.01). The strong prognostic relevance of MDM2 amplification for both DFS and OS was confirmed in multivariate analysis (P < 0.01 for both comparisons). Our results suggest that MDM2 gene amplification analysis provides additional prognostic information in surgically treated NSCLC patients.

  6. Extensive gene amplification and concerted evolution within the CPR family of cuticular proteins in mosquitoes.

    Science.gov (United States)

    Cornman, R Scott; Willis, Judith H

    2008-06-01

    Annotation of the Anopheles gambiae genome has revealed a large increase in the number of genes encoding cuticular proteins with the Rebers and Riddiford Consensus (the CPR gene family) relative to Drosophila melanogaster. This increase reflects an expansion of the RR-2 group of CPR genes, particularly the amplification of sets of highly similar paralogs. Patterns of nucleotide variation indicate that extensive concerted evolution is occurring within these clusters. The pattern of concerted evolution is complex, however, as sequence similarity within clusters is uncorrelated with gene order and orientation, and no comparable clusters occur within similarly compact arrays of the RR-1 group in mosquitoes or in either group in D. melanogaster. The dearth of pseudogenes suggests that sequence clusters are maintained by selection for high gene-copy number, perhaps due to selection for high expression rates. This hypothesis is consistent with the apparently parallel evolution of compact gene architectures within sequence clusters relative to single-copy genes. We show that RR-2 proteins from sequence-cluster genes have complex repeats and extreme amino-acid compositions relative to single-copy CPR proteins in An. gambiae, and that the amino-acid composition of the N-terminal and C-terminal sequence flanking the chitin-binding consensus region evolves in a correlated fashion.

  7. Selective Amplification of the Genome Surrounding Key Placental Genes in Trophoblast Giant Cells.

    Science.gov (United States)

    Hannibal, Roberta L; Baker, Julie C

    2016-01-25

    While most cells maintain a diploid state, polyploid cells exist in many organisms and are particularly prevalent within the mammalian placenta [1], where they can generate more than 900 copies of the genome [2]. Polyploidy is thought to be an efficient method of increasing the content of the genome by avoiding the costly and slow process of cytokinesis [1, 3, 4]. Polyploidy can also affect gene regulation by amplifying a subset of genomic regions required for specific cellular function [1, 3, 4]. This mechanism is found in the fruit fly Drosophila melanogaster, where polyploid ovarian follicle cells amplify genomic regions containing chorion genes, which facilitate secretion of eggshell proteins [5]. Here, we report that genomic amplification also occurs in mammals at selective regions of the genome in parietal trophoblast giant cells (p-TGCs) of the mouse placenta. Using whole-genome sequencing (WGS) and digital droplet PCR (ddPCR) of mouse p-TGCs, we identified five amplified regions, each containing a gene family known to be involved in mammalian placentation: the prolactins (two clusters), serpins, cathepsins, and the natural killer (NK)/C-type lectin (CLEC) complex [6-12]. We report here the first description of amplification at selective genomic regions in mammals and present evidence that this is an important mode of genome regulation in placental TGCs.

  8. Clinicopathologic significance of HER-2/neu protein expression and gene amplification in gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Shi-Yan Yan; Ying Hu; Jian-Gao Fan; Guo-Quan Tao; Yong-Ming Lu; Xu Cai; Bao-Hua Yu; Yi-Qun Du

    2011-01-01

    AIM: To study the HER-2/neu protein expression and gene amplification in gastric carcinoma and their relation. METHODS: One hundred and forty-five formalin-fixed and paraffin- embedded tumor tissue samples from Chinese gastric carcinoma patients were studied with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods. Clinicopathologic data about all patients were collected. RESULTS: The levels of HER-2 3+, HER-2 2+ and HER2 1+ were measurable in 6.9%, 8.3% and 17.2% of the samples, respectively. No HER-2 was stained in 67.6% of the samples. FISH showed that HER-2 gene was amplified in 18 samples, 10 HER-2 3+ samples, 5 HER-2 2+ samples, and 3 HER-2 1+ samples with IHC staining. HER-2 status was not correlated with the sex and age of patients, and tumor size, location or differentiation, but with the depth of invasion, TNM stage, lymph node and distant metastasis as well as histopathological classification of gastric cancer (P < 0.05). CONCLUSION: All samples with IHC as HER-2 expression should be analyzed with FISH. Detection of HER-2 gene amplification can assess the malignant biological behaviors and prognosis of gastric cancer.

  9. Optimization of laser capture microdissection and RNA amplification for gene expression profiling of prostate cancer

    Directory of Open Access Journals (Sweden)

    Vasmatzis George

    2007-03-01

    Full Text Available Abstract Background To discover prostate cancer biomarkers, we profiled gene expression in benign and malignant cells laser capture microdissected (LCM from prostate tissues and metastatic prostatic adenocarcinomas. Here we present methods developed, optimized, and validated to obtain high quality gene expression data. Results RNase inhibitor was included in solutions used to stain frozen tissue sections for LCM, which improved RNA quality significantly. Quantitative PCR assays, requiring minimal amounts of LCM RNA, were developed to determine RNA quality and concentration. SuperScript II™ reverse transcriptase was replaced with SuperScript III™, and SpeedVac concentration was eliminated to optimize linear amplification. The GeneChip® IVT labeling kit was used rather than the Enzo BioArray™ HighYield™ RNA transcript labeling kit since side-by-side comparisons indicated high-end signal saturation with the latter. We obtained 72 μg of labeled complementary RNA on average after linear amplification of about 2 ng of total RNA. Conclusion Unsupervised clustering placed 5/5 normal and 2/2 benign prostatic hyperplasia cases in one group, 5/7 Gleason pattern 3 cases in another group, and the remaining 2/7 pattern 3 cases in a third group with 8/8 Gleason pattern 5 cases and 3/3 metastatic prostatic adenocarcinomas. Differential expression of alpha-methylacyl coenzyme A racemase (AMACR and hepsin was confirmed using quantitative PCR.

  10. Oligonucleotide primers for targeted amplification of single-copy nuclear genes in apocritan Hymenoptera.

    Directory of Open Access Journals (Sweden)

    Gerrit Hartig

    Full Text Available BACKGROUND: Published nucleotide sequence data from the mega-diverse insect order Hymenoptera (sawflies, bees, wasps, and ants are taxonomically scattered and still inadequate for reconstructing a well-supported phylogenetic tree for the order. The analysis of comprehensive multiple gene data sets obtained via targeted PCR could provide a cost-effective solution to this problem. However, oligonucleotide primers for PCR amplification of nuclear genes across a wide range of hymenopteran species are still scarce. FINDINGS: Here we present a suite of degenerate oligonucleotide primer pairs for PCR amplification of 154 single-copy nuclear protein-coding genes from Hymenoptera. These primers were inferred from genome sequence data from nine Hymenoptera (seven species of ants, the honeybee, and the parasitoid wasp Nasonia vitripennis. We empirically tested a randomly chosen subset of these primer pairs for amplifying target genes from six Hymenoptera, representing the families Chrysididae, Crabronidae, Gasteruptiidae, Leucospidae, Pompilidae, and Stephanidae. Based on our results, we estimate that these primers are suitable for studying a large number of nuclear genes across a wide range of apocritan Hymenoptera (i.e., all hymenopterans with a wasp-waist and of aculeate Hymenoptera in particular (i.e., apocritan wasps with stingers. CONCLUSIONS: The amplified nucleotide sequences are (a with high probability from single-copy genes, (b easily generated at low financial costs, especially when compared to phylogenomic approaches, (c easily sequenced by means of an additionally provided set of sequencing primers, and (d suitable to address a wide range of phylogenetic questions and to aid rapid species identification via barcoding, as many amplicons contain both exonic and fast-evolving intronic nucleotides.

  11. Rapid PCR amplification protocols decrease the turn-around time for detection of antibiotic resistance genes in Gram-negative pathogens.

    Science.gov (United States)

    Geyer, Chelsie N; Hanson, Nancy D

    2013-10-01

    A previously designed end-point multiplex PCR assay and singleplex assays used to detect β-lactamase genes were evaluated using rapid PCR amplification methodology. Amplification times were 16-18 minutes with an overall detection time of 1.5 hours. Rapid PCR amplifications could decrease the time required to identify resistance mechanisms in Gram-negative organisms.

  12. Isolation of epithelial cells from tooth brush and gender identification by amplification of SRY gene

    Directory of Open Access Journals (Sweden)

    A Vikram Simha Reddy

    2011-01-01

    Full Text Available Introduction: This study determines the importance of tooth brush from which DNA can be isolated and used for sex determination in forensic analysis. Materials and Methods: A total of 30 samples were collected and stored at room temperature for different periods of time interval. The epithelial cells adhered to the bristles of tooth brush were collected and genomic DNA was extracted and quantified using Nanodrop 1000 spectrophotometer. Results: Gender identification was done by amplification of sex determining region on Y chromosome (SRY gene using real-time polymerase chain reaction and minimal amount of DNA (in pico grams with 100% sensitivity and 73.3% specificity, i.e., all male samples showed positive results and out of 15 female samples 4 showed false positive results, i.e wrongly identified as males. Conclusion: With this study, we conclude that PCR is a valuable and sensitive procedure where minute contamination may cause alteration in the result, i.e, 4 females showed false positive result. Minute amount of DNA in picograms, which was collected at different intervals is enough for amplification of SRY gene and tooth brush can be used as one of the very valuable sources of gender identification.

  13. High-Resolution Genomic and Expression Profiling Reveals 105 Putative Amplification Target Genes in Pancreatic Cancer

    Directory of Open Access Journals (Sweden)

    Eija H. Mahlamaki

    2004-09-01

    Full Text Available Comparative genomic hybridization (CGH studies have provided a wealth of information on common copy number aberrations in pancreatic cancer, but the genes affected by these aberrations are largely unknown. To identify putative amplification target genes in pancreatic cancer, we performed a parallel copy number and expression survey in 13 pancreatic cancer cell lines using a 12,232-clone cDNA microarray, providing an average resolution of 300 kb throughout the human genome. CGH on cDNA microarray allowed highly accurate mapping of copy number increases and resulted in identification of 24 independent amplicons, ranging in size from 130 kb to 11 Mb. Statistical evaluation of gene copy number and expression data across all 13 cell lines revealed a set of 105 genes whose elevated expression levels were directly attributable to increased copy number. These included genes previously reported to be amplified in cancer as well as several novel targets for copy number alterations, such as p21-activated kinase 4 (PAK4, which was previously shown to be involved in cell migration, cell adhesion, and anchorage-independent growth. In conclusion, our results implicate a set of 105 genes that is likely to be actively involved in the development and progression of pancreatic cancer.

  14. IDENTIFICATION, ISOLATION AND AMPLIFICATION OF BRCA1 GENE INVOLVED IN BREAST CANCER

    Directory of Open Access Journals (Sweden)

    Karaneh Eftekhari

    2014-02-01

    Full Text Available Cancer is a disease that begins in the cells ofthe body which is characterized by uncontrolled, uncoordinated and undesirable cell division. If a cell accumulates critical mutations in five or six of the proto-oncogenes, tumour suppressor genes and DNA repair genes are likely to result in a fully malignant cell, capable of forming a tumour. In this work we described the isolation and amplification of the BRCA1 gene. Primers were designed and synthesised later used to amplify the BRCA1 gene. The total new workflow includes all steps from purified DNA to data analysis, and includes PCR for all amplicons covering the gene, PCR cleanup, cycle sequencing, electrophoresis, and data analysis. To simplify workflows and decrease the time-to-result, we focused on the method “one sample, one assay” approach. The success of this workflow was the 24-well plate design, which contained prespotted PCR primers covering the gene and also included multiplex nontemplate controls. The workflow was developed using a Genetic Analyzer and bands were observed.

  15. Amplification of the E2F1 transcription factor gene in the HEL erythroleukemia cell line

    DEFF Research Database (Denmark)

    Saito, M; Helin, K; Valentine, M B;

    1995-01-01

    and overexpressed in HEL erythroleukemia cells and translocated to other chromosomes in several established human leukemia cell lines. This study provides the first evidence of gene amplification involving a member of the E2F family of transcription factors. We propose that E2F1 overexpression in erythroid......The E2F transcription factor plays an important regulatory role in cell proliferation, mediating the expression of genes whose products are essential for inducing resting cells to enter the cell cycle and synthesize DNA. To investigate the possible involvement of E2F in hematopoietic malignancies...... progenitors may stimulate abnormal cell proliferation by overriding negative regulatory signals mediated by tumor suppressor proteins such as pRb....

  16. Two-step amplification of the human PPT sequence provides specific gene expression in an immunocompetent murine prostate cancer model.

    Science.gov (United States)

    Dzojic, H; Cheng, W-S; Essand, M

    2007-03-01

    The recombinant prostate-specific PPT sequence comprises a prostate-specific antigen enhancer, a PSMA enhancer and a TARP promoter. It is transcriptionally active in human prostate cancer cells both in the presence and absence of testosterone. However, in experimental murine prostate cancer, it has no detectable transcriptional activity. Herein, we describe that the PPT sequence in combination with a two-step transcriptional amplification (TSTA) system becomes active also in murine prostate cancer cells. An adenovirus with TSTA-amplified PPT-controlled expression of the luciferase reporter gene, Ad[PPT/TSTA-Luc], has up to 100-fold higher prostate-specific transcriptional activity than a non-amplified PPT-based adenovirus, Ad[PPT-Luc], in human cells. In addition, Ad[PPT/TSTA-Luc] confers prostate-specific transgene expression in murine cells, with an activity that is approximately 23% of Ad[CMV-Luc] in the transgenic adenocarcinoma of the mouse prostate (TRAMP)-C2 cells. Moreover, to visualize luciferase expression in living mice a charge-coupled device camera was used. Ad[PPT/TSTA-Luc] yielded approximately 30-fold higher transgene expression than Ad[PPT-Luc] in LNCaP tumor xenografts. Importantly, Ad[PPT/TSTA-Luc] also showed activity in murine TRAMP-C2 tumors, whereas Ad[PPT-Luc] activity was undetectable. These results highlight that the recombinant PPT sequence is active in murine prostate cancer cells when augmented by a TSTA system. This finding opens up for preclinical studies with prostate-specific therapeutic gene expression in immunocompetent mice.

  17. Monosomy of chromosome 17 in breast cancer during interpretation of HER2 gene amplification

    Science.gov (United States)

    Brunelli, Matteo; Nottegar, Alessia; Bogina, Giuseppe; Caliò, Anna; Cima, Luca; Eccher, Albino; Vicentini, Caterina; Marcolini, Lisa; Scarpa, Aldo; Pedron, Serena; Brunello, Eleonora; Knuutila, Sakari; Sapino, Anna; Marchiò, Caterina; Bria, Emilio; Molino, Annamaria; Carbognin, Luisa; Tortora, Giampaolo; Jasani, Bharat; Miller, Keith; Merdol, Ibrahim; Zanatta, Lucia; Laurino, Licia; Wirtanen, Tiina; Zamboni, Giuseppe; Marconi, Marcella; Chilosi, Marco; Manfrin, Erminia; Martignoni, Guido; Bonetti, Franco

    2015-01-01

    Monosomy of chromosome 17 may affect the assessment of HER2 amplification. Notably, the prevalence ranges from 1% up to 49% due to lack of consensus in recognition. We sought to investigate the impact of monosomy of chromosome 17 to interpretation of HER2 gene status. 201 breast carcinoma were reviewed for HER2 gene amplification and chromosome 17 status. FISH analysis was performed by using double probes (LSI/CEP). Absolute gene copy number was also scored per each probe. HER2 FISH test was repeated on serial tissue sections, ranging in thickness from 3 to 20 µm. Ratio was scored and subsequently corrected by monosomy after gold control test using the aCGH method to overcome false interpretation due to artefactual nuclear truncation. HER2 immunotests was performed on all cases. 26/201 cases were amplified (13%). Single signals per CEP17 were revealed in 7/201 (3.5%) cases. Five out of 7 cases appeared monosomic with aCGH (overall, 5/201, 2.5%) and evidenced single signals in >60% of nuclei after second-look on FISH when matching both techniques. Among 5, one case showed amplification with a pattern 7/1 (HER2/CEP17>2) of copies (3+ at immunotest); three cases revealed single signals per both probes (LSI/CEP=1) and one case revealed a 3:1 ratio; all last 4 cases showed 0/1+ immunoscore. We concluded that: 1) monosomy of chromosome 17 may be observed in 2.5% of breast carcinoma; 2) monosomy of chromosome 17 due to biological reasons rather than nuclear truncation was observed when using the cut-off of 60% of nuclei harboring single signals; 3) the skewing of the ratio due to single centromeric 17 probe may lead to false positive evaluation; 4) breast carcinomas showing a 3:1 ratio (HER2/CEP17) usually show negative 0/1+ immunoscore and <6 gene copy number at FISH. PMID:26328251

  18. Sequencing the mouse Y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes.

    Science.gov (United States)

    Soh, Y Q Shirleen; Alföldi, Jessica; Pyntikova, Tatyana; Brown, Laura G; Graves, Tina; Minx, Patrick J; Fulton, Robert S; Kremitzki, Colin; Koutseva, Natalia; Mueller, Jacob L; Rozen, Steve; Hughes, Jennifer F; Owens, Elaine; Womack, James E; Murphy, William J; Cao, Qing; de Jong, Pieter; Warren, Wesley C; Wilson, Richard K; Skaletsky, Helen; Page, David C

    2014-11-06

    We sequenced the MSY (male-specific region of the Y chromosome) of the C57BL/6J strain of the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700 protein-coding genes. Only 2% of the MSY derives from the ancestral autosomes that gave rise to the mammalian sex chromosomes. Instead, all but 45 of the MSY's genes belong to three acquired, massively amplified gene families that have no homologs on primate MSYs but do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism between acquired X-Y homologs. The mouse MSY sequence presents opportunities for experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable model organism.

  19. CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast

    Science.gov (United States)

    Strucko, Tomas; Buron, Line Due; Jarczynska, Zofia Dorota; Nødvig, Christina Spuur; Mølgaard, Louise; Halkier, Barbara Ann; Mortensen, Uffe Hasbro

    2017-01-01

    Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploiting expression from the multicopy 2 μ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached. However, with 2 μ-based plasmid expression, the population of cells is very heterogeneous with respect to protein production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene amplification system, CASCADE, which enables construction of strains with defined gene copy numbers. One or more genes can be amplified simultaneously and the resulting strains can be stably propagated on selection-free medium. As proof-of-concept, we have successfully used CASCADE to increase heterologous production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside. PMID:28134264

  20. Homozygous Deletions and Recurrent Amplifications Implicate New Genes Involved in Prostate Cancer

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    Wennuan Liu

    2008-08-01

    Full Text Available Prostate cancer cell lines provide ideal in vitro systems for the identification and analysis of prostate tumor suppressors and oncogenes. A detailed characterization of the architecture of prostate cancer cell line genomes would facilitate the study of precise roles of various genes in prostate tumorigenesis in general. To contribute to such a characterization, we used the GeneChip 500K single nucleotide polymorphic (SNP array for analysis of genotypes and relative DNA copy number changes across the genome of 11 cell lines derived from both normal and cancerous prostate tissues. For comparison purposes, we also examined the alterations observed in the cell lines in tumor/normal pairs of clinical samples from 72 patients. Along with genome-wide maps of DNA copy number changes and loss of heterozygosity for these cell lines, we report previously unreported homozygous deletions and recurrent amplifications in prostate cancers in this study. The homozygous deletions affected a number of biologically important genes, including PPP2R2A and BNIP3L identified in this study and CDKN2A/CDKN2B reported previously. Although most amplified genomic regions tended to be large, amplifications at 8q24.21 were of particular interest because the affected regions are relatively small, are found in multiple cell lines, are located near MYC, an oncogene strongly implicated in prostate tumorigenesis, and are known to harbor SNPs that are associated with inherited susceptibility for prostate cancer. The genomic alterations revealed in this study provide an important catalog of positional information relevant to efforts aimed at deciphering the molecular genetic basis of prostate cancer.

  1. Specific amplification of iron receptor genes in Xylella fastidiosa strains from different hosts

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    Flávia Teresa Hansen Pacheco

    2006-01-01

    Full Text Available Bacterial production of siderophores may involve specific genes related to nonribosomal peptide and polyketide biosynthesis, which have not been fully identified in the genome of Xylella fastidiosa strain 9a5c. However, a search for siderophore-related genes in strain 9a5c indicated five membrane receptors, including siderophore, ferrichrome-iron and hemin receptors. All these biomolecules are thought to be associated with iron transport and utilization. Eighty isolates obtained from citrus orchards containing trees that developed citrus variegated chlorosis (CVC were screened for siderophore production. The results demonstrated that only 10 of the isolates did not produce siderophores. Additional strains obtained from coffee, almond, mulberry, elm, ragweed, periwinkle and grape also infected by X. fastidiosa were also shown by the chromeazurol bioassay to produce siderophores. In order to correlate siderophore production with the presence of siderophore-related genes, a polymerase chain reaction (PCR was developed using specific primers for the catechol-type ferric enterobactin receptor (pfeA and the hydroxamate-type ferrisiderophore receptor (fiuA genes of strain 9a5c. The PCR results confirmed our hypothesis by demonstrating that amplification products were detected in all strains except for those isolates that did not produce siderophores.

  2. Amplification of a Gene Related to Mammalian mdr Genes in Drug-Resistant Plasmodium falciparum

    Science.gov (United States)

    Wilson, Craig M.; Serrano, Adelfa E.; Wasley, Annemarie; Bogenschutz, Michael P.; Shankar, Anuraj H.; Wirth, Dyann F.

    1989-06-01

    The malaria parasite Plasmodium falciparum contains at least two genes related to the mammalian multiple drug resistance genes, and at least one of the P. falciparum genes is expressed at a higher level and is present in higher copy number in a strain that is resistant to multiple drugs than in a strain that is sensitive to the drugs.

  3. Specific amplification by PCR of rearranged genomic variable regions of immunoglobulin genes from mouse hybridoma cells.

    Science.gov (United States)

    Berdoz, J; Monath, T P; Kraehenbuhl, J P

    1995-04-01

    We have designed a novel strategy for the isolation of the rearranged genomic fragments encoding the L-VH-D-JH and L-V kappa/lambda-J kappa/lambda regions of mouse immunoglobulin genes. This strategy is based on the PCR amplification of genomic DNA from mouse hybridomas using multiple specific primers chosen in the 5'-untranslated region and in the intron downstream of the rearranged JH/J kappa/lambda sequences. Variable regions with intact coding sequences, including full-length leader peptides (L) can be obtained without previous DNA sequencing. Our strategy is based on a genomic template that produces fragments that do not need to be adapted for recombinant antibody expression, thus facilitating the generation of chimeric and isotype-switched immunoglobulins.

  4. Human-specific gene ARHGAP11B promotes basal progenitor amplification and neocortex expansion.

    Science.gov (United States)

    Florio, Marta; Albert, Mareike; Taverna, Elena; Namba, Takashi; Brandl, Holger; Lewitus, Eric; Haffner, Christiane; Sykes, Alex; Wong, Fong Kuan; Peters, Jula; Guhr, Elaine; Klemroth, Sylvia; Prüfer, Kay; Kelso, Janet; Naumann, Ronald; Nüsslein, Ina; Dahl, Andreas; Lachmann, Robert; Pääbo, Svante; Huttner, Wieland B

    2015-03-27

    Evolutionary expansion of the human neocortex reflects increased amplification of basal progenitors in the subventricular zone, producing more neurons during fetal corticogenesis. In this work, we analyze the transcriptomes of distinct progenitor subpopulations isolated by a cell polarity-based approach from developing mouse and human neocortex. We identify 56 genes preferentially expressed in human apical and basal radial glia that lack mouse orthologs. Among these, ARHGAP11B has the highest degree of radial glia-specific expression. ARHGAP11B arose from partial duplication of ARHGAP11A (which encodes a Rho guanosine triphosphatase-activating protein) on the human lineage after separation from the chimpanzee lineage. Expression of ARHGAP11B in embryonic mouse neocortex promotes basal progenitor generation and self-renewal and can increase cortical plate area and induce gyrification. Hence, ARHGAP11B may have contributed to evolutionary expansion of human neocortex.

  5. Simultaneous amplification of two bacterial genes: more reliable method of Helicobacter pylori detection in microbial rich dental plaque samples.

    Science.gov (United States)

    Chaudhry, Saima; Idrees, Muhammad; Izhar, Mateen; Butt, Arshad Kamal; Khan, Ayyaz Ali

    2011-01-01

    Polymerase Chain reaction (PCR) assay is considered superior to other methods for detection of Helicobacter pylori (H. pylori) in oral cavity; however, it also has limitations when sample under study is microbial rich dental plaque. The type of gene targeted and number of primers used for bacterial detection in dental plaque samples can have a significant effect on the results obtained as there are a number of closely related bacterial species residing in plaque biofilm. Also due to high recombination rate of H. pylori some of the genes might be down regulated or absent. The present study was conducted to determine the frequency of H. pylori colonization of dental plaque by simultaneously amplifying two genes of the bacterium. One hundred dental plaque specimens were collected from dyspeptic patients before their upper gastrointestinal endoscopy and presence of H. pylori was determined through PCR assay using primers targeting two different genes of the bacterium. Eighty-nine of the 100 samples were included in final analysis. With simultaneous amplification of two bacterial genes 51.6% of the dental plaque samples were positive for H. pylori while this prevalence increased to 73% when only one gene amplification was used for bacterial identification. Detection of H. pylori in dental plaque samples is more reliable when two genes of the bacterium are simultaneously amplified as compared to one gene amplification only.

  6. mdm-2 gene amplification in 3T3-L1 preadipocytes.

    Science.gov (United States)

    Berberich, S J; Litteral, V; Mayo, L D; Tabesh, D; Morris, D

    1999-05-01

    In this study the regulation of the murine double minute-2 (mdm-2) gene was examined in NIH 3T3-L1 preadipocytes. The 3T3-L1 cell line, under proper conditions, has the capacity to differentiate from fibroblasts into adipocytes [15]. A recent report demonstrated that mdm-2 overexpression could block myogenesis [12]. While examining the regulation of the mdm-2 gene during adipogenesis, it was discovered that 3T3-L1 cells possess a 36-fold elevation of mdm-2 mRNA relative to A31 cells, another immortalized Balb/c 3T3 fibroblast cell line that lacks the capacity to differentiate. Based on Southern blot analysis, the increase in mdm-2 mRNA was the result of a mdm-2 gene amplification. The level of Mdm-2 protein in undifferentiated 3T3-L1 cells was elevated relative to A31 fibroblasts and resulted from translation of mRNA transcripts initiating from the p53-independent P1 promoter. We also examined how mdm-2 and p53 levels changed as undifferentiated fibroblasts converted to adipocytes. While mdm-2 mRNA levels remained elevated, p53 mRNA, protein, and DNA-binding activity decreased. These results suggest that adipogenesis is unaffected by elevated Mdm-2 levels and that the overexpression of mdm-2 mRNA is predominantly p53 independent.

  7. Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler

    Science.gov (United States)

    TerMaat, Joel R.; Pienaar, Elsje; Whitney, Scott E.; Mamedov, Tarlan G.; Subramanian, Anuradha

    2013-01-01

    Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653 bp) and pUC19 β-lactamase (929 bp) in under 20 min. However, rapid integrated PCA–PCR was found to be problematic when attempted with the TM-1 gene (1509 bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers. PMID:19799938

  8. MLL gene amplification in acute myeloid leukemia and myelodysplastic syndromes is associated with characteristic clinicopathological findings and TP53 gene mutation.

    Science.gov (United States)

    Tang, Guilin; DiNardo, Courtney; Zhang, Liping; Ravandi, Farhad; Khoury, Joseph D; Huh, Yang O; Muzzafar, Tariq; Medeiros, L Jeffrey; Wang, Sa A; Bueso-Ramos, Carlos E

    2015-01-01

    MLL gene rearrangements are well-recognized aberrations in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). In contrast, MLL gene amplification in AML/MDS remains poorly characterized. Here, we report a series of 21 patients with myeloid neoplasms associated with MLL gene amplification from 1 institution. This series included 13 men and 8 women, with a median age of 64 years. Eleven patients presented as AML with myelodysplasia-related changes, 6 as therapy-related AML, and 4 as therapy-related MDS. All patients had a highly complex karyotype, including frequent -5/del(5q), -18, and -17/del(17p) abnormalities; 16 patients were hypodiploid. TP53 mutations were detected in all 12 patients tested, and 3 patients showed TP53 mutation before MLL amplification. Morphologically, the leukemic cells frequently showed cytoplasmic vacuoles, bilobed nuclei, and were associated with background dyspoiesis. Immunophenotypically, 15 patients had a myeloid and 4 had myelomonocytic immunophenotype. Laboratory coagulopathies were common; 7 patients developed disseminated intravascular coagulopathy, and 3 died of intracranial bleeding. All patients were refractory to therapy; the median overall survival was 1 month, after MLL gene amplification was detected. We concluded that AML/MDS with MLL gene amplification is likely a subset of therapy-related AML/MDS or AML with myelodysplasia-related changes, associated with distinct clinicopathological features, frequent disseminated intravascular coagulopathy, a highly complex karyotype, TP53 deletion/mutation, and an aggressive clinical course.

  9. Development of a Loop-Mediated Isothermal Amplification Assay Targeting the mpb64 Gene for Diagnosis of Intraocular Tuberculosis

    Science.gov (United States)

    Balne, Praveen Kumar; Barik, Manas Ranjan; Sharma, Savitri

    2013-01-01

    A loop-mediated isothermal amplification (LAMP) assay targeting the mpb64 gene for the diagnosis of intraocular tuberculosis was highly specific (100%), sensitive (85.7%), rapid, and easy to perform. The LAMP assay can be an alternative to conventional PCR for the diagnosis of ocular tuberculosis in resource-limited settings. PMID:23966513

  10. Molecular diagnosis of Kingella kingae pericarditis by amplification and sequencing of the 16S rRNA gene.

    Science.gov (United States)

    Matta, Matta; Wermert, Delphine; Podglajen, Isabelle; Sanchez, Olivier; Buu-Hoï, Annie; Gutmann, Laurent; Meyer, Guy; Mainardi, Jean-Luc

    2007-09-01

    Kingella kingae is a fastidious gram-negative bacillus that is considered an emerging pathogen in pediatric settings but remains less common in adults. Here we describe a case of pericarditis in an immunocompetent adult host. The microorganism was identified directly from the clinical sample by molecular techniques, i.e., 16S rRNA gene amplification and sequencing.

  11. AIB1 gene amplification and the instability of polyQ encoding sequence in breast cancer cell lines

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    Clarke Robert

    2006-05-01

    Full Text Available Abstract Background The poly Q polymorphism in AIB1 (amplified in breast cancer gene is usually assessed by fragment length analysis which does not reveal the actual sequence variation. The purpose of this study is to investigate the sequence variation of poly Q encoding region in breast cancer cell lines at single molecule level, and to determine if the sequence variation is related to AIB1 gene amplification. Methods The polymorphic poly Q encoding region of AIB1 gene was investigated at the single molecule level by PCR cloning/sequencing. The amplification of AIB1 gene in various breast cancer cell lines were studied by real-time quantitative PCR. Results Significant amplifications (5–23 folds of AIB1 gene were found in 2 out of 9 (22% ER positive cell lines (in BT-474 and MCF-7 but not in BT-20, ZR-75-1, T47D, BT483, MDA-MB-361, MDA-MB-468 and MDA-MB-330. The AIB1 gene was not amplified in any of the ER negative cell lines. Different passages of MCF-7 cell lines and their derivatives maintained the feature of AIB1 amplification. When the cells were selected for hormone independence (LCC1 and resistance to 4-hydroxy tamoxifen (4-OH TAM (LCC2 and R27, ICI 182,780 (LCC9 or 4-OH TAM, KEO and LY 117018 (LY-2, AIB1 copy number decreased but still remained highly amplified. Sequencing analysis of poly Q encoding region of AIB1 gene did not reveal specific patterns that could be correlated with AIB1 gene amplification. However, about 72% of the breast cancer cell lines had at least one under represented (3CAA(CAG9(CAACAG3(CAACAGCAG2CAA of the original cell line, a number of altered poly Q encoding sequences were found in the derivatives of MCF-7 cell lines. Conclusion These data suggest that poly Q encoding region of AIB1 gene is somatic unstable in breast cancer cell lines. The instability and the sequence characteristics, however, do not appear to be associated with the level of the gene amplification.

  12. Calcium Channel Genes Associated with Bipolar Disorder Modulate Lithium's Amplification of Circadian Rhythms

    Science.gov (United States)

    McCarthy, Michael J.; LeRoux, Melissa; Wei, Heather; Beesley, Stephen; Kelsoe, John R.; Welsh, David K.

    2015-01-01

    Bipolar disorder (BD) is associated with mood episodes and low amplitude circadian rhythms. Previously, we demonstrated that fibroblasts grown from BD patients show weaker amplification of circadian rhythms by lithium compared to control cells. Since calcium signals impact upon the circadian clock, and L-type calcium channels (LTCC) have emerged as genetic risk factors for BD, we examined whether loss of function in LTCCs accounts for the attenuated response to lithium in BD cells. We used fluorescent dyes to measure Ca2+ changes in BD and control fibroblasts after lithium treatment, and bioluminescent reporters to measure Per2∷luc rhythms in fibroblasts from BD patients, human controls, and mice while pharmacologically or genetically manipulating calcium channels. Longitudinal expression of LTCC genes (CACNA1C, CACNA1D and CACNB3) was then measured over 12-24 hr in BD and control cells. Our results indicate that independently of LTCCs, lithium stimulated intracellular Ca2+ less effectively in BD vs. control fibroblasts. In longitudinal studies, pharmacological inhibition of LTCCs or knockdown of CACNA1A, CACNA1C, CACNA1D and CACNB3 altered circadian rhythm amplitude. Diltiazem and knockdown of CACNA1C or CACNA1D eliminated lithium's ability to amplify rhythms. Knockdown of CACNA1A or CACNB3 altered baseline rhythms, but did not affect rhythm amplification by lithium. In human fibroblasts, CACNA1C genotype predicted the amplitude response to lithium, and the expression profiles of CACNA1C, CACNA1D and CACNB3 were altered in BD vs. controls. We conclude that in cells from BD patients, calcium signaling is abnormal, and that LTCCs underlie the failure of lithium to amplify circadian rhythms. PMID:26476274

  13. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene.

    Science.gov (United States)

    Brakstad, O G; Aasbakk, K; Maeland, J A

    1992-07-01

    Synthetic oligonucleotide primers of 21 and 24 bases, respectively, were used in the polymerase chain reaction (PCR) to amplify a sequence of the nuc gene, which encodes the thermostable nuclease of Staphylococcus aureus. A DNA fragment of approximately 270 bp was amplified from lysed S. aureus cells or isolated DNA. The PCR product was detected by agarose gel electrophoresis or Southern blot analysis by using a 33-mer internal nuc gene hybridization probe. With S. aureus cells the lower detection limit was less than 10 CFU, and with the isolated target the lower detection limit was 0.69 pg of DNA. The primers recognized 90 of 90 reference or clinical S. aureus strains. Amplification was not recorded when 80 strains representing 16 other staphylococcal species were tested or when 20 strains representing 9 different nonstaphylococcal species were tested. Some of the non-S. aureus staphylococci produced thermostable nucleases but were PCR negative. The PCR product was generated when in vitro-cultured S. aureus was used to prepare simulated clinical specimens of blood, urine, cerebrospinal fluid, or synovial fluid. No PCR product was generated when the sterile body fluids were tested. However, the sensitivity of the PCR was reduced when S. aureus in blood or urine was tested in comparison with that when bacteria in saline were tested. With the bacteria in blood, the detection limit of the PCR was 10(3) CFU. A positive PCR result was recorded when a limited number of clinical samples from wounds verified to be infected with S. aureus were tested, while the PCR product was not detected in materials from infections caused by other bacteria. Generation of PCR products was not affected by exposure of S. aureus to bactericidal agents, including cloxacillin and gentamicin, prior to testing, but was affected by exposure to UV radiation. The PCR for amplification of the nuc gene has potential for the rapid diagnosis of S. aureus infections by direct testing of clinical

  14. Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious hematopoietic necrosis virus

    Science.gov (United States)

    Arakawa, C.K.; Deering, R.E.; Higman, K.H.; Oshima, K.H.; O'Hara, P.J.; Winton, J.R.

    1990-01-01

    The polymerase chain reaction [PCR) was used to amplify a portion of the nucleoprotein [NI gene of infectious hematopoietic necrosis virus (IHNV). Using a published sequence for the Round Butte isolate of IHNV, a pair of PCR pnmers was synthesized that spanned a 252 nucleotide region of the N gene from residue 319 to residue 570 of the open reading frame. This region included a 30 nucleotide target sequence for a synthetic oligonucleotide probe developed for detection of IHNV N gene messenger RNA. After 25 cycles of amplification of either messenger or genomic RNA, the PCR product (DNA) of the expected size was easily visible on agarose gels stained with ethidium bromide. The specificity of the amplified DNA was confirmed by Southern and dot-blot analysis using the biotinylated oligonucleotide probe. The PCR was able to amplify the N gene sequence of purified genomic RNA from isolates of IHNV representing 5 different electropherotypes. Using the IHNV primer set, no PCR product was obtained from viral hemorrhagic septicemia virus RNA, but 2 higher molecular weight products were synthesized from hirame rhabdovirus RNA that did not hybridize with the biotinylated probe. The PCR could be efficiently performed with all IHNV genomic RNA template concentrations tested (1 ng to 1 pg). The lowest level of sensitivity was not determined. The PCR was used to amplify RNA extracted from infected cell cultures and selected tissues of Infected rainbow trout. The combination of PCR and nucleic acid probe promises to provide a detection method for IHNV that is rapid, h~ghly specific, and sensitive.

  15. Lager yeasts possess dynamic genomes that undergo rearrangements and gene amplification in response to stress.

    Science.gov (United States)

    James, Tharappel C; Usher, Jane; Campbell, Susan; Bond, Ursula

    2008-03-01

    A long-term goal of the brewing industry is to identify yeast strains with increased tolerance to the stresses experienced during the brewing process. We have characterised the genomes of a number of stress-tolerant mutants, derived from the lager yeast strain CMBS-33, that were selected for tolerance to high temperatures and to growth in high specific gravity wort. Our results indicate that the heat-tolerant strains have undergone a number of gross chromosomal rearrangements when compared to the parental strain. To determine if such rearrangements can spontaneously arise in response to exposure to stress conditions experienced during the brewing process, we examined the chromosome integrity of both the stress-tolerant strains and their parent during a single round of fermentation under a variety of environmental stresses. Our results show that the lager yeast genome shows tremendous plasticity during fermentation, especially when fermentations are carried out in high specific gravity wort and at higher than normal temperatures. Many localised regions of gene amplification were observed especially at the telomeres and at the rRNA gene locus on chromosome XII, and general chromosomal instability was evident. However, gross chromosomal rearrangements were not detected, indicating that continued selection in the stress conditions are required to obtain clonal isolates with stable rearrangements. Taken together, the data suggest that lager yeasts display a high degree of genomic plasticity and undergo genomic changes in response to environmental stress.

  16. Widespread hypomethylation occurs early and synergizes with gene amplification during esophageal carcinogenesis.

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    Hector Alvarez

    2011-03-01

    Full Text Available Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia, and adenocarcinoma, the combinatorial effect of these changes is unknown. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We find that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation synergizes with gene amplification and leads to significant upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation is observed at a restricted number of loci and, in combination with hemi-allelic deletions, leads to downregulatation of selected transcripts during multistep progression. We also observe that epigenetic regulation during epithelial carcinogenesis is not restricted to traditionally defined "CpG islands," but may also occur through a mechanism of differential methylation outside of these regions. Finally, validation of novel upregulated targets (CXCL1 and 3, GATA6, and DMBT1 in a larger independent panel of samples confirms the utility of integrative analysis in cancer biomarker discovery.

  17. The Mechanism by Which MYCN Amplification Confers an Enhanced Sensitivity to a PCNA-Derived Cell Permeable Peptide in Neuroblastoma Cells

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    Long Gu

    2015-12-01

    Full Text Available Dysregulated expression of MYC family genes is a hallmark of many malignancies. Unfortunately, these proteins are not amenable to blockade by small molecules or protein-based therapeutic agents. Therefore, we must find alternative approaches to target MYC-driven cancers. Amplification of MYCN, a MYC family member, predicts high-risk neuroblastoma (NB disease. We have shown that R9-caPep blocks the interaction of PCNA with its binding partners and selectively kills human NB cells, especially those with MYCN amplification, and we now show the mechanism. We found elevated levels of DNA replication stress in MYCN-amplified NB cells. R9-caPep exacerbated DNA replication stress in MYCN-amplified NB cells and NB cells with an augmented level of MYC by interfering with DNA replication fork extension, leading to Chk1 dependence and susceptibility to Chk1 inhibition. We describe how these effects may be exploited for treating NB.

  18. Preparation and Amplification of Colony of Goat Transgenic Fetal Fibroblast and Mammary Gland Epithelial Cell with Human Lactoferrin Gene

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yu-ling; LIU Feng-jun; ZHANG Yong

    2009-01-01

    [Objective] The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C1 which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0%, 50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture, neo gene was as screened gene, genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [Result] Compared with non-conditioned culture medium, 100% conditioned culture medium could greatly increase survived rate of single colony cells (FF: 53.33% vs. 10.00%; MGE: 33.33% vs. 6.67%). Compared with control, con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%), confluence time of amplification culture was significantly decreased (20-30 d). The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [Conclusion] The study provides a reliable method for separating transgenic cell, inserting and diagnosing ideal vector, and can save expense and time for transgenic animal production.

  19. Amplification and expression of genes from the 17q11 approximately q12 amplicon in breast cancer cells.

    Science.gov (United States)

    Luoh, Shiuh-Wen

    2002-07-01

    Amplification of a portion of 17q11 approximately q12 involving the human epidermal growth factor receptor-2 (HER-2/neu) gene is common in solid tumors. HER-2/neu amplification in breast cancer is associated with a poor prognosis and may predict response to therapeutic interventions. The molecular complexity and informative content of the 17q11 approximately q12 amplified DNA remain largely unknown. Study of available sequence information in public database allowed us to construct a contig in bacterial artificial chromosome that covers this region. We have identified a several hundred kilobase core segment from this region that is amplified in three out of three breast cancer cell lines with HER-2/neu amplification. There is striking correlation between amplification and overexpression of genes from this region. Amplified and overexpressed genes, in addition to HER-2/neu, may play functional roles in the pathogenesis of breast cancer and may serve as additional targets for therapy. Published by Elsevier Science Inc.

  20. Distinct phenotypic differences associated with differential amplification of receptor tyrosine kinase genes at 4q12 in glioblastoma.

    Directory of Open Access Journals (Sweden)

    Anna Burford

    Full Text Available Gene amplification at chromosome 4q12 is a common alteration in human high grade gliomas including glioblastoma, a CNS tumour with consistently poor prognosis. This locus harbours the known oncogenes encoding the receptor tyrosine kinases PDGFRA, KIT, and VEGFR2. These receptors are potential targets for novel therapeutic intervention in these diseases, with expression noted in tumour cells and/or associated vasculature. Despite this, a detailed assessment of their relative contributions to different high grade glioma histologies and the underlying heterogeneity within glioblastoma has been lacking. We studied 342 primary high grade gliomas for individual gene amplification using specific FISH probes, as well as receptor expression in the tumour and endothelial cells by immunohistochemistry, and correlated our findings with specific tumour cell morphological types and patterns of vasculature. We identified amplicons which encompassed PDGFRA only, PDGFRA/KIT, and PDGFRA/KIT/VEGFR2, with distinct phenotypic correlates. Within glioblastoma specimens, PDGFRA amplification alone was linked to oligodendroglial, small cell and sarcomatous tumour cell morphologies, and rare MGMT promoter methylation. A younger age at diagnosis and better clinical outcome in glioblastoma patients is only seen when PDGFRA and KIT are co-amplified. IDH1 mutation was only found when all three genes are amplified; this is a subgroup which also harbours extensive MGMT promoter methylation. Whilst PDGFRA amplification was tightly linked to tumour expression of the receptor, this was not the case for KIT or VEGFR2. Thus we have identified differential patterns of gene amplification and expression of RTKs at the 4q12 locus to be associated with specific phenotypes which may reflect their distinct underlying mechanisms.

  1. THE AMPLIFICATION AND EXPRESSION OF MDR1 GENE IN ADRIAMYCINE RESISTANT CELL LINE OF COLON CANCER CELL HR8348

    Institute of Scientific and Technical Information of China (English)

    周中军; 罗贤懋; 林晨; 陈凤

    1996-01-01

    P-glycoprotein plays an important role in highly drug resistant cells. But its high expression cannot be acheived by chemotherapy. In order to study the effect of P-glycoprotein on clinical tumors, wo ostablished a low ADM resistant colon cancer ceil line HR/ADM and determined the amplification and expression of mdr-1 gene. The GLC/ADM showed a resistant pattern similar to classical MDR and the transcription of mdr-1 gene determined by RT-PCR increased. The immunocytcchemical analysis showed strong positive staining with monoelonal antibozly. The gene amplification of mdr-l was dearly demonstrated by southern blot. Our results suggested that moderate expression of P-glycoprotein might be enough for a high resistant pattern.

  2. MYEOV : A candidate gene for DNA amplification events occurring centromeric to CCNDI in breast cancer

    NARCIS (Netherlands)

    Janssen, JWG; Cuny, M; Orsetti, B; Rodriguez, C; Valles, H; Bartram, CR; Schuuring, E; Theillet, C

    2002-01-01

    Rearrangements of chromosome 11q13 are frequently observed in human cancer. The 11q13 region harbors several chromosomal breakpoint clusters found in hematologic malignancies and exhibits frequent DNA amplification in carcinomas. DNA amplification patterns in breast tumors are consistent with the ex

  3. Detection of MYCN Gene Amplification in Neuroblastoma by Fluorescence In Situ Hybridization: A Pediatric Oncology Group Study

    Directory of Open Access Journals (Sweden)

    Prasad Mathew

    2001-01-01

    Full Text Available To assess the utility of fluorescence in situ hybridization (FISH for analysis of MYCN gene amplification in neuroblastoma, we compared this assay with Southern blot analysis using tumor specimens collected from 232 patients with presenting characteristics typical of this disease. The FISH technique identified MYCN amplification in 47 cases, compared with 39 by Southern blotting, thus increasing the total number of positive cases by 21%. The major cause of discordancy was a low fraction of tumor cells (≤30% replacement in clinical specimens, which prevented an accurate estimate of MYCN copy number by Southern blotting. With FISH, by contrast, it was possible to analyze multiple interphase nuclei of tumor cells, regardless of the proportion of normal peripheral blood, bone marrow, or stromal cells in clinical samples. Thus, FISH could be performed accurately with very small numbers of tumor cells from touch preparations of needle biopsies. Moreover, this procedure allowed us to discern the heterogeneous pattern of MYCN amplification that is characteristic of neuroblastoma. We conclude that FISH improves the detection of MYCN gene amplification in childhood neuroblastomas in a clinical setting, thus facilitating therapeutic decisions based on the presence or absence of this prognostically important biologic marker.

  4. DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila.

    Science.gov (United States)

    Liu, Jun; Zimmer, Kurt; Rusch, Douglas B; Paranjape, Neha; Podicheti, Ram; Tang, Haixu; Calvi, Brian R

    2015-10-15

    Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC.

  5. Complex oncogenic translocations with gene amplification are initiated by specific DNA breaks in lymphocytes.

    Science.gov (United States)

    Wright, Sarah M; Woo, Yong H; Alley, Travis L; Shirley, Bobbi-Jo; Akeson, Ellen C; Snow, Kathy J; Maas, Sarah A; Elwell, Rachel L; Foreman, Oded; Mills, Kevin D

    2009-05-15

    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. Whereas chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double-strand break repair in suppression of oncogenic genome instability, the genomic elements required for chromosome rearrangements, especially complex lesions, have not been elucidated. Using a mouse model of B-lineage lymphoma, characterized by complicon formation involving the immunoglobulin heavy chain (Igh) locus and the c-myc oncogene, we have now investigated the requirement for specific genomic segments as donors for complex rearrangements. We now show that specific DNA double-strand breaks, occurring within a narrow segment of Igh, are necessary to initiate complicon formation. By contrast, neither specific DNA breaks nor the powerful intronic enhancer Emu are required for complicon-independent oncogenesis. This study is the first to delineate mechanisms of complex versus simple instability and the first to identify specific chromosomal elements required for complex chromosomal aberrations. These findings will illuminate genomic cancer susceptibility and risk factors.

  6. BHK cell lines with increased rates of gene amplification are hypersensitive to ultraviolet light

    Energy Technology Data Exchange (ETDEWEB)

    Giulotto, E.; Bertoni, L.; Attolini, C.; Rainaldi, G.; Anglana, M. (Dipartimento di Genetica e Microbiologia Adriano Buzzati-Traverso, Pavia (Italy))

    1991-04-15

    Four cell lines (MP1, -4, -5, -7), isolated from baby hamster kidney cells after simultaneous selection with N-(phosphonacetyl)-L-aspartate and methotrexate, have previously been shown to amplify their DNA at an increased rate. We now show that all four lines are hypersensitive to killing by UV light and mitomycin C. At high doses of UV light or mitomycin C, the MP lines survived less than 10% or less than 5% as well as parental cells, respectively. After UV irradiation, inhibition of DNA and RNA synthesis was greater in MP than in parental cells, and recovery was slower or absent. A 2- to 3.5-fold increase in the frequency of UV-induced sister chromatid exchange was also seen in the four cell lines. In MP5, unscheduled DNA replication after treatment with UV light was only approximately 70% as great as in parental cells and the other MP lines. In MP4 and MP7 cells S phase was elongated. Although their individual properties confirm that the four cell lines are independent, their common properties suggest a relationship between tolerance of DNA damage and gene amplification.

  7. Amplification of hTERT and hTERC genes in leukemic cells with high expression and activity of telomerase.

    Science.gov (United States)

    Nowak, Tomasz; Januszkiewicz, Danuta; Zawada, Mariola; Pernak, Monika; Lewandowski, Krzysztof; Rembowska, Jolanta; Nowicka, Karina; Mankowski, Przemyslaw; Nowak, Jerzy

    2006-08-01

    Reactivation of telomerase plays an important role in carcinogenesis. Malignant cells almost always possess high activity and expression of telomerase. The aim of this study was to see whether there is any relationship between telomerase activity and expression and hTERT and hTERC gene amplification in acute lymphoblastic leukemia (ALL) and non-lymphoblastic leukemia (ANLL) cells. In addition telomere length was tested in leukemic cells at the time of diagnosis and during remission. Expression of the three components of telomerase (hTERT, hTERC and TP1) as well as telomerase activity was found both in ALL and ANLL cells. Telomerase activity was diminished in patients in remission. The leukemic cells showed considerable heterogeneity of terminal restriction fragments, that is telomere length. ALL cells showed a variable pattern of telomere length in contrast to ANLL cells which produced a predominantly short telomere pattern. Telomere length in the lymphocytes of leukemia patients was shorter in remission as compared to the time of diagnosis. FISH analysis revealed amplification of hTERT and hTERC genes in ALL and ANLL cells. Quantitative analysis showed that leukemic cells possess higher number of hTERT and hTERC copies than the normal PBL. Our results suggest that the activation of telomerase in leukemic cells is connected with amplification of hTERT and hTERC genes. The high expression and activity of telomerase found in leukemic cells may be partially explained by amplified hTERT and hTERC genes. Amplification of the telomerase genes seems to be a common event in carcinogenesis and may play a role in telomerase reactivation leading to cell immortalization.

  8. Comparison of the c-MET gene amplification between primary tumor and metastatic lymph nodes in non-small cell lung cancer.

    Science.gov (United States)

    Xu, Chun-Wei; Wang, Wen-Xian; Wu, Mei-Juan; Zhu, You-Cai; Zhuang, Wu; Lin, Gen; Du, Kai-Qi; Huang, Yun-Jian; Chen, Yan-Ping; Chen, Gang; Fang, Mei-Yu

    2017-09-01

    c-MET has recently been identified as a promising novel target in non-small cell lung cancer (NSCLC). We detected the consistency of c-MET gene amplification in metastatic lymph nodes and tumor tissues of NSCLC patients and discuss the clinical application value of c-MET gene amplification in metastatic lymph nodes. Real-time fluorescent quantitative PCR was used to test tumor tissues in 368 NSCLC patients and 178 paired metastatic lymph node samples. The amplification consistency in metastatic lymph nodes and tissue samples were compared and the correlation between c-MET gene amplification and the clinical characteristics of patients was analyzed. The c-MET gene amplification rate was 8.97% (33/368) in tumor tissues. Of the 178 paired cases, c-MET gene amplification was positive in 7.95% (15/178) of cancerous tissues and 18.54% (33/178) of metastatic lymph nodes. c-MET gene amplification was detected more frequently in metastatic lymph nodes than in primary cancerous tissue. When metastatic lymph nodes were used as surrogate samples of primary cancerous tissues, the sensitivity was 86.67% (13/15) and the specificity was 87.69% (143/163). Screening for c-MET gene amplification in lymph node metastases could determine which patients are eligible for tyrosine kinase inhibitor therapy. Lymph node metastasis can predict c-MET gene amplification in a primary tumor and guide the clinical use of c-MET gene targeted drugs. © 2017 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

  9. [Evaluation of c-myc and CCNE2 amplification in breast cancer with quantitative multi-gene fluorescence in-situ hybridization].

    Science.gov (United States)

    Li, Zhishuang; Meng, Qingyong; Yu, Qiong; Zhou, Zhiqiang; Li, Li

    2014-07-01

    To investigate c-myc and CCNE2 gene amplifications and their relationship in breast cancer. Sixty-six infiltrating ductal breast carcinomas with foci of ductal carcinoma in situ components collected from January 2005 to December 2007 were selected for tissue microarray and quantitative multi-gene FISH for c-myc and CCNE2 gene amplification, and the relationship with the clinicopathologic features was analyzed. Of the 66 cases, 18 (27.3%) showed c-myc amplification and 23 (34.8%) showed CCNE2 amplification. A strong correlation was found between c-myc and CCNE2 amplification (P c-myc and CCNE2 amplifications were all aneuploidy, and were HER2 positive (P c-myc amplification also showed higher Ki-67 index (P C-myc and CCNE2 amplifications are common events in breast cancer, and they often coexist. C-myc and CCNE2 genes may play critical roles in the pathogenesis and development of breast cancer through unique and overlapping signaling pathways.

  10. Amplification of Whole Tumor Genomes and Gene-by-Gene Mapping of Genomic Aberrations from Limited Sources of Fresh-Frozen and Paraffin-Embedded DNA

    Science.gov (United States)

    Bredel, Markus; Bredel, Claudia; Juric, Dejan; Kim, Young; Vogel, Hannes; Harsh, Griffith R.; Recht, Lawrence D.; Pollack, Jonathan R.; Sikic, Branimir I.

    2005-01-01

    Sufficient quantity of genomic DNA can be a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffin-embedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Phi29 amplified DNA from matched pairs of fresh-frozen and formalin-fixed/paraffin-embedded tumor samples with similar efficiency. The distortion in gene dosage representation in the amplified DNA was nonrandom and reproducibly involved distinct genomic loci. Regional amplification efficiency was significantly linked to regional GC content of the template genome. The biased gene representation in amplified tumor DNA could be effectively normalized by using amplified reference DNA. Our data suggest that genome-wide gene dosage alterations in clinical tumor samples can be reliably assessed from a few hundred tumor cells. Therefore, this amplification method should lend itself to high-throughput genetic analyses of limited sources of tumor, such as fine-needle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens. PMID:15858140

  11. The SRY-HMG box gene, SOX4, is a target of gene amplification at chromosome 6p in lung cancer

    NARCIS (Netherlands)

    Medina, P.P.; Castillo, S.D.; Blanco, S.; Sanz-Garcia, M.; Largo, C.; Alvarez, S.; Yokota, J.; Gonzalez-Neira, A.; Benitez, J.; Clevers, H.; Cigudosa, J.C.; Lazo, P.A.; Sanchez-Cespedes, M.

    2009-01-01

    The search for oncogenes is becoming increasingly important in cancer genetics because they are suitable targets for therapeutic intervention. To identify novel oncogenes, activated by gene amplification, we analyzed cDNA microarrays by high-resolution comparative genome hybridization and compared D

  12. Human identification from forensic materials by amplification of a human-specific sequence in the myoglobin gene.

    OpenAIRE

    Ono T; Miyaishi S; Yamamoto Y; Yoshitome K; Ishikawa T.; Ishizu H

    2001-01-01

    We developed a method for human identification of forensic biological materials by PCR-based detection of a human-specific sequence in exon 3 of the myoglobin gene. This human-specific DNA sequence was deduced from differences in the amino acid sequences of myoglobins between humans and other animal species. The new method enabled amplification of the target DNA fragment from 30 samples of human DNA, and the amplified sequences were identical with that already reported. Using this method, we ...

  13. Human telomerase gene amplification and high-risk human papillomavirus infection in women with cervical intra-epithelial neoplasia.

    Science.gov (United States)

    Takac, Iztok; Arko, D; Kodric, T; Poljak, M; Zagorac, A; Erjavec-Skerget, A; Kokalj-Vokac, N

    2009-01-01

    This study was designed to investigate whether a correlation exists between amplification of the human telomerase gene (human telomerase RNA component [TERC]) and high-risk human papillomavirus (HR-HPV) infection in 101 women with cervical intra-epithelial neoplasia (CIN). Eight patients (7.9%) had CIN 1, 24 (23.8%) had CIN 2 and 69 (68.3%) had CIN 3. TERC was amplified in 31.7% of all CIN patients. The difference in frequency of TERC amplification between patients with low-grade CIN (CIN 1) and those with high-grade CIN (CIN 2 and CIN 3) was not significant. HR-HPV infection was detected in 88.1% of all CIN cases and was significantly more frequent in patients with CIN 2 and CIN 3 than in patients with CIN 1. There was no significant difference in the frequency of HR-HPV infection between groups of patients with and without TERC amplification. In conclusion, this study found no correlation between TERC amplification and HR-HPV infection in patients with CIN.

  14. Clinical significance of hTERC and C-Myc genes amplification in a group of Egyptian patients with cancer cervix.

    Science.gov (United States)

    Eid, M M; Nossair, H M; Ismael, M T; Amira, G; Hosney, M M; Abdul Rahman, R

    2011-07-01

    Cervical cancer is the second most common cancer in women worldwide after breast cancer. Cervical cancer is a preventable disease. The implementation of cervical cancer screening programs has greatly decreased the morbidity and mortality, as precancerous lesions and early invasive cervical cancer could be detected and treated effectively. The detection of hTERC gene amplification was suggested as a possible diagnostic marker for use in routine cytological screening. The present study was designed to detect genomic gains of the hTERC and C-MYC genes using FISH technique and to investigate the relationship between genes amplification and the clinical data of the patients. The current study was carried out on twelve cases with cervical cancer at different grades (three cases were grade I, six cases were grade II and three cases were grade III). Interphase FISH analysis using LSI probe, Cervical Cancer probe hTERC (3q26) & C-MYC (8q24), was successfully performed on 12 patients with cancer cervix. Interphase FISH analysis revealed positive hTERC gene amplification in all cases of cancer cervix (100%). However C-MYC gene amplification was detected in four cases only (33.3%). Statistical analysis of the data revealed significant correlation between hTERC amplification and grading. Also, there was significant correlation between C-MYC amplification and grading and highly significant correlation between C-MYC amplification and hTERC amplification. On the other hand hTERC and C-MYC genes amplification showed an inverse correlation with the ages of the patients. The present study highlights the importance of using hTERC and C-MYC genes FISH probes for cases with cancer cervix or pre-malignant lesions as a sensitive technique. This method provides an easy and effective applicable approach which helps in the diagnosis and prognosis, as an increased copy number is associated with a more advanced grade that could be detected in the early stages of the disease.

  15. Comparison of different protocols for DNA preparation and PCR amplification of mitochondrial genes of tardigrades

    Directory of Open Access Journals (Sweden)

    Ralph O. SCHILL

    2007-09-01

    Full Text Available Phylogenetic relationships and molecular taxonomy within the Tardigrada have been given a lot of attention in recent years. Here I present the first comparison of different protocols for DNA preparation by investigating six commercial available DNA extraction kits and the CTAB method. Successful extraction of DNA from tardigrades depends strongly on the life-stage (embryo, adult, and on the condition of the specimens, respectively on the preservation (anhydrobiotic, ethanol. Although the extraction kits showed differences in the amount of extracted DNA, in all cases fresh tissue of live animals or embryos resulted in the best quality and quantity of DNA. A lesser amount of DNA was extractable from anhydrobiotic animals and embryos and the results of specimens fixed in ethanol were unsatisfactory. All used commercially available DNA extraction kits and PCR cocktails have been focused on vertebrate tissues, blood, cultured cells, bacteria and yeast. However, I used successfully the kits according to the manufacturer’s instruction without changes in the protocols for DNA extraction of tardigrades. Commercial kits provide a simple and convenient way to isolate pure genomic DNA of high-quality from tardigrades. Furthermore I tested eight different Taq polymerase enzymes for PCR amplification of mitochondrial genes of tardigrades. Each of the enzymes resulted in a PCR product, and even if the amount of the PCR products was quite different, it was possible to use it successful for direct sequencing. Summarizing, the successful PCR of the target DNA depends on the purity and quality of the DNA template and for this the species preservation is more critical than the extraction method or the PCR cocktail which can be optimized.

  16. Human telomerase RNA component (hTERC gene amplification detected by FISH in precancerous lesions and carcinoma of the larynx

    Directory of Open Access Journals (Sweden)

    Liu Yu

    2012-03-01

    Full Text Available Abstract Background Gain of 3q26 is frequently observed in squamous cell carcinomas of mucosal origin, including those originating in the head and neck region. The human telomerase RNA component (hTERC gene, which is located on chromosome 3q26, encodes for an RNA subunit of telomerase that maintains the length of telomeres through cellular divisions, and is activated in malignant diseases. The present study was designed to detect hTERC amplification in laryngeal lesions and evaluate whether this might serve as a supportive biomarker in histopathological analysis for in the diagnosis of laryngeal lesions. Methods Fluorescent in situ hybridization (FISH was applied on formalin-fixed paraffin-embedded blocks of 93 laryngeal specimens, including 14 normal epithelium (NE, 15 mild dysplasia (Md, 18 moderate dysplasia (MD, 16 severe dysplasia (SD, 9 carcinoma in situ (CIS, and 21 invasive carcinoma (IC. Results By histopathologic examination, hTERC amplification rates in NE, Md, MD, SD, CIS and IC cases were 0% (0/14, 13.33% (2/15, 72.22% (13/18, 81.25% (13/16, 100% (9/9 and 100% (21/21, respectively. Amplification of hTERC was significantly associated with histopathologic diagnosis (P Conclusions The hTERC amplification is important in the development of laryngeal squamous cell carcinoma (LSCC. FISH detection of hTERC amplification may provide an effective approach in conjunction with histopathologic evaluation for differential diagnosis of laryngeal lesions. Virtual Slides The virtual slide(s for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2226606266791985

  17. mRNA Expression of Vimentin Gene in Lens of Transgenic Mouse and DNA Amplification in Human Cataracts

    Institute of Scientific and Technical Information of China (English)

    YanLi; XienpingLiu; 等

    1995-01-01

    Purpose:To investigate the role of vimentin gene in cataractogenesis.Methods:The12.7kb chicken vimentin genes were microinjected into the male pronuclei of 918 fertilized mice eggs.841injected embryos were transferred into oviducts of pseudopregnant recipient females.of which 12pregnant mice gave birth to 49offsping mice.The integration and expression of exogenous gene in the offsping were analysed by Southern and Northern blot byhridizations,In the human senile cataract,the lens vimentin gene was analyzed with the chicken vi-mentin gene probe.Results:It showed that four of F1offspring were transgenic mice in which the chicken vimenttin gene was integrated in their genomes.The transgenic band was12kb,similar to the12.7kb chicken vimentin fragment injected.One2kbvi-mentin mRNAwas visualized on E2 mouse lens blot.which revealed that the chicken vimentin gene was efficiently expressed in this transgenic mouse.In the humansenile cataract lens,12kb BamHI-restricted vimentin fragments displayed a stronger hybridization signal than that of the control lens in Southern blot anal-ysis,It implies that the Formation of human senile cataract may be associated with the amplification of vimentin gene.Conclusions:We have successfully developed four transgenic mice bearing chicken vimentin gene and having mRNA expression which can be used for further study.It is to be observed if the normal lens cell function is affected by the expressed product and cataract occurs in our transgenic mice.The cause of the gene ampli-fication in human ctaract remains for further investigation.Eye Science 1995;11:113-116.

  18. Use of aiiA gene amplification for AHL-lactonase production from endophytic bacterium Enterobacter species.

    Science.gov (United States)

    Rajesh, P S; Rai, V Ravishankar

    2015-01-01

    AHL-lactonase has gained renewed interest due to biotechnological applications such as antiquorum sensing, antibiofilm strategies, biofouling, etc. In our study, the production of AHL-lactonase from endophytic bacteria Enterobacter aerogenes VT66 was optimized by response surface methodology (RSM) using central composite design (CCD) for four different cultural conditions. The relative activity of AHL-lactonase was correlated with amplification of aiiA homologous gene amplification with respect to cultural conditions. Statistical analysis by ANOVA of the quadratic regression model showed that the RSM model constructed is highly significant, as indicated by F-test with a low probability value (p(model) < 0.0001) and high regression coefficient (0.9997) as well as lower coefficient of variation (1.86%) indicate that suitability of variable parameters. The quadratic regression model of AHL-lactonase production in terms of relative activity was built and the optimal cultural conditions for maximum enzyme production were determined as 32.5 °C temperature, pH 7.0, 350 μM of substrate concentration and 33 h of incubation time. The enhanced AHL-lactonase yielded 1.33 fold increases in relative activity and it positively correlated with the amplification of aiiA gene.

  19. EPSPS Gene Amplification in Glyphosate-Resistant Italian Ryegrass (Lolium perenne ssp. multiflorum) Populations from Arkansas (United States).

    Science.gov (United States)

    Salas, Reiofeli A; Scott, Robert C; Dayan, Franck E; Burgos, Nilda R

    2015-07-01

    Glyphosate-resistant Italian ryegrass was detected in Arkansas (United States) in 2007. In 2014, 45 populations were confirmed resistant in eight counties across the state. The level of resistance and resistance mechanisms in six populations were studied to assess the severity of the problem and identify alternative management approaches. Dose-response bioassays, glyphosate absorption and translocation experiments, herbicide target (EPSPS) gene sequence analysis, and gene amplification assays were conducted. The dose causing 50% growth reduction (GR50) was 7-19 times higher for the resistant population than for the susceptible standard. Uptake and translocation of (14)C-glyphosate were similar in resistant and susceptible plants, and no mutation in the EPSPS gene known to be associated with resistance to glyphosate was detected. Resistant plants contained from 11- to >100-fold more copies of the EPSPS gene than the susceptible plants, whereas the susceptible plants had only one copy of EPSPS. Plants surviving the recommended dose of glyphosate contained at least 10 copies. The EPSPS copy number was positively related to glyphosate resistance level (r = 80). Therefore, resistance to glyphosate in these populations is due to multiplication of the target site. Resistance mechanisms could be location-specific. Suppressing the mechanism for gene amplification may overcome resistance.

  20. Isolation of epithelial cells from acrylic removable dentures and gender identification by amplification of SRY gene using real time PCR

    Directory of Open Access Journals (Sweden)

    Renjith George

    2010-01-01

    Full Text Available This study evaluates the usefulness of acrylic dentures as the source of DNA for forensic analysis. Thirty-eight samples (21 males and 17 females were collected and stored for different time periods. The epithelial cells adhered to the dentures were retrieved and the genomic DNA was extracted. All the samples yielded sufficient amount of DNA for analysis irrespective of the storage time. Gender determination was done by amplification of the sex determining region on the Y chromosome (SRY using real-time polymerase chain reaction with 100% accuracy, within minimal time. With this study, we conclude that saliva-stained acrylic dentures can act as a source of forensic DNA and co-amplification of SRY gene with other routine sex typing markers will give unambiguous gender identification.

  1. HIV-1包膜单基因Nest-PCR体系优化及包膜基因准种扩增%Optimization of HIV-1 envelope gene PCR amplification system and amplification of envelope gene quasispecies

    Institute of Scientific and Technical Information of China (English)

    全红; 刘松; 张昊天; 任彩云; 庄敏; 凌虹; 李妍

    2012-01-01

    目的 确定HIV-1包膜(envelope,env)单基因扩增(single genome amplification,SGA)的最优反应体系,获得env单基因扩增产物.方法 采用SGA及Nest-PCR扩增HIV-1 env全长基因,并对Nest-PCR反应体系中各成分量进行优化;在此基础上,适当稀释HIV-1感染者cDNA模板进行Nest-PCR,以获得PCR扩增产物阳性率不超过30%的模板稀释度.结果 SGA Nest-PCR扩增HIV-1 env全长基因,引物浓度为0.3 μmol/L,dNTP浓度为0.25 μmol/L时可获得特异性高的env全长基因;凝胶回收纯化及稀释第一轮PCR产物可有效提高特异性条带的产量.采用优化反应体系成功从一名HIV-1感染者体内扩增出18株env单基因序列.结论 优化了HIV-1 env基因SGA Nest-PCR的反应体系,并从HIV-1感染者获得了病毒包膜基因准种,为后续进行准种分析奠定了基础.%Objective To optimize the PCR conditions for single genome amplification (SGA) of HIV-1 envelope (env) gene. Methods The efficiency of the SGA-PCR was assessed based on the amplification results of env gene using the non-diluted cDNA template, the optimal dilution of cDNA template that may result in no more than 30% positive amplification was predicted. Results The elements of SGA and Nested-PCR such as the amounts of primers were 0. 3 μmol/L, dNTP were 0. 25 μmol/L, purification of products of the first-round PCR reactions and diluting cDNA templates increased the amount of interest products effectively. One HIV-1 infected individual after the env gene SGA, 18 interest products confirmed by sequencing env sequence. Conclusion Optimization of the HIV-1 env gene the SGA Nest-PCR reaction system, and the viral envelope gene quasispecies from HIV-1 infection, laid the foundation for subsequent analysis of the quasispecies.

  2. A phase II study of Epirubicin in oxaliplatin-resistant patients with metastatic colorectal cancer and TOP2A gene amplification

    DEFF Research Database (Denmark)

    Tarpgaard, Line S.; Qvortrup, Camilla; Nygård, Sune Boris;

    2016-01-01

    A gene amplification in their tumor cells. BACKGROUND: Epirubicin is an anthracycline that targets DNA topoisomerase 2-α enzyme encoded by the TOP2A gene. It is used for treatment of several malignancies, but currently not in CRC. TOP2A gene amplifications predict improved efficacy of epirubicin...... normal tissue. The TOP2A gene is located on chromosome 17 and when the TOP2A/CEN-17 ratio was applied to identify tumors with gene loss or amplifications, 10.5 % had a ratio ≥ 1.5 consistent with gene amplification and 2.6 % had a ratio ≤ 0.8 suggesting gene deletions. Based on these observations......, investigating the efficacy of epirubicin in patients with oxaliplatin refractory mCRC and with a cancer cell TOP2A/CEN-17 ratio ≥ 1.5. TOP2A gene amplification measured by fluorescence in situ hybridization. A total of 25 evaluable patients (15 + 10 in two steps) will be included (Simon's two-stage minimax...

  3. Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

    Science.gov (United States)

    Liu, Zhongle; Moran, Gary P.; Myers, Lawrence C.

    2016-01-01

    Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of ‘free,’ non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large ‘free’ pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the

  4. PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenome for biocatalysis.

    Science.gov (United States)

    Itoh, Nobuya; Isotani, Kentaro; Makino, Yoshihide; Kato, Masaki; Kitayama, Kouta; Ishimota, Tuyoshi

    2014-02-05

    The amplification of useful genes from metagenomes offers great biotechnological potential. We employed this approach to isolate alcohol dehydrogenase (adh) genes from Pseudomonas to aid in the synthesis of optically pure alcohols from various ketones. A PCR primer combination synthesized by reference to the adh sequences of known Pseudomonas genes was used to amplify full-length adh genes directly from 17 samples of DNA extracted from soil. Three such adh preparations were used to construct Escherichia coli plasmid libraries. Of the approximately 2800 colonies obtained, 240 putative adh-positive clones were identified by colony-PCR. Next, 23 functional adh genes named using the descriptors HBadh and HPadh were analyzed. The adh genes obtained via this metagenomic approach varied in their DNA and amino acid sequences. Expression of the gene products in E. coli indicated varying substrate specificity. Two representative genes, HBadh-1 and HPadh-24, expressed in E. coli and Pseudomonas putida, respectively, were purified and characterized in detail. The enzyme products of these genes were confirmed to be useful for producing anti-Prelog chiral alcohols.

  5. Heterogeneous HER2 gene amplification: impact on patient outcome and a clinically relevant definition.

    Science.gov (United States)

    Bartlett, Alastair I; Starcyznski, Jane; Robson, Tammy; Maclellan, Alex; Campbell, Fiona M; van de Velde, Cornelis J H; Hasenburg, Annette; Markopoulos, Christos; Seynaeve, Caroline; Rea, Daniel; Bartlett, John M S

    2011-08-01

    Heterogeneous expression or amplification is a challenge to HER2 diagnostics. A guideline defines heterogeneity as the presence of between 5% and 50% cells with HER2/CEP17 ratios of more than 2.20. We audited the frequency of such cells and their clinical impact in the results from 2 pathology laboratories combined with data from the TEAM [Tamoxifen vs Exemestane Adjuvant Multicentre] pathology study. HER2 reports were scanned and the percentages of amplified cells reported. Of 6,461 eligible cases, 754 (11.7%) exhibited 50% or more cells with ratios of more than 2.20, which is "amplified" by College of American Pathologists guidelines. Of the cases, 2,166 (33.5%) exhibited more than 5% but less than 50% of cells with HER2/CEP17 ratios of more than 2.20, or "heterogeneous amplification." No prognostic impact was observed when fewer than 30% of cells exhibited ratios of more than 2.20. All amplified cases with 30% to 50% of cells with ratios more than 2.20 were identified as such by United Kingdom guidelines. The percentage of tumor cells with HER2/CEP17 ratios more than 2.20 does not identify cases with heterogeneous amplification or poor outcome. A modified approach for identification of true heterogeneous amplification is suggested.

  6. Investigation of Gene Expression Correlating With Centrosome Amplification in Development and Progression of Breast Cancer

    Science.gov (United States)

    2004-09-01

    Endocrinology 142, 4006-4014. 9. Colditz, G. A., Stampfer , M. J., Willett, W. C., Hennekens, C. H., Rosner, B. 37. Li, J. J., Hou, X., Banerjee, S. K...overexpression induces centrosome amplification, mitotic spindle abnormalities, and aneuploidy Christopher J. Nelsen,Ŗ Ryoko Kuriyama,3𔃾 Betsy Hirsch,4𔃿

  7. SMAD5 Gene Expression, Rearrangements, Copy Number, Amplification at Fragile Site FRA5C in Human Hepatocellular Carcinoma

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    Drazen B. Zimonjic

    2003-09-01

    Full Text Available Signaling by the transforming growth factor (TGFfamily members is transduced from the cell surface to the nucleus by the Smad group of intracellular proteins. Because we detected alterations on the long arm of chromosome 5, we examined the status of the SMAD5 gene in human hepatocellular carcinoma (HCC cell lines and primary HCC. In 16 cell lines, chromosome alterations of chromosome 5 were observed in nine cell lines by fluorescence in situ hybridization (FISH, an increase in SMAD5 gene copy number relative to the ploidy level was found in eight lines. The breakpoints in unbalanced translocations and deletions frequently occurred near the SMAD5 locus, but apparently did not cause loss of SMAD5. In one cell line, where comparative genomic hybridization showed DNA copy number gain confined to the region 5831, we detected by FISH high-level amplification of the SMAD5 gene located within the fragile site FRA5C. Semiquantitative polymerase chain reaction did not reveal changes in SMAD5 DNA levels in 15 of 17 primary HCC specimens. In 17 HCC cell lines, SMAD5 mRNA levels were either maintained or upregulated by an increase in gene dosage or another mechanism. Collectively, our results show that SMAD5 undergoes copy number gain and increased expression, rather than loss of expression, therefore suggest that this gene does not act as a tumorsuppressor gene in HCC. The Hep-40 HCC cell line with high-level amplification and significant overexpression of SMAD5 may be useful in studying the interaction of SMAD5 with other genes.

  8. Double trouble: medical implications of genetic duplication and amplification in bacteria.

    Science.gov (United States)

    Craven, Sarah H; Neidle, Ellen L

    2007-06-01

    Gene amplification allows organisms to adapt to changing environmental conditions. This type of increased gene dosage confers selectable benefits, typically by augmenting protein production. Gene amplification is a reversible process that does not require permanent genetic change. Although transient, altered gene dosage has significant medical impact. Recent examples of amplification in bacteria, described here, affect human disease by modifying antibiotic resistance, the virulence of pathogens, vaccine efficacy and antibiotic biosynthesis. Amplification is usually a two-step process whereby genetic duplication (step one) promotes further increases in copy number (step two). Both steps have important evolutionary significance for the emergence of innovative gene functions. Recent genome sequence analyses illustrate how genome plasticity can affect the evolution and immunogenic properties of bacterial pathogens.

  9. Clinical significance of fluorescence in situ hybridization for detection of hTERC gene amplification in cervical cancer and precancerous tissues cases

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    Shuang LIU

    2012-06-01

    Full Text Available Objective  To detect the human telomerase RNA gene (hTERC amplification in cervical lesions, and explore its clinical significance. Methods  The tissues of the cervical lesions were collected from 195 patients, including 33 of chronic cervicitis, 34 of CINⅠ, 37 of CIN Ⅱ-Ⅲ, 30 of cervical squamous cell carcinoma, and 61 of cervica1 adenocarcinoma, and abnormal hTERC was detected with amplification of fluorescence in situhybridization (FISH. The relationship between hTERC gene amplification and clinicopathological parameters was analyzed. Results  Among the 195 patients, the positive rate of hTERC gene amplification was 3.03% (1/33, 29.41% (10/34, 72.97% (27/37, 100% (30/30, 91.8% (56/61 in chronic cervicitis, CINⅠ, CIN Ⅱ-Ⅲ, cervical squamous cell carcinoma and cervica1 adenocarcinoma respectively, and the results showed that hTERC amplification rate was significantly higher in group CIN Ⅱ-Ⅲthan in group CINⅠ(P 0.05. Conclusion  Detection of gene amplification by FISH technology can be used as a means for accurate diagnosis and prediction of the histologically difficult-to-diagnose lesion and for risk assessment after treatment of cervical precancerous lesions.

  10. Whole-genome amplification: a useful approach to characterize new genes in unculturable protozoan parasites such as Bonamia exitiosa.

    Science.gov (United States)

    Prado-Alvarez, Maria; Couraleau, Yann; Chollet, Bruno; Tourbiez, Delphine; Arzul, Isabelle

    2015-10-01

    Bonamia exitiosa is an intracellular parasite (Haplosporidia) that has been associated with mass mortalities in oyster populations in the Southern hemisphere. This parasite was recently detected in the Northern hemisphere including Europe. Some representatives of the Bonamia genus have not been well categorized yet due to the lack of genomic information. In the present work, we have applied Whole-Genome Amplification (WGA) technique in order to characterize the actin gene in the unculturable protozoan B. exitiosa. This is the first protein coding gene described in this species. Molecular analysis revealed that B. exitiosa actin is more similar to Bonamia ostreae actin gene-1. Actin phylogeny placed the Bonamia sp. infected oysters in the same clade where the herein described B. exitiosa actin resolved, offering novel information about the classification of the genus. Our results showed that WGA methodology is a promising and valuable technique to be applied to unculturable protozoans whose genomic material is limited.

  11. Amplification of a cytochrome P450 gene is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae.

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    Alin M Puinean

    2010-06-01

    Full Text Available The aphid Myzus persicae is a globally significant crop pest that has evolved high levels of resistance to almost all classes of insecticide. To date, the neonicotinoids, an economically important class of insecticides that target nicotinic acetylcholine receptors (nAChRs, have remained an effective control measure; however, recent reports of resistance in M. persicae represent a threat to the long-term efficacy of this chemical class. In this study, the mechanisms underlying resistance to the neonicotinoid insecticides were investigated using biological, biochemical, and genomic approaches. Bioassays on a resistant M. persicae clone (5191A suggested that P450-mediated detoxification plays a primary role in resistance, although additional mechanism(s may also contribute. Microarray analysis, using an array populated with probes corresponding to all known detoxification genes in M. persicae, revealed constitutive over-expression (22-fold of a single P450 gene (CYP6CY3; and quantitative PCR showed that the over-expression is due, at least in part, to gene amplification. This is the first report of a P450 gene amplification event associated with insecticide resistance in an agriculturally important insect pest. The microarray analysis also showed over-expression of several gene sequences that encode cuticular proteins (2-16-fold, and artificial feeding assays and in vivo penetration assays using radiolabeled insecticide provided direct evidence of a role for reduced cuticular penetration in neonicotinoid resistance. Conversely, receptor radioligand binding studies and nucleotide sequencing of nAChR subunit genes suggest that target-site changes are unlikely to contribute to resistance to neonicotinoid insecticides in M. persicae.

  12. Quantification and assessment of viability of Cryptococcus neoformans by LightCycler amplification of capsule gene mRNA.

    Science.gov (United States)

    Amjad, Muhammad; Kfoury, Najla; Cha, Raymond; Mobarak, Reem

    2004-12-01

    Cryptococcus neoformans is an opportunistic fungal pathogen. It infects the central nervous system causing meningitis, which is fatal if untreated, especially in AIDS and immunosuppressed patients. In this study a method of quantification and assessment of viability of C. neoformans by LightCycler RT-PCR amplification of the capsule gene mRNA is established. The sequence of primers and probes were derived from C. neoformans capsular CAP10 gene mRNA (GenBank accession number AF144574), and were species specific. Agarose gel electrophoresis analysis of LightCycler RT-PCR product showed a single band of 223 bp in length. In order to develop an internal control a 223 bp exon fragment of capsule mRNA was cloned in the pCR2.1 plasmid vector and RNA was generated by in vitro transcription. To determine the sensitivity of the assay, serial dilutions of in vitro-transcribed RNA with known concentrations and copy numbers, and serially diluted cultures of viable and nonviable C. neoformans were used. Under optimal conditions as little as 0.472 fg of capsule mRNA could be detected, corresponding to 1-10 c.f.u. ml(-1) of the sample. No amplification was observed from up to 10(5) heat/UV radiation-killed yeast cells and RNA of other bacterial and fungal pathogens and human genomic DNA or RNA. The amplification of capsule mRNA represents a sensitive, specific and quantitative means of detection of viable C. neoformans in clinical specimens and can be useful in the evaluation of the therapeutic efficacy of antifungal drugs in the treatment of C. neoformans meningitis.

  13. Altered regulation of DNA ligase IV activity by aberrant promoter DNA methylation and gene amplification in colorectal cancer.

    Science.gov (United States)

    Kuhmann, Christine; Li, Carmen; Kloor, Matthias; Salou, Mariam; Weigel, Christoph; Schmidt, Christopher R; Ng, Linda W C; Tsui, Wendy W Y; Leung, Suet Y; Yuen, Siu T; Becker, Natalia; Weichenhan, Dieter; Plass, Christoph; Schmezer, Peter; Chan, Tsun L; Popanda, Odilia

    2014-04-15

    Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.

  14. Amplification and expression of a salivary gland DNA puff gene in the prothoracic gland of Bradysia hygida (Diptera: Sciaridae).

    Science.gov (United States)

    Candido-Silva, Juliana Aparecida; Machado, Maiaro Cabral Rosa; Hartfelder, Klaus Hartmann; de Almeida, Jorge Cury; Paçó-Larson, Maria Luisa; Monesi, Nadia

    2015-03-01

    The DNA puff BhC4-1 gene, located in DNA puff C4 of Bradysiahygida, is amplified and expressed in the salivary gland at the end of the fourth larval instar as a late response to the increase in 20-hydroxyecdysone titer that triggers metamorphosis. Functional studies revealed that the mechanisms that regulate BhC4-1 expression in the salivary gland are conserved in transgenic Drosophila. These studies also led to the identification of a cis-regulatory module that drives developmentally regulated expression of BhC4-1-lacZ in the prothoracic gland cells of the ring gland, a compound organ which in Drosophila results from the fusion of the prothoracic glands, the corpus allatum and the corpus cardiacum. Here we have investigated the occurrence of BhC4-1 expression in B. hygida prothoracic glands. We report the identification of the B. hygida prothoracic gland and demonstrate that it releases ecdysone. Using RT-qPCR, western blots and immunolocalization experiments, we demonstrate that the BhC4-1 mRNA and the BhC4-1 protein are both expressed in the B. hygida prothoracic glands at the same time that DNA puff C4 is formed in the salivary gland. We also show that BhC4-1 is concomitantly amplified 4.8-fold in the prothoracic gland and 23-fold in the salivary gland. Our results reveal the occurrence of stage specific expression of a DNA puff gene in the prothoracic glands of B. hygida, and extend previous studies that have shown that DNA puff genes expression is not restricted to the salivary gland. In addition, the description of stage specific gene amplification in the prothoracic glands of B. hygida constitutes the first demonstration that gene amplification in Diptera might occur concomitantly in two different tissues in the same developmental stage.

  15. Rapid and specific detection of the thermostable direct hemolysin gene in Vibrio parahaemolyticus by loop-mediated isothermal amplification.

    Science.gov (United States)

    Nemoto, Jiro; Sugawara, Chiyo; Akahane, Kenji; Hashimoto, Keiji; Kojima, Tadashi; Ikedo, Masanari; Konuma, Hirotaka; Hara-Kudo, Yukiko

    2009-04-01

    Several investigators have reported that thermostable direct hemolysin (TDH) and TDH-related hemolysin are important virulence factors of Vibrio parahaemolyticus, but it has been difficult to detect these factors rapidly in seafood and other environmental samples. A novel nucleic acid amplification method, termed the loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity and rapidity under isothermal conditions, was applied. In this study, we designed tdh gene-specific LAMP primers for detection of TDH-producing V. parahaemolyticus. The specificity of this assay was evaluated with 32 strains of TDH-producing V. parahaemolyticus, one strain of TDH-producing Grimontia hollisae, 10 strains of TDH-nonproducing V. parahaemolyticus, and 94 strains of TDH-nonproducing bacteria, and the sensitivity was high enough to detect one cell per test. Moreover, to investigate the detection of TDH-producing V. parahaemolyticus in oysters, the LAMP assay was performed with enrichment culture in alkaline peptone water of oyster samples inoculated with TDH-producing V. parahaemolyticus and TDH-nonproducing V. parahaemolyticus and V. alginolyticus after enrichment in alkaline peptone water. These results suggest that the LAMP assay targeting tdh gene has high sensitivity and specificity and is useful to detect TDH-producing V. parahaemolyticus in oyster after enrichment.

  16. Rapid and sensitive detection of Listeria ivanovii by loop-mediated isothermal amplification of the smcL gene.

    Directory of Open Access Journals (Sweden)

    Yi Wang

    Full Text Available A loop-mediated isothermal amplification (LAMP assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six primers targeting eight regions on the species-specific gene smcL. The LAMP assay could be completed within 1 h at 64°C in a water bath. Amplification products were directly observed by the Loopamp Fluorescent Detection Reagent (FD or detected by agarose gel electrophoresis. Moreover, the LAMP reactions were also detected by real-time measurement of turbidity. The exclusivity of 77 non-L. ivanovii and the inclusivity of 17 L. ivanovii were both 100% in the assay. Sensitivity of the LAMP assay was 250 fg DNA and 16 CFU per reaction for detection of L. ivanovii in pure cultures and simulated human stool. The LAMP assay was 10 and 100-fold more sensitive than quantitative PCR (qPCR and conventional PCR assays,respectively. When applied to human stool samples spiked with low level (8 CFU/0.5 g of L. ivanovii strains, the new LAMP assay described here achieved positive detection after 6 hours enrichment. In conclusion, the new LAMP assay in this study can be used as a valuable, rapid and sensitive detection tool for the detection of L. ivanovii in field, medical and veterinary laboratories.

  17. Loop-mediated amplification of the Clavibacter michiganensis subsp. michiganensis micA gene is highly specific.

    Science.gov (United States)

    Yasuhara-Bell, Jarred; Kubota, Ryo; Jenkins, Daniel M; Alvarez, Anne M

    2013-12-01

    Loop-mediated amplification (LAMP) was used to specifically identify Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial canker of tomato. LAMP primers were developed to detect micA, a chromosomally stable gene that encodes a type II lantibiotic, michiganin A, which inhibits growth of other C. michiganensis subspecies. In all, 409 bacterial strains (351 C. michiganensis subsp. michiganensis and 58 non-C. michiganensis subsp. michiganensis) from a worldwide collection were tested with LAMP to determine its specificity. LAMP results were compared with genetic profiles established using polymerase chain reaction (PCR) amplification of seven genes (dnaA, ppaJ, pat-1, chpC, tomA, ppaA, and ppaC). C. michiganensis subsp. michiganensis strains produced eight distinct profiles. The LAMP reaction identified all C. michiganensis subsp. michiganensis strains and discriminated them from other C. michiganensis subspecies and non-Clavibacter bacteria. LAMP has advantages over immunodiagnostic and other molecular detection methods because of its specificity and isothermal nature, which allows for easy field application. The LAMP reaction is also not affected by as many inhibitors as PCR. This diagnostic tool has potential to provide an easy, one-step test for rapid identification of C. michiganensis subsp. michiganensis.

  18. Development of a rapid and specific loop-mediated isothermal amplification detection method that targets Marek's disease virus meq gene.

    Science.gov (United States)

    Wei, Xiuying; Shi, Xingming; Zhao, Yan; Zhang, Jing; Wang, Mei; Liu, Changjun; Cui, Hongyu; Hu, Shunlei; Quan, Yanming; Chen, Hongyan; Wang, Yunfeng

    2012-08-01

    A rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) method was developed and evaluated for the detection of Marek's disease virus (MDV) by amplification of conserved MDV meq gene sequences. LAMP is an innovative technique that allows the rapid detection of targeted nucleic acid sequences under isothermal conditions without the need for complex instrumentation. In this study, meq gene sequences were amplified successfully from different MDV strains by LAMP within 60min and no cross-reactivity was observed in a panel of related viruses that were associated with diseases of chickens. The detection limit of LAMP was 3.2 copies/million cells compared with 320 copies/million cells required for conventional PCR. Positive detection rates were assessed using either LAMP or PCR by examination of feather follicles that were collected from chickens infected experimentally with either strain J-1 (n=20) or strain Md5 (n=17), In addition to these samples, three isolates that were suspected to have been infected in the clinic were also tested. Results showed that the positive detection rate for LAMP was 95% (38/40), compared with 87.5% (35/40) and 90% (38/40) for strains J-1 and Md5 by PCR, respectively. These results indicated that the LAMP assay was more sensitive, rapid and specific than conventional PCR for the detection of MDV. This easy-to-perform technique will be useful for the detection of MDV and will aid in the establishment of disease control protocols.

  19. A titratable two-step transcriptional amplification strategy for targeted gene therapy based on ligand-induced intramolecular folding of a mutant human estrogen receptor

    DEFF Research Database (Denmark)

    Chen, Ian Y; Paulmurugan, Ramasamy; Nielsen, Carsten Haagen

    2014-01-01

    PURPOSE: The efficacy and safety of cardiac gene therapy depend critically on the level and the distribution of therapeutic gene expression following vector administration. We aimed to develop a titratable two-step transcriptional amplification (tTSTA) vector strategy, which allows modulation of ...

  20. AMPLIFICATION OF AZOSPIRILLUM SP. JG3 GLPD GENE FRAGMENT USING DEGENERATE PRIMERS GENERATED BY WEB-BASED TOOLS

    Directory of Open Access Journals (Sweden)

    Stalis Norma Ethica

    2013-12-01

    Full Text Available Primaclade and In Silico web-based tools were used as a strategy to obtain the correct-size PCR amplicon targeting a fragment of gene encoding glycerol-3-phosphate dehydrogenase (glpD of Azospirillum sp. JG3. The bacterial strains are soil, Gram-negative PGPR (Plant-Growth Promoting Rhizobacteria isolated from an agricultural land in Purwokerto, Central Java, Indonesia, which have ability to produce several commercial enzymes. The aim is to obtain a pair of reliable degenerate primers from a limited number of glpD sequences from other Azospirilla retrieved in GenBank using bioinformatics approach. We demonstrated degenerate primer design that led to successful PCR amplification corresponding to the targeted DNA fragment. Homology analysis showed that the obtained DNA fragment is 61% and 99% similar to sn-glycerol-3-phosphate dehydrogenase genes of Azospirillum brasilense and Stenotrophomonas maltophili respectively.

  1. Adaptive gene amplification as an intermediate step in the expansion of virus host range.

    Directory of Open Access Journals (Sweden)

    Greg Brennan

    2014-03-01

    Full Text Available The majority of recently emerging infectious diseases in humans is due to cross-species pathogen transmissions from animals. To establish a productive infection in new host species, viruses must overcome barriers to replication mediated by diverse and rapidly evolving host restriction factors such as protein kinase R (PKR. Many viral antagonists of these restriction factors are species specific. For example, the rhesus cytomegalovirus PKR antagonist, RhTRS1, inhibits PKR in some African green monkey (AGM cells, but does not inhibit human or rhesus macaque PKR. To model the evolutionary changes necessary for cross-species transmission, we generated a recombinant vaccinia virus that expresses RhTRS1 in a strain that lacks PKR inhibitors E3L and K3L (VVΔEΔK+RhTRS1. Serially passaging VVΔEΔK+RhTRS1 in minimally-permissive AGM cells increased viral replication 10- to 100-fold. Notably, adaptation in these AGM cells also improved virus replication 1000- to 10,000-fold in human and rhesus cells. Genetic analyses including deep sequencing revealed amplification of the rhtrs1 locus in the adapted viruses. Supplying additional rhtrs1 in trans confirmed that amplification alone was sufficient to improve VVΔEΔK+RhTRS1 replication. Viruses with amplified rhtrs1 completely blocked AGM PKR, but only partially blocked human PKR, consistent with the replication properties of these viruses in AGM and human cells. Finally, in contrast to AGM-adapted viruses, which could be serially propagated in human cells, VVΔEΔK+RhTRS1 yielded no progeny virus after only three passages in human cells. Thus, rhtrs1 amplification in a minimally permissive intermediate host was a necessary step, enabling expansion of the virus range to previously nonpermissive hosts. These data support the hypothesis that amplification of a weak viral antagonist may be a general evolutionary mechanism to permit replication in otherwise resistant host species, providing a molecular foothold

  2. From amplification to gene in thyroid cancer: A high-resolution mapped bacterial-artificial-chromosome resource for cancer chromosome aberrations guides gene discovery after comparative genome hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, X.N.; Gonsky, R.; Korenberg, J.R. [UCLA School of Medicine, Los Angeles, CA (United States). Cedars-Sinai Research Inst.; Knauf, J.A.; Fagin, J.A. [Univ. of Cincinnati, OH (United States). Div. of Endocrinology/Metabolism; Wang, M.; Lai, E.H. [Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Pharmacology; Chissoe, S. [Washington Univ. School of Medicine, St. Louis, MO (United States). Genome Sequencing

    1998-08-01

    Chromosome rearrangements associated with neoplasms provide a rich resource for definition of the pathways of tumorigenesis. The power of comparative genome hybridization (CGH) to identify novel genes depends on the existence of suitable markers, which are lacking throughout most of the genome. The authors now report a general approach that translates CGH data into higher-resolution genomic-clone data that are then used to define the genes located in aneuploid regions. They used CGH to study 33 thyroid-tumor DNAs and two tumor-cell-line DNAs. The results revealed amplifications of chromosome band 2p21, with less-intense amplification on 2p13, 19q13.1, and 1p36 and with least-intense amplification on 1p34, 1q42, 5q31, 5q33-34, 9q32-34, and 14q32. To define the 2p21 region amplified, a dense array of 373 FISH-mapped chromosome 2 bacterial artificial chromosomes (BACs) was constructed, and 87 of these were hybridized to a tumor-cell line. Four BACs carried genomic DNA that was amplified in these cells. The maximum amplified region was narrowed to 3--6 Mb by multicolor FISH with the flanking BACs, and the minimum amplicon size was defined by a contig of 420 kb. Sequence analysis of the amplified BAC 1D9 revealed a fragment of the gene, encoding protein kinase C epsilon (PKC{epsilon}), that was then shown to be amplified and rearranged in tumor cells. In summary, CGH combined with a dense mapped resource of BACs and large-scale sequencing has led directly to the definition of PKC{epsilon} as a previously unmapped candidate gene involved in thyroid tumorigenesis.

  3. Amplification of the cap20 pathogenicity gene and genetic characterization using different markers molecular in Colletotrichum gloeosporioides isolates

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    Danielli Barreto Maciel

    2010-12-01

    Full Text Available Studies were performed to analyze the genetic characterization using RFLP-ITS and Intron (primer EI1 markers and the amplification of the cap20 pathogenicity gene by PCR in Colletotrichum gloeosporioides isolates of different hosts plant. The genetic variability was accessed using RFLP-ITS and Intron markers and grouping by UPGMA method. Primers to cap20 gene were constructed using selected sequences of the GenBank (National Center of Biotechnology Information, http://www.ncbi.nlm.nih.gov with the Primer 3 program. The dendrograms analysis showed that the RFLP-ITS marker was more informative to separate the Colletotrichum sp, and that primer EI1 demonstrated greater genetic diversity. The amplification of the DNA of the Colletotrichum isolates to the cap20 gene with primers P1 and P2 indicated that this gene could present variations into C. gloeosporioides related with the host, and also that it was present in other Colletotrichum sp.Estudos foram realizados para analisar a caracterização genética usando marcadores de RFLP-ITS e ISSP e a amplicação do gene de patogenicidade cap20 por PCR em isolados de Colletotrichum gloeosporioides de diferentes hospedeiros. Primers para o gene cap20 foram construídos a partir de seqüências selecionadas do GenBank (National Center of Biotechnology Information, http://www.ncbi.nlm.nih.gov com o programa Primer 3. A análise dos dendrogramas revelou que o marcador RFLP-ITS foi mais informativo em separar as espécies de Colletotrichum, e que o primer EI1 evidenciou maior diversidade genética. A amplificação do DNA dos isolados de Colletotrichum para o gene cap20 com os primers P1 e P2 indicou que este gene pode apresentar variações dentro de C. gloeosporioides relacionada ao hospedeiro, e que também está presente em outras espécies de Colletotrichum.

  4. Improved gene amplification by cell-cycle engineering combined with the Cre-loxP system in Chinese hamster ovary cells.

    Science.gov (United States)

    Matsuyama, Rima; Tsutsui, Tomomi; Lee, Kyoung Ho; Onitsuka, Masayoshi; Omasa, Takeshi

    2015-12-01

    The dihydrofolate reductase gene amplification system is widely used in Chinese hamster ovary (CHO) cells for the industrial production of therapeutic proteins. To enhance the efficiency of conventional gene amplification systems, we previously presented a novel method using cell-cycle checkpoint engineering. Here, we constructed high-producing and stable cells by the conditional expression of mutant cell division cycle 25 homolog B (CDC25B) using the Cre-loxP system. A bispecific antibody-producing CHO DG44-derived cell line was transfected with floxed mutant CDC25B. After inducing gene amplification in the presence of 250 nM methotrexate, mutant CDC25B sequence was removed by Cre recombinase protein expression. Overexpression of the floxed mutant CDC25B significantly enhanced the efficiency of transgene amplification and productivity. Moreover, the specific production rate of the isolated clone CHO Cre-1 and Cre-2 were approximately 11-fold and 15-fold higher than that of mock-transfected clone CHO Mock-S. Chromosomal aneuploidy was increased by mutant CDC25B overexpression, but Cre-1 and Cre-2 did not show any changes in chromosome number during long-term cultivation, as is the case with CHO Mock-S. Our results suggest that high-producing and stable cells can be constructed by conditionally controlling a cell-cycle checkpoint integrated in conventional gene amplification systems.

  5. HER2/neu (c-erbB-2) gene amplification and protein expression are rare in uterine cervical neoplasia: a tissue microarray study of 814 archival specimens

    DEFF Research Database (Denmark)

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Stephen;

    2009-01-01

    Published studies have reported widely variable incidence of HER2/neu (c-erbB-2) protein expression and HER2/neu (c-erbB-2) gene amplification in cervical carcinoma. We examined tissue microarrays (TMAs) constructed from 814 formaldehyde-fixed paraffin-embedded archival specimens of cervical...... and invasive cervical carcinoma specimens. When present, Her-2/neu positivity is more commonly seen in higher grades of cervical dysplasia and in carcinoma. However, this large TMA study shows that HER2/neu oncoprotein expression and HER2/neu gene amplification overall are uncommon events in cervical neoplasia...... intraepithelial neoplasia (CIN)1 (n = 262), CIN2 (n = 230), CIN3 (n = 186) and invasive carcinoma (n = 136), for HER2/neu protein expression by immunohistochemistry (IHC) and for HER2/neu gene amplification by chromogenic in situ hybridization (CISH). We found moderate or strong immunohistochemical positivity...

  6. Gene expression profiling of RNA extracted from FFPE tissues: NuGEN technologies' whole-transcriptome amplification system.

    Science.gov (United States)

    Turner, Leah; Heath, Joe Don; Kurn, Nurith

    2011-01-01

    Gene expression profiling of RNA isolated from formalin fixed, paraffin-embedded (FFPE) tissue samples has been historically challenging. Yet FFPE samples are sought-after because of the in-depth retrospective records typically associated with them rendering these samples a valuable resource for translational medicine studies. Extensive degradation, chemical modifications, and cross-linking have made it difficult to isolate RNA of sufficient quality required for large-scale gene expression profiling studies. NuGEN Technologies' WT-Ovation™ FFPE System linearly amplifies RNA from FFPE samples through a robust and simple whole-transcriptome approach using as little as 50 ng total RNA isolated from FFPE samples. The amplified material may be labeled with validated kits and/or protocols from NuGEN for analysis on any of the major gene expression microarray platforms, including: Affymetrix, Agilent, and Illumina gene expression arrays. Results compare well with those obtained using RNA from fresh-frozen samples. RNA quality from FFPE samples varies significantly and neither sample age nor sample size analysis via gel electrophoresis or the Agilent Bioanalyzer system accurately predict materials suitable for amplification. Therefore, NuGEN has validated a correlative qPCR-based analytical method for the RNA derived from FFPE samples which effectively predicts array results. The NuGEN approach enables fast and successful analysis of samples previously thought to be too degraded for gene expression analysis.

  7. Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitive proteins

    Science.gov (United States)

    Tang, Nicholas C.; Chilkoti, Ashutosh

    2016-04-01

    Most genes are synthesized using seamless assembly methods that rely on the polymerase chain reaction (PCR). However, PCR of genes encoding repetitive proteins either fails or generates nonspecific products. Motivated by the need to efficiently generate new protein polymers through high-throughput gene synthesis, here we report a codon-scrambling algorithm that enables the PCR-based gene synthesis of repetitive proteins by exploiting the codon redundancy of amino acids and finding the least-repetitive synonymous gene sequence. We also show that the codon-scrambling problem is analogous to the well-known travelling salesman problem, and obtain an exact solution to it by using De Bruijn graphs and a modern mixed integer linear programme solver. As experimental proof of the utility of this approach, we use it to optimize the synthetic genes for 19 repetitive proteins, and show that the gene fragments are amenable to PCR-based gene assembly and recombinant expression.

  8. Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitive proteins.

    Science.gov (United States)

    Tang, Nicholas C; Chilkoti, Ashutosh

    2016-04-01

    Most genes are synthesized using seamless assembly methods that rely on the polymerase chain reaction (PCR). However, PCR of genes encoding repetitive proteins either fails or generates nonspecific products. Motivated by the need to efficiently generate new protein polymers through high-throughput gene synthesis, here we report a codon-scrambling algorithm that enables the PCR-based gene synthesis of repetitive proteins by exploiting the codon redundancy of amino acids and finding the least-repetitive synonymous gene sequence. We also show that the codon-scrambling problem is analogous to the well-known travelling salesman problem, and obtain an exact solution to it by using De Bruijn graphs and a modern mixed integer linear programme solver. As experimental proof of the utility of this approach, we use it to optimize the synthetic genes for 19 repetitive proteins, and show that the gene fragments are amenable to PCR-based gene assembly and recombinant expression.

  9. Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosomes 3. Evidence for the role of chromosome rearrangement in gene amplification

    Energy Technology Data Exchange (ETDEWEB)

    Stallings, R.L.; Munk, A.C.; Longmire, J.L.; Hildebrand, C.E.; Crawford, B.D.

    1984-12-01

    Cadmium resistant (Cd/sup r/) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cd/sup r/ variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster x mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. The authors speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cd/sup r/ hamster cell lines. 34 references, 3 figures, 1 table.

  10. Rapid screening of innate immune gene expression in zebrafish using reverse transcription - multiplex ligation-dependent probe amplification

    Directory of Open Access Journals (Sweden)

    Spaink Herman P

    2011-06-01

    Full Text Available Abstract Background With the zebrafish increasingly being used in immunology and infectious disease research, there is a need for efficient molecular tools to evaluate immune gene expression in this model species. RT-MLPA (reverse transcription - multiplex ligation-dependent probe amplification provides a sensitive and reproducible method, in which fluorescently labelled amplification products of unique lengths are produced for a defined set of target transcripts. The method employs oligonucleotide probes that anneal to adjacent sites on a target sequence and are then joined by a heat-stable ligase. Subsequently, multiplex PCR with universal primers gives rise to amplicons that can be analyzed with standard sequencing equipment and relative quantification software. Allowing the simultaneous quantification of around 40 selected markers in a one-tube assay, RT-MLPA is highly useful for high-throughput screening applications. Findings We employed a dual-colour RT-MLPA probe design for chemical synthesis of probe pairs for 34 genes involved in Toll-like receptor signalling, transcriptional activation of the immune response, cytokine and chemokine production, and antimicrobial defence. In addition, six probe pairs were included for reference genes unaffected by infections in zebrafish. First, we established assay conditions for adult zebrafish infected with different strains of Mycobacterium marinum causing acute and chronic disease. Addition of competitor oligonucleotides was required to achieve peak heights in a similar range for genes with different expression levels. For subsequent analysis of embryonic samples it was necessary to adjust the amounts of competitor oligonucleotides, as the expression levels of several genes differed to a large extent between adult and embryonic tissues. Assay conditions established for one-day-old Salmonella typhimurium-infected embryos could be transferred without further adjustment to five-day-old M. marinum

  11. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.

    Science.gov (United States)

    Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu

    2014-10-01

    Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings.

  12. Molecular detection of field isolates of Turkey Eimeria by polymerase chain reaction amplification of the cytochrome c oxidase I gene.

    Science.gov (United States)

    Rathinam, T; Gadde, U; Chapman, H D

    2015-07-01

    Oocysts of Eimeria spp. were isolated from litter samples obtained from 30 commercial turkey farms. Genomic DNA was extracted from clean oocysts, and polymerase chain amplification of the species-specific cytochrome c oxidase subunit I (COI) gene was performed for five species of turkey Eimeria. The species tested were Eimeria adenoeides, Eimeria meleagrimitis, Eimeria meleagridis, Eimeria dispersa, and Eimeria gallopavonis. All DNA samples were positive for E. meleagrimitis, nine were positive for E. adenoeides, two were positive for E. dispersa, and none for E. meleagridis and E. gallopavonis. E. meleagrimitis occurred as a single species in 21 (70 %) of the farms while 9 (30 %) farms had a mixed species with E. meleagrimitis and E. adenoeides and 2 (7 %) were triple positive with E. meleagrimitis, E. adenoeides, and E. dispersa. This is the first account of the field prevalence of turkey Eimeria species using molecular methods.

  13. Loop-Mediated Isothermal Amplification Assay Targeting the MOMP Gene for Rapid Detection of Chlamydia psittaci Abortus Strain

    Directory of Open Access Journals (Sweden)

    Guo-Zhen Lin, Fu-Ying Zheng, Ji-Zhang Zhou, Guang-Hua Wang, Xiao-An Cao, Xiao-Wei Gong and Chang-Qing Qiu*

    2012-05-01

    Full Text Available For rapid detection of the Chlamydia psittaci abortus strain, a loop-mediated isothermal amplification (LAMP assay was developed and evaluated in this study. The primers for the LAMP assay were designed on the basis of the main outer membrane protein (MOMP gene sequence of C. psittaci. Analysis showed that the assay could detect the abortus strain of C. psittaci with adequate specificity. The sensitivity of the test was the same as that of the nested-conventional PCR and higher than that of chick embryo isolation. Testing of 153 samples indicated that the LAMP assay could detect the genome of the C. psittaci abortus strain effectively in clinical samples. This assay is a useful tool for rapid diagnosis of C. psittaci infection in sheep, swine and cattle.

  14. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella.

    Science.gov (United States)

    Rahn, K; De Grandis, S A; Clarke, R C; McEwen, S A; Galán, J E; Ginocchio, C; Curtiss, R; Gyles, C L

    1992-08-01

    Amplification of nucleotide sequences within the invA gene of Salmonella typhimurium was evaluated as a means of detecting Salmonella. A collection of 630 strains of Salmonella comprising over 100 serovars, including the 20 most prevalent serovars isolated from animals and humans in Canada, was examined. Controls consisted of 142 non-Salmonella strains comprising 21 genera of bacteria. Cultures were screened by inoculating a single colony of bacteria directly into a polymerase chain reaction (PCR) mixture which contained a pair of primers specific for the invA gene. The specific PCR product was a 284 bp DNA fragment which was visualized in 2% agarose gels. With the exception of two S. litchfield and two S. senftenberg strains, all Salmonella strains were detected. In contrast, none of the non-Salmonella strains yielded the specific amplification product. Non-specific amplification of a few non-Salmonella strains resulted in a product that was distinctly different in size from the specific 284 bp product. Specificity of amplification was further confirmed by demonstration of hybridization of a 32P-labelled invA gene fragment only to the specific 284 bp product. The detection of 99.4% of Salmonella strains tested and the failure to specifically amplify DNA from non-Salmonella strains confirm that the invA gene contains sequences unique to Salmonella and demonstrate that this gene is a suitable PCR target, with potential diagnostic applications.

  15. Characterisation of amplification patterns and target genes at chromosome 11q13 in CCND1-amplified sporadic and familial breast tumours.

    Science.gov (United States)

    Holm, Karolina; Staaf, Johan; Jönsson, Göran; Vallon-Christersson, Johan; Gunnarsson, Haukur; Arason, Adalgeir; Magnusson, Linda; Barkardottir, Rosa B; Hegardt, Cecilia; Ringnér, Markus; Borg, Ake

    2012-06-01

    Amplification of chromosomal region 11q13, containing the cell cycle regulatory gene CCND1, is frequently found in breast cancer and other malignancies. It is associated with the favourable oestrogen receptor (ER)-positive breast tumour phenotype, but also with poor prognosis and treatment failure. 11q13 spans almost 14 Mb and contains more than 200 genes and is affected by various patterns of copy number gains, suggesting complex mechanisms and selective pressure during tumour progression. In this study, we used 32 k tiling BAC array CGH to analyse 94 CCND1-amplified breast tumours from sporadic, hereditary, and familial breast cancers to fine map chromosome 11q13. A set containing 281 CCND1-non-amplified breast tumours was used for comparisons. We used gene expression data to further validate the functional effect of gene amplification. We identified six core regions covering 11q13.1-q14.1 that were amplified in different combinations. The major core contained CCND1, whereas two cores were found proximal of CCND1 and three distal. The majority of the CCND1-amplified tumours were ER-positive and classified as luminal B. Furthermore, we found that CCND1 amplification is associated with a more aggressive phenotype within histological grade 2 tumours and luminal A subtype tumours. Amplification was equally prevalent in familial and sporadic tumours, but strikingly rare in BRCA1- and BRCA2-mutated tumours. We conclude that 11q13 includes many potential target genes in addition to CCND1.

  16. Relationship of Amplification and Expression of the C-MYC Gene with Survival among Gastric Cancer Patients.

    Science.gov (United States)

    Khaleghian, Malihea; Shakoori, Abbas; Razavi, Amirnader Emami; Azimi, Cyrus

    2015-01-01

    During the past decades, the incidence and mortality rate of stomach cancer has demonstrated a great decrease in the world, but it is still one of the most common and fatal cancers especially among men worldwide, including Iran. The MYC proto-oncogene, which is located at 8q24.1, regulates 15% of genes and is activated in 20% of all human tumors. MYC amplification and overexpression of its protein product has been reported in 15-30% of gastric neoplasias. The aim of this investigation was to find the relative efficacy of CISH (chromogenic in situ hybridization) or IHC (immunohistochemistry) in diagnosis and prognosis of gastric cancer, as well as the relationship of amplification and expression of C-MYC gene with patient survival. In this cross-sectional study, 102 samples of gastric cancer were collected from patients who had undergone primary surgical resection at the Cancer Institute Hospital, Tehran University of Medical Sciences, from July 2009 to March 2014. All samples were randomly selected from those who were diagnosed with gastric adenocarcinomas. CISH and IHC methods were performed on all of them. Patients were classified into two groups. The first consisted of stage I and II cases, and the second of stage III and IV. Survival tests for both groups was carried out with referrnce to CISH test reults. Group II (stage III and IV) with CISH+ featured lower survival than those with CISH- (p=0.233), but group I (stage I and II) patients demonstrated no significant variation with CISH+ or CISH- (p=0.630). Kaplan-Meier for both groups was carried out with IHC test findings and showed similar results. This data revealed that both diffuse and intestinal types of gastric cancer occurred significantly more in men than women. Our data also showed that CISH+ patients (43%) were more frequent in comparison with IHC+ patients (14.7%). For planning treatment of gastric cancer patients, by focusing on expanding tumors, which is the greatest concern of the surgeons and

  17. A phase II study of Epirubicin in oxaliplatin-resistant patients with metastatic colorectal cancer and TOP2A gene amplification

    DEFF Research Database (Denmark)

    Tarpgaard, Line S.; Qvortrup, Camilla; Nygård, Sune Boris

    2016-01-01

    UNLABELLED: ᅟ: The overall purpose of this study is to provide proof of concept for introducing the anthracycline epirubicin as an effective, biomarker-guided treatment for metastatic colorectal cancer (mCRC) patients who are refractory to treatment with oxaliplatin-based chemotherapy and have TOP2......A gene amplification in their tumor cells. BACKGROUND: Epirubicin is an anthracycline that targets DNA topoisomerase 2-α enzyme encoded by the TOP2A gene. It is used for treatment of several malignancies, but currently not in CRC. TOP2A gene amplifications predict improved efficacy of epirubicin...... in patients with breast cancer and thus could be an alternative option for patients with CRC and amplified TOP2A gene. We have previously analysed the frequency of TOP2A gene aberrations in CRC and found that 46.6 % of these tumors had TOP2A copy gain and 2.0 % had loss of TOP2A when compared to adjacent...

  18. Determination of variants in the 3'-region of the Tyrosinase gene requires locus specific amplification.

    NARCIS (Netherlands)

    Chaki, M.; Mukhopadhyay, A.; Ray, K.

    2005-01-01

    Mutations in the Tyrosinase gene (TYR, 11q14-q21) cause oculocutaneous albinism type 1 (OCA1). The 3'-region of the TYR shows 98.55% sequence identity with a pseudogene, known as Tyrosinase-Like Gene (TYRL, 11p11.2-cen). A large number of publicly available nucleotide variants of TYR in this region

  19. A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes.

    Science.gov (United States)

    Shi, Xianzong; Jarvis, Donald L

    2006-09-15

    Rapid amplification of cDNA ends (RACE) is widely used to determine the 5'- and 3'-terminal nucleotide sequences of genes. Many different RACE methods have been developed to meet various requirements, but none addresses the difficult problems that arise when trying to isolate the ends of extremely guanine plus cytosine (GC)-rich genes. In this study, we found that we were unable to isolate the correct 5' or 3' end of an insect gene, which appeared to include extremely GC-rich sequences, using current RACE methods. Thus, we developed a new RACE method that can be used for this purpose. This new method entails first-strand cDNA synthesis at 70 degrees C with Thermo-X reverse transcriptase in the presence of homoectoine, followed by a polymerase chain reaction with 98 degrees C denaturation steps and Phusion DNA polymerase in the presence of 1M betaine and 5% dimethyl sulfoxide (DMSO). The use of these conditions yielded 5'- and 3'-RACE products that were approximately 80% GC over 213 and 162bp, respectively, and included shorter internal regions of 82 to 89% GC.

  20. Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin.

    Science.gov (United States)

    Libermann, T A; Nusbaum, H R; Razon, N; Kris, R; Lax, I; Soreq, H; Whittle, N; Waterfield, M D; Ullrich, A; Schlessinger, J

    Epidermal growth factor (EGF), through interaction with specific cell surface receptors, generates a pleiotropic response that, by a poorly defined mechanism, can induce proliferation of target cells. Subversion of the EGF mitogenic signal through expression of a truncated receptor may be involved in transformation by the avian erythroblastosis virus (AEV) oncogene v-erb-B, suggesting that similar EGF receptor defects may be found in human neoplasias. Overexpression of EGF receptors has been reported on the epidermoid carcinoma cell line A431, in various primary brain tumours and in squamous carcinomas. In A431 cells the receptor gene is amplified. Here we show that 4 of 10 primary brain tumours of glial origin which express levels of EGF receptors that are higher than normal also have amplified EGF receptor genes. Amplified receptor genes were not detected in the other brain tumours examined. Further analysis of EGF receptor defects may show that such altered expression and amplification is a particular feature of certain human tumours.

  1. Genomic amplification of the human telomerase gene (hTERC associated with human papillomavirus is related to the progression of uterine cervical dysplasia to invasive cancer

    Directory of Open Access Journals (Sweden)

    Liu Hongqian

    2012-10-01

    Full Text Available Abstract Background Human papillomavirus (HPV infection plays an etiological role in the development of cervical dysplasia and cancer. Amplification of human telomerase gene (hTERC and over expression of telomerase were found to be associated with cervical tumorigenesis. This study was performed to analyze genomic amplification of hTERC gene, telomerase activity in association with HPV infection in different stages of cervical intraepithelial neoplasia (CIN and cervical cancer. We were studying the role of hTERC in the progression of uterine cervical dysplasia to invasive cancer, and proposed an adjunct method for cervical cancer screening. Methods Exfoliated cervical cells were collected from 114 patients with non neoplastic lesion (NNL, n=27, cervical intraepithelial neoplasia (CIN1, n=26, CIN2, n=16, CIN3, n=24 and cervical carcinoma (CA, n=21, and analyzed for amplification of hTERC with two-color fluorescence in situ hybridization (FISH probe and HPV-DNA with Hybrid Capture 2. From these patients, 53 were taken biopsy to analyze telomerase activity by telomeric repeat amplification protocol (TRAP and expression of human telomerase reverse transcriptase (hTERT, with immunohistochemistry (IHC. All biopsies were clinically confirmed by phathologists. Results Amplification of hTERC was significantly associated with the histologic diagnoses (p Conclusions hTERC ampliffication can be detected with FISH technique on exfoliated cervical cells. Amplification of hTERC and HPV infection are associated with more progressive CIN3 and CA. The testing of hTERC amplification might be a supplementary to cytology screening and HPV test, especially high-risk patients. Virtual slides The virtual slide(s for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1857134686755648.

  2. Detection of denitrification genes by in situ rolling circle amplification - fluorescence in situ hybridization (in situ RCA-FISH) to link metabolic potential with identity inside bacterial cells

    DEFF Research Database (Denmark)

    Hoshino, Tatsuhiko; Schramm, Andreas

    2010-01-01

    A target-primed in situ rolling circle amplification (in situ RCA) protocol was developed for detection of single-copy genes inside bacterial cells and optimized with Pseudomonas stutzeri, targeting nitrite and nitrous oxide reductase genes (nirS and nosZ). Two padlock probes were designed per gene......). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA-FISH was demonstrated on activated sludge by the differential detection of two types of nirS-defined denitrifiers; one of them was identified...

  3. Amplification of the telomerase RNA component gene as a new genetic marker for disease progression and prognosis in esophageal squamous cell carcinoma.

    Science.gov (United States)

    Wang, J-D; Ma, J; Wang, F-Y; Peng, L-B; Wang, X; Shi, S-S; Ma, H-H; Lu, Z-F; Lu, G-M; Zhou, X-J

    2013-01-01

    Amplification of the human telomerase RNA component (TERC) gene was found in esophageal squamous cell carcinoma (ESCC). However, its roles in the progression and prognosis of ESCC have not been well understood. The amplification of TERC in normal mucosa, low-grade and high-grade intraepithelial neoplasia, and invasive ESCC samples were evaluated using a fluorescence in situ hybridization assay. The amplification of TERC invariably occurred in high-grade intraepithelial neoplasia and invasive ESCC, partially occurred in low-grade intraepithelial neoplasia specimens, and seldom occurred in normal mucosa. The average signal ratio of TERC to chromosome 3 centromere-specific probe (TERC/CSP3) was 1.00 ± 0.01 (average ± standard deviation) in normal mucosas, 1.01 ± 0.08 in low-grade intraepithelial neoplasias, 1.39 ± 0.26 in high-grade intraepithelial neoplasias, and 1.56 ± 0.41 in invasive ESCC. High TERC/CSP3 ratio was positively associated with lymph node metastasis (P = 0.005) and advanced tumor stage (P = 0.045). Patients with high amplification of TERC had poor survival (P = 0.01). The amplification of TERC could be used as a new genomic marker for disease progression and prognosis of ESCC. The amplified TERC gene may be a potential therapeutic target for ESCC.

  4. A novel gene amplification causes upregulation of the PatAB ABC transporter and fluoroquinolone resistance in Streptococcus pneumoniae.

    Science.gov (United States)

    Baylay, Alison J; Ivens, Alasdair; Piddock, Laura J V

    2015-01-01

    Overexpression of the ABC transporter genes patA and patB confers efflux-mediated fluoroquinolone resistance in Streptococcus pneumoniae and is also linked to pneumococcal stress responses. Although upregulation of patAB has been observed in many laboratory mutants and clinical isolates, the regulatory mechanisms controlling expression of these genes are unknown. In this study, we aimed to identify the cause of high-level constitutive overexpression of patAB in M184, a multidrug-resistant mutant of S. pneumoniae R6. Using a whole-genome transformation and sequencing approach, we identified a novel duplication of a 9.2-kb region of the M184 genome which included the patAB genes. This duplication did not affect growth and was semistable with a low segregation rate. The expression levels of patAB in M184 were much higher than those that could be fully explained by doubling of the gene dosage alone, and inactivation of the first copy of patA had no effect on multidrug resistance. Using a green fluorescent protein reporter system, increased patAB expression was ascribed to transcriptional read-through from a tRNA gene upstream of the second copy of patAB. This is the first report of a large genomic duplication causing antibiotic resistance in S. pneumoniae and also of a genomic duplication causing antibiotic resistance by a promoter switching mechanism.

  5. Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Nakano, Ryuichi; Nakano, Akiyo; Ishii, Yoshikazu; Ubagai, Tsuneyuki; Kikuchi-Ueda, Takane; Kikuchi, Hirotoshi; Tansho-Nagakawa, Shigeru; Kamoshida, Go; Mu, Xiaoqin; Ono, Yasuo

    2015-03-01

    Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of β-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory.

  6. CAT基因突变热点区域的PCR扩增方法%Optimization of PCR Amplification Parameters for Mutational Hotspots in the Human Catalase Gene

    Institute of Scientific and Technical Information of China (English)

    李毅; 赵华; 赵红宇; 章锦才

    2009-01-01

    Objective To study the speciality and sensitivity of PCR for mutational hotspots in the human catalase gene. Methods Blood samples were taken from volunteers for genomic DNA preparation. Polymerase chain reaction (PCR) amplification of sixteen gene segments encompassing mutational hotspots in the human catalase was carried out. Touchdown and Hot-start PCR was imple-mented to improve the efficiency of gene amplification. Results High and constant amplification effi-ciency is obtained using touchdown and hot-start PCR. Conclusion The stable and reproducible PCR amplification parameters for mutational hotspots in the human catalase gene were optimized.%目的 探讨过氧化氢酶基因突变热点区域PCR扩增方法 ,提高PCR反应的特异性和灵敏度,有助于快速检测CAT基因相关疾病.方法 从人静脉血液标本提取人血液基因组DNA,设计引物扩增特定的CAT基因片段,联合应用热启动PCR和降落PCR技术.结果 建立了重复性好,分辨率高的PCR反应体系.结论 建立了适用于CAT基因突变热点区域的PCR反应体系,有助于快速检测CAT基因相关痰病.

  7. Regulation of Pattern Formation and Gene Amplification During Drosophila Oogenesis by the miR-318 microRNA

    DEFF Research Database (Denmark)

    Ge, Wanzhong; Deng, Qiannan; Guo, Ting

    2015-01-01

    and laid eggs with abnormal morphology. Removal of miR-318 disrupted the dorsal-anterior follicle cell patterning, resulting in abnormal dorsal appendages. miR-318 mutant females also produced thin and fragile eggshells, due to impaired chorion gene amplification. We provide evidence that the ecdysone...

  8. Amplification of chromosome 2:Lq22.3 harboring trefoil factor family genes in liver fluke related cholangiocarcinoma is associated with poor prognosis

    Institute of Scientific and Technical Information of China (English)

    Kanuengnuch Muenphon; Temduang Limpaiboon; Patcharee Jearanaikoon; Chawalit Pairojkul; Banchob Sripa; Vajarabhongsa Bhudhisawasdi

    2006-01-01

    AIM: To determine allelic imbalance on chromosomal region 21q22-qter including trefoil factor family genes (TFF) in cholangiocarcinoma (CCA) patients and analyze the correlation between allelic imbalances and clinicopathological parameters.METHODS: Quantitative PCR amplification was performed on four microsatellite markers and trefoil factor family genes (TFF1, TFF2, and TFF3) using a standard curve and SYBR Green Ⅰ dye method. The relative copy number was determined by DNA copy number of tested locus to reference locus. The relative copy number was interpreted as deletion or amplification by comparison with normal reference range. Associations between allelic imbalance and clinicopathological parameters of CCA patients were evaluated by x2-tests.Kaplan-Meier method was used to analyze survival.RESULTS: The frequencies of amplification at D21S1890,D21S1893, and TFF3 were 32.5%, 30.0%, and 28.7%,respectively. Patients who had amplification at regions covering D21S1893, D21S1890, and TFF showed poor prognosis, whereas patients who had deletion showed favorable prognosis (mean: 51.7 wk vs 124.82 wk,P = 0.012). Multivariate Cox regression analysis revealed that amplification of D21S1893, D21S1890 and TFF,blood vessel invasion, and staging were associated with poor prognosis.CONCLUSION: D21S1893-D21S1890 region may harbor candidate genes especially TFF and serine protease family, which might be involved in tumor invasion and metastasis contributing to poor survival. The amplification in this region may be used as a prognostic marker in the treatment of CCA patients.

  9. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

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    Chiemi Noguchi

    Full Text Available Amplification of the dihydrofolate reductase gene (Dhfr by methotrexate (Mtx exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR and a matrix attachment region (MAR, which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  10. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

    Science.gov (United States)

    Noguchi, Chiemi; Araki, Yoshio; Miki, Daisuke; Shimizu, Noriaki

    2012-01-01

    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  11. Modified PCR methods for 3' end amplification from serial analysis of gene expression (SAGE) tags.

    Science.gov (United States)

    Xu, Wang-Jie; Wang, Zhao-Xia; Qiao, Zhong-Dong

    2009-05-01

    Serial analysis of gene expression (SAGE) is a powerful technique to study gene expression at the genome level. However, a disadvantage of the shortness of SAGE tags is that it prevents further study of SAGE library data, thus limiting extensive application of the SAGE method in gene expression studies. However, this problem can be solved by extension of the SAGE tags to 3' cDNAs. Therefore, several methods based on PCR have been developed to generate a 3' longer fragment cDNA corresponding to a SAGE tag. The list of modified methods is extensive, and includes rapid RT-PCR analysis of unknown SAGE tags (RAST-PCR), generation of longer cDNA fragments from SAGE tags for gene identification (GLGI), a high-throughput GLGI procedure, reverse SAGE (rSAGE), two-step analysis of unknown SAGE tags (TSAT-PCR), etc. These procedures are constantly being updated because they have characteristics and advantages that can be shared. Development of these methods has promoted the widespread use of the SAGE technique, and has accelerated the speed of studies of large-scale gene expression.

  12. Gene identification using exon amplification on human chromosome 18q21: implications for bipolar disorder.

    Science.gov (United States)

    Chen, H; Huo, Y; Patel, S; Zhu, X; Swift-Scanlan, T; Reeves, R H; DePaulo, R; Ross, C A; McInnis, M G

    2000-09-01

    We previously reported linkage between bipolar disorder and a region on human chromosome (HC) 18q21. To identify genes in this region, exon trapping was performed on cosmids isolated from an HC18-specific cosmid library (LL18NC02) using 47 sequence tagged site (STS) markers from 18q21 as hybridization probes. A total of 285 unique sequences (exons) were obtained from 850 sequenced clones. Homology searching of the databases using NCBI's BLAST algorithms revealed that 31 exons have identity to known genes and/or ESTs, seven are identical to regions of finished genomic sequences in the 18q21 region, 20 have significant similarity (>30% sequence identity) to genes from human and/or other species, 19 were repetitive sequences, and 208 sequences (72%) are novel. Seventy per cent of the trapped sequences were predicted to be derived from genes using library screening and RT-PCR analyses. This represents an initial stage in characterizing genes in a susceptibility region for further study in bipolar disorder or other diseases that map to this region.

  13. Ancestral genes can control the ability of horizontally acquired loci to confer new traits.

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    H Deborah Chen

    2011-07-01

    Full Text Available Horizontally acquired genes typically function as autonomous units conferring new abilities when introduced into different species. However, we reasoned that proteins preexisting in an organism might constrain the functionality of a horizontally acquired gene product if it operates on an ancestral pathway. Here, we determine how the horizontally acquired pmrD gene product activates the ancestral PmrA/PmrB two-component system in Salmonella enterica but not in the closely related bacterium Escherichia coli. The Salmonella PmrD protein binds to the phosphorylated PmrA protein (PmrA-P, protecting it from dephosphorylation by the PmrB protein. This results in transcription of PmrA-dependent genes, including those conferring polymyxin B resistance. We now report that the E. coli PmrD protein can activate the PmrA/PmrB system in Salmonella even though it cannot do it in E. coli, suggesting that these two species differ in an additional component controlling PmrA-P levels. We establish that the E. coli PmrB displays higher phosphatase activity towards PmrA-P than the Salmonella PmrB, and we identified a PmrB subdomain responsible for this property. Replacement of the E. coli pmrB gene with the Salmonella homolog was sufficient to render E. coli resistant to polymyxin B under PmrD-inducing conditions. Our findings provide a singular example whereby quantitative differences in the biochemical activities of orthologous ancestral proteins dictate the ability of a horizontally acquired gene product to confer species-specific traits. And they suggest that horizontally acquired genes can potentiate selection at ancestral loci.

  14. Rapid, highly sensitive and highly specific gene detection by combining enzymatic amplification and DNA chip detection simultaneously

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    Koji Hashimoto

    2016-05-01

    Full Text Available We have developed a novel gene detection method based on the loop-mediated isothermal amplification (LAMP reaction and the DNA dissociation reaction on the same DNA chip surface to achieve a lower detection limit, broader dynamic range and faster detection time than are attainable with a conventional DNA chip. Both FAM- and thiol-labeled DNA probe bound to the complementary sequence accompanying Dabcyl was immobilized on the gold surface via Au/thiol bond. The LAMP reaction was carried out on the DNA probe fixed gold surface. At first, Dabcyl molecules quenched the FAM fluorescence. According to the LAMP reaction, the complementary sequence with Dabcyl was competitively reacted with the amplified targeted sequence. As a result, the FAM fluorescence increased owing to dissociation of the complementary sequence from the DNA probe. The simultaneous reaction of LAMP and DNA chip detection was achieved, and 103 copies of the targeted gene were detected within an hour by measuring fluorescence intensity of the DNA probe.

  15. Multiplex ligation-dependent probe amplification for rapid detection of deletions and duplications in the dystrophin gene

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective:Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked disorders caused by mutations in the dystrophin gene. The majority of recognized mutations are copy number changes of individual exons. The objective of the present study was to assess the multiplex ligation-dependent probe amplification (MLPA) effects of detection of gene mutations. Methods: Samples of 20 control males and 80 males and their mothers referred to our diagnostic facility on the clinical suspicion of DMD or BMD were tested by MLPA and multiplex PCR. Results: The mean DQs for all peak of 20 control male samples was 1.02 (range from 0.83 to 1.21) by MLPA. Deletions or duplications were identified in 6 out of 31 families that had been previously tested as negative by multiplex PCR. One case of complex rearrangement involving a duplication of two regions: dupEX3-9 and dupEX 17-41 were found by MLPA. Conclusions: MLPA is a highly sensitive method and rapid alternative to multiplex PCR for detection of DMD and BMD.

  16. Mtp-40 and alpha antigen gene fragment amplification for the detection of Mycobacterium tuberculosis in Colombian clinical specimens

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    Rosalba Alfonso

    2002-12-01

    Full Text Available In this study, the use of Mtp-40 and alpha antigen polymerase chain reaction (PCR amplification fragments for the precise tuberculosis (TB diagnosis was evaluated. One hundred and ninety two different samples were obtained from 113 patients with suspected TB. Mtp-40 and alpha antigen protein genes were amplified by the PCR technique and compared to both the "gold standard" (culture test, as well as the clinical parameters (including a clinical record and X-ray film exam in 113 patients. Thirty-eight of the 113 patients had a presumptive clinical diagnosis of TB; 74% being detected by PCR technique, 58% by culture and 44% by direct microscopic visualization. Weconclude that it is possible to use PCR as a suitable technique for the detection of any mycobacteria by means of the alpha antigen product, or the specific infection of Mycobacterium tuberculosis by means of the mtp-40 gene. This might be a good supporting tool in difficult clinical TB diagnosis and pauci-bacillary cases.

  17. Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum.

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    Rok Lenarčič

    Full Text Available The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes.

  18. Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples

    Energy Technology Data Exchange (ETDEWEB)

    Braker, G.; Witzel, K.P. [Max-Planck-Inst. fuer Limnologie, Ploen (Germany); Fesefeldt, A. [Univ. Kiel (Germany). Inst. fuer Allgemeine Mikrobiologie

    1998-10-01

    A system was developed for the detection of denitrifying bacteria by the application of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128); the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained wit h nondenitrifying bacteria or with strains of the other nir type. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying the generally amplifying primer combination for each nir gene developed in this study to total DNA preparations from aquatic habitats.

  19. Amplification and sequencing of a sulfur-rich 10kd prolamin gene from rice seeds

    Institute of Scientific and Technical Information of China (English)

    YUJingjuan; AOGuangming

    1994-01-01

    Nutritious value of seed storage protein is low due to deficiency in essential amino acid contents. Cereals are mainly deficient in lysine and legumes in sulfur-containing amino acids ( methionine and cysteine). So far, several sufur-rich seed protein genes have been isolated and the essential amino acid contents of seed proteins were increased in transgenic tobacco and Brassica napus.

  20. Gene Amplification Is A Mechanism For Rapid Weed Evolution To Herbicide Resistance

    Science.gov (United States)

    The herbicide glyphosate became widely used in the U.S. and other parts of the world following the introduction of glyphosate-resistant crops. These crops were created by introduction of a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the herbicide target site. Increased use of ...

  1. Amplification, expression, and packaging of a foreign gene by giardiavirus in Giardia lamblia.

    Science.gov (United States)

    Yu, D C; Wang, A L; Wang, C C

    1996-12-01

    Giardia lamblia is an intestinal protozoan parasite and one of the earliest eukaryotic divergents. The trophozoite multiplies via asexual binary fission and lacks all natural means of lateral gene transfer. A system is developed here for long-term expression of a foreign gene in this organism by exploiting recombinant virions derived from the giardiavirus (GLV), a double-stranded RNA virus that infects many Giardia isolates. An in vitro transcript of the cloned GLV cDNA, comprising the firefly luciferase-encoding region flanked by 5' and 3' fragments of GLV positive-strand RNA, was electroporated into GLV-infected trophozoites. Luciferase activity in electroporated cells peaked on day 2 at levels 6 orders of magnitude above background. Expression of this foreign gene remained at 80% of its peak level after 30 days in the absence of selective pressure. The chimeric RNA was replicated as double-stranded RNA and packaged into virus-like particles. The recombinant virions were partially purified from the wild-type helper virus by CsCl equilibrium density-gradient centrifugation and used to superinfect Giardia trophozoites. At multiplicities of infection of 100 or higher, these chimeric virions were able to initiate new rounds of expression of luciferase activity in the superinfected cells. Thus, the engineered virion can be successfully used to introduce and efficiently express a heterologous gene in this eukaryotic microorganism.

  2. Novel subtractive transcription-based amplification of mRNA (STAR method and its application in search of rare and differentially expressed genes in AD brains

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    Walker P Roy

    2006-11-01

    Full Text Available Abstract Background Alzheimer's disease (AD is a complex disorder that involves multiple biological processes. Many genes implicated in these processes may be present in low abundance in the human brain. DNA microarray analysis identifies changed genes that are expressed at high or moderate levels. Complementary to this approach, we described here a novel technology designed specifically to isolate rare and novel genes previously undetectable by other methods. We have used this method to identify differentially expressed genes in brains affected by AD. Our method, termed Subtractive Transcription-based Amplification of mRNA (STAR, is a combination of subtractive RNA/DNA hybridization and RNA amplification, which allows the removal of non-differentially expressed transcripts and the linear amplification of the differentially expressed genes. Results Using the STAR technology we have identified over 800 differentially expressed sequences in AD brains, both up- and down- regulated, compared to age-matched controls. Over 55% of the sequences represent genes of unknown function and roughly half of them were novel and rare discoveries in the human brain. The expression changes of nearly 80 unique genes were further confirmed by qRT-PCR and the association of additional genes with AD and/or neurodegeneration was established using an in-house literature mining tool (LitMiner. Conclusion The STAR process significantly amplifies unique and rare sequences relative to abundant housekeeping genes and, as a consequence, identifies genes not previously linked to AD. This method also offers new opportunities to study the subtle changes in gene expression that potentially contribute to the development and/or progression of AD.

  3. A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

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    Yanagihara Kazuyoshi

    2009-06-01

    Full Text Available Abstract Background Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS, which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. Methods DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. Results DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20 of gastric cancer cell lines (8–18-fold amplification and 4.7% (4/86 of primary gastric tumors (8–50-fold amplification. KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild

  4. Amplification of the flgE gene provides evidence for the existence of a Brazilian borreliosis

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    Elenice Mantovani

    2012-06-01

    Full Text Available INTRODUCTION: The symptoms of Brazilian borreliosis resemble the clinical manifestations of Lyme disease (LD. However, there are differences between the two in terms of epidemiological and laboratory findings. Primers usually employed to diagnose LD have failed to detect Borrelia strains in Brazil. OBJECTIVE: We aimed to identify the Brazilian Borrelia using a conserved gene that synthesizes the flagellar hook (flgE of Borrelia burgdorferi sensu lato. METHOD: Three patients presenting with erythema migrans and positive epidemiological histories were recruited for the study. Blood samples were collected, and the DNA was extracted by commercial kits. RESULTS: The gene flgE was amplified from DNA of all selected patients. Upon sequencing, these positive samples revealed 99% homology to B. burgdorferi flgE. CONCLUSION: These results support the existence of borreliosis in Brazil. However, it is unclear whether this borreliosis is caused by a genetically modified B. burgdorferi sensu stricto or by a new species of Borrelia spp.

  5. Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Crawford, B.D.; Enger, M.D.; Griffith, B.B.; Griffith, J.K.; Hanners, J.L.; Longmire, J.L.; Munk, A.C.; Stallings, R.L.; Tesmer, J.G.; Walters, R.A.; Hildebrand, C.E.

    1985-02-01

    The authors describe here the derivation, characterization, and use of clonal cadmium-resistance (Cd/sup r) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylaminde gel electrophoresis, the authors showed that the stable Cd/sup r/ phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 M, coordinate amplifications of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which the authors have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cd/sup r/ variants expressing abundant MT and their corresponding Cd/sup s/ parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expressions of the isometallothioneins. 59 references, 8 figures.

  6. A phase II study of Epirubicin in oxaliplatin-resistant patients with metastatic colorectal cancer and TOP2A gene amplification.

    Science.gov (United States)

    Tarpgaard, Line S; Qvortrup, Camilla; Nygård, Sune B; Nielsen, Signe L; Andersen, Diana R; Jensen, Niels Frank; Stenvang, Jan; Detlefsen, Sönke; Brünner, Nils; Pfeiffer, Per

    2016-02-11

    The overall purpose of this study is to provide proof of concept for introducing the anthracycline epirubicin as an effective, biomarker-guided treatment for metastatic colorectal cancer (mCRC) patients who are refractory to treatment with oxaliplatin-based chemotherapy and have TOP2A gene amplification in their tumor cells. Epirubicin is an anthracycline that targets DNA topoisomerase 2-α enzyme encoded by the TOP2A gene. It is used for treatment of several malignancies, but currently not in CRC. TOP2A gene amplifications predict improved efficacy of epirubicin in patients with breast cancer and thus could be an alternative option for patients with CRC and amplified TOP2A gene. We have previously analysed the frequency of TOP2A gene aberrations in CRC and found that 46.6% of these tumors had TOP2A copy gain and 2.0% had loss of TOP2A when compared to adjacent normal tissue. The TOP2A gene is located on chromosome 17 and when the TOP2A/CEN-17 ratio was applied to identify tumors with gene loss or amplifications, 10.5% had a ratio ≥ 1.5 consistent with gene amplification and 2.6% had a ratio ≤ 0.8 suggesting gene deletions. Based on these observations and the knowledge gained from treatment of breast cancer patients, we have initiated a prospective clinical, phase II protocol using epirubicin (90 mg/m2 iv q 3 weeks) in mCRC patients, who are refractory to treatment with oxaliplatin. The study is an open label, single arm, phase II study, investigating the efficacy of epirubicin in patients with oxaliplatin refractory mCRC and with a cancer cell TOP2A/CEN-17 ratio ≥ 1.5. TOP2A gene amplification measured by fluorescence in situ hybridization. A total of 25 evaluable patients (15 + 10 in two steps) will be included (Simon's two-stage minimax design). Every nine weeks, response is measured by computed tomography imaging and evaluated according to RECIST 1.1. The primary end-point of the study is progression-free survival. Eudract no. 2013-001648-79.

  7. Genes confer similar robustness to environmental, stochastic, and genetic perturbations in yeast.

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    Ben Lehner

    Full Text Available Gene inactivation often has little or no apparent consequence for the phenotype of an organism. This property-enetic (or mutational robustness-is pervasive, and has important implications for disease and evolution, but is not well understood. Dating back to at least Waddington, it has been suggested that mutational robustness may be related to the requirement to withstand environmental or stochastic perturbations. Here I show that global quantitative data from yeast are largely consistent with this idea. Considering the effects of mutations in all nonessential genes shows that genes that confer robustness to environmental or stochastic change also buffer the effects of genetic change, and with similar efficacy. This means that selection during evolution for environmental or stochastic robustness (also referred to as canalization may frequently have the side effect of increasing genetic robustness. A dynamic environment may therefore promote the evolution of phenotypic complexity. It also means that "hub" genes in genetic interaction (synthetic lethal networks are generally genes that confer environmental resilience and phenotypic stability.

  8. Double-hairpin molecular-beacon-based amplification detection for gene diagnosis linked to cancer.

    Science.gov (United States)

    Xu, Huo; Zhang, Rongbo; Li, Feng; Zhou, Yingying; Peng, Ting; Wang, Xuedong; Shen, Zhifa

    2016-09-01

    A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related KRAS gene detection based on the one-to-two stoichiometry. During target DNA detection, DHMB can execute signal transduction even if no any exogenous element is involved. Unlike the conventional molecular beacon based on the one-to-one interaction, one target DNA not only hybridizes with one DHMB and opens its hairpin but also promotes the interaction between two DHMBs, causing the separation of two fluorophores from quenchers. This leads to an enhanced fluorescence signal. As a result, the target KRAS gene is able to be detected within a wide dynamic range from 0.05 to 200 nM with the detection limit of 50 pM, indicating a dramatic improvement compared with traditional molecular beacons. Moreover, the point mutations existing in target DNAs can be easily screened. The potential application for target species in real samples was indicated by the analysis of PCR amplicons of DNAs from the DNA extracted from SW620 cell. Besides becoming a promising candidate probe for molecular biology research and clinical diagnosis of genetic diseases, the DHMB is expected to provide a significant insight into the design of DNA probe-based homogenous sensing systems. Graphical Abstract A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related gene KRAS detection based on the one-to-two stoichiometry. Without the help of any exogenous probe, the point mutation is easily screened, and the target DNA can be quantified down to 50 pM, indicating a dramatic improvement compared with traditional molecular beacons.

  9. Amplifications of NCOA3 gene in colorectal cancers in a Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhi Li; Zheng-Yu Fang; Yi Ding; Wan-Tong Yao; Yang Yang; Zhong-Qing Zhu; Wen Wang; Qin-xian Zhang

    2012-01-01

    AIM:To investigate the copy number variation of NAC03 gene in colorectal cancer (CRC) and its correlation with tumor progression.METHODS:A total of 142 samples of case-matched CRC tissues and adjacent normal tissues were obtained from patients undergoing bowel resection.Quantitative real-time polymerase chain reaction method was used to investigate the copy number variations of NCOA3 as well as gene expression in the collected tissues.RESULTS:Copy number gains of NCOA3 were detected in 39 CRC samples (27.5%) and were correlated with tumor progression (x2 =6.42,P =0.0112).Moreover,there was a positive correlation between copy number gain and mRNA over-expression of NCOA3 in CRCs (P =0.0023).Expression level of NCOA3 mRNA was also enhanced in the CRC samples with unaltered copy numbers (3.85 ± 1.23 vs 2.71 ± 0.64,P < 0.01).CONCLUSION:Sporadic colorectal cancers exhibit different mechanisms of NCOA3 regulation.

  10. Rapid and sensitive detection of Plesiomonas shigelloides by loop-mediated isothermal amplification of the hugA gene.

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    Shuang Meng

    Full Text Available Plesiomonas shigelloides is one of the causative agents of human gastroenteritis, with increasing number of reports describing such infections in recent years. In this study, the hugA gene was chosen as the target to design loop-mediated isothermal amplification (LAMP assays for the rapid, specific, and sensitive detection of P. shigelloides. The performance of the assay with reference plasmids and spiked human stools as samples was evaluated and compared with those of quantitative PCR (qPCR. No false-positive results were observed for the 32 non-P. shigelloides strains used to evaluate assay specificity. The limit of detection for P. shigelloides was approximately 20 copies per reaction in reference plasmids and 5×10(3 CFU per gram in spiked human stool, which were more sensitive than the results of qPCR. When applied in human stool samples spiked with 2 low levels of P. shigelloides, the LAMP assays achieved accurate detection after 6-h enrichment. In conclusion, the LAMP assay developed in this study is a valuable method for rapid, cost-effective, and simple detection of P. shigelloides in basic clinical and field laboratories in the rural areas of China.

  11. Human identification from forensic materials by amplification of a human-specific sequence in the myoglobin gene.

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    Ono T

    2001-06-01

    Full Text Available We developed a method for human identification of forensic biological materials by PCR-based detection of a human-specific sequence in exon 3 of the myoglobin gene. This human-specific DNA sequence was deduced from differences in the amino acid sequences of myoglobins between humans and other animal species. The new method enabled amplification of the target DNA fragment from 30 samples of human DNA, and the amplified sequences were identical with that already reported. Using this method, we were able to distinguish human samples from those of 21 kinds of animals: the crab-eating monkey, horse, cow, sheep, goat, pig, wild boar, dog, raccoon dog, cat, rabbit, guinea pig, hamster, rat, mouse, whale, chicken, pigeon, turtle, frog, and tuna. However, we were unable to distinguish between human and gorilla samples. This method enabled us to detect the target sequence from 25 pg of human DNA, and the target DNA fragment from blood stored at 37 degrees C for 6 months, and from bloodstains heated at 150 degrees C for 4 h or stored at room temperature for 26 years. Herein we also report a practical application of the method for human identification of a bone fragment.

  12. Gene changes in Duchenne muscular dystrophy: Comparison of multiplex PCR and multiplex ligation-dependent probe amplification techniques

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    Kohli Sudha

    2010-01-01

    Full Text Available Background: Duchenne muscular dystrophy (DMD is a common X-linked recessive neuromuscular disorder, affecting 1 in 3,500 live male births. About 65% of cases are caused by deletions; ~5% to 8%, by duplication; and the remaining, by point mutations of the dystrophin gene. The frequency of complex rearrangements (double-deletion and non-contiguous duplications is reported to be 4%. Aim: In this study, we examined the usefulness of multiplex ligation-dependent probe amplification (MLPA for screening of deletion and duplication mutations in a group of DMD/ BMD (Becker muscular dystrophy patients from India. Patients and Methods: We analyzed 180 patients referred from all over India, by both multiplex PCR technique (22 exons and MLPA (all 79 exons. Results and Conclusion: By multiplex PCR, deletions were detected in 90 (50% patients. MLPA studies in these cases detected 3 additional deletions, 16 (8.9% duplications and 2 point mutations. MLPA is useful to verify absence of deletions/ duplications in all 79 exons. This sets the stage to look for point mutations using RNA- or DNA-based tests because of the availability of the drug PTC124. Also, the extent of the deletions and duplications could be more accurately defined by MLPA. The delineation of the precise extent of deletion helps in deciding whether exon-skipping technique would be useful as therapy.

  13. A protein-tagging system for signal amplification in gene expression and fluorescence imaging.

    Science.gov (United States)

    Tanenbaum, Marvin E; Gilbert, Luke A; Qi, Lei S; Weissman, Jonathan S; Vale, Ronald D

    2014-10-23

    Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.

  14. Sex identification based on AMEL gene PCR amplification from blue sheep (Pseudois nayaur) fecal DNA samples.

    Science.gov (United States)

    Liu, X; Yang, Y Y; Wang, X M; Liu, Z S; Wang, Z H; Ding, Y Z

    2015-08-07

    The use of noninvasive genetic sampling to identify the sex of wild animals is an extremely valuable and important tool in molecular ecology and wildlife conservation. Sex determination using the amelogenin gene has been conducted in many species because only a single pair of primers is required to amplify both X- and Y-linked alleles. However, this method has not been used in field research with the feces of wildlife. In this study, we applied this method to 222 fecal samples from wild blue sheep (Pseudois nayaur) using amelogenin primers (SE47/SE48) after testing the effectiveness of sex determination using tissue samples and fecal samples from blue sheep of known sex. We found this method to be highly reliable (80.2%) for blue sheep. Amelogenin can be used to identify the sex of wild animals using fecal samples.

  15. KIT amplification and gene mutations in acral/mucosal melanoma in Korea.

    Science.gov (United States)

    Yun, Jina; Lee, Jeeyun; Jang, Jiryeon; Lee, Eui Jin; Jang, Kee Taek; Kim, Jung Han; Kim, Kyoung-Mee

    2011-06-01

    Mucosal and acral melanomas have demonstrated different genetic alterations and biological behavior compared with more common cutaneous melanomas. It was recently reported that gain-of-function KIT mutations and/or copy number increases are more common in mucosal and acral melanomas. Thus, we studied the frequency and pattern of KIT aberrations in mucosal and acral melanomas in Korea. We analyzed 97 patients who were pathologically confirmed with mucosal or acral melanoma between 1997 and 2010 at Samsung Medical Center. Of the 97 melanoma patients, 92 were screened for mutations in KIT exons 11, 13, 17, and 18, BRAF and NRAS genes. KIT copy number was assessed by quantitative, real-time PCR. Of the 97 patients, 55 (56.7%) were mucosal, 40 (41.2%) were acral melanoma, and two were of unknown primary origin. Among seven cases with KIT mutation, five (60.0%) occurred in exon 11, one (20.0%) in exon 17, and one (20.0%) in exon 13. Point mutations were the most common, resulting in substitutions in exon 11 (K558R, T574A, L576P, and V559A), exon 13 (N655K), and exon 17 (N822K). A novel Thr574Ala (c.1720A>G) KIT mutation, which has not been reported in melanoma or other tumor types, was identified in one genital melanoma case. Of the 97 mucosal or acral melanoma specimens, 49 were tested for KIT gene copy number changes using quantitative PCR. Increased KIT copy number was identified in 15 patients: seven (40%) of 20 acral melanomas and eight (31%) of 26 mucosal melanomas. Our study implicates that a significant proportion of acral and mucosal melanomas have KIT mutations in Asian population.

  16. Species identification of Asini Corii Collas (donkey glue) by PCR amplification of cytochrome b gene.

    Science.gov (United States)

    Kumeta, Yukie; Maruyama, Takuro; Asama, Hiroshi; Yamamoto, Yutaka; Hakamatsuka, Takashi; Goda, Yukihiro

    2014-01-01

    Asini Corii Collas (ACC; donkey glue) is a crude drug used to promote hematopoiesis and arrest bleeding. Because adulteration of the drug with substances from other animals such as horses, cattle, and pigs has been found, we examined PCR methods based on the sequence of the cytochrome b gene for source species identification. Two strategies for extracting DNA from ACC were compared, and the ion-exchange resin procedure was revealed to be more suitable than the silica-based one. Using DNA extracted from ACC by the ion-exchange resin procedure, PCR methods for species-specific detection of donkey, horse, cattle, and pig substances were established. When these species-specific PCR methods were applied to ACC, amplicons were obtained only by the donkey-specific PCR. Cattle-specific PCR detected as little as 0.1% admixture of cattle glue in the ACC. These results suggest that the species-specific PCR methods established in this study would be useful for simple and easy detection of adulteration of ACC.

  17. Involvement of facultative apomixis in inheritance of EPSPS gene amplification in glyphosate-resistant Amaranthus palmeri.

    Science.gov (United States)

    Ribeiro, Daniela N; Pan, Zhiqiang; Duke, Stephen O; Nandula, Vijay K; Baldwin, Brian S; Shaw, David R; Dayan, Franck E

    2014-01-01

    The inheritance of glyphosate resistance in two Amaranthus palmeri populations (R1 and R2) was examined in reciprocal crosses (RC) and second reciprocal crosses (2RC) between glyphosate-resistant (R) and -susceptible (S) parents of this dioecious species. R populations and Female-R × Male-S crosses contain higher 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copy numbers than the S population. EPSPS expression, EPSPS enzyme activity, EPSPS protein quantity, and level of resistance to glyphosate correlated positively with genomic EPSPS relative copy number. Transfer of resistance was more influenced by the female than the male parent in spite of the fact that the multiple copies of EPSPS are amplified in the nuclear genome. This led us to hypothesize that this perplexing pattern of inheritance may result from apomictic seed production in A. palmeri. We confirmed that reproductively isolated R and S female plants produced seeds, indicating that A. palmeri can produce seeds both sexually and apomictically (facultative apomixis). This apomictic trait accounts for the low copy number inheritance in the Female-S × Male-R offsprings. Apomixis may also enhance the stability of the glyphosate resistance trait in the R populations in the absence of reproductive partners.

  18. Identification of endotrypanum species from a sloth, a squirrel and Lutzomyia sandflies in ecuador by PCR amplification and sequencing of the mini-exon gene.

    Science.gov (United States)

    Katakura, Ken; Mimori, Tatsuyuki; Furuya, Masato; Uezato, Hiroshi; Nonaka, Shigeo; Okamoto, Munehiro; Gomez L, Eduardo A; Hashiguchi, Yoshihisa

    2003-05-01

    PCR amplification and nucleotide sequencing of the mini-exon gene revealed that four strains isolated from a sloth (Choloepus hoffmanni), a squirrel (Sciurus granatensis) and two sandflies (Lutzomyia hartmanni) in Ecuador were indistinguishable from Endotrypanum monterogeii. Another strain isolated from Lu. hartmanni showed the high sequence similarity to E. schaudinni. Since three of these strains have been previously identified as Leishmania (Viannia) equatorensis, the results demonstrate that L. (V.) equatorensis is genetically closely related to the genus Endotrypanum. The present study also indicates that Endotrypanum species are distributed in arboreal animals and sandflies in Ecuador, and that mini-exon gene amplification is useful for epidemiological studies of Leishmania and Endotrypanum in the New World.

  19. CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast

    DEFF Research Database (Denmark)

    Strucko, Tomas; Buron, Line Due; Jarczynska, Zofia Dorota

    2017-01-01

    Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploi...... production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside....

  20. Isothermal Amplification of Nucleic Acids.

    Science.gov (United States)

    Zhao, Yongxi; Chen, Feng; Li, Qian; Wang, Lihua; Fan, Chunhai

    2015-11-25

    Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

  1. Rapid amplification of cDNA ends (RACE) improves the PCR-based isolation of immunoglobulin variable region genes from murine and human lymphoma cells and cell lines.

    Science.gov (United States)

    Doenecke, A; Winnacker, E L; Hallek, M

    1997-10-01

    The isolation of rearranged immunoglobulin (Ig) variable region (V) genes is usually performed by PCR with consensus primers binding to conserved regions within the V sequences. However, the isolation of Ig genes by this method is hampered in 15-35% by technical difficulties, mostly mismatches of oligonucleotide primers to V sequences. In order to obtain DNA sequences from V heavy chain (VH) genes which could not be amplified with consensus primers, we used a modified PCR technique, the rapid amplification of cDNA ends (RACE) PCR in combination with new heavy chain constant region primers for the isolation of human and murine VH genes. In comparison, consensus primer PCR with different sets of previously published oligonucleotide primers was used. Both methods were applied to isolate VH genes from murine B cell lymphoma (A20 and BCL1), myeloma (NS1) and hybridoma (SP6) cell lines and from freshly isolated human chronic lymphocytic leukemia and lymphoma cells. RACE PCR allowed the amplification and subsequent cloning of the complete VH gene in all cases. In contrast, consensus primer PCR failed to isolate the VH sequence of the murine A20 cell line; this was explained by a mismatch of consensus primers with VH sequences. When both PCR methods amplified VH sequences, the DNA sequences obtained were identical. Taken together, RACE PCR represents a reliable and versatile method for the isolation of VH genes from human and murine lymphoma cells, in particular if consensus primer PCR fails.

  2. The clinical pathological characteristics and prognosis of FGFR1 gene amplification in non-small-cell lung cancer: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Xie FJ

    2016-01-01

    Full Text Available Fa-Jun Xie,1,2 Hong-Yang Lu,1,3 Qiu-Qing Zheng,3 Jing Qin,1,3 Yun Gao,3 Yi-Ping Zhang,1,3 Xun Hu,2 Wei-Min Mao3,4 1Department of Medical Oncology, Zhejiang Cancer Hospital, 2Cancer Institute (Key Laboratory for Cancer Intervention and Prevention, China National Ministry of Education, Zhejiang Provincial Key Laboratory of Molecular Biology in Medical Sciences, Second Affiliated Hospital, Zhejiang University School of Medicine,3Zhejiang Key Laboratory of Diagnosis and Treatment Technology on Thoracic Oncology (Lung and Esophagus, Hangzhou, 4Department of Thoracic Surgery, Zhejiang Cancer Hospital, Hangzhou, People’s Republic of China Abstract: FGFR1 amplification is recognized as a novel therapy target for non-small-cell lung cancer (NSCLC, especially in squamous cell carcinoma (SCC. However, the association between FGFR1 amplification and the clinicopathological characteristics of NSCLC remains controversial. We performed a meta-analysis of 17 eligible studies to examine the correlation between FGFR1 gene amplification and clinicopathological characteristics. FGFR1 amplification was closely related to these clinicopathological features, including sex (odds ratio [OR] 2.05, 95% confidence interval [CI] 1.50–2.80, smoking (OR 3.31, 95% CI 2.02–5.44, and histology (OR 3.60, 95% CI 2.82–4.59. FGFR1 amplification was associated with shorter overall survival, and no significant heterogeneity existed between studies (I2=3.8%. We should note that publication bias may partly account for these results, but our findings remained significant after the trim-and-fill method (hazard ratio 1.22, 95% CI 1.06–1.40. However, no significant correlation was found with poor disease-free survival (hazard ratio 1.43, 95% CI 0.96–2.12. In conclusion, this study showed that FGFR1 amplification was significantly associated with sex, smoking, and histology. FGFR1 amplification could be a marker of poor prognosis in NSCLC patients, especially in SCC patients

  3. Yeast functional screen to identify genes conferring salt stress tolerance in Salicornia europaea

    Directory of Open Access Journals (Sweden)

    Yoshiki eNakahara

    2015-10-01

    Full Text Available Salinity is a critical environmental factor that adversely affects crop productivity. Halophytes have evolved various mechanisms to adapt to saline environments. Salicornia europaea L. is one of the most salt-tolerant plant species. It does not have special salt-secreting structures like a salt gland or salt bladder, and is therefore a good model for studying the common mechanisms underlying plant salt tolerance. To identify candidate genes encoding key proteins in the mediation of salt tolerance in S. europaea, we performed a functional screen of a cDNA library in yeast. The library was screened for genes that allowed the yeast to grow in the presence of 1.3 M NaCl. We obtained three full-length S. europaea genes that confer salt tolerance. The genes are predicted to encode (1 a novel protein highly homologous to thaumatin-like proteins, (2 a novel coiled-coil protein of unknown function, and (3 a novel short peptide of 32 residues. Exogenous application of a synthetic peptide corresponding to the 32 residues improved salt tolerance of Arabidopsis. The approach described in this report provides a rapid assay system for large-scale screening of S. europaea genes involved in salt stress tolerance and supports the identification of genes responsible for such mechanisms. These genes may be useful candidates for improving crop salt tolerance by genetic transformation.

  4. Toll-like receptors gene polymorphisms may confer increased susceptibility to breast cancer development.

    Science.gov (United States)

    Theodoropoulos, George E; Saridakis, Vasilios; Karantanos, Theodoros; Michalopoulos, Nikolaos V; Zagouri, Flora; Kontogianni, Panagiota; Lymperi, Maria; Gazouli, Maria; Zografos, George C

    2012-08-01

    Toll-like receptor (TLR) activation may be an important event in tumor cell immune evasion. TLR2 and TLR4 gene polymorphisms have been related to increased susceptibility to cancer development in various organs. 261 patients and 480 health individuals were investigated for genotype and allelic frequencies of a 22-bp nucleotide deletion (-196 to -174del) in the promoter of TLR2 gene as well as two polymorphisms causing amino acid substitutions (Asp299Gly and Thr399Ile) in TLR4 gene. As far as (-196 to -174del) in TLR2 gene is concerned ins/del and del/del genotypes and del allele were significantly more frequent in breast cancer patients compared to healthy controls. Considering Asp299Gly replacement of TLR4 gene, Gly carriers (Asp/Gly & Gly/Gly genotype) and Gly allele were overrepresented among the breast cancer cases. The -174 to -196del of TLR2 gene and Asp299Gly of TLR4 gene polymorphisms may confer an increased susceptibility to breast cancer development.

  5. Simultaneous detection of human papillomavirus integration and c-MYC gene amplification in cervical lesions: an emerging marker for the risk to progression.

    Science.gov (United States)

    Gimenes, Fabrícia; Souza, Raquel Pantarotto; de Abreu, André Luelsdorf Pimenta; Pereira, Monalisa Wolski; Consolaro, Marcia Edilaine Lopes; da Silva, Vânia Ramos Sela

    2016-04-01

    The persistence of high-risk oncogenic human papillomavirus (HR-HPV) infection and its integration into the host genome are key steps in the induction of malignant alterations. c-MYC chromosome region is a frequent localization for HPV insertion that has been observed in chromosome band 8q24 by fluorescence in situ hybridization (FISH). We report the HPV viral integration and amplification patterns of the c-MYC gene in cytological smears with FISH as a potential biomarker for the progression of squamous intraepithelial lesions (SIL). HPV detection and genotyping by polymerase chain reaction (PCR) and FISH analysis by "Vysis Cervical FISH Probe" kit (ABBOTT Molecular Inc.) were performed in 37 cervical samples including 8 NILM, 7 ASC-US, 7 LSIL, 3 ASC-H, 7 HSIL and 5 SCC. The results show concordance between FISH and PCR techniques for HPV detection. The majority of the samples contained HR-HPV, the majority being -16 and -18 genotypes. HPV integration as determined by FISH was most frequent in high-risk lesions. The c-MYC gene amplification was found only in HPV-positive samples and was detected primarily in high-risk lesions and in cells with an integrated form of HPV. HPV integration and c-MYC gene amplification detected by FISH could be an important biomarker for use in clinical practice to determine SIL with a risk of progression.

  6. Rapid, simple and sensitive detection of Q fever by loop-mediated isothermal amplification of the htpAB gene.

    Directory of Open Access Journals (Sweden)

    Lei Pan

    Full Text Available BACKGROUND: Q fever is the most widespread zoonosis, and domestic animals are the most common sources of transmission. It is not only difficult to distinguish from other febrile diseases because of the lack of specific clinical manifestations in humans, but it is also difficult to identify the disease in C. burnetii-carrying animals because of the lack of identifiable features. Conventional serodiagnosis requires sera from the acute and convalescent stages of infection, which are unavailable at early diagnosis. Nested PCR and real-time PCR require equipment. In this study, we developed a Loop-Mediated Isothermal Amplification (LAMP assay to identify C. burnetii rapidly and sensitively. METHODS: A universal LAMP primer set was designed to detect the repeated sequence IS1111a of the htpAB gene of C. burnetii using PrimerExplorer V4 software. The sensitivity of the LAMP assay was evaluated using known quantities of recombined reference plasmids containing the targeted genes. The specificity of the developed LAMP assay was determined using 26 members of order Rickettsiae and 18 other common pathogens. The utility of the LAMP assay was further compared with real time PCR by the examination 24 blood samples including 6 confirmed and 18 probable Q fever cases, which diagnosed by IFA serological assessment and real time PCR. In addition, 126 animal samples from 4 provinces including 97 goats, 7 cattle, 18 horses, 3 marmots and 1 deer were compared by these two methods. RESULTS: The limits of detection of the LAMP assay for the htpAB gene were 1 copy per reaction. The specificity of the LAMP assay was 100%, and no cross-reaction was observed among the bacteria used in the study. The positive rate of unknown febrile patients was 33.3%(95%CI 30.2%-36.4% for the LAMP assay and 8.3%(95%CI 7.4%-9.2% for the real time PCR(P<0.05. Similarly, the total positive rate of animals was 7.9%(95%CI 7.1%-8.7% for the LAMP assay and 0.8%(95%CI 0.7%-0.9%for the real time

  7. Two genes conferring resistance to Pythium stalk rot in maize inbred line Qi319.

    Science.gov (United States)

    Song, Feng-Jing; Xiao, Ming-Gang; Duan, Can-Xing; Li, Hong-Jie; Zhu, Zhen-Dong; Liu, Bao-Tao; Sun, Su-Li; Wu, Xiao-Fei; Wang, Xiao-Ming

    2015-08-01

    Stalk rots are destructive diseases in maize around the world, and are most often caused by the pathogen Pythium, Fusarium and other fungi. The most efficient management for controlling stalk rots is to breed resistant cultivars. Pythium stalk rot can cause serious yield loss on maize, and to find the resistance genes from the existing germplasm is the basis to develop Pythium-resistance hybrid lines. In this study, we investigated the genetic resistance to Pythium stalk rot in inbred line Qi319 using F2 and F2:3 population, and found that the resistance to Pythium inflatum in Qi319 was conferred by two independently inherited dominant genes, RpiQI319-1 and RpiQI319-2. Linkage analysis uncovered that the RpiQI319-1 co-segregated with markers bnlg1203, and bnlg2057 on chromosome 1, and that the RpiQI319-2 locus co-segregated with markers umc2069 and bnlg1716 on chromosome 10. The RpiQI319-1 locus was further mapped into a ~500-kb interval flanked by markers SSRZ33 and SSRZ47. These results will facilitate marker-assisted selection of Pythium stalk rot-resistant cultivars in maize breeding. To our knowledge, this is the first report on the resistance to P. inflatum in the inbred line Qi319, and is also the first description of two independently inherited dominant genes conferring the resistance of Pythium stalk rot in maize.

  8. Additive susceptibility to insulin-dependent diabetes conferred by HLA-DQB1 and insulin genes.

    Science.gov (United States)

    She, J X; Bui, M M; Tian, X H; Muir, A; Wakeland, E K; Zorovich, B; Zhang, L P; Liu, M C; Thomson, G; Maclaren, N K

    1994-01-01

    Several genomic polymorphisms at the insulin (INS) gene and its flanking regions were analyzed in 197 unrelated Caucasian patients affected by insulin-dependent diabetes (IDDM) and 159 ethnically matched, normal controls ascertained from the South-Eastern United States. We found that the frequency of homozygotes for the common variant at the insulin gene was significantly increased in the diabetic population (RR = 2.0, p INS gene. We determined the HLA-DQB1 genotypes by denaturing gradient gel electrophoresis (DGGE) and/or sequence-specific primers (SSP) techniques to assess the possible interactions between INS and HLA. DQB1*0302 had the strongest predisposing effect on IDDM susceptibility (RR = 9.3) and DQB1*0602 the strongest protective effect (RR = 0.02). However, a significant predisposing effect of DQB1*0201 could be demonstrated only after removal of the effects of DQB1*0302 and DQB1*0602. Analyses of the genotypes revealed that all genotypes containing 0602 were protective and that the heterozygous genotype 0201/0302 and homozygous genotype 0302/0302 confer the highest risk (RR = 20.9 and 12.9 respectively). However, heterozygous genotypes 0302/X (X excludes 0201, 0302 and 0602) have a significantly lower predisposing risk. Similarly, there is heterogeneity in risk between predisposing 0201/0201 homozygous individuals and protective 0201/X individuals. When subjects were stratified by HLA genotypes, the relative risks conferred by INS did not vary, thus suggesting that the susceptibility effects conferred by HLA and INS are additive rather than interactive.

  9. Characterization of Abcc4 gene amplification in stepwise-selected mouse J774 macrophages resistant to the topoisomerase II inhibitor ciprofloxacin.

    Science.gov (United States)

    Marquez, Béatrice; Ameye, Geneviève; Vallet, Coralie M; Tulkens, Paul M; Poirel, Hélène A; Van Bambeke, Françoise

    2011-01-01

    Exposure of J774 mouse macrophages to stepwise increasing concentrations of ciprofloxacin, an antibiotic inhibiting bacterial topoisomerases, selects for resistant cells that overexpress the efflux transporter Abcc4 (Marquez et al. [2009] Antimicrob. Agents Chemother. 53: 2410-2416), encoded by the Abcc4 gene located on Chromosome 14qE4. In this study, we report the genomic alterations occurring along the selection process. Abcc4 expression progressively increased upon selection rounds, with exponential changes observed between cells exposed to 150 and 200 µM of ciprofloxacin, accompanied by a commensurate decrease in ciprofloxacin accumulation. Molecular cytogenetics experiments showed that this overexpression is linked to Abcc4 gene overrepresentation, grading from a partial trisomy of Chr 14 at the first step of selection (cells exposed to 100 µM ciprofloxacin), to low-level amplifications (around three copies) of Abcc4 locus on 1 or 2 Chr 14 (cells exposed to 150 µM ciprofloxacin), followed by high-level amplification of Abcc4 as homogeneous staining region (hsr), inserted on 3 different derivative Chromosomes (cells exposed to 200 µM ciprofloxacin). In revertant cells obtained after more than 60 passages of culture without drug, the Abcc4 hsr amplification was lost in approx. 70% of the population. These data suggest that exposing cells to sufficient concentrations of an antibiotic with low affinity for eukaryotic topoisomerases can cause major genomic alterations that may lead to the overexpression of the transporter responsible for its efflux. Gene amplification appears therefore as a mechanism of resistance that can be triggered by non-anticancer agents but contribute to cross-resistance, and is partially and slowly reversible.

  10. Molecular mapping and characterization of two genes conferring resistance to Phytophthora sojae in a soybean landrace PI 567139B

    Science.gov (United States)

    Phytophthora root and stem rot (PRR), caused by the soil-borne oomycete pathogen Phytophthora sojae, is one of the most destructive diseases of soybean. PRR can be effectively controlled by race-specific genes conferring resistance to P. sojae (Rps). However, the Rps genes are usually non-durable, a...

  11. Amplification of the Full-Length PAMP Gene and Difference of the mRNA Expression Among Three Lean Pig Breeds

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To understand the function of porcine adipocyte-special membrane protein (PAMP) gene and the difference of fat deposition ability among various lean pig breeds, a full-length porcine adipocyte-special membrane protein (PAMP) gene was successfully amplified using reverse transcription polymerase chain reaction (RT-PCR) and 5'-rapid amplification of cDNA end (5'-RACE). The open reading frame was 1 587 bp encoding 529 amino acids. The nucleotide sequence of the full-length PAMP gene was deposited in the GenBank under the accession number EF433431. The PAMP gene mRNA expression was analyzed on three lean pig breeds by quantitative reverse transcription polymerase chain reaction (QRT-PCR). The PAMP gene mRNA levels in YHM (Yorkshire × Hampshire × Meishan) pig and DLY (Duroc×Landrance× Yorkshire) pig were about 0.82 and 0.38 times of that in SW (Shanxi-White) pig, respectively.

  12. The correlation between dual-color chromogenic in situ hybridization and fluorescence in situ hybridization in assessing HER2 gene amplification in breast cancer

    DEFF Research Database (Denmark)

    Pedersen, Marianne; Rasmussen, Birgitte Bruun

    2009-01-01

    obtained by the 2 methods showed excellent correlation (correlation coefficient 0.95). In conclusion, it is possible by dual-color CISH method to demonstrate HER2 genes and chromosome 17 genes, in the same tissue section and reliably assess HER2 status. The CISH method is a very promising alternative...... invasive breast carcinomas, both FISH and CISH detected HER2 amplification in 24 cases and nonamplification was detected in 47 cases. One case showed a discrepancy between FISH and CISH. The concordance between CISH and FISH was found to be almost perfect (98.6%). The correlation between the HER2 ratios...

  13. PL1 fusion gene: a novel visual selectable marker gene that confers tolerance to multiple abiotic stresses in transgenic tomato.

    Science.gov (United States)

    Jin, Feng; Li, Shu; Dang, Lijie; Chai, Wenting; Li, Pengli; Wang, Ning Ning

    2012-10-01

    Visual selectable markers, including the purple color caused by the accumulation of anthocyanins, have been proposed for use as antibiotic-free alternatives. However, the excessive accumulation of anthocyanins seriously inhibits the growth and development of transgenic plants. In our study, the AtDWF4 promoter from Arabidopsis and the tomato LeANT1 gene, encoding a MYB transcription factor, were used to construct the PL1 fusion gene to test whether it could be used as a visual selectable marker gene for tomato transformation. All the PL1 transgenic shoots exhibited intense purple color on shoot induction medium. In the transgenic tomato plants, PL1 was highly expressed in the cotyledons, but expressed only slightly in the true leaves and other organs. The expression of PL1 had no significantly adverse effects on the growth or development of the transgenic tomato plants, and conferred tolerance to multiple abiotic stresses in them. With the “cut off green shoots” method, multiple independent 35S::GFP transgenic tomato lines were successfully obtained using PL1 as the selectable marker gene. These results suggest that PL1 has potential application of visual selectable marker gene for tomato transformation.

  14. Amplification of the Insulin-Like Growth Factor 1 Receptor Gene Is a Rare Event in Adrenocortical Adenocarcinomas: Searching for Potential Mechanisms of Overexpression

    Directory of Open Access Journals (Sweden)

    Tamaya Castro Ribeiro

    2014-01-01

    Full Text Available Context. IGF1R overexpression appears to be a prognostic biomarker of metastatic pediatric adrenocortical tumors. However, the molecular mechanisms that are implicated in its upregulation remain unknown. Aim. To investigate the potential mechanisms involved in IGF1R overexpression. Patients and Methods. We studied 64 adrenocortical tumors. IGF1R copy number variation was determined in all patients using MLPA and confirmed using real time PCR. In a subgroup of 32 patients, automatic sequencing was used to identify IGF1R allelic variants and the expression of microRNAs involved in IGF1R regulation by real time PCR. Results. IGF1R amplification was detected in an adrenocortical carcinoma that was diagnosed in a 46-year-old woman with Cushing’s syndrome and virilization. IGF1R overexpression was demonstrated in this case. In addition, gene amplification of other loci was identified in this adrenocortical malignant tumor, but no IGF1R copy number variation was evidenced in the remaining cases. Automatic sequencing revealed three known polymorphisms but they did not correlate with its expression. Expression of miR-100, miR-145, miR-375, and miR-126 did not correlate with IGF1R expression. Conclusion. We demonstrated amplification and overexpression of IGF1R gene in only one adrenocortical carcinoma, suggesting that these combined events are uncommon. In addition, IGF1R polymorphisms and abnormal microRNA expression did not correlate with IGF1R upregulation in adrenocortical tumors.

  15. Development and application of loop-mediated isothermal amplification methods targeting the seM gene for detection of Streptococcus equi subsp. equi.

    Science.gov (United States)

    Hobo, Seiji; Niwa, Hidekazu; Oku, Kazuomi

    2012-03-01

    Loop-mediated isothermal amplification (LAMP) constitutes a potentially valuable diagnostic tool for rapid diagnosis of contagious diseases. In this study, we developed a novel LAMP method (seM-LAMP) to detect the seM gene of Streptococcus equi subsp. equi (S. equi), the causative agent of strangles in equids. The seM-LAMP successfully amplified the target sequence of the seM gene at 63°C within 60 min. The sensitivity of the seM-LAMP was slightly lower than the 2nd reaction of the seM semi-nested PCR. To evaluate the species specificity of the seM-LAMP, we tested 100 S. equi and 189 non-S. equi strains. Significant amplification of the DNA originating from S. equi was observed within 60 min incubation, but no amplification of non-S. equi DNA occurred. The results were identical to those of seM semi-nested PCR. To investigate the clinical usefulness of the methods, the seM-LAMP and the seM semi-nested PCR were used to screen 590 nasal swabs obtained during an outbreak of strangles. Both methods showed that 79 and 511 swabs were S. equi positive and negative, respectively, and the results were identical to those of the culture examination. These results indicate that the seM-LAMP is potentially useful for the reliable routine diagnosis of Streptococcus equi subsp. equi infections.

  16. Multiplexed real-time PCR amplification of tlh, tdh and trh genes in Vibrio parahaemolyticus and its rapid detection in shellfish and Gulf of Mexico water.

    Science.gov (United States)

    Rizvi, Amy V; Bej, Asim K

    2010-10-01

    In this study, we have developed a SYBR Green I-based real-time multiplexed PCR assay for the detection of Vibrio parahaemolyticus in Gulf of Mexico water (gulf water), artificially seeded and natural oysters targeting three hemolysin genes, tlh, tdh and trh in a single reaction. Post-amplification melt-temperature analysis confirmed the amplification of all three targeted genes with high specificity. The detection sensitivity was 10 cfu (initial inoculum) in 1 ml of gulf water or oyster tissue homogenate, following 5 h enrichment. The results showed 58% of the oysters to be positive for tlh, indicating the presence of V. parahaemolyticus; of which 21% were positive for tdh; and 0.7% for trh, signifying the presence of pathogenic strains. The C(t) values showed that oyster tissue matrix had some level of inhibition, whereas the gulf water had negligible effect on PCR amplification. The assay was rapid (approximately 8 h), specific and sensitive, meeting the ISSC guidelines. Rapid detection using real-time multiplexed PCR will help reduce V. parahaemolyticus-related disease outbreaks, thereby increasing consumer confidence and economic success of the seafood industry.

  17. Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp

    Directory of Open Access Journals (Sweden)

    Hall Matthew J

    2007-06-01

    Full Text Available Abstract Background Signal-Mediated Amplification of RNA Technology (SMART is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus. Results Here we report that the SMART assay allowed us to differentiate between infected and non-infected host cultures. Expression of the cyanophage strain S-PM2 portal vertex gene (g20 was detected from infected host Synechococcus sp. WH7803 cells. Using the SMART assay, we demonstrated that g20 mRNA peaked 240 – 360 minutes post-infection, allowing us to characterise this as a mid to late transcript. g20 DNA was also detected, peaking 10 hours post-infection, coinciding with the onset of host lysis. Conclusion The SMART assay is based on isothermal nucleic acid amplification, allowing the detection of specific sequences of DNA or RNA. It was shown to be suitable for differentiating between virus-infected and non-infected host cultures and for the detection of virus gene expression: the first reported use of this technology for such applications.

  18. Development of Multiplex-Mismatch Amplification Mutation-PCR Assay for Simultaneous Detection of Campylobacter jejuni and Mutation in gyrA Gene Related to Fluoroquinolone Resistance.

    Science.gov (United States)

    Cui, Mingquan; Wu, Chenbin; Zhang, Peng; Wu, Congming

    2016-11-01

    Campylobacter jejuni, a foodborne pathogen, is the major cause of enteritis in humans worldwide, however, its increasing resistance to fluoroquinolones reported recently is of a major concern. In the present study, multiplex-mismatch amplification mutation assay-polymerase chain reaction (MMAMA-PCR) was developed for the first time with the aim to quickly identify C. jejuni and to detect the single nucleotide mutation (C-257 to T) frequently observed in gyrA gene, associated with the acquisition of resistance to fluoroquinolones. In this assay, mismatch amplification mutation primers for the detection of gyrA mutation in C. jejuni were coupled with primers for the hip gene encoding for hippuricase and 16S rRNA gene of C. jejuni, respectively, in the multiplex PCR assay. The specificity and accuracy of this method were analyzed by the use of 78 C. jejuni strains with previously confirmed resistance phenotypes and the mutation (C-257 to T) in gyrA gene, as well as 107 clinical isolates of various bacterial species, including 29 C. jejuni isolates. This study indicates that MMAMA-PCR is a promising assay for the rapid identification of C. jejuni with a specific mutation in gyrA gene, responsible for the resistance to fluoroquinolones.

  19. Incidence of and factors associated with false positives in laboratory diagnosis of norovirus infection by amplification of the RNA-dependent RNA polymerase gene.

    Directory of Open Access Journals (Sweden)

    Fang-Ru Lin

    Full Text Available BACKGROUND: Conventional reverse transcription-polymerase chain reaction (RT-PCR amplification of the RNA-dependent RNA polymerase (RdRp gene remains a used method for the rapid detection of norovirus (NV in clinical laboratories. The incidence of and factors associated with false positives in this assay have not been previously evaluated. METHODS/PRINCIPAL FINDINGS: After an NV outbreak caused by the GII.4 Sydney strain in 2012, we reanalysed 250 stool samples positive for NV by RdRp gene detection. True positives were confirmed in 154 (61.6% samples by successful amplification and sequencing confirmation of the viral protein 1 gene. Of the remaining 96 samples that underwent RT-PCR for the RdRp gene, 34 samples yielded PCR products of the expected length. However, the sequences of the amplicons belonged to the human genome, with 91-97% matched nucleotide sequences, indicating false positives. Multivariate analysis of the clinical features of the patients identified a positive stool culture for bacteria (adjusted odds ratio [aOR] 9.07, 95% adjusted confidence interval [aCI] 2.17-37.92, P = .003 and the use of parenteral antibiotics (aOR 5.55, 95% aCI 1.21-24.73, P = .027 as significant and independent factors associated with false positives. CONCLUSION: Conventional RT-PCR targeting the RdRp gene of NV can lead to false positives in patients with bacterial enterocolitis by incidental amplification of DNA from a human source.

  20. Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: Methionine sulfoximine resistance.

    Science.gov (United States)

    Yu, Da Young; Noh, Soo Min; Lee, Gyun Min

    2016-08-10

    To investigate the feasibility of glutamine synthetase (GS)-mediated gene amplification in HEK293 cells for the high-level stable production of therapeutic proteins, HEK293E cells were transfected by the GS expression vector containing antibody genes and were selected at various methionine sulfoximine (MSX) concentrations in 96-well plates. For a comparison, CHOK1 cells were transfected by the same GS expression vector and selected at various MSX concentrations. Unlike CHOK1 cells, HEK293E cells producing high levels of antibodies were not selected at all. For HEK293E cells, the number of wells with the cell pool did not decrease with an increase in the concentration of MSX up to 500μM MSX. A q-RT-PCR analysis confirmed that the antibody genes in the HEK293E cells, unlike the CHOK1 cells, were not amplified after increasing the MSX concentration. It was found that the GS activity in HEK293E cells was much higher than that in CHOK1 cells (P<0.05). In a glutamine-free medium, the GS activity of HEK293E cells was approximately 4.8 times higher than that in CHOK1 cells. Accordingly, it is inferred that high GS activity of HEK293E cells results in elevated resistance to MSX and therefore hampers GS-mediated gene amplification by MSX. Thus, in order to apply the GS-mediated gene amplification system to HEK293 cells, the endogenous GS expression level in HEK293 cells needs to be minimized by knock-out or down-regulation methods.

  1. Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9.

    Science.gov (United States)

    Kanitkar, Yogendra H; Stedtfeld, Robert D; Steffan, Robert J; Hashsham, Syed A; Cupples, Alison M

    2016-01-08

    Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures.

  2. Analyses of numerical aberrations of chromosome 17 and tp53 gene deletion/amplification in human oral squamous cell carcinoma using dual-color fluorescence in situ hybridization

    Directory of Open Access Journals (Sweden)

    Noemi MESZAROS

    2010-05-01

    Full Text Available In Romania, oral and facial cancers represent approximately 5% of all cancers. Deactivation and unregulated expression of oncogenes and tumor suppressor genes may be involved in the pathogenesis of oral squamous cell carcinoma. The genomic change results in numerical and structural chromosomal alterations, particularly in chromosomes 3, 9, 11 and 17. The aim of our study was to identify numerical aberrations of chromosome 17, deletion or amplification of p53 gene and to reveal correlations between abnormalities of chromosome 17and of p53 gene with TNM status and grading in 15 subjects with oral squamous cell carcinoma. 80 % of cases presented chromosome 17 polysomy and only 20% of cases had chromosome 17 monosomy. 46.6 % of samples revealed p53 gene amplification and 33.3 % of them p53 deletion. Polysomy of chromosome 17 was also detected in tumor-adjacent epithelia. The degree of the cytogenetic abnormality did not correlate with the stage of the disease, the histological differentiation of oral squamous cell carcinoma and lymph node metastasis. Molecular cytogenetic techniques, using fluorescence in situ hybridization with chromosome-specific DNA probes, facilitate the confirmation of presumed chromosomal aberrations with high sensitivity and specificity.

  3. Amplification of HER2 and TOP2A and deletion of TOP2A genes in breast cancer investigated by new FISH probes

    DEFF Research Database (Denmark)

    Olsen, Karen E; Knudsen, Helle; Rasmussen, Birgitte

    2004-01-01

    New strategies for improving treatment of patients with breast carcinoma have focused on the HER2 oncoprotein with regard to response to traditional therapy regimes and the effect of a new drug specifically directed against the protein. Furthermore, the status of the topoisomerase IIalpha (TOP2A......). In addition, the HercepTest was evaluated as a screening tool for choosing cases for FISH investigation of TOP2A gene aberrations. The HercepTest score 3+ identified HER2 gene amplification in 27 of 30 amplified tumours (sensitivity of 0.90) with a false-negative rate of 0.10 and a false-positive rate of 0.......06. TOP2A gene amplification or deletion was found in 20 cases. Sixteen (80%) of these carcinomas were in the HercepTest 3+ group, but four tumours had alterations in the TOP2A gene with normal HER2 status. Traditionally, in the FISH technique the result has been based on counting 60 cells. However, we...

  4. A novel resistance gene, lnu(H), confers resistance to lincosamides inriemerella anatipestiferCH-2.

    Science.gov (United States)

    Luo, Hong-Yan; Liu, Ma-Feng; Wang, Ming-Shu; Zhao, Xin-Xin; Jia, Ren-Yong; Chen, Shun; Sun, Kun-Feng; Yang, Qiao; Wu, Ying; Chen, Xiao-Yue; Biville, Francis; Zou, Yuan-Feng; Jing, Bo; Cheng, An-Chun; Zhu, De-Kang

    2017-08-23

    The Gram-negative bacteria Riemerella anatipestifer CH-2 is resistant to lincosamide (the MIC value of lincomycin is 128 µg/ml). The G148_1775 gene of R. anatipestifer CH-2, designated lnu(H), encodes a 260-amino-acid protein with ≤ 41% identity to other reported lincosamide nucleotidyltransferases. The E. coli Rosetta (DE3) containing pBAD24-lnu(H) plasmid showed 4- and 2-fold increases in lincomycin and clindamycin MICs, respectively. A kinetic assay of the purified Lnu(H) enzyme for lincomycin and clindamycin showed that the protein could inactive lincosamides. Mass spectrometry analysis results demonstrated that the Lnu(H) enzyme catalyzed adenylation of lincosamides. In addition, the lnu(H) gene deletion strain exhibited 512- and 32-fold decreases in lincomycin and clindamycin MICs, respectively. Wild-type level of lincosamide resistance could be restored by complementation with a shuttle plasmid carrying the lnu(H) gene. The transformant ATCC 11845 (lnu(H)) acquired by natural transformation also exhibited high-level lincosamide resistance. Moreover, of the R. anatipestifer field isolates, 32% (56/175) were positive for the lnu(H) gene by PCR. In conclusion, Lnu(H) is a novel lincosamide nucleotidyltransferase, which inactivates lincomycin and clindamycin by nucleotidylation, thus conferring high-level of lincosamide resistance to R. anatipestifer CH-2. Copyright © 2017. Published by Elsevier B.V.

  5. The wheat Lr34 multipathogen resistance gene confers resistance to anthracnose and rust in sorghum.

    Science.gov (United States)

    Schnippenkoetter, Wendelin; Lo, Clive; Liu, Guoquan; Dibley, Katherine; Chan, Wai Lung; White, Jodie; Milne, Ricky; Zwart, Alexander; Kwong, Eunjung; Keller, Beat; Godwin, Ian; Krattinger, Simon G; Lagudah, Evans

    2017-11-01

    The ability of the wheat Lr34 multipathogen resistance gene (Lr34res) to function across a wide taxonomic boundary was investigated in transgenic Sorghum bicolor. Increased resistance to sorghum rust and anthracnose disease symptoms following infection with the biotrophic pathogen Puccinia purpurea and the hemibiotroph Colletotrichum sublineolum, respectively, occurred in transgenic plants expressing the Lr34res ABC transporter. Transgenic sorghum lines that highly expressed the wheat Lr34res gene exhibited immunity to sorghum rust compared to the low-expressing single copy Lr34res genotype that conferred partial resistance. Pathogen-induced pigmentation mediated by flavonoid phytoalexins was evident on transgenic sorghum leaves following P. purpurea infection within 24-72 h, which paralleled Lr34res gene expression. Elevated expression of flavone synthase II, flavanone 4-reductase and dihydroflavonol reductase genes which control the biosynthesis of flavonoid phytoalexins characterized the highly expressing Lr34res transgenic lines 24-h post-inoculation with P. purpurea. Metabolite analysis of mesocotyls infected with C. sublineolum showed increased levels of 3-deoxyanthocyanidin metabolites were associated with Lr34res expression, concomitant with reduced symptoms of anthracnose. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  6. A novel gene trap method using terminator-REMI and 3' rapid amplification of cDNA ends (RACE) in Dictyostelium.

    Science.gov (United States)

    Takeda, Kosuke; Saito, Tamao; Tanaka, Tomoyuki; Morio, Takahiro; Maeda, Mineko; Tanaka, Yoshimasa; Ochiai, Hiroshi

    2003-07-17

    We describe a novel restriction enzyme-mediated integration (REMI) method for gene trapping in Dictyostelium based on the use of a terminator-deficient vector. The vector has a blasticidin deaminase (bsr) gene as a selectable marker but lacks a terminator containing a poly(A) addition signal (AATAAA). Thus, the vector was expected to integrate into the coding region of a gene to create a fusion transcript flanked by the 3' proximal region of the trapped gene. The trapped gene can be identified by simply amplifying the fusion transcript by 3' rapid amplification of cDNA ends (3'-RACE). In the analysis of 35 integration events into known genes, the vectors were found to be integrated 20 times in close proximity to the 3' ends of the genes and in the direction of transcription. This strictly localized insertion seemed to be mediated by negative selection via the surveillance system referred to nonsense-mediated mRNA decay. In contrast, in 15 events the vector integrated in the opposite direction to transcription and at random positions throughout the coding sequence. Analysis of the trapped 3' sequences showed that the transcription of the fusion gene terminated prematurely without the apparent use of an endogenous terminator; nevertheless the transcript did exhibit a poly(A) tail. Based on these results, we designated the method terminator-REMI. Using this method, we have generated a library of tagged Dictyostelium clones from which we have thus far isolated 242 developmental mutants.

  7. Determination of the relative expression levels of rubisco small subunit genes in Arabidopsis by rapid amplification of cDNA ends.

    Science.gov (United States)

    Yoon, M; Putterill, J J; Ross, G S; Laing, W A

    2001-04-15

    Multigene families are common in higher organisms. However, due to the close similarities between members, it is often difficult to assess the individual contribution of each gene to the overall expression of the family. In Arabidopsis thaliana, there are four genes encoding the small subunits (SSU) of ribulose-1.5-bisphosphate carboxylase oxygenase (rubisco) whose nucleotide sequences are up to 98.4% identical. In order to overcome the technical limitations associated with gene-specific probes (or primers) commonly used in existing methods, we developed a new gene expression assay based on the RACE (rapid amplification of cDNA ends) technique with a single pair of primers. With this RACE gene expression assay, we were able to determine the relative transcript levels between four Arabidopsis SSU genes. We found that the relative SSU gene expression differed significantly between plants grown at different temperatures. Our observation raises the possibility that an adaptation of rubisco to the environment may be achieved through the specific synthesis of the SSU proteins, which is determined by the relative expression levels between the SSU genes.

  8. Rapid detection of Candida albicans in clinical samples by DNA amplification of common regions from C. albicans-secreted aspartic proteinase genes.

    Science.gov (United States)

    Flahaut, M; Sanglard, D; Monod, M; Bille, J; Rossier, M

    1998-02-01

    Laboratory diagnosis based on genomic amplification methods such as PCR may provide an alternative and more sensitive method than conventional culture for the early detection of deep-seated candidiasis, an increasing cause of morbidity and mortality among immunocompromised patients. A novel method of DNA extraction from clinical samples based on treatment with proteinase K and isolation of DNA on a silica membrane was developed. The targets used for DNA amplification were the Candida albicans-secreted aspartic proteinase (SAP) genes, a multiple-gene family of at least seven members in C. albicans. A single pair of primers was designed in order to detect six of these SAP genes and, subsequently, to increase the sensitivity of the test. Detection of the PCR product by enzyme-linked immunosorbent assay was found to be as sensitive as Southern blotting with an SAP-labeled probe. The sensitivity of the assay was 1 cell/ml from serially diluted Candida cultures and 1 to 4 cells/ml from seeded blood specimens. The sensitivity and specificity of the present assay were tested in a retrospective study performed blindly with 156 clinical samples and were 100 and 98%, respectively, compared with the results of culture. For the subset of blood culture samples (n = 124), the sensitivity and the specificity were 100%. The two false-positive PCR samples came from patients treated with azole antifungal agents, indicating that PCR was probably able to detect damaged organisms that could not be recovered by culture.

  9. Identification of goose (Anser anser) and mule duck (Anasplatyrhynchos x Cairina moschata) foie gras by multiplex polymerase chain reaction amplification of the 5S RDNA gene.

    Science.gov (United States)

    Rodríguez, M A; García, T; González, I; Asensio, L; Fernández, A; Lobo, E; Hernández, P E; Martín, R

    2001-06-01

    Polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene has been used for the identification of goose and mule duck foie gras. Two species-specific reverse primers were designed and used in a multiplex reaction, together with a forward universal primer, to amplify specific fragments of the 5S rDNA in each species. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose and mule duck foie gras samples. This genetic marker can be useful for detecting fraudulent substitution of the duck liver for the more expensive goose liver.

  10. DNA Extraction from Formalin-fixed and Paraffin-embedded Tissues by Triton X-100 for Effective Amplification of EGFR Gene by Polymerase Chain Reaction

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-feng; DU Zhen-wu; WU Mei; ZHANG Yu-cheng; JIANG Yang; ZHANG Gui-zhen

    2012-01-01

    For first-line non-small-cell lung cancer(NSCLC) therapy,detecting mutation status of the epidermal growth factor receptor(EGFR) gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor(TKI) therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded(FFPE) tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction(PCR) are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100(pH=10.0) was performed by heating at 95 C for 30min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,.19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1%Triton X was about 250-500 base pairs(bp).However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.

  11. Prognostic significance of ESR1 gene amplification, mRNA/protein expression and functional profiles in high-risk early breast cancer: a translational study of the Hellenic Cooperative Oncology Group (HeCOG.

    Directory of Open Access Journals (Sweden)

    George Pentheroudakis

    Full Text Available BACKGROUND: Discrepant data have been published on the incidence and prognostic significance of ESR1 gene amplification in early breast cancer. PATIENTS AND METHODS: Formalin-fixed paraffin-embedded tumor blocks were collected from women with early breast cancer participating in two HeCOG adjuvant trials. Messenger RNA was studied by quantitative PCR, ER protein expression was centrally assessed using immunohistochemistry (IHC and ESR1 gene copy number by dual fluorescent in situ hybridization probes. RESULTS: In a total of 1010 women with resected node-positive early breast adenocarcinoma, the tumoral ESR1/CEP6 gene ratio was suggestive of deletion in 159 (15.7%, gene gain in 551 (54.6% and amplification in 42 cases (4.2%, with only 30 tumors (3% harboring five or more ESR1 copies. Gene copy number ratio showed a significant, though weak correlation to mRNA and protein expression (Spearman's Rho <0.23, p = 0.01. ESR1 clusters were observed in 9.5% (57 gain, 38 amplification of cases. In contrast to mRNA and protein expression, which were favorable prognosticators, gene copy number changes did not obtain prognostic significance. When ESR1/CEP6 gene ratio was combined with function (as defined by ER protein and mRNA expression in a molecular classifier, the Gene Functional profile, it was functional status that impacted on prognosis. In univariate analysis, patients with functional tumors (positive ER protein expression and gene ratio normal or gain/amplification fared better than those with non-functional tumors with ESR1 gain (HR for relapse or death 0.49-0.64, p = 0.003. Significant interactions were observed between gene gain/amplification and paclitaxel therapy (trend for DFS benefit from paclitaxel only in patients with ESR1 gain/amplification, p = 0.066 and Gene Functional profile with HER2 amplification (Gene Functional profile prognostic only in HER2-normal cases, p = 0.029. CONCLUSIONS: ESR1 gene deletion and

  12. Chromosomal locations of the maize (Zea mays L.) HtP and rt genes that confer resistance to Exserohilum turcicum

    National Research Council Canada - National Science Library

    Ogliari, Juliana Bernardi; Guimarães, Marco Antônio; Camargo, Luis Eduardo Aranha

    2007-01-01

    ...) L30HtPHtPRtRt and L30htphtpRtRt and the L40htphtprtrt line which contrast regarding the presence of the recently described dominant HtP and the recessive rt genes that confer resistance to Exserohilum turcicum...

  13. Map - vs. homology - based cloning for the recessive gene ol-2 conferring resistance to tomato powdery mildew

    NARCIS (Netherlands)

    Pavan, S.N.C.; Zheng, Z.; Borisova, M.; Berg, van den P.M.M.M.; Lotti, C.; Giovanni, de C.; Lindhout, P.; Jong, de J.H.; Ricciardi, L.; Visser, R.G.F.; Bai, Y.

    2008-01-01

    The recessive gene ol-2 confers papilla-associated and race-non-specific resistance to tomato powdery mildew caused by Oidium neolycopersici. In order to facilitate marker assisted selection (MAS) in practical breeding programmes, we identified two simple sequence repeat (SSR) markers and one

  14. Circular rapid amplification of cDNA ends for high-throughput extension cloning of partial genes.

    Science.gov (United States)

    Fu, Glenn K; Wang, Jonathan T; Yang, Junming; Au-Young, Janice; Stuve, Laura L

    2004-07-01

    The rapid amplification of cDNA ends (RACE) procedure is a widely used PCR-based method to clone the cDNA ends of mRNA transcripts. Current RACE methods often produce a high background of nonspecific PCR products, which can exclude the identification of the target cDNA of interest. We describe here an improved RACE procedure using circular cDNA templates and demonstrate the successful extension cloning of 4406 cDNAs.

  15. Validation of whole genome amplification for analysis of the p53 tumor suppressor gene in limited amounts of tumor samples.

    Science.gov (United States)

    Hasmats, Johanna; Green, Henrik; Solnestam, Beata Werne; Zajac, Pawel; Huss, Mikael; Orear, Cedric; Validire, Pierre; Bjursell, Magnus; Lundeberg, Joakim

    2012-08-24

    Personalized cancer treatment requires molecular characterization of individual tumor biopsies. These samples are frequently only available in limited quantities hampering genomic analysis. Several whole genome amplification (WGA) protocols have been developed with reported varying representation of genomic regions post amplification. In this study we investigate region dropout using a φ29 polymerase based WGA approach. DNA from 123 lung cancers specimens and corresponding normal tissue were used and evaluated by Sanger sequencing of the p53 exons 5-8. To enable comparative analysis of this scarce material, WGA samples were compared with unamplified material using a pooling strategy of the 123 samples. In addition, a more detailed analysis of exon 7 amplicons were performed followed by extensive cloning and Sanger sequencing. Interestingly, by comparing data from the pooled samples to the individually sequenced exon 7, we demonstrate that mutations are more easily recovered from WGA pools and this was also supported by simulations of different sequencing coverage. Overall this data indicate a limited random loss of genomic regions supporting the use of whole genome amplification for genomic analysis.

  16. The potential role of incorporating real-time PCR and DNA sequencing for amplification and detection of 16S rRNA gene signatures in neonatal sepsis.

    Science.gov (United States)

    Midan, Dina A; Abo El Fotoh, Wafaa Moustafa M; El Shalakany, Abeer H

    2017-06-01

    This study aimed to explore whether 16S rRNA gene amplification by real time PCR and sequencing could serve as genetic-based methods in rapid and accurate diagnosis of neonatal sepsis. This case control study was conducted on 40 neonates suffering from sepsis like manifestations recruited from the neonatal intensive care unit of Menoufia university hospital over a period of 6 months. Their blood samples were used for paired analysis of bacterial growth using BACTEC 9050 instrument and real time PCR assay with subsequent DNA sequencing for bacterial species identification. The detection rate of culture proven sepsis was 70%. By using real time 16S r RNA PCR amplification method, the detection of bacteria was improved to 80%. Real time PCR revealed sensitivity, specificity, positive predictive value and negative predictive value of [100%, 66.7%, 87.5% and 100%] respectively. Compared to culture, the 16S rRNA real time PCR demonstrated a high negative value for ruling out neonatal sepsis. There was significant statistical difference between the PCR positive and negative cases as regards the hematological sepsis score. The results demonstrated the ability of DNA sequencing to recognize 4 pathogens which were negative by blood culture. The time consumed to detect sepsis using blood culture was up to 5 days while it took up to 16 h only by PCR and sequencing methods. 16S rRNA gene amplification by real time PCR and sequence analysis could be served as ideal and reliable genetic-based methods to diagnose and rule out sepsis with provision of additional data that cannot be obtained by routine laboratory tests with a shorter turnaround time than those with culture-based protocols.

  17. Impact of sample acquisition and linear amplification on gene expression profiling of lung adenocarcinoma: laser capture micro-dissection cell-sampling versus bulk tissue-sampling

    Directory of Open Access Journals (Sweden)

    Wigle Dennis A

    2009-03-01

    Full Text Available Abstract Background The methods used for sample selection and processing can have a strong influence on the expression values obtained through microarray profiling. Laser capture microdissection (LCM provides higher specificity in the selection of target cells compared to traditional bulk tissue selection methods, but at an increased processing cost. The benefit gained from the higher tissue specificity realized through LCM sampling is evaluated in this study through a comparison of microarray expression profiles obtained from same-samples using bulk and LCM processing. Methods Expression data from ten lung adenocarcinoma samples and six adjacent normal samples were acquired using LCM and bulk sampling methods. Expression values were evaluated for correlation between sample processing methods, as well as for bias introduced by the additional linear amplification required for LCM sample profiling. Results The direct comparison of expression values obtained from the bulk and LCM sampled datasets reveals a large number of probesets with significantly varied expression. Many of these variations were shown to be related to bias arising from the process of linear amplification, which is required for LCM sample preparation. A comparison of differentially expressed genes (cancer vs. normal selected in the bulk and LCM datasets also showed substantial differences. There were more than twice as many down-regulated probesets identified in the LCM data than identified in the bulk data. Controlling for the previously identified amplification bias did not have a substantial impact on the differences identified in the differentially expressed probesets found in the bulk and LCM samples. Conclusion LCM-coupled microarray expression profiling was shown to uniquely identify a large number of differentially expressed probesets not otherwise found using bulk tissue sampling. The information gain realized from the LCM sampling was limited to differential analysis

  18. Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode.

    Science.gov (United States)

    Lin, Jingyu; Mazarei, Mitra; Zhao, Nan; Zhu, Junwei J; Zhuang, Xiaofeng; Liu, Wusheng; Pantalone, Vincent R; Arelli, Prakash R; Stewart, Charles N; Chen, Feng

    2013-12-01

    Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence-related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full-length cDNAs of GmSAMT1 from a SCN-resistant soybean line and from a SCN-susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli-expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μM. To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN-susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.

  19. Fluorescence immunophenotyping and interphase cytogenetics (FICTION) detects BCL6 abnormalities, including gene amplification, in most cases of nodular lymphocyte-predominant Hodgkin lymphoma.

    Science.gov (United States)

    Bakhirev, Alexei G; Vasef, Mohammad A; Zhang, Qian-Yun; Reichard, Kaaren K; Czuchlewski, David R

    2014-04-01

    BCL6 translocations are a frequent finding in B-cell lymphomas of diverse subtypes, including some cases of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). However, reliable analysis of BCL6 rearrangements using fluorescence in situ hybridization is difficult in NLPHL because of the relative paucity of neoplastic cells. Combined immunofluorescence microscopy and fluorescence in situ hybridization, or fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION), permits targeted analysis of neoplastic cells. To better define the spectrum of BCL6 abnormalities in NLPHL using FICTION analysis. We performed an optimized FICTION analysis of 24 lymph nodes, including 11 NLPHL, 5 follicular hyperplasia with prominent progressive transformation of germinal centers, and 8 follicular hyperplasia without progressive transformation of germinal centers. BCL6 rearrangement was identified in 5 of 11 cases of NLPHL (46%). In addition, BCL6 gene amplification, with large clusters of BCL6 signals in the absence of chromosome 3 aneuploidy, was detected in 3 of 11 cases of NLPHL (27%). One NLPHL showed extra copies of BCL6 present in conjunction with multiple copies of chromosome 3. Altogether, we detected BCL6 abnormalities in 9 of 11 cases of NLPHL (82%). None of the progressive transformation of germinal centers or follicular hyperplasia cases showed BCL6 abnormalities by FICTION. To our knowledge, this is the first report of BCL6 gene amplification in NLPHL. Our optimized protocol for FICTION permits detection of cytogenetic abnormalities in most NLPHL cases and may represent a useful ancillary diagnostic technique.

  20. Gene amplification, ABC transporters and cytochrome P450s: unraveling the molecular basis of pyrethroid resistance in the dengue vector, Aedes aegypti.

    Directory of Open Access Journals (Sweden)

    Vassiliki Bariami

    Full Text Available BACKGROUND: Pyrethroid insecticides are widely utilized in dengue control. However, the major vector, Aedes aegypti, is becoming increasingly resistant to these insecticides and this is impacting on the efficacy of control measures. The near complete transcriptome of two pyrethroid resistant populations from the Caribbean was examined to explore the molecular basis of this resistance. PRINCIPAL FINDINGS: Two previously described target site mutations, 1016I and 1534C were detected in pyrethroid resistant populations from Grand Cayman and Cuba. In addition between two and five per cent of the Ae. aegypti transcriptome was differentially expressed in the resistant populations compared to a laboratory susceptible population. Approximately 20 per cent of the genes over-expressed in resistant mosquitoes were up-regulated in both Caribbean populations (107 genes. Genes with putative monooxygenase activity were significantly over represented in the up-regulated subset, including five CYP9 P450 genes. Quantitative PCR was used to confirm the higher transcript levels of multiple cytochrome P450 genes from the CYP9J family and an ATP binding cassette transporter. Over expression of two genes, CYP9J26 and ABCB4, is due, at least in part, to gene amplification. SIGNIFICANCE: These results, and those from other studies, strongly suggest that increases in the amount of the CYP9J cytochrome P450s are an important mechanism of pyrethroid resistance in Ae. aegypti. The genetic redundancy resulting from the expansion of this gene family makes it unlikely that a single gene or mutation responsible for pyrethroid resistance will be identified in this mosquito species. However, the results from this study do pave the way for the development of new pyrethroid synergists and improved resistance diagnostics. The role of copy number polymorphisms in detoxification and transporter genes in providing protection against insecticide exposure requires further investigation.

  1. A cfr-like gene cfr(C) conferring linezolid resistance is common in Clostridium difficile.

    Science.gov (United States)

    Candela, Thomas; Marvaud, Jean-Christophe; Nguyen, Tiep Khac; Lambert, Thierry

    2017-09-01

    Clostridium difficile T10 and Clostridium bolteae 90B3 were co-resistant to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A (PhLOPSA) and harbored an unreported cfr-like determinant that may alter the 23S rRNA by m(8)A2503 methylation. The cfr-like cfr(C) gene was cloned in C. difficile 630Δerm in which it conferred PhLOPSA resistance. In C. bolteae 90B3: (i) qRT-PCR analysis indicated that cfr(C) was similarly expressed in the absence or presence of either chloramphenicol or clindamycin or linezolid; and (ii) cfr(C) was part of a putative 24 kb-transposon, which generated a detectable circular intermediate. An element differing by a single nucleotide was found in C. difficile DA00203 from GenBank data, consistent with a recent horizontal transfer. In silico analysis showed cfr(C) in 19 out of 274 C. difficile genomes. This gene was also detected by PCR analysis in 9 out of 80 C. difficile from our laboratory strain collection according to resistance to linezolid and florfenicol. The fact that cfr(C) was mainly confined in C. difficile within polymorphic environments indicates this microorganism is a reservoir for PhLOPSA resistance. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  2. Duplications of the neuropeptide receptor gene VIPR2 confer significant risk for schizophrenia.

    LENUS (Irish Health Repository)

    Vacic, Vladimir

    2011-03-24

    Rare copy number variants (CNVs) have a prominent role in the aetiology of schizophrenia and other neuropsychiatric disorders. Substantial risk for schizophrenia is conferred by large (>500-kilobase) CNVs at several loci, including microdeletions at 1q21.1 (ref. 2), 3q29 (ref. 3), 15q13.3 (ref. 2) and 22q11.2 (ref. 4) and microduplication at 16p11.2 (ref. 5). However, these CNVs collectively account for a small fraction (2-4%) of cases, and the relevant genes and neurobiological mechanisms are not well understood. Here we performed a large two-stage genome-wide scan of rare CNVs and report the significant association of copy number gains at chromosome 7q36.3 with schizophrenia. Microduplications with variable breakpoints occurred within a 362-kilobase region and were detected in 29 of 8,290 (0.35%) patients versus 2 of 7,431 (0.03%) controls in the combined sample. All duplications overlapped or were located within 89 kilobases upstream of the vasoactive intestinal peptide receptor gene VIPR2. VIPR2 transcription and cyclic-AMP signalling were significantly increased in cultured lymphocytes from patients with microduplications of 7q36.3. These findings implicate altered vasoactive intestinal peptide signalling in the pathogenesis of schizophrenia and indicate the VPAC2 receptor as a potential target for the development of new antipsychotic drugs.

  3. 番茄抗花叶病毒基因类似序列扩增研究%Studies on Gene Similar Sequence Amplification of Tomato Mosaic Virus

    Institute of Scientific and Technical Information of China (English)

    李敏

    2011-01-01

    [目的]研究番茄抗花叶病毒基因类似序列扩增.[方法]以选育和生产上广泛使用的20个番茄品种为试材,对其进行了田间和实验室抗花叶病毒病(ToMV)筛选、基因类似序列扩增、RAPD聚类分析及特异引物PCR扩增.[结果]美味樱桃番茄、中杂9号、旱红宝、W2624、OH-2-2-11、黄圣果和美国番茄对ToMV具有较强的抗性;根据聚类分析,受试品种可分为3类,从中选出9个抗性和非抗性品种进行抗ToMV,基因类似序列分析,其中非抗性和购买无抗性番茄品种未扩增出任何谱带,而抗性品种扩增出约300 bp的谱带,初步认为该谱带为抗ToMV类似序列基因.[结论]为培育抗花叶病毒病的番茄品种提供了理论依据.%[ Objective ] The aim was to study the gene similar sequence amplification of tomato mosaic virus. [ Method ] Taking 20 tomato varieties cultivating in breeding and production widely as tested materials,tomato mosaic virus (ToMV) was screened in field and laboratory,furthennore RAPD cluster analysis and gene amplification experiment were carried out,and specific primers of PCR amplification was carried outfinally. [ Result] Dilicioas cherry tomato,China tomato hybrid 9 ,early red jewel tomato,W262 -4,Oh -2 -2 - 11 ,yellow holy tomato and American tomato had stronger resistance to ToMV. According to the result of RAPD cluster analysis,the 20 tomato cultivars could be divided into three types, from which 9 varieties inclnding resistant and nonresistant varieties were selected amplify gene similar sequence. None band was found from those nonresistant varieties,while a band of about 300 bp was found from those resistant varieties,so the band was regarded as anti-ToMV gene similar sequence. [ Conclusion ] The reseach provides theoretical basis for breeding the tomato varieties which are resistant to ToMV.

  4. The Arabidopsis NPR1 gene confers broad-spectrum disease resistance in strawberry.

    Science.gov (United States)

    Silva, Katchen Julliany P; Brunings, Asha; Peres, Natalia A; Mou, Zhonglin; Folta, Kevin M

    2015-08-01

    Although strawberry is an economically important fruit crop worldwide, production of strawberry is limited by its susceptibility to a wide range of pathogens and the lack of major commercial cultivars with high levels of resistance to multiple pathogens. The objective of this study is to ectopically express the Arabidopsis thaliana NPR1 gene (AtNPR1) in the diploid strawberry Fragaria vesca L. and to test transgenic plants for disease resistance. AtNPR1 is a key positive regulator of the long-lasting broad-spectrum resistance known as systemic acquired resistance (SAR) and has been shown to confer resistance to a number of pathogens when overexpressed in Arabidopsis or ectopically expressed in several crop species. We show that ectopic expression of AtNPR1 in strawberry increases resistance to anthracnose, powdery mildew, and angular leaf spot, which are caused by different fungal or bacterial pathogens. The increased resistance is related to the relative expression levels of AtNPR1 in the transgenic plants. In contrast to Arabidopsis plants overexpressing AtNPR1, which grow normally and do not constitutively express defense genes, the strawberry transgenic plants are shorter than non-transformed controls, and most of them fail to produce runners and fruits. Consistently, most of the transgenic lines constitutively express the defense gene FvPR5, suggesting that the SAR activation mechanisms in strawberry and Arabidopsis are different. Nevertheless, our results indicate that overexpression of AtNPR1 holds the potential for generation of broad-spectrum disease resistance in strawberry.

  5. Multiplex Ligation-Dependent Probe Amplification Analysis of GATA4 Gene Copy Number Variations in Patients with Isolated Congenital Heart Disease

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    Valentina Guida

    2010-01-01

    Full Text Available GATA4 mutations are found in patients with different isolated congenital heart defects (CHDs, mostly cardiac septal defects and tetralogy of Fallot. In addition, GATA4 is supposed to be the responsible gene for the CHDs in the chromosomal 8p23 deletion syndrome, which is recognized as a malformation syndrome with clinical symptoms of facial anomalies, microcephaly, mental retardation, and congenital heart defects. Thus far, no study has been carried out to investigate the role of GATA4 copy number variations (CNVs in non-syndromic CHDs. To explore the possible occurrence of GATA4 gene CNVs in isolated CHDs, we analyzed by multiplex ligation-dependent probe amplification (MLPA a cohort of 161 non-syndromic patients with cardiac anomalies previously associated with GATA4 gene mutations. The patients were mutation-negative for GATA4, NKX2.5, and FOG2 genes after screening with denaturing high performance liquid chromatography. MLPA analysis revealed that normalized MLPA signals were all found within the normal range values for all exons in all patients, excluding a major contribution of GATA4 gene CNVs in CHD pathogenesis.

  6. Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blaCTX-M genes in Enterobacteriaceae.

    Science.gov (United States)

    Monstein, H-J; Ostholm-Balkhed, A; Nilsson, M V; Nilsson, M; Dornbusch, K; Nilsson, L E

    2007-12-01

    Extended-spectrum beta-lactamases (ESBLs) are often mediated by (bla-)SHV, (bla)TEM and (bla)CTX-M genes in Enterobacteriaceae and other Gram-negative bacteria. Numerous molecular typing methods, including PCR-based assays, have been developed for their identification. To reduce the number of PCR amplifications needed we have developed a multiplex PCR assay which detects and discriminates between (bla-)SHV, (bla)TEM and (bla)CTX-M PCR amplicons of 747, 445 and 593 bp, respectively. This multiplex PCR assay allowed the identification of (bla-)SHV, (bla)TEM and (bla)CTX-M genes in a series of clinical isolates of Enterobacteriaceae with previously characterised ESBL phenotype. The presence of (bla)SHV, (bla)TEM and (bla)CTX-M genes was confirmed by partial DNA sequence analysis. Apparently, the universal well-established CTX-M primer pair used here to reveal plasmid-encoded (bla)CTX-M genes would also amplify the chromosomally located K-1 enzyme gene in all Klebsiella oxytoca strains included in the study.

  7. Experimental evolution of resistance to artemisinin combination therapy results in amplification of the mdr1 gene in a rodent malaria parasite.

    Directory of Open Access Journals (Sweden)

    Louise A Rodrigues

    Full Text Available BACKGROUND: Lacking suitable alternatives, the control of malaria increasingly depends upon Artemisinin Combination Treatments (ACT: resistance to these drugs would therefore be disastrous. For ACTs, the biology of resistance to the individual components has been investigated, but experimentally induced resistance to component drugs in combination has not been generated. METHODOLOGY/PRINCIPAL FINDINGS: We have used the rodent malaria parasite Plasmodium chabaudi to select in vivo resistance to the artesunate (ATN+mefloquine (MF version of ACT, through prolonged exposure of parasites to both drugs over many generations. The selection procedure was carried out over twenty-seven consecutive sub-inoculations under increasing ATN+MF doses, after which a genetically stable resistant parasite, AS-ATNMF1, was cloned. AS-ATNMF1 showed increased resistance to ATN+MF treatment and to artesunate or mefloquine administered separately. Investigation of candidate genes revealed an mdr1 duplication in the resistant parasites and increased levels of mdr1 transcripts and protein. There were no point mutations in the atpase6 or ubp1genes. CONCLUSION: Resistance to ACTs may evolve even when the two drugs within the combination are taken simultaneously and amplification of the mdr1 gene may contribute to this phenotype. However, we propose that other gene(s, as yet unidentified, are likely to be involved.

  8. Deletion/duplication mutation screening of TP53 gene in patients with transitional cell carcinoma of urinary bladder using multiplex ligation-dependent probe amplification.

    Science.gov (United States)

    Bazrafshani, Mohammad Reza R; Nowshadi, Pouriaali A; Shirian, Sadegh; Daneshbod, Yahya; Nabipour, Fatemeh; Mokhtari, Maral; Hosseini, Fatemehsadat; Dehghan, Somayeh; Saeedzadeh, Abolfazl; Mosayebi, Ziba

    2016-02-01

    Bladder cancer is a molecular disease driven by the accumulation of genetic, epigenetic, and environmental factors. The aim of this study was to detect the deletions/duplication mutations in TP53 gene exons using multiplex ligation-dependent probe amplification (MLPA) method in the patients with transitional cell carcinoma (TCC). The achieved formalin-fixed paraffin-embedded tissues from 60 patients with TCC of bladder were screened for exonal deletions or duplications of every 12 TP53 gene exons using MLPA. The pathological sections were examined by three pathologists and categorized according to the WHO scoring guideline as 18 (30%) grade I, 22 (37%) grade II, 13 (22%) grade III, and 7 (11%) grade IV cases of TCC. None mutation changes of TP53 gene were detected in 24 (40%) of the patients. Furthermore, mutation changes including, 15 (25%) deletion, 17 (28%) duplication, and 4 (7%) both deletion and duplication cases were observed among 60 samples. From 12 exons of TP53 gene, exon 1 was more subjected to exonal deletion. Deletion of exon 1 of TP53 gene has occurred in 11 (35.4%) patients with TCC. In general, most mutations of TP53, either deletion or duplication, were found in exon 1, which was statistically significant. In addition, no relation between the TCC tumor grade and any type of mutation were observed in this research. MLPA is a simple and efficient method to analyze genomic deletions and duplications of all 12 exons of TP53 gene. The finding of this report that most of the mutations of TP53 occur in exon 1 is in contrast to that of the other reports suggesting that exons 5-8 are the most (frequently) mutated exons of TP53 gene. The mutations of exon 1 of TP53 gene may play an important role in the tumorogenesis of TCC.

  9. 肿瘤细胞回文序列介导的基因扩增机制研究进展%Research progress on the mechanism of gene amplification mediated by palindromes in tumor genome

    Institute of Scientific and Technical Information of China (English)

    刘佳玮; 徐晖; 张海员; 白静; 傅松滨; 周春水

    2014-01-01

    基因组不稳定是肿瘤细胞的特征性标志,基因扩增是一种特殊的基因组不稳定性的表现形式,其在激活原癌基因引发癌症的过程中起着重要的作用.基因扩增将导致细胞加速生长增殖或耐药生长等表型.人们已经广泛注意到其在癌症的诊断和治疗等方面的重要临床意义.但是,人们对基因扩增的分子机制的认识仍然十分有限,本文综述了回文序列介导的基因扩增的分子机制,提出在DNA断点处以无模板形式、新合成小回文序列可能是一部分肿瘤细胞中基因扩增的关键机制,为进一步探索基因扩增的分子机制提供理论参考.%Gene amplification is a common chromosomal aberration which is often the result of tumor genome instability,and plays a crucial role in the process of activating oncogenes and leading to cancers.As gene amplification often is evident in advanced stages of cancer,its clinical importance in diagnosis and treatment has been popularly recognized.However,the underlying mechanisms governing gene amplification are still not fully understood.Herein,we reviewed one of the well-defined gene amplification mechanisms:DNA double-strand breaks-triggered,palindromes or short DNA inverted repeatsmediated gene amplification model,which was established and validated in a variety of research organisms including T.thermophila,fission yeast,budding yeast and rodent cells.Based on the recent published reports,we put forward a new model for the palindromes mediated gene amplification mechanism,that is de novo synthesis of small palindromic sequences (usually several hundred base pairs in length or shorter) at the DNA breakpoints in a template-free manner is the key determinant for gene amplification in certain tumor genomes.Elucidating the potential mechanism and involved enzymes for the regulation and creation of novel palindromic sequences should shed more light onto the palindromes mediated gene amplification and the

  10. Discordant cellular response to pre-surgical letrozole in bilateral synchronous ER+ breast cancers with a KRAS mutation or FGFR1 gene amplification

    Science.gov (United States)

    Balko, Justin M.; Mayer, Ingrid A.; Sanders, Melinda E.; Miller, Todd W.; Kuba, Maria G.; Meszoely, Ingrid M.; Wagle, Nikhil; Garraway, Levi A.; Arteaga, Carlos L.

    2013-01-01

    We describe herein a patient presenting with bilateral ER+ breast tumors who was enrolled in a clinical trial exploring molecular aberrations associated with hormone-refractory tumor cell proliferation. Short-term (2-week) hormonal therapy with the aromatase inhibitor letrozole substantially reduced proliferation as measured by Ki67 immunohistochemistry in one tumor, while the second was essentially unchanged. Extensive molecular and genetic workup of the two tumors yielded divergent lesions in the two tumors: an activating KRAS mutation in the responsive tumor, and an amplification of the FGFR1 locus in the treatment-refractory tumor. These findings provide an insight to possible mechanisms of resistance to antiestrogen therapy in ER+ breast cancers. First, they illustrate the necessity of clinically approved assays to identify FGFR1 gene amplification, which occur in ~5% of breast tumors and have been linked to antiestrogen resistance. It is quite possible that the addition of FGFR inhibitors to ER-targeted therapy will yield a superior antitumor effect and improved patient outcome. Second, they suggest that the role of activating mutations in RAS, although rare in breast cancer, may need to be explored in the context of ER+ breast tumors. PMID:22879364

  11. Target-induced reconfiguration of DNA probes for recycling amplification and signal-on electrochemical detection of hereditary tyrosinemia type I gene.

    Science.gov (United States)

    Dou, Baoting; Yang, Cuiyun; Chai, Yaqin; Yuan, Ruo; Xiang, Yun

    2015-09-01

    By coupling target DNA-induced reconfiguration of the dsDNA probes with enzyme-assisted target recycling amplification, we describe the development of a signal-on electrochemical sensing approach for sensitive detection of hereditary tyrosinemia type I gene. The dsDNA probes are self-assembled on the sensing electrode, and the addition of the target DNA reconfigures and switches the dsDNA probes into active substrates for exonuclease III, which catalytically digests the probes and leads to cyclic reuse of the target DNA. The target DNA recycling and the removal of one of the ssDNA from the dsDNA probes by exonuclease III result in the formation of many hairpin structures on the sensor surface, which brings the electroactive methylene blue labels into proximity with the electrode and produces a significantly amplified current response for sensitive detection of the target gene down to 0.24 pM. This method is also selective to discriminate single-base mismatch and can be employed to detect the target gene in human serum samples. With the demonstration for the detection of the target gene, we expect the developed method to be a universal sensitive sensing platform for the detection of different nucleic acid sequences.

  12. Deletion of gene encoding methyltransferase (gidB) confers high-level antimicrobial resistance in Salmonella.

    Science.gov (United States)

    Mikheil, Dareen M; Shippy, Daniel C; Eakley, Nicholas M; Okwumabua, Ogi E; Fadl, Amin A

    2012-04-01

    The glucose-inhibited division gene (gid)B, which resides in the gid operon, was thought to have a role in the modulation of genes similar to that of gidA. Recent studies have indicated that GidB is a methyltransferase enzyme that is involved in the methylation of the 16S ribosomal RNA (rRNA) in Escherichia coli. In this study, we investigated the role of GidB in susceptibility to antibiotics and the overall biology of Salmonella. A gidB isogenic mutant of Salmonella was constructed and subsequently characterized under different conditions. Our data indicated that growth and invasion characteristics of the gidB mutant were similar to those of the wild type (WT). The gidB mutant was outgrown by the WT in a competitive growth assay, indicating a compromised overall bacterial fitness. Under the stress of nalidixic acid, the gidB mutant's motility was significantly reduced. Similarly, the mutant showed a filamentous morphology and smaller colony size compared with the rod-shaped and large colonies of the WT in the presence of nalidixic acid. Most importantly, deletion of gidB conferred high-level resistance to the aminoglycoside antibiotics streptomycin and neomycin. A primer extension assay determined the methylation site for the WT to be at G527 of the 16S rRNA. A lack of methylation in the mutant indicated that GidB is required for this methylation. Taken together, these data indicate that the GidB enzyme has a significant role in the alteration of antibiotic susceptibility and the modulation of growth and morphology under stress conditions in Salmonella.

  13. The wheat durable, multipathogen resistance gene Lr34 confers partial blast resistance in rice.

    Science.gov (United States)

    Krattinger, Simon G; Sucher, Justine; Selter, Liselotte L; Chauhan, Harsh; Zhou, Bo; Tang, Mingzhi; Upadhyaya, Narayana M; Mieulet, Delphine; Guiderdoni, Emmanuel; Weidenbach, Denise; Schaffrath, Ulrich; Lagudah, Evans S; Keller, Beat

    2016-05-01

    The wheat gene Lr34 confers durable and partial field resistance against the obligate biotrophic, pathogenic rust fungi and powdery mildew in adult wheat plants. The resistant Lr34 allele evolved after wheat domestication through two gain-of-function mutations in an ATP-binding cassette transporter gene. An Lr34-like fungal disease resistance with a similar broad-spectrum specificity and durability has not been described in other cereals. Here, we transformed the resistant Lr34 allele into the japonica rice cultivar Nipponbare. Transgenic rice plants expressing Lr34 showed increased resistance against multiple isolates of the hemibiotrophic pathogen Magnaporthe oryzae, the causal agent of rice blast disease. Host cell invasion during the biotrophic growth phase of rice blast was delayed in Lr34-expressing rice plants, resulting in smaller necrotic lesions on leaves. Lines with Lr34 also developed a typical, senescence-based leaf tip necrosis (LTN) phenotype. Development of LTN during early seedling growth had a negative impact on formation of axillary shoots and spikelets in some transgenic lines. One transgenic line developed LTN only at adult plant stage which was correlated with lower Lr34 expression levels at seedling stage. This line showed normal tiller formation and more importantly, disease resistance in this particular line was not compromised. Interestingly, Lr34 in rice is effective against a hemibiotrophic pathogen with a lifestyle and infection strategy that is different from obligate biotrophic rusts and mildew fungi. Lr34 might therefore be used as a source in rice breeding to improve broad-spectrum disease resistance against the most devastating fungal disease of rice.

  14. Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis.

    Science.gov (United States)

    Hirasawa, Takashi; Yoshikawa, Katsunori; Nakakura, Yuki; Nagahisa, Keisuke; Furusawa, Chikara; Katakura, Yoshio; Shimizu, Hiroshi; Shioya, Suteaki

    2007-08-01

    During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.

  15. Amplification of cellular oncogenes in solid tumors

    Directory of Open Access Journals (Sweden)

    Ozkan Bagci

    2015-01-01

    Full Text Available The term gene amplification refers to an increase in copy number of a gene. Upregulation of gene expression through amplification is a general mechanism to increase gene dosage. Oncogene amplifications have been shown in solid human cancers and they are often associated with progression of cancer. Defining oncogene amplification is useful since it is used as a prognostic marker in clinical oncology nowadays, especially v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (HER2 targeted agents are used in breast cancer patients with high level of HER2 overexpression as a therapeutic approach. However, patients without HER2 overexpression do not appear to benefit from these agents. We concluded that determination of oncogene amplification in solid tumors is an important factor in treatment of human cancers with many unknowns. We have referred to PubMed and some databases to prepare this article.

  16. Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

    Directory of Open Access Journals (Sweden)

    Puisieux Alain

    2003-10-01

    Full Text Available Abstract Background Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. Results We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC in peripheral blood mononuclear cells; the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Conclusion Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

  17. Identification of planorbids from Venezuela by polymerase chain reaction amplification and restriction fragment length polymorphism of internal transcriber spacer of the RNA ribosomal gene

    Directory of Open Access Journals (Sweden)

    Caldeira Roberta L

    2000-01-01

    Full Text Available Snails of the genus Biomphalaria from Venezuela were subjected to morphological assessment as well as polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP analysis. Morphological identification was carried out by comparison of characters of the shell and the male and female reproductive apparatus. The PCR-RFLP involved amplification of the internal spacer region ITS1 and ITS2 of the RNA ribosomal gene and subsequent digestion of this fragment by the restriction enzymes DdeI, MnlI, HaeIII and MspI. The planorbids were compared with snails of the same species and others reported from Venezuela and present in Brazil, Cuba and Mexico. All the enzymes showed a specific profile for each species, that of DdeI being the clearest. The snails were identified as B. glabrata, B. prona and B. kuhniana.

  18. Map - vs. homology - based cloning for the recessive gene ol-2 conferring resistance to tomato powdery mildew

    OpenAIRE

    Pavan, S.N.C.; Zheng, Z.; Borisova, M.; Berg, van den, G.J.; Lotti, C.; Giovanni, , da Bergamo; Lindhout, P.; de Jong; Ricciardi, L.; Visser, R.G.F.; Bai, Y.

    2008-01-01

    The recessive gene ol-2 confers papilla-associated and race-non-specific resistance to tomato powdery mildew caused by Oidium neolycopersici. In order to facilitate marker assisted selection (MAS) in practical breeding programmes, we identified two simple sequence repeat (SSR) markers and one cleaved amplified polymorphic sequence (CAPS) marker which are linked to the resistance locus and co-dominantly inherited. Aiming to provide a base for ol-2 positional cloning, we used a large segregatin...

  19. Gene amplification and immunohistochemical expression of ERBB2 and EGFR in cervical carcinogenesis. Correlation with cell-cycle markers and HPV presence.

    Science.gov (United States)

    Conesa-Zamora, Pablo; Torres-Moreno, Daniel; Isaac, María A; Pérez-Guillermo, Miguel

    2013-10-01

    Although the members of the epidermal growth factor receptor family ERBB2 and EGFR are important therapeutic targets in the treatment of malignant neoplasias, little is known about their role in cervical carcinogenesis. Our objective was to evaluate the dysfunction of ERBB2 and EGFR at the gene copy number and protein expression level in neoplastic lesions of the uterine cervix with the aim of obtaining information about its role in cervical carcinogenesis and their possible use as therapeutic targets in these diseases. We studied gene amplification and protein expression of ERBB2 and EGFR and their relationship with Ki67, p16 and p53 and HPV presence in 22 normal/benign (N/B) cervices, 20 low-grade squamous intraepithelial lesions (LSILs), 70 high-grade SILs (HSILs) and 32 invasive squamous cervical carcinomas (ISCCs). No cases showed selective amplification of ERBB2 or EGFR but corresponding chromosome-specific probes displayed chromosome 17 and 7 polyploidy associated with the grade of the lesion (plesions (N/B plus LSIL 21.4% vs. HSIL plus ISCC 45.5%; p=0.007). No association was observed between EGFR expression and that of cell-cycle markers or HPV presence. Increased copy number of EGFR and ERBB2 is due to polyploidy of 7 and 17 chromosomes, this being a phenomenon associated with lesion severity and with an increase in the expression of cell-cycle markers. EGFR, but not ERBB2, is expressed in precursor lesions of squamous cervical neoplasia and is related to the neoplastic progression but not to proliferation marker expression and therefore ERBB2 and this calls into question the usefulness of ERBB2 as a therapeutic target.

  20. Correlation of HER2 overexpression with gene amplification and its relation to chromosome 17 aneuploidy: a 5-year experience with invasive ductal and lobular carcinomas.

    Science.gov (United States)

    Nassar, Aziza; Khoor, Andras; Radhakrishnan, Reshmitha; Radhakrishnan, Anu; Cohen, Cynthia

    2014-01-01

    The HER2 oncogene shows expression or amplification, or both, in approximately 15% to 20% of breast cancers and has been associated with poor prognosis and a response to trastuzumab therapy. HER2 gene status determines the eligibility of breast cancer patients for trastuzumab therapy and a large fraction (41-56%) of these patients respond to targeted therapy. Several studies have related the increased expression of HER2 to an increased copy number of chromosome 17, rather than amplification of the HER2 gene. We compared the results of immunohistochemistry and fluorescence in situ hybridization in both invasive ductal and invasive lobular carcinomas, to determine the frequency of chromosome 17 aneuploidy associated with discordant results. In total, 390 invasive ductal carcinomas and 180 invasive lobular carcinomas diagnosed from January 2000 to December 2005 were included in the study only if results were available for immunohistochemistry (HercepTest; DAKO, Carpinteria, California) and fluorescence in situ hybridization (PathVysion HER2 DNA Probe Kit; Abbott Laboratories, Des Plaines, Illinois). Tumors classified as invasive ductal carcinomas were graded according to the Bloom-Richardson grading system. Correlation between the results of immunohistochemistry and fluorescence in situ hybridization was performed for all categories. Among invasive ductal carcinomas, 29% (115/390) showed chromosome 17 aneuploidy, mostly associated with grade 3/HER2 2+ (45%) or grade 2/HER2 3+ (55%) that were not amplified. Also, 34% (12/35) of invasive lobular carcinomas showed chromosome 17 aneuploidy; approximately one-third of these cases were HER2 2+ (33%) and HER2 3+ (37%) that were not amplified. Discordance between the results of immunohistochemistry and fluorescence in situ hybridization in both ductal and lobular carcinomas is largely associated with chromosome 17 aneuploidy.

  1. Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of the tdh and trh genes of Vibrio parahaemolyticus and related Vibrio species.

    Science.gov (United States)

    Yamazaki, Wataru; Kumeda, Yuko; Misawa, Naoaki; Nakaguchi, Yoshitsugu; Nishibuchi, Mitsuaki

    2010-02-01

    Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.

  2. Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

    Directory of Open Access Journals (Sweden)

    Feiwu Li

    2014-08-01

    Full Text Available The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

  3. Resistance to powdery mildew in the pea cultivar Xucai 1 is conferred by the gene er1

    Institute of Scientific and Technical Information of China (English)

    Suli Sun; Zhongyi Wang; Haining Fu; Canxing Duan; Xiaoming Wang; Zhendong Zhu

    2015-01-01

    Powdery mildew, caused by Erysiphe pisi D.C., is a major constraint to pea production worldwide. The pea cultivar Xucai 1 has shown high resistance to E. pisi under greenhouse and field conditions. The objectives of this study were to identify and characterize genes conferring resistance to powdery mildew in Xucai 1. Three crosses, Qizhen 76 × Xucai 1,Bawan 6 × Xucai 1, and Xucai 1 × Bawan 6, were made to generate populations for genetic analysis. The resistance to E. pisi and segregation ratios in the F1, F2, and F2:3populations suggested a single recessive gene conferring the resistance of Xucai 1. Bulked segregant analysis was used to map the resistance gene using two F2 populations. The resistance gene was close to markers AD60 and c5 DNAmet on linkage group VI with genetic distances of9.9 c M and 15.4 c M in the Xucai 1 × Bawan 6 F2 population and 8.7 c M and 8.1 c M in the Qizhen 76 × Xucai 1 F2 population, respectively, suggesting that the resistance gene was an er1 allele. This hypothesis was confirmed by comparison of the c DNA sequences of the Ps MLO1 gene between the parents and the Ps MLO1 wild type. Three distinct types of transcripts in Xucai 1, characterized by a 129-bp deletion and 155- and 220-bp insertions,were detected, consistent with the structure of the er1-2 allele. We concluded that resistance in Xucai 1 was conferred by er1-2 and that its linked markers will be useful in pea breeding programs.

  4. An improved method for detection of Edwardsiella tarda by loop-mediated isothermal amplification by targeting the EsrB gene

    Institute of Scientific and Technical Information of China (English)

    XIE Guosi; ZHANG Qingli; HAN Nana; SHI Chengyin; WANG Xiuhua; LIU Qinghui; HUANG Jie

    2012-01-01

    Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses.An improved method for quick and accurate detection of E.tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene (EsrBLAMP).In this method,the Mg2+ concentration,reaction temperature,and reaction time were optimized to 8 mmol/L,61℃,and 40 min,respectively.The detection limit with the EsrB gene was as low as 10 copies,which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR).The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene (hemolysin-LAMP) for detection ofE.tarda.The EsrB-LAMP was also highly specific to E.tarda and had no cross-reaction with 13 other strains of bacteria.The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture.Taken together,the improved EsrB-LAMP diagnostic protocol has the potential for detection ofE.tarda from indoor and outdoor samples.

  5. Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset

    Directory of Open Access Journals (Sweden)

    Nayereh Nouri

    2014-01-01

    Full Text Available Background: The Duchenne muscular dystrophy (DMD gene is located in the short arm of the X chromosome (Xp21. It spans 2.4 Mb of the human genomic DNA and is composed of 79 exons. Mutations in the Dystrophin gene result in DMD and Becker muscular dystrophy. In this study, the efficiency of multiplex ligation-dependent probe amplification (MLPA over multiplex polymerase chain reaction (PCR assays in an Iranian population was investigated. Materials and Methods: Multiplex PCR assays and MLPA analysis were carried out in 74 patients affected with DMD. Results: Multiplex PCR detected deletions in 51% of the patients with DMD. MLPA analysis could determine all the deletions detected by the multiplex PCR. Additionally, MLPA was able to identify one more deletion and duplication in patients without detectable mutations by multiplex PCR. Moreover, MLPA precisely determined the exact size of the deletions. Conclusion: Although MLPA analysis is more sensitive for detection of deletions and duplications in the dystrophin gene, multiplex PCR might be used for the initial analysis of the boys affected with DMD in the Iranian population as it was able to detect 95% of the rearrangements in patients with DMD.

  6. A PCR-based sex determination method for possible application in caprine gender selection by simultaneous amplification of the Sry and Aml-X genes.

    Science.gov (United States)

    Phua, Alice Choon Yen; Abdullah, Ramli Bin; Mohamed, Zulqarnain

    2003-08-01

    Sex determination of livestock is performed to achieve the objectives of livestock breeding programmes. Techniques for sex determination have evolved from karyotyping to detecting Y-specific antigens and recently to the polymerase chain reaction (PCR), which appears to be the most sensitive, accurate, rapid and reliable method to date. In this study, a PCR-based sex determination method for potential application in goat breeding programmes was developed. Primers were designed to amplify a portion of the X amelogenin gene (Aml-X) on the X chromosome to give a 300 bp product and Sry gene on the Y chromosome to give a 116 bp product. PCR optimization was performed using DNA template extracted from a whole blood sample of Jermasia goats (German Fawn x Katjang) of both sexes. It was possible to identify the sex chromosomes by amplifying both male- and female-specific genes simultaneously in a duplex reaction with males yielding two bands and females yielding one band. The Aml-X primer set, which served as an internal control primer, did not interfere with amplification of the Y-specific sequence even when a low amount of DNA (1 ng) was used. The duplex reaction subjected to a blind test showed 100% (14/14) concordance, proving its accuracy and reliability. The primer sets used were found to be highly specific and were suitable for gender selection of goats.

  7. Rapid, Sensitive Detection of Bartonella quintana by Loop-Mediated Isothermal Amplification of the groEL Gene

    Science.gov (United States)

    Hu, Shoukui; Niu, Lina; Luo, Lijuan; Song, Xiuping; Sun, Jimin; Liu, Qiyong

    2016-01-01

    Trench fever, caused by Bartonella quintana, is recognized as a re-emerging and neglected disease. Rapid and sensitive detection approaches are urgently required to monitor and help control B. quintana infections. Here, loop-mediated isothermal amplification (LAMP), which amplifies target DNA at a fixed temperature with high sensitivity, specificity and rapidity, was employed to detect B. quintana. Thirty-six strains, including 10 B. quintana, 13 other Bartonella spp., and 13 other common pathogens, were applied to verify and evaluate the LAMP assay. The specificity of the LAMP assay was 100%, and the limit of detection was 125 fg/reaction. The LAMP assay was compared with qPCR in the examination of 100 rhesus and 20 rhesus-feeder blood samples; the diagnostic accuracy was found to be 100% when LAMP was compared to qPCR, but the LAMP assay was significantly more sensitive (p < 0.05). Thus, LAMP methodology is a useful for diagnosis of trench fever in humans and primates, especially in low-resource settings, because of its rapid, sensitive detection that does not require sophisticated equipment. PMID:27916953

  8. Rapid, Sensitive Detection of Bartonella quintana by Loop-Mediated Isothermal Amplification of the groEL Gene

    Directory of Open Access Journals (Sweden)

    Shoukui Hu

    2016-12-01

    Full Text Available Trench fever, caused by Bartonella quintana, is recognized as a re-emerging and neglected disease. Rapid and sensitive detection approaches are urgently required to monitor and help control B. quintana infections. Here, loop-mediated isothermal amplification (LAMP, which amplifies target DNA at a fixed temperature with high sensitivity, specificity and rapidity, was employed to detect B. quintana. Thirty-six strains, including 10 B. quintana, 13 other Bartonella spp., and 13 other common pathogens, were applied to verify and evaluate the LAMP assay. The specificity of the LAMP assay was 100%, and the limit of detection was 125 fg/reaction. The LAMP assay was compared with qPCR in the examination of 100 rhesus and 20 rhesus-feeder blood samples; the diagnostic accuracy was found to be 100% when LAMP was compared to qPCR, but the LAMP assay was significantly more sensitive (p < 0.05. Thus, LAMP methodology is a useful for diagnosis of trench fever in humans and primates, especially in low-resource settings, because of its rapid, sensitive detection that does not require sophisticated equipment.

  9. Relation Among DNA Content, N-myc Gene Amplification and the Clinical Prognosis%小儿神经母细胞瘤DNA含量、N-myc基因扩增与临床预后的关系

    Institute of Scientific and Technical Information of China (English)

    刘获; 吴建新; 呼守生; 马汝柏; 林久治

    1995-01-01

    本文比较分析了18例神经母细胞瘤的DNA含量、N-myc基因扩增、临床分期、原发部位等因素与预后的关系.结果表明,有N-myc基因扩增的死亡率(7/10)明显高于无扩增的(1/8);DNA含量增高与肿瘤的恶性度成正比;临床分期与N-myc基因扩增情况相结合与病死率的关系极为密切.%In this study, 18 children with neuroblastoma were analysed on the relation among DNA content, N-myc gene amplification and the clinical staging. The results reveal; the mortality of patients with N-myc gene amplification (7/10) was higher than those without N-myc gene amplification (1/8); DNA content was related to malignancy of the tumor, combination of N-myc gene amplification and the clinical staging kept close link with the prognosis.

  10. The Batten disease gene CLN3 confers resistance to endoplasmic reticulum stress induced by tunicamycin

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Dan, E-mail: danw@bjmu.edu.cn [Department of Medical Genetics, Peking University Health Science Center, No 38 Xueyuan Road, Haidian district, Beijing 100191 (China); Liu, Jing; Wu, Baiyan [Department of Medical Genetics, Peking University Health Science Center, No 38 Xueyuan Road, Haidian district, Beijing 100191 (China); Tu, Bo; Zhu, Weiguo [Department of Biochemistry and Molecular Biology, Peking University Health Science Center, No 38 Xueyuan Road, Haidian district, Beijing 100191 (China); Luo, Jianyuan, E-mail: jluo@som.umaryland.edu [Department of Medical Genetics, Peking University Health Science Center, No 38 Xueyuan Road, Haidian district, Beijing 100191 (China); Department of Medical and Research Technology, School of Medicine, University of Maryland, Baltimore 21201 (United States)

    2014-04-25

    Highlights: • The work reveals a protective properties of CLN3 towards TM-induced apoptosis. • CLN3 regulates expression of the GRP78 and the CHOP in response to the ER stress. • CLN3 plays a specific role in the ERS response. - Abstract: Mutations in CLN3 gene cause juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), an early-onset neurodegenerative disorder that is characterized by the accumulation of ceroid lipofuscin within lysosomes. The function of the CLN3 protein remains unclear and is presumed to be related to Endoplasmic reticulum (ER) stress. To investigate the function of CLN3 in the ER stress signaling pathway, we measured proliferation and apoptosis in cells transfected with normal and mutant CLN3 after treatment with the ER stress inducer tunicamycin (TM). We found that overexpression of CLN3 was sufficient in conferring increased resistance to ER stress. Wild-type CLN3 protected cells from TM-induced apoptosis and increased cell proliferation. Overexpression of wild-type CLN3 enhanced expression of the ER chaperone protein, glucose-regulated protein 78 (GRP78), and reduced expression of the proapoptotic protein CCAAT/-enhancer-binding protein homologous protein (CHOP). In contrast, overexpression of mutant CLN3 or siRNA knockdown of CLN3 produced the opposite effect. Together, our data suggest that the lack of CLN3 function in cells leads to a failure of management in the response to ER stress and this may be the key deficit in JNCL that causes neuronal degeneration.

  11. Amplification of the aroA gene from Escherichia coli results in tolerance to the herbicide glyphosate.

    OpenAIRE

    Rogers, S G; Brand, L A; Holder, S B; Sharps, E S; Brackin, M J

    1983-01-01

    The predominant cellular target of the herbicide glyphosate is thought to be the enzyme 5-enolpyruvylshikimate-3-phosphoric acid synthase (EPSP synthase). As a means of biologically testing this finding, we cloned a segment of DNA from Escherichia coli that encodes this enzyme. Clones carrying the gene for EPSP synthase were identified by genetic complementation. Cells that contain a multicopy plasmid carrying the EPSP synthase gene overproduce the enzyme 5- to 17-fold and exhibit at least an...

  12. Risk-Conferring Glutamatergic Genes and Brain Glutamate Plus Glutamine in Schizophrenia

    Directory of Open Access Journals (Sweden)

    Juan R. Bustillo

    2017-06-01

    Full Text Available BackgroundThe proton magnetic resonance spectroscopy (1H-MRS signals from glutamate (or the combined glutamate and glutamine signal—Glx have been found to be greater in various brain regions in people with schizophrenia. Recently, the Psychiatric Genetics Consortium reported that several common single-nucleotide polymorphisms (SNPs in glutamate-related genes confer increased risk of schizophrenia. Here, we examined the relationship between presence of these risk polymorphisms and brain Glx levels in schizophrenia.Methods1H-MRS imaging data from an axial, supraventricular tissue slab were acquired in 56 schizophrenia patients and 67 healthy subjects. Glx was measured in gray matter (GM and white matter (WM regions. The genetic data included six polymorphisms genotyped across an Illumina 5M SNP array. Only three of six glutamate as well as calcium-related SNPs were available for examination. These included three glutamate-related polymorphisms (rs10520163 in CLCN3, rs12704290 in GRM3, and rs12325245 in SLC38A7, and three calcium signaling polymorphisms (rs1339227 in RIMS1, rs7893279 in CACNB2, and rs2007044 in CACNA1C. Summary risk scores for the three glutamate and the three calcium polymorphisms were calculated.ResultsGlx levels in GM positively correlated with glutamate-related genetic risk score but only in younger (≤36 years schizophrenia patients (p = 0.01. Glx levels did not correlate with calcium risk scores. Glx was higher in the schizophrenia group compared to levels in controls in GM and WM regardless of age (p < 0.001.ConclusionElevations in brain Glx are in part, related to common allelic variants of glutamate-related genes known to increase the risk for schizophrenia. Since the glutamate risk scores did not differ between groups, some other genetic or environmental factors likely interact with the variability in glutamate-related risk SNPs to contribute to an increase in brain Glx early in the illness.

  13. A new morphologically distinct avian malaria parasite that fails detection by established polymerase chain reaction-based protocols for amplification of the cytochrome B gene.

    Science.gov (United States)

    Zehtindjiev, Pavel; Križanauskienė, Asta; Bensch, Staffan; Palinauskas, Vaidas; Asghar, Muhammad; Dimitrov, Dimitar; Scebba, Sergio; Valkiūnas, Gediminas

    2012-06-01

    Plasmodium polymorphum n. sp. (Haemosporida, Plasmodiidae) was found in the skylark, Alauda arvensis (Passeriformes: Alaudidae), during autumnal migration in southern Italy. This organism is illustrated and described based on the morphology of its blood stages. The most distinctive feature of this malaria parasite is the clear preference of its blood stages (trophozoites, meronts, and gametocytes) for immature red blood cells, including erythroblasts. Based on preference of erythrocytic meronts for immature red blood cells, P. polymorphum is most similar to species of the subgenus Huffia . This parasite can be readily distinguished from all other bird malaria parasites, including Plasmodium ( Huffia ) spp., due to preferential development and maturation of its gametocytes in immature red blood cells, a unique character for avian Plasmodium spp. In addition, the margins of nuclei in blood stages of P. polymorphum are markedly smooth and distinct; this is also a distinct diagnostic feature of this parasite. Plasmodium polymorphum has been recorded only in the skylark; it is probably a rare parasite, whose host range and geographical distribution remain unclear. Microscopic examination detected a light infection of Plasmodium relictum (lineage GRW11, parasitemia of mitochondrial cytochrome b gene (cyt b ) of P. polymorphum from the microscopically positive sample by using published and newly designed primers for DNA amplification of avian Plasmodium spp. The light parasitemia of P. relictum was easily detectable using several polymerase chain reaction (PCR)-based assays, but P. polymorphum was undetectable in all applied assays. Quantitative PCR also showed the presence of light parasitemia (0.06%) of the lineage GRW11 in this sample. This supports the conclusion that the morphologically distinct parasite observed along with P. relictum and predominant in the sample is genetically dissimilar from the lineage GRW11 based on cyt b sequence. In samples with co

  14. 副溶血弧菌tdh基因LAMP检测技术的建立%Establishment of Loop-Mediated Isothermal Amplification for Detecting Vibrio Parahaemolyticus tdh Gene

    Institute of Scientific and Technical Information of China (English)

    程晓艳; 刘庆慧; 黄倢

    2012-01-01

    Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification technology which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. In this paper, LAMP was applied for rapid detection of Vibrio parahaemolyticus. According to the sequence of the specific tdh gene of Vibrio parahaemolyticus, a set of primers was designed to amplify the specifial DNA sequences by LAMP. Moreover, the reaction conditions were optimized. The results showed the optimum LAMP assay for the rapid detection of Vibrio paiakaemolyticus was performed at 60.8 ℃ for 45 min. No crossreaction was found with other common bacteria which showed a good specificity. The LAMP assay had an detection limit of 35.5 fg/tube, which was 10 times lower than that of PCR assay. The LAMP assay could be finished within an hour requiring only a regular laboratory water bath for reaction. The detection of the strains isolated from scallop also proved the LAMP assay reliability.%环等温扩增技术(Loop-mediated isothermal amplification,LAMP)是一种在等温条件下高特异性、高效、快速地扩增靶基因的DNA扩增技术.根据副溶血弧菌特异性的毒力基因tdh保守序列,本文设计1套特异性引物对该基因进行环等温扩增,同时对反应条件进行优化,建立携带tdh基因的致病性副溶血弧菌的LAMP快速检测技术.结果表明,LAMP最适反应在60.8℃恒温、45 min内完成,与其它常见的细菌无交叉反应.LAMP方法的细菌DNA最低检出限为35.5 fg/反应管,灵敏度较PCR方法高10倍.对扇贝样品中分离的菌株的检测表明,LAMP方法对检测致病性副溶血弧菌具有良好的可靠性.

  15. Hyd5 gene-based detection of the major gushing-inducing Fusarium spp. in a loop-mediated isothermal amplification (LAMP) assay.

    Science.gov (United States)

    Denschlag, Carla; Vogel, Rudi F; Niessen, Ludwig

    2012-06-01

    Fusarium graminearum and the closely related F. culmorum were found to be associated with over foaming of bottled beer (gushing) when contaminated brewing malt is used. The presence of highly surface active hydrophobins produced by these fungi upon growth on wheat or barley in the field or during malting may affect bubble formation and stability in gushing beers and other carbonated beverages. Aiming for a method for the rapid and user friendly analysis of unmalted and malted cereals during quality control in the brewing industry, a loop-mediated isothermal amplification (LAMP) assay for the detection of Fusarium spp. capable of producing the gushing inducing hydrophobin Hyd5p was set up. A set of primers was designed towards a 221 bp region within the hyd5 gene of F. culmorum. The LAMP product was verified by sequencing a 150 bp portion. Testing specificity with purified DNA from 99 different fungal species as well as barley and wheat showed that DNA synthesis only occurred during LAMP when DNA of the closely related species F. graminearum, F. culmorum, F. cerealis and F. lunulosporum were used as template. In-tube indirect detection of DNA amplification was applied using manganese-quenched calcein as fluorescence indicator for pyrophosphate produced during DNA synthesis. The assay had a detection limit of 0.74 pg of purified target DNA which corresponds 20 copy numbers per reaction within 30 minutes using a simple heating block. Analysis of Fusarium infected cereals revealed that the assay was able to detect F. graminearum at a level of 0.5% of infected grains in uninfected barley by analysis of surface washings without further sample preparation. Results show that the hyd5 based LAMP assay can be a rapid, useful and sensitive tool for quality control in the brewing and malting industry.

  16. Chromosomal localization of the murine RFC-1 gene encoding a folate transporter and its amplification in an antifolate resistant variant overproducing the transporter.

    Science.gov (United States)

    Roy, K; Chiao, J H; Spengler, B A; Tolner, B; Yang, C H; Biedler, J L; Sirotnak, F M

    1998-08-01

    83 cells. These results document the amplification at the site of a putative HSR in an L1210 cell variant of the RFC-1 gene regulating expression of the one-carbon, reduced folate transporter.

  17. Identification of Tunisian Leishmania spp. by PCR amplification of cysteine proteinase B (cpb) genes and phylogenetic analysis.

    Science.gov (United States)

    Chaouch, Melek; Fathallah-Mili, Akila; Driss, Mehdi; Lahmadi, Ramzi; Ayari, Chiraz; Guizani, Ikram; Ben Said, Moncef; Benabderrazak, Souha

    2013-03-01

    Discrimination of the Old World Leishmania parasites is important for diagnosis and epidemiological studies of leishmaniasis. We have developed PCR assays that allow the discrimination between Leishmania major, Leishmania tropica and Leishmania infantum Tunisian species. The identification was performed by a simple PCR targeting cysteine protease B (cpb) gene copies. These PCR can be a routine molecular biology tools for discrimination of Leishmania spp. from different geographical origins and different clinical forms. Our assays can be an informative source for cpb gene studying concerning drug, diagnostics and vaccine research. The PCR products of the cpb gene and the N-acetylglucosamine-1-phosphate transferase (nagt) Leishmania gene were sequenced and aligned. Phylogenetic trees of Leishmania based cpb and nagt sequences are close in topology and present the classic distribution of Leishmania in the Old World. The phylogenetic analysis has enabled the characterization and identification of different strains, using both multicopy (cpb) and single copy (nagt) genes. Indeed, the cpb phylogenetic analysis allowed us to identify the Tunisian Leishmania killicki species, and a group which gathers the least evolved isolates of the Leishmania donovani complex, that was originated from East Africa. This clustering confirms the African origin for the visceralizing species of the L. donovani complex.

  18. Enumeration of total bacteria and bacteria with genes for proteolytic activity in pure cultures and in environmental samples by quantitative PCR mediated amplification.

    Science.gov (United States)

    Bach, H-J; Tomanova, J; Schloter, M; Munch, J C

    2002-05-01

    Real-time quantitative PCR assays were developed for the absolute quantification of different groups of bacteria in pure cultures and in environmental samples. 16S rRNA genes were used as markers for eubacteria, and genes for extracellular peptidases were used as markers for potentially proteolytic bacteria. For the designed 16S rDNA TaqMan assay, specificity of the designed primer-probe combination for eubacteria, a high amplification efficiency over a wide range of starting copy numbers and a high reproducibility is demonstrated. Cell concentrations of Bacillus cereus, B. subtilis and Pseudomonas fluorescens in liquid culture were monitored by TaqMan-PCR using the 16S rDNA target sequence of Escherichia coli as external standard for quantification. Results agree with plate counts and microscopic counts of DAPI stained cells. The significance of 16S rRNA operon multiplicity to the quantification of bacteria is discussed.Furthermore, three sets of primer pair together with probe previously designed for targeting different classes of bacterial extracellular peptidases were tested for their suitability for TaqMan-PCR based quantification of proteolytic bacteria. Since high degeneracy of the probes did not allow accurate quantification, SybrGreen was used instead of molecular probes to visualize and quantify PCR products during PCR. The correlation between fluorescence and starting copy number was of the same high quality as for the 16S rDNA TaqMan assay for all the three peptidase gene classes. The detected amount of genes for neutral metallopeptidase of B. cereus, for subtilisin of B. subtilis and for alkaline metallopeptidase of P. fluorescens corresponded exactly to the numbers of bacteria investigated by the 16S rDNA targeting assay. The developed assays were applied for the quantification of bacteria in soil samples.

  19. Loop-Mediated Isothermal Amplification of the sefA Gene for Rapid Detection of Salmonella Enteritidis and Salmonella Gallinarum in Chickens.

    Science.gov (United States)

    Gong, Jiansen; Zhuang, Linlin; Zhu, Chunhong; Shi, Shourong; Zhang, Di; Zhang, Linji; Yu, Yan; Dou, Xinhong; Xu, Bu; Wang, Chengming

    2016-04-01

    Salmonella spp. pose a threat to both human and animal health, with more than 2600 serovars having been reported to date. Salmonella serovars are usually identified by slide agglutination tests, which are labor intensive and time consuming. In an attempt to develop a more rapid screening method for the major poultry Salmonella serovars, we developed a loop-mediated isothermal amplification (LAMP) assay, which directly detected the sefA gene, a fimbrial operon gene existing in several specific serovars of Salmonella enterica including the major poultry serovars, namely Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) and Salmonella enterica serovar Gallinarum (Salmonella Gallinarum). With the 177 bacterial strains we tested, positive reactions were only observed with 85 strains of serovar Salmonella Enteritidis and Salmonella Gallinarum. The detection limit of the LAMP assay was 4 CFU/reaction with genomic DNAs of Salmonella Enteritidis (ATCC 13076) from pure culture and 400 CFU/ reaction with DNA extracted from spiked chicken feces. The LAMP assay was more sensitive than conventional culture, especially without enrichment, in detecting Salmonella Enteritidis (CMCC 50041) in the spiked fecal samples. The results show the sefA LAMP method is a rapid, sensitive, specific, and practical method for directly detection of Salmonella Enteritidis and Salmonella Gallinarum in chickens. The sefA LAMP assay can potentially serve as new on-site diagnostics in the poultry industry.

  20. Specific amplification of gene encoding N-terminal region of catalase-peroxidase protein (KatG-N) for diagnosis of disseminated MAC disease in HIV patients.

    Science.gov (United States)

    Latawa, Romica; Singh, Krishna Kumar; Wanchu, Ajay; Sethi, Sunil; Sharma, Kusum; Sharma, Aman; Laal, Suman; Verma, Indu

    2014-10-01

    Disseminated Mycobacterium avium-intracellulare complex (MAC) infection is considered as severe complication of advanced HIV/AIDS disease. Currently available various laboratory investigations have not only limited ability to discriminate between MAC infection and tuberculosis but are also laborious and time consuming. The aim of this study was, therefore, to design a molecular-based strategy for specific detection of MAC and its differentiation from Mycobacterium tuberculosis (M. tb) isolated from the blood specimens of HIV patients. A simple PCR was developed based on the amplification of 120-bp katG-N gene corresponding to the first 40 amino acids of N-terminal catalase-peroxidase (KatG) protein of Mycobacterium avium that shows only ~13% sequence homology by clustal W alignment to N-terminal region of M. tb KatG protein. This assay allowed the accurate and rapid detection of MAC bacteremia, distinguishing it from M. tb in a single PCR reaction without any need for sequencing or hybridization protocol to be performed thereafter. This study produced enough evidence that a significant proportion of Indian HIV patients have disseminated MAC bacteremia, suggesting the utility of M. avium katG-N gene PCR for early detection of MAC disease in HIV patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Dissection of two soybean QTL conferring partial resistance to Phytophthora sojae through sequence and gene expression analysis

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    Wang Hehe

    2012-08-01

    Full Text Available Abstract Background Phytophthora sojae is the primary pathogen of soybeans that are grown on poorly drained soils. Race-specific resistance to P. sojae in soybean is gene-for-gene, although in many areas of the US and worldwide there are populations that have adapted to the most commonly deployed resistance to P. sojae ( Rps genes. Hence, this system has received increased attention towards identifying mechanisms and molecular markers associated with partial resistance to this pathogen. Several quantitative trait loci (QTL have been identified in the soybean cultivar ‘Conrad’ that contributes to the expression of partial resistance to multiple P. sojae isolates. Results In this study, two of the Conrad QTL on chromosome 19 were dissected through sequence and expression analysis of genes in both resistant (Conrad and susceptible (‘Sloan’ genotypes. There were 1025 single nucleotide polymorphisms (SNPs in 87 of 153 genes sequenced from Conrad and Sloan. There were 304 SNPs in 54 genes sequenced from Conrad compared to those from both Sloan and Williams 82, of which 11 genes had SNPs unique to Conrad. Eleven of 19 genes in these regions analyzed with qRT-PCR had significant differences in fold change of transcript abundance in response to infection with P. sojae in lines with QTL haplotype from the resistant parent compared to those with the susceptible parent haplotype. From these, 8 of the 11 genes had SNPs in the upstream, untranslated region, exon, intron, and/or downstream region. These 11 candidate genes encode proteins potentially involved in signal transduction, hormone-mediated pathways, plant cell structural modification, ubiquitination, and basal resistance. Conclusions These findings may indicate a complex defense network with multiple mechanisms underlying these two soybean QTL conferring resistance to P. sojae. SNP markers derived from these candidate genes can contribute to fine mapping of QTL and marker assisted breeding for

  2. Rapid, simple, and sensitive detection of the ompB gene of spotted fever group rickettsiae by loop-mediated isothermal amplification

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    Pan Lei

    2012-10-01

    Full Text Available Abstract Background Spotted fever caused spotted fever group rickettsiae (SFGR is prevalent throughout China. In this study, we describe a rapid, simple, and sensitive loop-mediated isothermal amplification (LAMP assay targeting the ompB gene of spotted fever group rickettsiae ideal for application in China. The LAMP assay has the potential to detect spotted fever group rickettsiae early in infection and could therefore serve as an alternative to existing methods. Methods A set of universal primers which are specific 7 common species of spotted fever group rickettsiae in China were designed using PrimerExplorer V4 software based on conserved sequences of ompB gene. The sensitivity, specificity and reproducibility of the LAMP were evaluated. The LAMP assay for detecting SFGR was compared with conventional PCR assays for sensitivity and specificity in early phase blood samples obtained from 11 infected human subjects. Results The sensitivity of the LAMP assay was five copies per reaction (25 μL total volume, and the assay did not detect false-positive amplification across 42 strains of 27 members of the order Rickettsiales and 17 common clinical pathogens. The LAMP assay was negative to typhus group rickettsiae including R. prowazekii and R. typhi for no available conserved sequences of ompB was obtained for designing primers. To evaluate the clinical applicability of the LAMP assay, a total of 11 clinical samples, 10 samples confirmed serologically (3 cases, ecologically (1 case, by real-time polymerase chain reaction (PCR; 2 cases, ecologically and by real-time PCR (1 case, and serologically and by real-time PCR (3 cases were analyzed by the ompB LAMP assay. Data were validated using a previously established nested PCR protocol and real-time PCR. A positive LAMP result was obtained for 8 of the 10 confirmed cases (sensitivity, 73%; specificity, 100%, while none of these samples were positive by nested PCR (sensitivity, 0%; specificity, 100

  3. Sex determination of cattle meat by polymerase chain reaction amplification of the amelogenin (AMELX/AMELY gene

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    P. Gokulakrishnan

    Full Text Available Aim: The objective of this study was to develop a simplified, efficient, and accurate protocol for sexing of cattle meat based on the amelogenin (AMELX/AMELY gene using PCR technique which is superior to earlier work in terms of band patterns. Materials and Methods: Based on the amelogenin gene located on the conservation region of X and Y chromosomes, a pair of primers was designed and the system of PCR was established to amplify a 241-bp fragment from the X chromosome in female cattle, and a 241-bp fragment from X chromosome and 178-bp fragment from the Y chromosome in male cattle, respectively. The accuracy and specificity of the primers was assessed using DNA template extracted from cattle meat samples of known sex. The protocol was subjected to a blind test showed 100% concordance, proving its accuracy and reliability. Results: PCR products of cattle meat samples after electrophoresis showed two bands (241, 178-bp for tissue from male while female tissue resulted in only one (241-bp band. Conclusions: Our findings show that the PCR assay based on the amelogenin gene is reliable for sex determination in cattle meat. [Vet World 2012; 5(9.000: 526-529

  4. Multiplex PCR-based simultaneous amplification of selectable marker and reporter genes for the screening of genetically modified crops.

    Science.gov (United States)

    Randhawa, Gurinder Jit; Chhabra, Rashmi; Singh, Monika

    2009-06-24

    The development and commercialization of genetically modified (GM) crops with enhanced insect and herbicide resistance, abiotic stress tolerance, and improved nutritional quality has expanded dramatically. Notwithstanding the huge potential benefits of GM crops, the perceived environmental risks associated with these crops need to be addressed in proper perspective. One critical concern is the adventitious presence or unintentional mixing of GM seed in non-GM seed lots, which can seriously affect the global seed market. It would therefore be necessary though a challenging task to develop reliable, efficient, and economical assays for GM detection, identification, and quantification in non-GM seed lots. This can be systematically undertaken by preliminary screening for control elements and selectable or scorable (reporter) marker genes. In this study, simplex and multiplex polymerase chain reaction (PCR) assays individually as well as simultaneously amplifying the commonly used selectable marker genes, i.e., aadA, bar, hpt, nptII, pat encoding, respectively, for aminoglycoside-3'-adenyltransferase, Streptococcus viridochromogenes phosphinothricin-N-acetyltransferase, hygromycin phosphotransferase, neomycin phosphotransferase, Streptococcus hygroscopicus phosphinothricin-N-acetyltransferase, and a reporter gene uidA encoding beta-d-glucuronidase, were developed as a reliable tool for qualitative screening of GM crops. The efficiency of the assays was also standardized in the test samples prepared by artificial mixing of transgenic seed samples in different proportions. The developed multiplex PCR assays will be useful in verifying the GM status of a sample irrespective of the crop and GM trait.

  5. Resistance to Downy Mildew in Lettuce 'La Brillante' is Conferred by Dm50 Gene and Multiple QTL.

    Science.gov (United States)

    Simko, Ivan; Ochoa, Oswaldo E; Pel, Mathieu A; Tsuchida, Cayla; Font I Forcada, Carolina; Hayes, Ryan J; Truco, Maria-Jose; Antonise, Rudie; Galeano, Carlos H; Michelmore, Richard W

    2015-09-01

    Many cultivars of lettuce (Lactuca sativa L.) are susceptible to downy mildew, a nearly globally ubiquitous disease caused by Bremia lactucae. We previously determined that Batavia type cultivar 'La Brillante' has a high level of field resistance to the disease in California. Testing of a mapping population developed from a cross between 'Salinas 88' and La Brillante in multiple field and laboratory experiments revealed that at least five loci conferred resistance in La Brillante. The presence of a new dominant resistance gene (designated Dm50) that confers complete resistance to specific isolates was detected in laboratory tests of seedlings inoculated with multiple diverse isolates. Dm50 is located in the major resistance cluster on linkage group 2 that contains at least eight major, dominant Dm genes conferring resistance to downy mildew. However, this Dm gene is ineffective against the isolates of B. lactucae prevalent in the field in California and the Netherlands. A quantitative trait locus (QTL) located at the Dm50 chromosomal region (qDM2.2) was detected, though, when the amount of disease was evaluated a month before plants reached harvest maturity. Four additional QTL for resistance to B. lactucae were identified on linkage groups 4 (qDM4.1 and qDM4.2), 7 (qDM7.1), and 9 (qDM9.2). The largest effect was associated with qDM7.1 (up to 32.9% of the total phenotypic variance) that determined resistance in multiple field experiments. Markers identified in the present study will facilitate introduction of these resistance loci into commercial cultivars of lettuce.

  6. Site-directed mutagenesis of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti confers insecticide insensitivity.

    Science.gov (United States)

    Vaughan, A; Rocheleau, T; ffrench-Constant, R

    1997-11-01

    Insecticide resistance is a serious problem facing the effective control of insect vectors of disease. Insensitive acetylcholinesterase (AChE) confers resistance to organophosphorus (OP) and carbamate insecticides and is a widespread resistance mechanism in vector mosquitoes. Although the point mutations that underlie AChE insensitivity have been described from Drosophila, the Colorado potato beetle, and house flies, no resistance associated mutations have been documented from mosquitoes to date. We are therefore using a cloned acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti as a model in which to perform site directed mutagenesis in order to understand the effects of potential resistance associated mutations. The same resistance associated amino-acid replacements as found in other insects also confer OP and carbamate resistance to the mosquito enzyme. Here we describe the levels of resistance conferred by different combinations of these mutations and the effects of these mutations on the kinetics of the AChE enzyme. Over-expression of these constructs in baculovirus will facilitate purification of each of the mutant enzymes and a more detailed analysis of their associated inhibition kinetics.

  7. High-resolution fine mapping of ps-2, a mutated gene conferring functional male sterility in tomato due to non-dehiscent anthers

    NARCIS (Netherlands)

    Gorguet, B.J.M.; Schipper, E.H.; Heusden, van A.W.; Lindhout, P.

    2006-01-01

    Functional male sterility is an important trait for the production of hybrid seeds. Among the genes coding for functional male sterility in tomato is the positional sterility gene ps-2. ps-2 is monogenic recessive, confers non-dehiscent anthers and is the most suitable for practical uses. In order

  8. A second gene at the tomato Cf-4 locus confers resistance to Cladosporium fulvum through recognition of a novel avirulence determinant

    NARCIS (Netherlands)

    Nijkamp, HJJ; Takken, Frank L.W.; Thomas, Colwyn M.; Joosten, Matthieu H.A.J.; Golstein, Catherine; Westerink, Nienke; Hille, Jacques; Nijkamp, H. John J.; Wit, Pierre J.G.M. de; Jones, Jonathan D.G.

    1999-01-01

    The tomato Cf-4 and Cf-9 genes confer resistance to the leaf mould pathogen Cladosporium fulvum and map at a complex locus on the short arm of chromosome 1. It was previously shown that the gene encoding Cf-4, which recognizes the Avr4 avirulence determinant, is one of five tandemly duplicated homol

  9. High-resolution fine mapping of ps-2, a mutated gene conferring functional male sterility in tomato due to non-dehiscent anthers

    NARCIS (Netherlands)

    Gorguet, B.J.M.; Schipper, E.H.; Heusden, van A.W.; Lindhout, P.

    2006-01-01

    Functional male sterility is an important trait for the production of hybrid seeds. Among the genes coding for functional male sterility in tomato is the positional sterility gene ps-2. ps-2 is monogenic recessive, confers non-dehiscent anthers and is the most suitable for practical uses. In order t

  10. Isolation and characterisation of the human lung NK-2 receptor gene using rapid amplification of cDNA ends.

    Science.gov (United States)

    Graham, A; Hopkins, B; Powell, S J; Danks, P; Briggs, I

    1991-05-31

    Functional cDNA clones for human NK-2 receptor were isolated from human lung RNA using a polymerase chain reaction (PCR) based method (RACE-PCR). In this method the cDNA was isolated as 5' end and 3'-end fragments; the entire cDNA was obtained by RNA-PCR. The sequence derived was 398 amino acids in length encoding an open-reading frame that was highly homologous to both the bovine and rat NK-2 receptor. The entire human cDNA sequence was cloned into a mammalian expression vector and mRNA was synthesised by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesised mRNA. The most potent of the three tachykinin peptides tested was neurokinin A. We have screened a human cosmid library and isolated a clone which contains the entire NK-2 receptor gene. The gene contains five exons and we have determined the complete sequence of the exons and the intron-exon junctions.

  11. Low Prevalence of TP53 Mutations and MDM2 Amplifications in Pediatric Rhabdomyosarcoma

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    Simona Ognjanovic

    2012-01-01

    Full Text Available The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer. The reported prevalence of mutations in rhabdomyosarcoma (RMS varies widely, with recent larger studies suggesting that TP53 mutations in pediatric RMS may be extremely rare. Overexpression of MDM2 also attenuates p53 function. We have performed TP53 mutation/MDM2 amplification analyses in the largest series analyzed thus far, including DNA isolated from 37 alveolar and 38 embryonal RMS tumor samples obtained from the Cooperative Human Tissue Network (CHTN. Available samples were frozen tumor tissues (N=48 and histopathology slides. TP53 mutations in exons 4–9 were analyzed by direct sequencing in all samples, and MDM2 amplification analysis was performed by differential PCR on a subset of 22 samples. We found only one sample (1/75, 1.3% carrying a TP53 mutation at codon 259 (p.D259Y and no MDM2 amplification. Two SNPs in the TP53 pathway, associated with accelerated tumor onset in germline TP53 mutation carriers, (TP53 SNP72 (rs no. 1042522 and MDM2 SNP309 (rs no. 2279744, were not found to confer earlier tumor onset. In conclusion, we confirm the extremely low prevalence of TP53 mutations/MDM2 amplifications in pediatric RMS (1.33% and 0%, respectively. The possible inactivation of p53 function by other mechanisms thus remains to be elucidated.

  12. Distribution of genes conferring combined resistance to tetracycline and minocycline among group B streptococcal isolates from humans and various animals.

    Science.gov (United States)

    Schwarz, S; Wibawan, I W; Lämmler, C

    1994-11-01

    Forty-nine tetracycline and minocycline resistant streptococci of serological group B isolated from humans, cattle, pigs and nutrias were investigated for the presence of genes conferring this combined resistance. Southern blot hybridization of EcoRI-digested chromosomal DNA of the bacteria revealed for 39 of the cultures a hybridization signal with tet(M), for four of the cultures a hybridization signal with tet(O) and for none of the cultures a hybridization signal with the tet(Q) gene probe. The restriction endonuclease digested and blotted DNA of six tetracycline and minocycline resistant group B streptococci did not hybridize with any of the available gene probes. The tet(M) gene probes recognized complementary sequences of EcoRI fragments of approximately 10.5 kb and 21.5 kb, the tet(O) gene probe hybridized with fragments of approximately 19 kb. The hybridization of the tet(M) gene probe in two different patterns appeared to be related to the origin of the cultures.

  13. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean

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    Bingfu eGuo

    2015-10-01

    Full Text Available Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at four-fold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops.

  14. Mismatch repair genes of Streptococcus pneumoniae: HexA confers a mutator phenotype in Escherichia coli by negative complementation.

    Science.gov (United States)

    Prudhomme, M; Méjean, V; Martin, B; Claverys, J P

    1991-11-01

    DNA repair systems able to correct base pair mismatches within newly replicated DNA or within heteroduplex molecules produced during recombination are widespread among living organisms. Evidence that such generalized mismatch repair systems evolved from a common ancestor is particularly strong for two of them, the Hex system of the gram-positive Streptococcus pneumoniae and the Mut system of the gram-negative Escherichia coli and Salmonella typhimurium. The homology existing between HexA and MutS and between HexB and MutL prompted us to investigate the effect of expressing hex genes in E. coli. Complementation of mutS or mutL mutations, which confer a mutator phenotype, was assayed by introducing on a multicopy plasmid the hexA and hexB genes, under the control of an inducible promoter, either individually or together in E. coli strains. No decrease in mutation rate was conferred by either hexA or hexB gene expression. However, a negative complementation effect was observed in wild-type E. coli cells: expression of hexA resulted in a typical Mut- mutator phenotype. hexB gene expression did not increase the mutation rate either individually or in conjunction with hexA. Since expression of hexA did not affect the mutation rate in mutS mutant cells and the hexA-induced mutator effect was recA independent, it is concluded that this effect results from inhibition of the Mut system. We suggest that HexA, like its homolog MutS, binds to mismatches resulting from replication errors, but in doing so it protects them from repair by the Mut system. In agreement with this hypothesis, an increase in mutS gene copy number abolished the hexA-induced mutator phenotype. HexA protein could prevent repair either by being unable to interact with Mut proteins or by producing nonfunctional repair complexes.

  15. Development and evaluation of a loop-mediated isothermal amplification (LAMP assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene

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    Hongwei Ji

    2012-08-01

    Full Text Available This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae. A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63ºC for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV, and Pseudorabies virus. The detection limit of the conventional PCR was 10² CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

  16. Molecular characterization of Cryptosporidium xiaoi in goat kids in Bangladesh by nested PCR amplification of 18S rRNA gene

    Institute of Scientific and Technical Information of China (English)

    AMAM; Zonaed; Siddiki; Sohana; Akter; Mina; Zinat; Farzana; Bibi; Ayesa; Rasel; Das; Mohammad; Alamgir; Hossain

    2015-01-01

    Objective:To investigate the prevalence of Cryptosporidium spp.in goat kids in selected areas of Bangladesh and to elucidate the potential zoonotic hazards.Methods:In the present study,we have used Ziehl-Neelsen staining and nested PCR approach to identify and characterize the Cryptosporidium sp.from diarrhoeic feces of goat kids.A total of 100 diarrhoeic feces samples were collected from Chittagong region in Southern Bangladesh.For nested PCR analysis,specific primers for amplification of 581 base pair fragments of 18 S rRNA gene were used.Results:A total of 15%and 3%samples were found positive in microscopic study and in nested PCR analysis respectively.Phylogenetic analysis of sequence data showed similarity with that of Cryptosporidium xiaoi recorded from sheep and goat.Conclusions:To our knowledge,this is the first report of Cryptosporidium xiaoi responsible for diarrhoea in goat kids in Bangladesh.Further study can highlight their zoonotic significance along with genetic diversity in other host species inside the country.

  17. Rapid and sensitive detection of Dasheen mosaic virus infecting elephant foot yam by reverse transcription loop mediated isothermal amplification of coat protein gene.

    Science.gov (United States)

    Kamala, S; Makeshkumar, T

    2015-09-15

    Dasheen mosaic virus (DsMV), the pathogen causing mosaic disease of elephant foot yam (Amorphophallus paeoniifoilius) is disseminated mainly through vegetative propagation of the tubers. For the rapid and sensitive detection of the virus, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay based on the coat protein gene has been developed. A final concentration of 5.4 mM magnesium sulphate and 0.7 M betaine in the reaction mixture was found to be optimum for getting characteristic ladder like bands of the amplified product after gel electrophoresis. The reaction was set at 65°C for 50 min followed by reaction termination at 86°C for 5 min in a water bath. The sensitivity of the assay was found to be 100 times higher than that of RT-PCR. The virus was indexed successfully from tubers of elephant foot yam. In tube detection of the DsMV was carried out using fluorescence detection reagents. The assay was validated with field samples from various regions of Kerala state, India. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter.

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    Aaron J Stevens

    Full Text Available We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5' end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.

  19. G-Quadruplex Structures and CpG Methylation Cause Drop-Out of the Maternal Allele in Polymerase Chain Reaction Amplification of the Imprinted MEST Gene Promoter

    Science.gov (United States)

    Cree, Simone L.; Gibb, Andrew; Miller, Allison L.; Doudney, Kit; Aitchison, Alan; Eccles, Michael R.; Joyce, Peter R.; Filichev, Vyacheslav V.; Kennedy, Martin A.

    2014-01-01

    We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5′ end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings. PMID:25437198

  20. A Loop-Mediated Isothermal Amplification Assay Targeting 16S rRNA Gene for Rapid Detection of Anaplasma phagocytophilum Infection in Sheep and Goats.

    Science.gov (United States)

    Wang, Jinhong; Zhang, Yan; Wang, Xiaoxing; Cui, Yanyan; Yan, Yaqun; Wang, Rongjun; Jian, Fuchun; Zhang, Longxian; Ning, Changshen

    2017-04-01

    Anaplasma phagocytophilum is a zoonotic pathogen and the causative agent of human granulocytic anaplasmosis in humans and tick-borne fever in various kinds of animals. In the present study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of A. phagocytophilum was developed using primers specific to 16S rRNA gene of this organism. The LAMP assay was performed at 65 C for 60 min and terminated at 80 C for 10 min. The optimal reaction conditions under which no cross-reaction was observed with other closely related tick-borne parasites ( Anaplasma bovis , Anaplasma ovis , Theileria luwenshuni, Babesia motasi, and Schistosoma japonicum ) were established. The assay exhibited much higher sensitivity compared with conventional polymerase chain reaction (PCR) (1 copy vs. 1,000 copies). To evaluate the applicability of the LAMP assay, 94 field samples of sheep blood were analyzed for A. phagocytophilum infection by using LAMP, nested PCR, and conventional PCR assays at the same time. A positive LAMP result was obtained from 53 (56.4%) of the 94 samples, whereas only 12 (12.8%) and 3 (3.2%) tested positive by nested and conventional PCR, respectively. In conclusion, this LAMP assay is a specific, sensitive, and rapid method for the detection of A. phagocytophilum in sheep/goats.

  1. 应用双重原位杂交检测胃癌HER2基因扩增%Amplification of HER2 gene in gastric carcinoma detected by dual in-situ hybridization

    Institute of Scientific and Technical Information of China (English)

    冼丽芳; 黄香婷; 文剑明; 叶玉清

    2012-01-01

    Objective To explore the status of HER2 gene amplification and its product HER2 protein expression in gastric carcinoma, so as to aid in patient selection for anti-HER2 targeted chemotherapy.Methods Eighty-five cases of gastric carcinoma biopsy tissues were collected.The status of HER2 gene amplification was detected by dual in situ hybridization (dual-ISH).And HER2 protein was detected by immunohistochemistry.Results HER2 gene amplification was detected in 10/85 ( 11.8% )cases of gastric carcinoma,and no amplification was detected in 75/85 (88.2% ) cases.In the 10 cases with HER2 amplification,HER2 immunoreaction scorings of 3 +,2 + and 0/1 + were present in 7,2 and 1 cases,respectively.In the 75 cases without HER2 amplification,HER2 immunoreaction scorings of 3 +,2 + and 0/1 + were present in 0,18 (24.0% ) and 57 (76.0% ) cases,respectively.Histologically,most gastric carcinoma with amplification of HER2 gene was moderately differentiated tubular adenocarcinoma.Conclusions HER2 gene dual-ISH technique is a reliable and objective method for detecting HER2 gene amplification in gastric carcinoma biopsy. Clinically, only few gastric carcinomas show HER2 gene amplification and are suitable candidates for anti-HER2 targeted chemotherapy.%目的 探讨胃癌HER2基因扩增状态及其基因产物蛋白表达,以协助临床筛选抗HER2靶向治疗患者.方法 收集胃癌活检标本共85例,进行HER2基因扩增和蛋白表达检测.HER2基因扩增状态采用双重原位杂交检测(HER2 dual-ISH detection),而HER2基因蛋白采用免疫组织化学[全程在全自动染色机(Ventana)]进行检测.结果 85例胃癌病例中,有HER2基因扩增10例,扩增率为11.8%,无扩增75例,占88.2%.在有HER2基因扩增的10例病例中,7例HER2蛋白免疫组织化学为3+,2例为2+,1例为-/1+;75例无基因扩增的病例,HER2蛋白免疫组织化学3+为0例、2+为18例(24.0%),-/1+为57例(76.0%).HER2基因扩增与HER2

  2. Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene.

    Science.gov (United States)

    Pinhanelli, V C; Costa, P N M; Silva, G; Aguiar, D M; Silva, C M L; Fachin, A L; Marins, M

    2015-12-22

    Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME.

  3. PCR amplification and high throughput sequencing of immunoglobulin heavy chain genes from formalin-fixed paraffin-embedded human biopsies.

    Science.gov (United States)

    Tabibian-Keissar, Hilla; Schibby, Ginette; Michaeli, Miri; Rakovsky-Shapira, Aviya; Azogui-Rosenthal, Noemie; Dunn-Walters, Deborah K; Rosenblatt, Kinneret; Mehr, Ramit; Barshack, Iris

    2013-02-01

    The use of high throughput sequencing (HTS) technologies in biomedicine is expanding in a variety of fields in recent years. The 454 system is an HTS platform that is ideally suited to characterize B cell receptor (BCR) repertoires by sequencing of immunoglobulin (Ig) genes, as it is able to sequence stretches of several hundred nucleotides. Most studies that used this platform for antibody repertoire analyses have started from fresh or frozen tissues or peripheral blood samples, and rely on starting with optimal quality DNA. In this paper we demonstrate that BCR repertoire analysis can be done using DNA from formalin-fixed paraffin-embedded (FFPE) human tissue samples. The heterogeneity of BCR repertoires we obtained confirms the plausibility of HTS of DNA from FFPE specimens. The establishment of experimental protocols and computational tools that enable sequence data analysis from the low quality DNA of FFPE tissues is important for enabling research, as it would enable the use of the rich source of preserved samples in clinical biobanks and biopsy archives.

  4. Construction and screening of metagenomic libraries derived from enrichment cultures: generation of a gene bank for genes conferring alcohol oxidoreductase activity on Escherichia coli.

    Science.gov (United States)

    Knietsch, Anja; Waschkowitz, Tanja; Bowien, Susanne; Henne, Anke; Daniel, Rolf

    2003-03-01

    Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C(2) to C(4)) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any

  5. Common variants of the PINK1 and PARL genes do not confer genetic susceptibility to schizophrenia in Han Chinese.

    Science.gov (United States)

    Li, Xiao; Zhang, Wen; Zhang, Chen; Yi, Zhenghui; Zhang, Deng-Feng; Gong, Wei; Tang, Jinsong; Wang, Dong; Lu, Weihong; Chen, Xiaogang; Fang, Yiru; Yao, Yong-Gang

    2015-04-01

    Schizophrenia is a prevalent psychiatric disorder with a complex etiology. Mitochondrial dysfunction has been frequently reported in schizophrenia. Phosphatase and tension homologue-induced kinase 1 (PINK1) and presenilin-associated rhomboid-like protease (PARL) are mitochondrial proteins, and genetic variants of these two genes may confer genetic susceptibility to schizophrenia by influencing mitochondrial function. In this study, we conducted a two-stage genetic association study to test this hypothesis. We genotyped 4 PINK1 and 5 PARL genetic variants and evaluated the potential association of the 9 SNPs with schizophrenia in two independent case-control cohorts of 2510 Han Chinese individuals. No positive association of common genetic variants of the PINK1 and PARL genes with schizophrenia was identified in our samples after Bonferroni correction. Re-analysis of the newly updated Psychiatric Genetics Consortium (PGC) data sets confirmed our negative result. Intriguingly, one PINK1 SNP (rs10916832), which showed a marginally significant association in only Hunan samples (P = 0.032), is associated with the expression of a schizophrenia susceptible gene KIF17 according to the expression quantitative trait locus (eQTL) analysis. Our study indicated that common genetic variants of the PINK1 and PARL genes are unlikely to be involved in schizophrenia. Further studies are essential to characterize the role of the PINK1 and PARL genes in schizophrenia.

  6. Identification of regulated genes conferring resistance to high concentrations of glyphosate in a new strain of Enterobacter.

    Science.gov (United States)

    Fei, Yun-Yan; Gai, Jun-Yi; Zhao, Tuan-Jie

    2013-12-01

    Glyphosate is a widely used herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity. Most plants and microbes are sensitive to glyphosate. However, transgenic-resistant crops that contain a modified epsps obtained from the resistant microbes have been commercially successful and therefore, new resistance genes and their adaptive regulatory mechanisms are of great interest. In this study, a soil-borne, glyphosate-resistant bacterium was selected and identified as Enterobacter. The EPSPS in this strain was found to have been altered to a resistant one. A total of 42 differentially expressed genes (DEGs) in the glyphosate were screened using microarray techniques. Under treatment, argF, sdhA, ivbL, rrfA-H were downregulated, whereas the transcripts of speA, osmY, pflB, ahpC, fusA, deoA, uxaC, rpoD and a few ribosomal protein genes were upregulated. Data were verified by quantitative real-time PCR on selected genes. All transcriptional changes appeared to protect the bacteria from glyphosate and associated osmotic, acidic and oxidative stresses. Many DEGs may have the potential to confer resistance to glyphosate alone, and some may be closely related to the shikimate pathway, reflecting the complex gene interaction network for glyphosate resistance.

  7. bHLH106 Integrates Functions of Multiple Genes through Their G-Box to Confer Salt Tolerance on Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Aftab Ahmad

    Full Text Available An activation-tagging methodology was applied to dedifferentiated calli of Arabidopsis to identify new genes involved in salt tolerance. This identified salt tolerant callus 8 (stc8 as a gene encoding the basic helix-loop-helix transcription factor bHLH106. bHLH106-knockout (KO lines were more sensitive to NaCl, KCl, LiCl, ABA, and low temperatures than the wild-type. Back-transformation of the KO line rescued its phenotype, and over-expression (OX of bHLH106 in differentiated plants exhibited tolerance to NaCl. Green fluorescent protein (GFP fused with bHLH106 revealed that it was localized to the nucleus. Prepared bHLH106 protein was subjected to electrophoresis mobility shift assays against E-box sequences (5'-CANNTG-3'. The G-box sequence 5'-CACGTG-3' had the strongest interaction with bHLH106. bHLH106-OX lines were transcriptomically analyzed, and resultant up- and down-regulated genes selected on the criterion of presence of a G-box sequence. There were 198 genes positively regulated by bHLH106 and 36 genes negatively regulated; these genes possessed one or more G-box sequences in their promoter regions. Many of these genes are known to be involved in abiotic stress response. It is concluded that bHLH106 locates at a branching point in the abiotic stress response network by interacting directly to the G-box in genes conferring salt tolerance on plants.

  8. Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes

    Science.gov (United States)

    Kariya, Satomi; Kurokawa, Junji

    2014-01-01

    Screening of gene-specific amplicons from metagenomes (S-GAM) has tremendous biotechnological potential. We used this approach to isolate alcohol dehydrogenase (adh) genes from metagenomes based on the Leifsonia species adh gene (lsadh), the enzyme product of which can produce various chiral alcohols. A primer combination was synthesized by reference to homologs of lsadh, and PCR was used to amplify nearly full-length adh genes from metagenomic DNAs. All adh preparations were fused with lsadh at the terminal region and used to construct Escherichia coli plasmid libraries. Of the approximately 2,000 colonies obtained, 1,200 clones were identified as adh positive (∼60%). Finally, 40 adh genes, Hladh-001 to Hladh-040 (for homologous Leifsonia adh), were identified from 223 clones with high efficiency, which were randomly sequenced from the 1,200 clones. The Hladh genes obtained via this approach encoded a wide variety of amino acid sequences (8 to 99%). After screening, the enzymes obtained (HLADH-012 and HLADH-021) were confirmed to be superior to LSADH in some respects for the production of anti-Prelog chiral alcohols. PMID:25085492

  9. The A395T mutation in ERG11 gene confers fluconazole resistance in Candida tropicalis causing candidemia.

    Science.gov (United States)

    Tan, Jingwen; Zhang, Jinqing; Chen, Wei; Sun, Yi; Wan, Zhe; Li, Ruoyu; Liu, Wei

    2015-04-01

    The mechanism of fluconazole resistance in Candida tropicalis is still unclear. Recently, we isolated a fluconazole-resistant strain of C. tropicalis from the blood specimen of a patient with candidemia in China. In vitro antifungal susceptibility of the isolate was determined by using CLSI M27-A3 and E-test methods. The sequence of ERG11 gene was then analyzed, and the three-dimensional model of Erg11p encoded by ERG11 gene was also investigated. The sequencing of ERG11 gene revealed the mutation of A395T in this fluconazole-resistant isolate of C. tropicalis, resulting in the Y132F substitution in Erg11p. Sequence alignment and three-dimensional model comparison of Erg11ps showed high similarity between fluconazole-susceptible isolates of C. tropicalis and Candida albicans. The comparison of the three-dimensional models of Erg11ps demonstrated that the position of the Y132F substitution in this isolate of C. tropicalis is identical to the isolate of C. albicans with fluconazole resistance resulting from Y132F substitution in Erg11p. Hence, we ascertain that the Y132F substitution of Erg11p caused by A395T mutation in ERG11 gene confers the fluconazole resistance in C. tropicalis.

  10. H2A.Z-mediated localization of genes at the nuclear periphery confers epigenetic memory of previous transcriptional state.

    Directory of Open Access Journals (Sweden)

    Donna Garvey Brickner

    2007-04-01

    Full Text Available Many genes are recruited to the nuclear periphery upon transcriptional activation. The mechanism and functional significance of this recruitment is unclear. We find that recruitment of the yeast INO1 and GAL1 genes to the nuclear periphery is rapid and independent of transcription. Surprisingly, these genes remain at the periphery for generations after they are repressed. Localization at the nuclear periphery serves as a form of memory of recent transcriptional activation, promoting reactivation. Previously expressed GAL1 at the nuclear periphery is activated much more rapidly than long-term repressed GAL1 in the nucleoplasm, even after six generations of repression. Localization of INO1 at the nuclear periphery is necessary and sufficient to promote more rapid activation. This form of transcriptional memory is chromatin based; the histone variant H2A.Z is incorporated into nucleosomes within the recently repressed INO1 promoter and is specifically required for rapid reactivation of both INO1 and GAL1. Furthermore, H2A.Z is required to retain INO1 at the nuclear periphery after repression. Therefore, H2A.Z-mediated localization of recently repressed genes at the nuclear periphery represents an epigenetic state that confers memory of transcriptional activation and promotes reactivation.

  11. An improved method for transformation of lettuce by Agrobacterium tumefaciens with a gene that confers freezing resistance

    Directory of Open Access Journals (Sweden)

    Pileggi Marcos

    2001-01-01

    Full Text Available An efficient method for constructing transgenic lettuce cultivars by Agrobacterium tumefaciens was described by Torres et al., 1993. In the present work, an improvement of the above procedure is described and applied to transform the cultivar Grand Rapids with a mutated P5CS gene. The major modifications were concerned with turning more practical the transformation and regeneration protocols. Also we tried to improve transformation steps by increasing injured area in explants and prolonging co-cultivation with Agrobacteria (in larger concentration. A more significant selective pressure was used against non-transformed plants and bacteria. In these work we were concerned to obtain T1 and T2 seeds. The P5CS gene codes for a delta¹-pyrroline-5-carboxylate synthetase, a bifunctional enzyme that catalyzes two steps of proline biosynthesis in plants (Zhang et al., 1995; Peng et al., 1996, while the mutated gene is insensitive to feedback inhibition by proline. The potential benefit of this gene is to confer water stress resistance (drought, salt, cold due to increased intracellular levels of proline that works like an osmoprotectant. In this work could obtain and characterize transgenic lettuce lineages which are resistant to freezing temperature.

  12. Genome-wide screening for genes whose deletions confer sensitivity to mutagenic purine base analogs in yeast

    Directory of Open Access Journals (Sweden)

    Kozmin Stanislav G

    2005-06-01

    Full Text Available Abstract Background N-hydroxylated base analogs, such as 6-hydroxylaminopurine (HAP and 2-amino-6-hydroxylaminopurine (AHA, are strong mutagens in various organisms due to their ambiguous base-pairing properties. The systems protecting cells from HAP and related noncanonical purines in Escherichia coli include specialized deoxyribonucleoside triphosphatase RdgB, DNA repair endonuclease V, and a molybdenum cofactor-dependent system. Fewer HAP-detoxification systems have been identified in yeast Saccharomyces cerevisiae and other eukaryotes. Cellular systems protecting from AHA are unknown. In the present study, we performed a genome-wide search for genes whose deletions confer sensitivity to HAP and AHA in yeast. Results We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA. We identified novel genes involved in the genetic control of base analogs sensitivity, including genes controlling purine metabolism, cytoskeleton organization, and amino acid metabolism. Conclusion We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP. Three of them also protect from AHA.

  13. Host-induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat.

    Science.gov (United States)

    Cheng, Wei; Song, Xiu-Shi; Li, He-Ping; Cao, Le-Hui; Sun, Ke; Qiu, Xiao-Li; Xu, Yu-Bin; Yang, Peng; Huang, Tao; Zhang, Jing-Bo; Qu, Bo; Liao, Yu-Cai

    2015-12-01

    Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co-expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T3 to T5 generations. Confocal microscopy revealed profoundly restricted mycelia in Fg-infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down-regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host-induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.

  14. A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus

    Directory of Open Access Journals (Sweden)

    Chen Wenbin

    2008-07-01

    Full Text Available Abstract Background The emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes. Results Bioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97% cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y. Conclusion Our one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.

  15. Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

    Science.gov (United States)

    Yang, Qi; Franco, Christopher M M; Zhang, Wei

    2015-10-01

    Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity.

  16. Novel Cytomegalovirus UL54 DNA Polymerase Gene Mutations Selected In Vitro That Confer Brincidofovir Resistance

    Science.gov (United States)

    Ercolani, Ronald J.; Lanier, E. Randall

    2016-01-01

    Eight in vitro selection experiments under brincidofovir pressure elicited the known cytomegalovirus DNA polymerase amino acid substitutions N408K and V812L and the novel exonuclease domain substitutions D413Y, E303D, and E303G, which conferred ganciclovir and cidofovir resistance with 6- to 11-fold resistance to brincidofovir or 17-fold when E303G was combined with V812L. The new exonuclease domain I resistance mutations selected under brincidofovir pressure add to the single instance previously reported and show the expected patterns of cross-resistance. PMID:27044553

  17. MsZEP, a novel zeaxanthin epoxidase gene from alfalfa (Medicago sativa), confers drought and salt tolerance in transgenic tobacco.

    Science.gov (United States)

    Zhang, Zhiqiang; Wang, Yafang; Chang, Leqin; Zhang, Tong; An, Jie; Liu, Yushi; Cao, Yuman; Zhao, Xia; Sha, Xuyang; Hu, Tianming; Yang, Peizhi

    2016-02-01

    The zeaxanthin epoxidase gene ( MsZEP ) was cloned and characterized from alfalfa and validated for its function of tolerance toward drought and salt stresses by heterologous expression in Nicotiana tabacum. Zeaxanthin epoxidase (ZEP) plays important roles in plant response to various environment stresses due to its functions in ABA biosynthetic and the xanthophyll cycle. To understand the expression characteristics and the biological functions of ZEP in alfalfa (Medicago sativa), a novel gene, designated as MsZEP (KM044311), was cloned, characterized and overexpressed in Nicotiana tabacum. The open reading frame of MsZEP contains 1992 bp nucleotides and encodes a 663-amino acid polypeptide. Amino acid sequence alignment indicated that deduced MsZEP protein was highly homologous to other plant ZEP sequences. Phylogenetic analysis showed that MsZEP was grouped into a branch with other legume plants. Real-time quantitative PCR revealed that MsZEP gene expression was clearly tissue-specific, and the expression levels were higher in green tissues (leaves and stems) than in roots. MsZEP expression decreased in shoots under drought, cold, heat and ABA treatment, while the expression levels in roots showed different trends. Besides, the results showed that nodules could up-regulate the MsZEP expression under non-stressful conditions and in the earlier stage of different abiotic stress. Heterologous expression of the MsZEP gene in N. tabacum could confer tolerance to drought and salt stress by affecting various physiological pathways, ABA levels and stress-responsive genes expression. Taken together, these results suggested that the MsZEP gene may be involved in alfalfa responses to different abiotic stresses and nodules, and could enhance drought and salt tolerance of transgenic tobacco by heterologous expression.

  18. PCR amplification of a multi-copy mitochondrial gene (cox3) improves detection of Cytauxzoon felis infection as compared to a ribosomal gene (18S).

    Science.gov (United States)

    Schreeg, Megan E; Marr, Henry S; Griffith, Emily H; Tarigo, Jaime L; Bird, David M; Reichard, Mason V; Cohn, Leah A; Levy, Michael G; Birkenheuer, Adam J

    2016-07-30

    Cytauxzoon felis is a tick-transmitted protozoan parasite that infects felids. Clinical disease caused by acute C. felis infection rapidly progresses in domestic cats, leading to high morbidity and mortality. Accurately diagnosing cytauxzoonosis as soon as possible during acute infection would allow for earlier initiation of antiprotozoal therapy which could lead to higher survival rates. Molecular detection of parasite rRNA genes (18S) by PCR has previously been shown to be a sensitive method of diagnosing C. felis infections. Based on evidence from related apicomplexan species, we hypothesized that C. felis mitochondrial genes would exist at higher copy numbers than 18S and would be a more sensitive diagnostic target. In this study we have designed a PCR assay targeting the C. felis mitochondrial gene cytochrome c oxidase subunit III (cox3). Herein we demonstrate that (1) the cox3 PCR can detect as low as 1 copy of DNA target and can detect C. felis in samples with known mitochondrial sequence heterogeneity, (2) cox3 copy number is increased relative to 18S in blood and tissue samples from acutely infected cats, and (3) the cox3 PCR is more sensitive than 18S PCR for detection of C. felis during early infections.

  19. Loop-mediated isothermal amplification assay targeting the blaCTX-M9 gene for detection of extended spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

    Science.gov (United States)

    Thirapanmethee, Krit; Pothisamutyothin, Kanokporn; Nathisuwan, Surakit; Chomnawang, Mullika T; Wiwat, Chanpen

    2014-12-01

    Extended-spectrum β-lactamases (ESBLs) produced by Enterobacteriaceae are one of the resistance mechanisms to most β-lactam antibiotics. ESBLs are currently a major problem in both hospitals and community settings worldwide. Rapid and reliable means of detecting ESBL-producing bacteria is necessary for identification, prevention and treatment. Loop-mediated isothermal amplification (LAMP) is a technique that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. This study was aimed to develop a convenient, accurate and inexpensive method for detecting ESBL-producing bacteria by a LAMP technique. ESBLs-producing Escherichia coli and Klebsiella pneumoniae were isolated from a tertiary hospital in Bangkok, Thailand and reconfirmed by double-disk synergy test. A set of four specific oligonucleotide primers of LAMP for detection of bla(CTX-M9) gene was designed based on bla(CTX-M9) from E. coli (GenBank Accession No. AJ416345). The LAMP reaction was amplified under isothermal temperature at 63°C for 60 min. Ladder-like patterns of band sizes from 226 bp of the bla(CTX-M9) DNA target was observed. The LAMP product was further analyzed by restriction digestion with MboI and TaqI endonucleases. The fragments generated were approximately 168, 177 and 250 bp in size for MboI digestion and 165, 193, 229, 281 and 314 bp for TaqI digestion, which is in agreement with the predicted sizes. The sensitivity of the LAMP technique to bla(CTX-M9) was greater than that of the PCR method by at least 10,000-fold. These results showed that the LAMP primers specifically amplified only the bla(CTX-M9) gene. Moreover, the presence of LAMP amplicon was simply determined by adding SYBR Green I in the reaction. In conclusion, this technique for detection of ESBLs is convenient, reliable and easy to perform routinely in hospitals or laboratory units in developing countries.

  20. A novel method for the purification of DNA by capturing nucleic acid and magnesium complexes on non-woven fabric filters under alkaline conditions for the gene diagnosis of tuberculosis by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Fukasawa, Tadashi; Oda, Naozumi; Wada, Yasunao; Tamaru, Aki; Fukushima, Yukari; Nakajima, Chie; Suzuki, Yasuhiko

    2010-07-01

    A novel method for purifying DNA from clinical samples based on the complex formation of DNA and magnesium ion (Mg(2+)) was developed for the detection of Mycobacterium tuberculosis. The formation of a DNA-Mg(2+) complex under alkaline conditions was observed by analyzing electrophoretic mobility reduction of DNA on agarose gel. The DNA-Mg(2+) complex increases the efficacy of DNA recovery from the sample solution on polyethylene terephthalate non-woven fabric (PNWF) filters. Among the various divalent metal cations, only Mg(2+) was associated with this effect. The applicability of DNA recovered on the PNWF filter was examined for the gene amplification method; loop-mediated isothermal amplification (LAMP). DNA on the PNWF filter could be used for the amplification of specific DNA fragments without elution from the filter. Using this method, DNA was directly purified from M. tuberculosis spiked sputum and examined by LAMP assay, showing a high sensitivity in comparison to the commercially available DNA extraction kit. These results indicated that the method developed in this study is useful for rapid gene diagnosis of tuberculosis.

  1. Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana.

    Science.gov (United States)

    Ghag, Siddhesh B; Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2014-06-01

    Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive diseases of banana (Musa spp.). Because no credible control measures are available, development of resistant cultivars through genetic engineering is the only option. We investigated whether intron hairpin RNA (ihpRNA)-mediated expression of small interfering RNAs (siRNAs) targeted against vital fungal genes (velvet and Fusarium transcription factor 1) in transgenic banana could achieve effective resistance against Foc. Partial sequences of these two genes were assembled as ihpRNAs in suitable binary vectors (ihpRNA-VEL and ihpRNA-FTF1) and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium-mediated genetic transformation. Eleven transformed lines derived from ihpRNA-VEL and twelve lines derived from ihpRNA-FTF1 were found to be free of external and internal symptoms of Foc after 6-week-long greenhouse bioassays. The five selected transgenic lines for each construct continued to resist Foc at 8 months postinoculation. Presence of specific siRNAs derived from the two ihpRNAs in transgenic banana plants was confirmed by Northern blotting and Illumina sequencing of small RNAs derived from the transgenic banana plants. The present study represents an important effort in proving that host-induced post-transcriptional ihpRNA-mediated gene silencing of vital fungal genes can confer efficient resistance against debilitating pathogens in crop plants.

  2. Functional Identification and Characterization of Genes Cloned from Halophyte Seashore Paspalum Conferring Salinity and Cadmium Tolerance

    Directory of Open Access Journals (Sweden)

    Yu eChen

    2016-02-01

    Full Text Available Salinity-affected and heavy metal-contaminated soils limit the growth of glycophytic plants. Identifying genes responsible for superior tolerance to salinity and heavy metals in halophytes has great potential for use in developing salinity- and Cd-tolerant glycophytes. The objective of this study was to identify salinity- and Cd-tolerance related genes in seashore paspalum (Paspalum vaginatum, a halophytic perennial grass species, using the yeast cDNA expression library screening method. Based on the Gateway-compatible vector system, a high quality entry library was constructed, which contained 9.9×106 clones with an average inserted fragments length of 1.48 kb representing a 100% full-length rate. The yeast expression libraries were screened in a salinity-sensitive and a Cd-sensitive yeast mutant. The screening yielded 32 salinity-tolerant clones harboring 18 salinity-tolerance genes and 20 Cd-tolerant clones, including 5 Cd-tolerance genes. qPCR analysis confirmed that most of the 18 salinity-tolerance and 5 Cd-tolerance genes were up-regulated at the transcript level in response to salinity or Cd stress in seashore paspalum. Functional analysis indicated that salinity-tolerance genes from seashore paspalum could be mainly involved in photosynthetic metabolism, antioxidant systems, protein modification, iron transport, vesicle traffic, and phospholipid biosynthesis. Cd-tolerance genes from seashore paspalum could be associated with regulating pathways involved in phytochelatin synthesis, HSFA4-relsted stress protection, CYP450 complex and sugar metabolism. The 18 salinity-tolerance genes and 5 Cd-tolerance genes could be potentially used as candidate genes for genetic modification of glycophytic grass species to improve salinity and Cd tolerance and for further analysis of molecular mechanisms regulating salinity and Cd tolerance.

  3. Functional Identification and Characterization of Genes Cloned from Halophyte Seashore Paspalum Conferring Salinity and Cadmium Tolerance

    Science.gov (United States)

    Chen, Yu; Chen, Chuanming; Tan, Zhiqun; Liu, Jun; Zhuang, Lili; Yang, Zhimin; Huang, Bingru

    2016-01-01

    Salinity-affected and heavy metal-contaminated soils limit the growth of glycophytic plants. Identifying genes responsible for superior tolerance to salinity and heavy metals in halophytes has great potential for use in developing salinity- and Cd-tolerant glycophytes. The objective of this study was to identify salinity- and Cd-tolerance related genes in seashore paspalum (Paspalum vaginatum), a halophytic perennial grass species, using yeast cDNA expression library screening method. Based on the Gateway-compatible vector system, a high-quality entry library was constructed, which contained 9.9 × 106 clones with an average inserted fragment length of 1.48 kb representing a 100% full-length rate. The yeast expression libraries were screened in a salinity-sensitive and a Cd-sensitive yeast mutant. The screening yielded 32 salinity-tolerant clones harboring 18 salinity-tolerance genes and 20 Cd-tolerant clones, including five Cd-tolerance genes. qPCR analysis confirmed that most of the 18 salinity-tolerance and five Cd-tolerance genes were up-regulated at the transcript level in response to salinity or Cd stress in seashore paspalum. Functional analysis indicated that salinity-tolerance genes from seashore paspalum could be involved mainly in photosynthetic metabolism, antioxidant systems, protein modification, iron transport, vesicle traffic, and phospholipid biosynthesis. Cd-tolerance genes could be associated with regulating pathways that are involved in phytochelatin synthesis, HSFA4-related stress protection, CYP450 complex, and sugar metabolism. The 18 salinity-tolerance genes and five Cd-tolerance genes could be potentially used as candidate genes for genetic modification of glycophytic grass species to improve salinity and Cd tolerance and for further analysis of molecular mechanisms regulating salinity and Cd tolerance. PMID:26904068

  4. A novel stress-induced sugarcane gene confers tolerance to drought, salt and oxidative stress in transgenic tobacco plants.

    Directory of Open Access Journals (Sweden)

    Kevin Begcy

    Full Text Available BACKGROUND: Drought is a major abiotic stress that affects crop productivity worldwide. Sugarcane can withstand periods of water scarcity during the final stage of culm maturation, during which sucrose accumulation occurs. Meanwhile, prolonged periods of drought can cause severe plant losses. METHODOLOGY/PRINCIPAL FINDINGS: In a previous study, we evaluated the transcriptome of drought-stressed plants to better understand sugarcane responses to drought. Among the up-regulated genes was Scdr1 (sugarcane drought-responsive 1. The aim of the research reported here was to characterize this gene. Scdr1 encodes a putative protein containing 248 amino acids with a large number of proline (19% and cysteine (13% residues. Phylogenetic analysis showed that ScDR1is in a clade with homologs from other monocotyledonous plants, separate from those of dicotyledonous plants. The expression of Scdr1 in different varieties of sugarcane plants has not shown a clear association with drought tolerance. CONCLUSIONS/SIGNIFICANCE: The overexpression of Scdr1 in transgenic tobacco plants increased their tolerance to drought, salinity and oxidative stress, as demonstrated by increased photosynthesis, water content, biomass, germination rate, chlorophyll content and reduced accumulation of ROS. Physiological parameters, such as transpiration rate (E, net photosynthesis (A, stomatal conductance (gs and internal leaf CO(2 concentration, were less affected by abiotic stresses in transgenic Scdr1 plants compared with wild-type plants. Overall, our results indicated that Scdr1 conferred tolerance to multiple abiotic stresses, highlighting the potential of this gene for biotechnological applications.

  5. A novel stress-induced sugarcane gene confers tolerance to drought, salt and oxidative stress in transgenic tobacco plants.

    Science.gov (United States)

    Begcy, Kevin; Mariano, Eduardo D; Gentile, Agustina; Lembke, Carolina G; Zingaretti, Sonia Marli; Souza, Glaucia M; Menossi, Marcelo

    2012-01-01

    Drought is a major abiotic stress that affects crop productivity worldwide. Sugarcane can withstand periods of water scarcity during the final stage of culm maturation, during which sucrose accumulation occurs. Meanwhile, prolonged periods of drought can cause severe plant losses. In a previous study, we evaluated the transcriptome of drought-stressed plants to better understand sugarcane responses to drought. Among the up-regulated genes was Scdr1 (sugarcane drought-responsive 1). The aim of the research reported here was to characterize this gene. Scdr1 encodes a putative protein containing 248 amino acids with a large number of proline (19%) and cysteine (13%) residues. Phylogenetic analysis showed that ScDR1is in a clade with homologs from other monocotyledonous plants, separate from those of dicotyledonous plants. The expression of Scdr1 in different varieties of sugarcane plants has not shown a clear association with drought tolerance. The overexpression of Scdr1 in transgenic tobacco plants increased their tolerance to drought, salinity and oxidative stress, as demonstrated by increased photosynthesis, water content, biomass, germination rate, chlorophyll content and reduced accumulation of ROS. Physiological parameters, such as transpiration rate (E), net photosynthesis (A), stomatal conductance (gs) and internal leaf CO(2) concentration, were less affected by abiotic stresses in transgenic Scdr1 plants compared with wild-type plants. Overall, our results indicated that Scdr1 conferred tolerance to multiple abiotic stresses, highlighting the potential of this gene for biotechnological applications.

  6. Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance

    Directory of Open Access Journals (Sweden)

    Yanmei Shi

    2015-12-01

    Full Text Available Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.

  7. An AFLP marker linked to the Pm-1 gene that confers resistance to Podosphaera xanthii race 1 in Cucumis melo

    Directory of Open Access Journals (Sweden)

    Ana Paula Matoso Teixeira

    2008-01-01

    Full Text Available Brazil produced 330,000 metric tons of melons in 2005, principally in the Northeast region where one of the most important melon pathogens is the powdery mildew fungus Podosphaera xanthii. The disease is controlled mainly by incorporating single dominant resistance genes into commercial hybrids. We report on linkage analysis of the Pm-1 resistance gene, introgressed from the AF125Pm-1 Cantalupensis Charentais-type breeding line into the yellow-fleshed melon (Group Inodorus breeding line AF426-S by backcrossing to produce the resistant line AF426-R, and the amplified fragment length polymorphism (AFLP marker M75/H35_155 reported to be polymorphic between AF426-S and AF426-R. Segregation analysis of M75/H35_155 using a backcross population of 143 plants derived from [AF426-R x AF426-S] x AF426-S and screened for resistance to P. xanthii race 1 produced a recombination frequency of 4.9%, indicating close linkage between M75/H35_155 and Pm-1. Using the same segregating population, the M75/H35_155 marker had previously been reported to be distantly linked to Prv¹, a gene conferring resistance to papaya ringspot virus-type W. Since M75/H35_155 is linked to Prv¹ at a distance of 40.9 cM it is possible that Pm-1 and Prv¹ are also linked.

  8. A simple method for screening of plant NBS-LRR genes that confer a hypersensitive response to plant viruses and its application for screening candidate pepper genes against Pepper mottle virus.

    Science.gov (United States)

    Tran, Phu-Tri; Choi, Hoseong; Kim, Saet-Byul; Lee, Hyun-Ah; Choi, Doil; Kim, Kook-Hyung

    2014-06-01

    Plant NBS-LRR genes are abundant and have been increasingly cloned from plant genomes. In this study, a method based on agroinfiltration and virus inoculation was developed for the simple and inexpensive screening of candidate R genes that confer a hypersensitive response to plant viruses. The well-characterized resistance genes Rx and N, which confer resistance to Potato virus X (PVX) and tobamovirus, respectively, were used to optimize a transient expression assay for detection of hypersensitive response in Nicotiana benthamiana. Infectious sap of PVX and Tobacco mosaic virus were used to induce hypersensitive response in Rx- and N-infiltrated leaves, respectively. The transient expression of the N gene induced local hypersensitive response upon infection of another tobamovirus, Pepper mild mottle virus, through both sap and transcript inoculation. When this method was used to screen 99 candidate R genes from pepper, an R gene that confers hypersensitive response to the potyvirus Pepper mottle virus was identified. The method will be useful for the identification of plant R genes that confer resistance to viruses. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Introgression and pyramiding into common bean market class fabada of genes conferring resistance to anthracnose and potyvirus.

    Science.gov (United States)

    Ferreira, Juan José; Campa, Ana; Pérez-Vega, Elena; Rodríguez-Suárez, Cristina; Giraldez, Ramón

    2012-03-01

    Anthracnose and bean common mosaic (BCM) are considered major diseases in common bean crop causing severe yield losses worldwide. This work describes the introgression and pyramiding of genes conferring genetic resistance to BCM and anthracnose local races into line A25, a bean genotype classified as market class fabada. Resistant plants were selected using resistance tests or combining resistance tests and marker-assisted selection. Lines A252, A321, A493, Sanilac BC6-Are, and BRB130 were used as resistance sources. Resistance genes to anthracnose (Co-2 ( C ), Co-2 ( A252 ) and Co-3/9) and/or BCM (I and bc-3) were introgressed in line A25 through six parallel backcrossing programs, and six breeding lines showing a fabada seed phenotype were obtained after six backcross generations: line A1258 from A252; A1231 from A321; A1220 from A493; A1183 and A1878 from Sanilac BC6-Are; and line A2418 from BRB130. Pyramiding of different genes were developed using the pedigree method from a single cross between lines obtained in the introgression step: line A1699 (derived from cross A1258 × A1220), A2438 (A1220 × A1183), A2806 (A1878 × A2418), and A3308 (A1699 × A2806). A characterization based on eight morpho-agronomic traits revealed a limited differentiation among the obtained breeding lines and the recurrent line A25. However, using a set of seven molecular markers linked to the loci used in the breeding programs it was possible to differentiate the 11 fabada lines. Considering the genetic control of the resistance in resistant donor lines, the observed segregations in the last backcrossing generation, the reaction against the pathogens, and the expression of the molecular markers it was also possible to infer the genotype conferring resistance in the ten fabada breeding lines obtained. As a result of these breeding programs, genetic resistance to three anthracnose races controlled by genes included in clusters Co-2 and Co-3/9, and genetic resistance to BCM controlled

  10. DNA microarray revealed and RNAi plants confirmed key genes conferring low Cd accumulation in barley grains

    DEFF Research Database (Denmark)

    Sun, Hongyan; Chen, Zhong-Hua; Chen, Fei

    2015-01-01

    accumulation and tolerance between the two contrasting barley genotypes: W6nk2 (a low-grain-Cd-accumulating and Cd-sensitive genotype) and Zhenong8 (a high-grain-Cd-accumulating and tolerant genotype). A DNA microarray analysis detected large-scale changes of gene expression in response to Cd stress...... with a substantial difference between the two genotypes. Cd stress led to higher expression of genes involved in transport, carbohydrate metabolism and signal transduction in the low-grain-Cd-accumulating genotype. Novel transporter genes such as zinc transporter genes were identified as being associated with low Cd......Background Understanding the mechanism of low Cd accumulation in crops is crucial for sustainable safe food production in Cd-contaminated soils. Results Confocal microscopy, atomic absorption spectrometry, gas exchange and chlorophyll fluorescence analyses revealed a distinct difference in Cd...

  11. Modified cellulose synthase gene from Arabidopsis thaliana confers herbicide resistance to plants

    Science.gov (United States)

    Somerville, Chris R.; Scheible, Wolf

    2007-07-10

    Cellulose synthase ("CS"), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl)phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  12. A Novel erm(44) Gene Variant from a Human Staphylococcus saprophyticus Isolate Confers Resistance to Macrolides and Lincosamides but Not Streptogramins.

    Science.gov (United States)

    Strauss, Christian; Hu, Yanmin; Coates, Anthony; Perreten, Vincent

    2017-01-01

    A novel erm(44) gene variant, erm(44)v, has been identified by whole-genome sequencing in a Staphylococcus saprophyticus isolate from the skin of a healthy person. It has the particularity to confer resistance to macrolides and lincosamides but not to streptogramin B when expressed in S. aureus The erm(44)v gene resides on a 19,400-bp genomic island which contains phage-associated proteins and is integrated into the chromosome of S. saprophyticus.

  13. Different Mechanisms Confer Gradual Control and Memory at Nutrient- and Stress-Regulated Genes in Yeast

    OpenAIRE

    Rienzo, Alessandro; Poveda-Huertes, Daniel; Aydin, Selcan; Buchler, Nicolas E.; Pascual-Ahuir, Amparo; Proft, Markus

    2015-01-01

    Cells respond to environmental stimuli by fine-tuned regulation of gene expression. Here we investigated the dose-dependent modulation of gene expression at high temporal resolution in response to nutrient and stress signals in yeast. The GAL1 activity in cell populations is modulated in a well-defined range of galactose concentrations, correlating with a dynamic change of histone remodeling and RNA polymerase II (RNAPII) association. This behavior is the result of a heterogeneous induction d...

  14. PCR Amplification System of DNA Barcoding Genes ITS, ITS2 and rbcL from Xanthium%DNA条形码基因ITS·ITS2及rbcL在苍耳属可采用相同的PCR条件

    Institute of Scientific and Technical Information of China (English)

    胡伟毅; 汪连军; 盛志超

    2013-01-01

    [目的]为植物 DNA条形码标准基因筛选研究提供参考,减少植物 DNA条形码研究中的工作量。[方法]对7种苍耳属植物 ITS、ITS2及 rbcL基因采用相同的扩增条件(95℃4 min;[35 cycles:94℃30 s;52℃45 s;72℃45 s];72℃10 min;4℃保存)。[结果]3种DNA条形码基因同时成功扩增。[结论]这说明植物 DNA条形码基因中 ITS、ITS2及 rbcL的PCR条件存在合并可能性。%[Objective] This study aimed to provide reference and reduce the workload for screening standard DNA barcoding genes of plants. [Method] Three DNA bar-coding genes ITS, ITS2 and rbcL were amplified from seven Xanthium species un-der the same PCR condition: PCR amplification was started with initial denaturation at 95 ℃ for 4 min, fol owed by 35 cycles of denaturation at 94 ℃ for 30 s, anneal-ing at 52 ℃ for 45 s, and extension at 72 ℃ for 45 s; the amplification was com-pleted by holding the reaction mixture at 72 ℃ for 10 min to al ow complete exten-sion of PCR, and the PCR products were stored at 4 ℃. [Result] Three DNA bar-coding genes ITS, ITS2 and rbcL were al amplified successful y. [Conclusion] This study indicates that PCR amplification conditions for DNA barcoding genes ITS, ITS2 and rbcL in plants may be consistent.

  15. Preparing a re-sequencing DNA library of 2 cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven’t benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several methods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their applications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a ligation by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2 as target regions, specifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation ’synthesis by sequencing technology’ Illumina/Solexa Genome Analyzer. Bioinformatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection

  16. Preparing a re-sequencing DNA library of 2 cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions

    Institute of Scientific and Technical Information of China (English)

    SU YeYang; LIN Lin; TIAN Geng; CHEN Chen; LIU Tao; XU Xingya; QI XinPeng; ZHANG XiuQing; YANG HuanMing

    2009-01-01

    To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing ser-vice, with subsequent bioinformatic software and laboratory methods developed to expand their ap-plications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven't benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several meth-ods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their ap-plications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a liga-tion by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2 as target regions, spe-cifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation 'synthesis by sequencing technology' IlluminalSolexa Genome Analyzer. Bioin-formatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection

  17. Chromosomal locations of the maize (Zea mays L. HtP and rt genes that confer resistance to Exserohilum turcicum

    Directory of Open Access Journals (Sweden)

    Juliana Bernardi Ogliari

    2007-01-01

    Full Text Available We used 125 microsatellite markers to genotype the maize (Zea mays L. near isogenic lines (NIL L30HtPHtPRtRt and L30htphtpRtRt and the L40htphtprtrt line which contrast regarding the presence of the recently described dominant HtP and the recessive rt genes that confer resistance to Exserohilum turcicum. Five microsatellite markers revealed polymorphisms between the NIL and were considered candidate linked markers for the HtP resistance gene. Linkage was confirmed by bulked segregant sample (BSS analysis of 32 susceptible and 34 resistant plants from a BC1F1 population derived from the cross (L30HtPHtPRtRt x L40htphtprtrt x L40htphtprtrt. The bnlg198 and dupssr25 markers, both located on maize chromosome 2L (bin 2.08, were polymorphic between bulks. Linkage distances were estimated based on co-segregation data of the 32 susceptible plants and indicated distances of 28.7 centimorgans (cM between HtP and bnlg198 and 23.5 cM between HtP and dupssr25. The same set of susceptible plants was also genotyped with markers polymorphic between L30HtPHtPRtRt and L40htphtprtrt in order to find markers linked to the rt gene. Marker bnlg197, from chromosome 3L (bin 3.06, was found linked to rt at a distance of 9.7 cM. This is the first report on the chromosomal locations of these newly described genes.

  18. Screening for Escherichia coli K-12 genes conferring glyoxal resistance or sensitivity by transposon insertions.

    Science.gov (United States)

    Lee, Changhan; Kim, Jihong; Kwon, Minsuk; Lee, Kihyun; Min, Haeyoung; Kim, Seong Hun; Kim, Dongkyu; Lee, Nayoung; Kim, Jiyeun; Kim, Doyun; Ko, Changmin; Park, Chankyu

    2016-09-01

    Glyoxal (GO) belongs to the reactive electrophilic species generated in vivo in all organisms. In order to identify targets of GO and their response mechanisms, we attempted to screen for GO-sensitive mutants by random insertions of TnphoA-132. The genes responsible for GO susceptibility were functionally classified as the following: (i) tRNA modification; trmE, gidA and truA, (ii) DNA repair; recA and recC, (iii) toxin-antitoxin; mqsA and (iv) redox metabolism; yqhD and caiC In addition, an insertion in the crp gene, encoding the cAMP responsive transcription factor, exhibits a GO-resistant phenotype, which is consistent with the phenotype of adenylate cyclase (cya) mutant showing GO resistance. This suggests that global regulation involving cAMP is operated in a stress response to GO. To further characterize the CRP-regulated genes directly associated with GO resistance, we created double mutants deficient in both crp and one of the candidate genes including yqhD, gloA and sodB The results indicate that these genes are negatively regulated by CRP as confirmed by real-time RT-PCR. We propose that tRNA as well as DNA are the targets of GO and that toxin/antitoxin, antioxidant and cAMP are involved in cellular response to GO.

  19. Diagnóstico de Mycoplasma genitalium por amplificación de los genes MgPa y ARN ribosomal 16S Diagnosis of Mycoplasma genitalium by MgPa and rRNA 16S gene amplification

    Directory of Open Access Journals (Sweden)

    Carmen Fernández-Molina

    2008-10-01

    Full Text Available OBJETIVO: El microorganismo Mycoplasma genitalium se ha relacionado con la uretritis no gonocócica (UNG. La técnica de PCR se ha convertido en el principal método de detección de este patógeno. En consecuencia, debe aplicarse un método de diagnóstico mediante la amplificación de fragmentos de ADN por la técnica PCR. MATERIAL Y MÉTODOS: Se seleccionaron los cebadores MGF-MGR y MgPaF-MgPaR, complementarios de los genes de ARNr 16S y MgPa de M. genitalium, respectivamente. Se efectuaron ensayos de especificidad y sensibilidad y se estudiaron muestras clínicas. RESULTADOS: La PCR con cada grupo de cebadores utilizado fue específica sólo para M. genitalium y la sensibilidad fue mayor con el grupo de cebadores MGF-MGR. En el estudio de 34 muestras clínicas, 18.5% fue positivo a M. genitalium y se encontró un mayor número de muestras positivas al utilizar los cebadores MgPaF-MgPaR. CONCLUSIONES: Debe aplicarse en la práctica clínica el diagnóstico de M. genitalium mediante la amplificación del ADN por PCR en los pacientes con UNG.OBJECTIVE: Mycoplasma genitalium has been associated with nongonococcal urethritis (NGU. Diagnosis by PCR has become the primary detection method for this organism. Thus, diagnosis by DNA amplification using the PCR technique should be utilized. MATERIAL AND METHODS: GMF/GMR and MgpF/MgpR primer pairs, complementary to the M. genitalium 16S rRNA and MgPa genes, respectively, were selected. Specificity and sensibility assays were conducted and clinical samples were studied. RESULTS: The PCR with each primer pair was specific only for M. genitalium, and the sensibility was higher with the GMF/GMR primers. In the study of 34 clinical samples, 18,5% were positive for M. genitalium, with more positive samples when the MgpF/MgpR primers were used. CONCLUSIONS: DNA amplification by PCR should be applied in clinical practice to the diagnosis of M. genitalium in patients with NGU should using.

  20. Coordinated Regulation of Anthocyanin Biosynthesis Genes Confers Varied Phenotypic and Spatial-Temporal Anthocyanin Accumulation in Radish (Raphanus sativus L.

    Directory of Open Access Journals (Sweden)

    Everlyne M'mbone Muleke

    2017-07-01

    Full Text Available Anthocyanins are natural pigments that have important functions in plant growth and development. Radish taproots are rich in anthocyanins which confer different taproot colors and are potentially beneficial to human health. The crop differentially accumulates anthocyanin during various stages of growth, yet molecular mechanisms underlying this differential anthocyanin accumulation remains unknown. In the present study, transcriptome analysis was used to concisely identify putative genes involved in anthocyanin biosynthesis in radish. Spatial-temporal transcript expressions were then profiled in four color variant radish cultivars. From the total transcript sequences obtained through illumina sequencing, 102 assembled unigenes, and 20 candidate genes were identified to be involved in anthocyanin biosynthesis. Fifteen genomic sequences were isolated and sequenced from radish taproot. The length of these sequences was between 900 and 1,579 bp, and the unigene coverage to all of the corresponding cloned sequences was more than 93%. Gene structure analysis revealed that RsF3′H is intronless and anthocyanin biosynthesis genes (ABGs bear asymmetrical exons, except RsSAM. Anthocyanin accumulation showed a gradual increase in the leaf of the red radish and the taproot of colored cultivars during development, with a rapid increase at 30 days after sowing (DAS, and the highest content at maturity. Spatial-temporal transcriptional analysis of 14 genes revealed detectable expressions of 12 ABGs in various tissues at different growth levels. The investigation of anthocyanin accumulation and gene expression in four color variant radish cultivars, at different stages of development, indicated that total anthocyanin correlated with transcript levels of ABGs, particularly RsUFGT, RsF3H, RsANS, RsCHS3 and RsF3′H1. Our results suggest that these candidate genes play key roles in phenotypic and spatial-temporal anthocyanin accumulation in radish through

  1. Overexpression of AmRosea1 Gene Confers Drought and Salt Tolerance in Rice

    Science.gov (United States)

    Dou, Mingzhu; Fan, Sanhong; Yang, Suxin; Huang, Rongfeng; Yu, Huiyun; Feng, Xianzhong

    2016-01-01

    Ectopic expression of the MYB transcription factor of AmROSEA1 from Antirrhinum majus has been reported to change anthocyanin and other metabolites in several species. In this study, we found that overexpression of AmRosea1 significantly improved the tolerance of transgenic rice to drought and salinity stresses. Transcriptome analysis revealed that a considerable number of stress-related genes were affected by exogenous AmRosea1 during both drought and salinity stress treatments. These affected genes are involved in stress signal transduction, the hormone signal pathway, ion homeostasis and the enzymes that remove peroxides. This work suggests that the AmRosea1 gene is a potential candidate for genetic engineering of crops. PMID:28025485

  2. Overexpression of AmRosea1 Gene Confers Drought and Salt Tolerance in Rice

    Directory of Open Access Journals (Sweden)

    Mingzhu Dou

    2016-12-01

    Full Text Available Ectopic expression of the MYB transcription factor of AmROSEA1 from Antirrhinum majus has been reported to change anthocyanin and other metabolites in several species. In this study, we found that overexpression of AmRosea1 significantly improved the tolerance of transgenic rice to drought and salinity stresses. Transcriptome analysis revealed that a considerable number of stress-related genes were affected by exogenous AmRosea1 during both drought and salinity stress treatments. These affected genes are involved in stress signal transduction, the hormone signal pathway, ion homeostasis and the enzymes that remove peroxides. This work suggests that the AmRosea1 gene is a potential candidate for genetic engineering of crops.

  3. MicroRNAs Suppress NB Domain Genes in Tomato That Confer Resistance to Fusarium oxysporum

    Science.gov (United States)

    Ouyang, Shouqiang; Park, Gyungsoon; Atamian, Hagop S.; Han, Cliff S.; Stajich, Jason E.; Kaloshian, Isgouhi; Borkovich, Katherine A.

    2014-01-01

    MicroRNAs (miRNAs) suppress the transcriptional and post-transcriptional expression of genes in plants. Several miRNA families target genes encoding nucleotide-binding site–leucine-rich repeat (NB-LRR) plant innate immune receptors. The fungus Fusarium oxysporum f. sp. lycopersici causes vascular wilt disease in tomato. We explored a role for miRNAs in tomato defense against F. oxysporum using comparative miRNA profiling of susceptible (Moneymaker) and resistant (Motelle) tomato cultivars. slmiR482f and slmiR5300 were repressed during infection of Motelle with F. oxysporum. Two predicted mRNA targets each of slmiR482f and slmiR5300 exhibited increased expression in Motelle and the ability of these four targets to be regulated by the miRNAs was confirmed by co-expression in Nicotiana benthamiana. Silencing of the targets in the resistant Motelle cultivar revealed a role in fungal resistance for all four genes. All four targets encode proteins with full or partial nucleotide-binding (NB) domains. One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen. The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response. Taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs. PMID:25330340

  4. Can Genomic Amplification of Human Telomerase Gene and C-MYC in Liquid-Based Cytological Specimens Be Used as a Method for Opportunistic Cervical Cancer Screening?

    Science.gov (United States)

    Gao, Kun; Eurasian, Menglan; Zhang, Jieqing; Wei, Yuluan; Zheng, Qian; Ye, Hongtao; Li, Li

    2015-01-01

    To evaluate the effectiveness of five methods including the ThinPrep cytological test (TCT), liquid-based cytology, the human papillomavirus (HPV) test, detection of the TERC and C-MYC genes and visual inspection with acetic acid/Lugol's iodine (VIA/VILI) for opportunistic cervical cancer screening, and to explore whether genomic amplification of the human telomerase gene and C-MYC in liquid-based cytological specimens can be used as a method for opportunistic cervical cancer screening. Data were collected prospectively from 1,010 consecutive patients who visited the gynecology clinic and agreed to participate in opportunistic cervical cancer screening at our institution from November 2010 to July 2011. The five methods mentioned above were used for the screening in all cases. The histopathological diagnosis served as the gold standard for the evaluation. A comparison between the five screening methods for the diagnosis of high-grade cervical intraepithelial neoplasia (CIN II and III) was performed for their sensitivity, specificity, false-positive rate, false-negative rate, accuracy rate, positive likelihood ratio and negative likelihood ratio. A comprehensive comparison of the different combination programs for screening was performed according to the analysis of the receiver operating characteristic (ROC) curve area. The accuracy of the five screening methods for the diagnosis of high-grade CIN (CIN II and III) was compared in the different age groups. A joint model for the diagnosis using different combinations of the five methods was developed according to the analysis by the SAS 8.0 software. The model was used to evaluate the accuracy of the different combination programs for the diagnosis of high-grade CIN, and the results were confirmed by the histopathological examination. The sensitivity and specificity of the single screen method (TCT, HPV test, detection of the TERC and C-MYC genes, and VIA/VILI method) for CIN II was 80.9, 70.2, 72.3, 76.6, and 72

  5. Cooperative interactions between CBP and TORC2 confer selectivity to CREB target gene expression

    DEFF Research Database (Denmark)

    Ravnskjær, Kim; Kester, Henri; Liu, Yi;

    2007-01-01

    , but have minimal effects on CRE-dependent transcription. Here, we show that the latent cytoplasmic coactivator TORC2 mediates target gene activation in response to cAMP signaling by associating with CBP/p300 and increasing its recruitment to a subset of CREB target genes. TORC2 is not activated in response...... to stress signals, however; and in its absence, P-CREB is unable to stimulate CRE-dependent transcription, due to a block in CBP recruitment. The effect of TORC2 on CBP/p300 promoter occupancy appears pivotal because a gain of function mutant CREB polypeptide with increased affinity for CBP restored CRE...

  6. Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross-amplification with fish feces

    Science.gov (United States)

    Methods focused on members of the genus Bacteroides have been increasingly utilized in microbial source tracking studies for identifying and quantifying sources of non-point fecal contamination. We present results using real-time PCR to show significant cross-amplification of a human-specific Bacter...

  7. Massive Amplification at an Unselected Locus Accompanies Complex Chromosomal Rearrangements in Yeast

    Directory of Open Access Journals (Sweden)

    Agnès Thierry

    2016-05-01

    Full Text Available Gene amplification has been observed in different organisms in response to environmental constraints, such as limited nutrients or exposure to a variety of toxic compounds, conferring them with specific phenotypic adaptations via increased expression levels. However, the presence of multiple gene copies in natural genomes has generally not been found in the absence of specific functional selection. Here, we show that the massive amplification of a chromosomal locus (up to 880 copies per cell occurs in the absence of any direct selection, and is associated with low-order amplifications of flanking segments in complex chromosomal alterations. These results were obtained from mutants with restored phenotypes that spontaneously appeared from genetically engineered strains of the yeast Saccharomyces cerevisiae suffering from severe fitness reduction. Grossly extended chromosomes (macrotene were formed, with complex structural alterations but sufficient stability to propagate unchanged over successive generations. Their detailed molecular analysis, including complete genome sequencing, identification of sequence breakpoints, and comparisons between mutants, revealed novel mechanisms causing their formation, whose combined action underlies the astonishing dynamics of eukaryotic chromosomes and their consequences.

  8. Altered DNA Binding and Amplification of Human Breast Cancer Suppressor Gene BRCA1 Induced by a Novel Antitumor Compound, [Ru(η6-p-phenylethacrynateCl2(pta

    Directory of Open Access Journals (Sweden)

    Adisorn Ratanaphan

    2012-10-01

    Full Text Available The ruthenium-based complex [Ru(η6-p-phenylethacrynateCl2(pta] (pta = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane, termed ethaRAPTA, is an interesting antitumor compound. The elucidation of the molecular mechanism of drug activity is central to the drug development program. To this end, we have characterized the ethaRAPTA interaction with DNA, including probing the sequence specific modified DNA structural stability and DNA amplification using the breast cancer suppressor gene 1 (BRCA1 of human breast and colon adenocarcinoma cell lines as models. The preference of ethaRAPTA base binding is in the order A > G > T > C. Once modified, the ethaRAPTA-induced BRCA1 structure has higher thermal stability than the modified equivalents of its related compound, RAPTA-C. EthaRAPTA exhibits a higher efficiency than RAPTA-C in inhibiting BRCA1 amplification. With respect to both compounds, the inhibition of BRCA1 amplification is more effective in an isolated system than in cell lines. These data provide evidence that will help to understand the process of elucidating the pathways involved in the response induced by ethaRAPTA.

  9. Modified cellulose synthase gene from 'Arabidopsis thaliana' confers herbicide resistance to plants

    Energy Technology Data Exchange (ETDEWEB)

    Somerville, Chris R.; Scieble, Wolf

    2000-10-11

    Cellulose synthase ('CS'), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  10. Barley Genes as Tools to Confer Abiotic Stress Tolerance in Crops.

    Science.gov (United States)

    Gürel, Filiz; Öztürk, Zahide N; Uçarlı, Cüneyt; Rosellini, Daniele

    2016-01-01

    Barley is one of the oldest cultivated crops in the world with a high adaptive capacity. The natural tolerance of barley to stress has led to increasing interest in identification of stress responsive genes through small/large-scale omics studies, comparative genomics, and overexpression of some of these genes by genetic transformation. Two major categories of proteins involved in stress tolerance are transcription factors (TFs) responsible from the re-programming of the metabolism in stress environment, and genes encoding Late Embryogenesis Abundant (LEA) proteins, antioxidant enzymes, osmolytes, and transporters. Constitutive overexpression of several barley TFs, such as C-repeat binding factors (HvCBF4), dehydration-responsive element-binding factors (HvDREB1), and WRKYs (HvWRKY38), in transgenic plants resulted in higher tolerance to drought and salinity, possibly by effectively altering the expression levels of stress tolerance genes due to their higher DNA binding affinity. Na(+)/H(+) antiporters, channel proteins, and lipid transporters can also be the strong candidates for engineering plants for tolerance to salinity and low temperatures.

  11. Barley genes as a tool to confer abiotic stress tolerance in crops

    Directory of Open Access Journals (Sweden)

    Filiz Gürel

    2016-08-01

    Full Text Available Barley is one of the oldest cultivated crops in the world with a high adaptive capacity. The natural tolerance of barley to stress has led to increasing interest in identification of stress responsive genes through small/large-scale omics studies, comparative genomics, and overexpression of some of these genes by genetic transformation. Two major categories of proteins involved in stress tolerance are transcription factors (TFs responsible from the re-programming of the metabolism in stress environment, and genes encoding Late Embryogenesis Abundant (LEA proteins, antioxidant enzymes, osmolytes and transporters. Constitutive overexpression of several barley TFs, such as C-repeat binding factors (HvCBF4, dehydration-responsive element-binding factors (HvDREB1 and WRKYs (HvWRKY38, in transgenic plants resulted in higher tolerance to drought and salinity, possibly by effectively altering the expression levels of stress tolerance genes due to their higher DNA binding affinity. Na+/H+ antiporters, channel proteins, and lipid transporters can also be the strong candidates for engineering plants for tolerance to salinity and low temperatures.

  12. Risk conferred by FokI polymorphism of vitamin D receptor (VDR gene for essential hypertension

    Directory of Open Access Journals (Sweden)

    N Swapna

    2011-01-01

    Full Text Available Background : The vitamin D receptor (VDR gene serves as a good candidate gene for susceptibility to several diseases. The gene has a critical role in regulating the renin-angiotensin system (RAS influencing the regulation of blood pressure. Hence determining the association of VDR polymorphisms with essential hypertension is expected to help in the evaluation of risk for the condition. Aim : The aim of this study was to evaluate association between VDR Fok I polymorphism and genetic susceptibility to essential hypertension. Materials and Methods : Two hundred and eighty clinically diagnosed hypertensive patients and 200 normotensive healthy controls were analyzed for Fok I (T/C [rs2228570] polymorphism by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP analysis. Genotype distribution and allele frequencies in patients and controls, and odds ratios (ORs were calculated to predict the risk for developing hypertension by the individuals of different genotypes. Results : The genotype distribution and allele frequencies of Fok I (T/C [rs2228570] VDR polymorphism differed significantly between patients and controls (χ2 of 18.0; 2 degrees of freedom; P = 0.000. FF genotype and allele F were at significantly greater risk for developing hypertension and the risk was elevated for both the sexes, cases with positive family history and habit of smoking. Conclusions : Our data suggest that VDR gene Fok I polymorphism is associated with the risk of developing essential hypertension

  13. Plant-Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer ability

    Directory of Open Access Journals (Sweden)

    Satoko eNonaka

    2014-12-01

    Full Text Available Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC deaminase, which cleaves the ethylene precursor ACC into α-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant–Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium.

  14. Genetic mapping of two genes conferring resistance to powdery mildew in common bean (Phaseolus vulgaris L.).

    Science.gov (United States)

    Pérez-Vega, Elena; Trabanco, Noemí; Campa, Ana; Ferreira, Juan José

    2013-06-01

    Powdery mildew (PM) is a serious disease in many legume species, including the common bean (Phaseolus vulgaris L.). This study investigated the genetic control behind resistance reaction to PM in the bean genotype, Cornell 49242. The results revealed evidence supporting a qualitative mode of inheritance for resistance and the involvement of two independent genes in the resistance reaction. The location of these resistance genes was investigated in a linkage genetic map developed for the XC RIL population. Contingency tests revealed significant associations for 28 loci out of a total of 329 mapped loci. Fifteen were isolated or formed groups with less than two loci. The thirteen remaining loci were located at three regions in linkage groups Pv04, Pv09, and Pv11. The involvement of Pv09 was discarded due to the observed segregation in the subpopulation obtained from the Xana genotype for the loci located in this region. In contrast, the two subpopulations obtained from the Xana genotype for the BM161 locus, linked to the Co-3/9 anthracnose resistance gene (Pv04), and from the Xana genotype for the SCAReoli locus, linked to the Co-2 anthracnose resistance gene (Pv11), exhibited monogenic segregations, suggesting that both regions were involved in the genetic control of resistance. A genetic dissection was carried out to verify the involvement of both regions in the reaction to PM. Two resistant recombinant lines were selected, according to their genotypes, for the block of loci included in the Co-2 and Co-3/9 regions, and they were crossed with the susceptible parent, Xana. Linkage analysis in the respective F2 populations supported the hypothesis that a dominant gene (Pm1) was located in the linkage group Pv11 and another gene (Pm2) was located in the linkage group Pv04. This is the first report showing the localization of resistance genes against powdery mildew in Phaseolus vulgaris and the results offer the opportunity to increase the efficiency of breeding

  15. A cfr-Like Gene from Clostridium difficile Confers Multiple Antibiotic Resistance by the Same Mechanism as the cfr Gene

    DEFF Research Database (Denmark)

    Hansen, Lykke H; Vester, Birte

    2015-01-01

    The Cfr RNA methyltransferase causes multiple resistances to peptidyl transferase inhibitors by methylation of A2503 23S rRNA. Many cfr-like gene sequences in the databases code for unknown functions. This study confirms that a Cfr-like protein from a Peptoclostridium difficile (formerly Clostrid......The Cfr RNA methyltransferase causes multiple resistances to peptidyl transferase inhibitors by methylation of A2503 23S rRNA. Many cfr-like gene sequences in the databases code for unknown functions. This study confirms that a Cfr-like protein from a Peptoclostridium difficile (formerly...... Clostridium difficile) strain does function as a Cfr protein. The enzyme is expressed in Escherichia coli and shows elevated MICs for five classes of antibiotics. A primer extension stop indicates a modification at A2503 in 23S rRNA....

  16. Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Korfhagen, T.R.; Glasser, S.W.; Wert, S.E.; Bruno, M.D.; Daugherty, C.C.; McNeish, J.D.; Stock, J.L.; Potter, S.S.; Whitsett, J.A. (Cincinnati College of Medicine, OH (USA))

    1990-08-01

    Pulmonary surfactant is produced in late gestation by developing type II epithelial cells lining the alveolar epithelium of the lung. Lack of surfactant at birth is associated with respiratory distress syndrome in premature infants. Surfactant protein C (SP-C) is a highly hydrophobic peptide isolated from pulmonary tissue that enhances the biophysical activity of surfactant phospholipids. Like surfactant phospholipid, SP-C is produced by epithelial cells in the distal respiratory epithelium, and its expression increases during the latter part of gestation. A chimeric gene containing 3.6 kilobases of the promoter and 5{prime}-flanking sequences of the human SP-C gene was used to express diphtheria toxin A. The SP-C-diphtheria toxin A fusion gene was injected into fertilized mouse eggs to produce transgenic mice. Affected mice developed respiratory failure in the immediate postnatal period. Morphologic analysis of lungs from affected pups showed variable but severe cellular injury confined to pulmonary tissues. Ultrastructural changes consistent with cell death and injury were prominent in the distal respiratory epithelium. Proximal components of the tracheobronchial tree were not severely affected. Transgenic animals were of normal size at birth, and structural abnormalities were not detected in nonpulmonary tissues. Lung-specific diphtheria toxin A expression controlled by the human SP-C gene injured type II epithelial cells and caused extensive necrosis of the distal respiratory epithelium. The absence of type I epithelial cells in the most severely affected transgenic animals supports the concept that developing type II cells serve as precursors to type I epithelial cells.

  17. Gene deficiency and pharmacological inhibition of soluble epoxide hydrolase confers resilience to repeated social defeat stress.

    Science.gov (United States)

    Ren, Qian; Ma, Min; Ishima, Tamaki; Morisseau, Christophe; Yang, Jun; Wagner, Karen M; Zhang, Ji-Chun; Yang, Chun; Yao, Wei; Dong, Chao; Han, Mei; Hammock, Bruce D; Hashimoto, Kenji

    2016-03-29

    Depression is a severe and chronic psychiatric disease, affecting 350 million subjects worldwide. Although multiple antidepressants have been used in the treatment of depressive symptoms, their beneficial effects are limited. The soluble epoxide hydrolase (sEH) plays a key role in the inflammation that is involved in depression. Thus, we examined here the role of sEH in depression. In both inflammation and social defeat stress models of depression, a potent sEH inhibitor, TPPU, displayed rapid antidepressant effects. Expression of sEH protein in the brain from chronically stressed (susceptible) mice was higher than of control mice. Furthermore, expression of sEH protein in postmortem brain samples of patients with psychiatric diseases, including depression, bipolar disorder, and schizophrenia, was higher than controls. This finding suggests that increased sEH levels might be involved in the pathogenesis of certain psychiatric diseases. In support of this hypothesis, pretreatment with TPPU prevented the onset of depression-like behaviors after inflammation or repeated social defeat stress. Moreover, sEH KO mice did not show depression-like behavior after repeated social defeat stress, suggesting stress resilience. The sEH KO mice showed increased brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor TrkB in the prefrontal cortex, hippocampus, but not nucleus accumbens, suggesting that increased BDNF-TrkB signaling in the prefrontal cortex and hippocampus confer stress resilience. All of these findings suggest that sEH plays a key role in the pathophysiology of depression, and that epoxy fatty acids, their mimics, as well as sEH inhibitors could be potential therapeutic or prophylactic drugs for depression.

  18. Polymorphisms of angiotensin-converting enzyme 2 gene confer a risk to lone atrial fibrillation in Chinese male patients

    Institute of Scientific and Technical Information of China (English)

    WANG Shu-xia; TAO Tao; FU Zhi-qing; XIE Xiang-zhu; WANG Hao; WANG Yu-tang

    2013-01-01

    Background Growing epidemiologic evidence has indicated that genetics can predispose individuals to the occurrence of lone atrial fibrillation (AF).The angiotensin-converting enzyme 2 (ACE2) gene has been established to be associated with hypertension and left ventricular hypertrophy.The objective of our study was to investigate the association of ACE2 gene polymorphisms with lone AF.Methods A total of 265 consecutive lone AF patients and 289 healthy controls were successfully investigated.The polymorphisms rs2106809 and rs2285666 were genotyped by polymerase chain reaction (PCR) and direct sequencing.A Logistic regression model was used to determine the odds ratio (OR) and 95% confidence intervals (CI) of variations of ACE2 for lone AF.Results The T allele of rs2106809 conferred an increased risk for lone AF (OR 1.24,95% CI 1.01-1.52,P=0.03) in males after adjustment for conventional risk factors.SNP at rs2285666 in males was not significantly different between AF patients and controls.No association was found between the two polymorphisms in the female population with lone AF.After (36.3±4.5) months of follow-up,the end point data were obtained:death (cardiac and noncardiac),ischemic stroke,and heart failure.In the male subgroup,the associations between rs2106809 T male carriers and combined end points including ischemic stroke,heart failure,and death in our study were of significance (OR 3.6,95% CI 1.0-13.1,P=0.04).Conclusions The results indicate that polymorphism at ACE2 gene is associated with male lone AF in a Chinese Han population.Lone AF males who carry the rs2106809 T allele are associated with adverse cardiac events.

  19. Extended life-span conferred by cotransporter gene mutations in Drosophila.

    Science.gov (United States)

    Rogina, B; Reenan, R A; Nilsen, S P; Helfand, S L

    2000-12-15

    Aging is genetically determined and environmentally modulated. In a study of longevity in the adult fruit fly, Drosophila melanogaster, we found that five independent P-element insertional mutations in a single gene resulted in a near doubling of the average adult life-span without a decline in fertility or physical activity. Sequence analysis revealed that the product of this gene, named Indy (for I'm not dead yet), is most closely related to a mammalian sodium dicarboxylate cotransporter-a membrane protein that transports Krebs cycle intermediates. Indy was most abundantly expressed in the fat body, midgut, and oenocytes: the principal sites of intermediary metabolism in the fly. Excision of the P element resulted in a reversion to normal life-span. These mutations may create a metabolic state that mimics caloric restriction, which has been shown to extend life-span.

  20. Serine/threonine kinase gene Stpk-V, a key member of powdery mildew resistance gene Pm21, confers powdery mildew resistance in wheat.

    Science.gov (United States)

    Cao, Aizhong; Xing, Liping; Wang, Xiaoyun; Yang, Xueming; Wang, Wei; Sun, Yulei; Qian, Chen; Ni, Jinlong; Chen, Yaping; Liu, Dajun; Wang, Xiue; Chen, Peidu

    2011-05-10

    Powdery mildew resistance gene Pm21, located on the chromosome 6V short arm of Haynaldia villosa and transferred to wheat as a 6VS·6AL translocation (T6VS·6AL), confers durable and broad-spectrum resistance to wheat powdery mildew. Pm21 has become a key gene resource for powdery mildew resistance breeding all over the world. In China, 12 wheat varieties containing Pm21 have been planted on more than 3.4 million hectares since 2002. Pm21 has been intractable to molecular genetic mapping because the 6VS does not pair and recombine with the 6AS. Moreover, all known accessions of H. villosa are immune to powdery mildew fungus. Pm21 is still defined by cytogenetics as a locus. In the present study, a putative serine and threonine protein kinase gene Stpk-V was cloned and characterized with an integrative strategy of molecular and cytogenetic techniques. Stpk-V is located on the Pm21 locus. The results of a single cell transient expression assay showed that Stpk-V could decrease the haustorium index dramatically. After the Stpk-V was transformed into a susceptible wheat variety Yangmai158, the characterized transgenic plants showed high and broad-spectrum powdery mildew resistance similar to T6VS·6AL. Silencing of the Stpk-V by virus-induced gene silencing in both T6VS·6AL and H. villosa resulted in their increased susceptibility. Stpk-V could be induced by Bgt and exogenous H(2)O(2), but it also mediated the increase of endogenous H(2)O(2), leading to cell death and plant resistance when the plant was attacked by Bgt.

  1. Accessory Genes Confer a High Replication Rate to Virulent Feline Immunodeficiency Virus

    OpenAIRE

    Troyer, Ryan M.; Thompson, Jesse; Elder, John H.; VandeWoude, Sue

    2013-01-01

    Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between tw...

  2. Distress of ostracism: oxytocin receptor gene polymorphism confers sensitivity to social exclusion

    OpenAIRE

    McQuaid, Robyn J.; McInnis, Opal A.; Matheson, Kimberly; Anisman, Hymie

    2015-01-01

    A single-nucleotide polymorphism on the oxytocin receptor gene (OXTR), rs53576, involving a guanine (G) to adenine (A) substitution has been associated with altered prosocial features. Specifically, individuals with the GG genotype (i.e. the absence of the polymorphism) display beneficial traits including enhanced trust, empathy and self-esteem. However, because G carriers might also be more socially sensitive, this may render them more vulnerable to the adverse effects of a negative social s...

  3. Ghd2, a CONSTANS-like gene, confers drought sensitivity through regulation of senescence in rice

    Science.gov (United States)

    Liu, Juhong; Shen, Jianqiang; Xu, Yan; Li, Xianghua; Xiao, Jinghua; Xiong, Lizhong

    2016-01-01

    CONSTANS (CO)-like genes have been intensively investigated for their roles in the regulation of photoperiodic flowering, but very limited information has been reported on their functions in other biological processes. Here, we found that a CO-like gene, Ghd2 (Grain number, plant height, and heading date2), which can increase the yield potential under normal growth condition just like its homologue Ghd7, is involved in the regulation of leaf senescence and drought resistance. Ghd2 is expressed mainly in the rice (Oryza sativa) leaf with the highest level detected at the grain-filling stage, and it is down-regulated by drought stress conditions. Overexpression of Ghd2 resulted in significantly reduced drought resistance, while its knockout mutant showed the opposite phenotype. The earlier senescence symptoms and the transcript up-regulation of many senescence-associated genes (SAGs) in Ghd2-overexpressing transgenic rice plants under drought stress conditions indicate that Ghd2 plays essential roles in accelerating drought-induced leaf senescence in rice. Moreover, developmental and dark-induced leaf senescence was accelerated in the Ghd2-overexpressing rice and delayed in the ghd2 mutant. Several SAGs were confirmed to be regulated by Ghd2 using a transient expression system in rice protoplasts. Ghd2 interacted with several regulatory proteins, including OsARID3, OsPURα, and three 14-3-3 proteins. OsARID3 and OsPURα showed expression patterns similar to Ghd2 in rice leaves, with the highest levels at the grain-filling stage, whereas OsARID3 and the 14-3-3 genes responded differently to drought stress conditions. These results indicate that Ghd2 functions as a regulator by integrating environmental signals with the senescence process into a developmental programme through interaction with different proteins. PMID:27638689

  4. Identification of Staphylococcus aureus, S. intermedius and S. hyicus by PCR amplification of coa and nuc genes Identificação de Staphylococcus aureus, S. intermedius e S. hyicus através de seqüências dos genes coa e nuc

    Directory of Open Access Journals (Sweden)

    Wladimir Padilha da Silva

    2003-11-01

    Full Text Available Sixty-five strains of coagulase positive staphylococci (Staphylococcus aureus, S. intermedius and S. hyicus were identified at species level by PCR amplification of the coa gene, specific for S. aureus, and of the nuc gene, specific for S. intermedius and for S. hyicus.Sessenta e cinco cepas de estafilococos coagulase positiva foram identificadas em nível de espécie, através da amplificação, por PCR, de seqüências do gene coa, específicas para S. aureus, e do gene nuc, específicas para S. intermedius e para S. hyicus.

  5. Cymbidium mosaic virus coat protein gene in antisense confers resistance to transgenic Nicotiana occidentalis.

    Science.gov (United States)

    Lim, S H; Ko, M K; Lee, S J; La, Y J; Kim, B D

    1999-12-31

    The nucleotide sequence of the 3'-terminal region of the Korean isolate of cymbidium mosaic virus (CyMV-Ca) from a naturally infected cattleya was determined. The sequence contains an open reading frame (ORF) coding for the viral coat protein (CP) at the 3'-end and three other ORFs (triple gene block or movement protein) of CyMV. The CP gene encodes a polypeptide chain of 220 amino acids with a molecular mass of 23,760 Da. The deduced CP sequence showed a strong homology with those of two CyMVs reported. A construct of the CyMV-Ca CP gene in the antisense orientation in the plant expression vector pMBP1 was transferred via Agrobacterium tumefaciens-mediated transformation into Nicotiana occidentalis which is a propagation host of CyMV. The T1 progeny of the transgenic plants were inoculated with CyMV and found to be highly resistant to CyMV infection.

  6. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

    Directory of Open Access Journals (Sweden)

    Fangquan Wang

    2016-05-01

    Full Text Available Rice black-streaked dwarf virus (RBSDV belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA. By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  7. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene confers drought tolerance in maize (Zea mays L.).

    Science.gov (United States)

    Lu, Yao; Li, Yajun; Zhang, Jiachang; Xiao, Yitao; Yue, Yuesen; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

    2013-01-01

    Abscisic acid (ABA) is a key component of the signaling system that integrates plant adaptive responses to abiotic stress. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene (LOS5) in maize markedly enhanced the expression of ZmAO and aldehyde oxidase (AO) activity, leading to ABA accumulation and increased drought tolerance. Transgenic maize (Zea mays L.) exhibited the expected reductions in stomatal aperture, which led to decreased water loss and maintenance of higher relative water content (RWC) and leaf water potential. Also, transgenic maize subjected to drought treatment exhibited lower leaf wilting, electrolyte leakage, malondialdehyde (MDA) and H(2)O(2) content, and higher activities of antioxidative enzymes and proline content compared to wild-type (WT) maize. Moreover, overexpression of LOS5 enhanced the expression of stress-regulated genes such as Rad 17, NCED1, CAT1, and ZmP5CS1 under drought stress conditions, and increased root system development and biomass yield after re-watering. The increased drought tolerance in transgenic plants was associated with ABA accumulation via activated AO and expression of stress-related gene via ABA induction, which sequentially induced a set of favorable stress-related physiological and biochemical responses.

  8. KIT mutations confer a distinct gene expression signature in core binding factor leukaemia.

    Science.gov (United States)

    Lück, Sonja C; Russ, Annika C; Du, Juan; Gaidzik, Verena; Schlenk, Richard F; Pollack, Jonathan R; Döhner, Konstanze; Döhner, Hartmut; Bullinger, Lars

    2010-03-01

    Core binding factor (CBF) leukaemias, characterized by either inv(16)(p13.1q22) or t(8;21)(q22;q22), constitute acute myeloid leukaemia (AML) subgroups with favourable prognosis. However, 40-50% of patients relapse, emphasizing the need for risk-adapted treatment approaches. In this regard, studying secondary genetic aberrations, such as mutations of the KIT gene, is of great interest, particularly as they can be targeted by receptor tyrosine kinase inhibitors (TKI). However, so far little is known about the biology underlying KIT-mutated CBF leukaemias. We analysed gene expression profiles of 83 CBF AML cases with known KIT mutation status in order to gain novel insights in KIT-mutated CBF pathogenesis. KIT-mutated cases were characterized by deregulation of genes belonging to the NFkB signalling complex suggesting impaired control of apoptosis. Notably, a subgroup of KIT wildtype cases was also characterized by the KIT mutation signature due to yet unknown aberrations. Our data suggest that this CBF leukaemia subgroup might profit from TKI therapy, however, the relevance of the KIT mutation-associated signature remains to be validated prior to clinical implementation. Nevertheless, the existence of such a signature supports the notion of relevant biological differences in CBF leukaemia and might serve as diagnostic tool in the future.

  9. MicroRNA-155 confers encephalogenic potential to Th17 cells by promoting effector gene expression

    Science.gov (United States)

    Hu, Ruozhen; Huffaker, Thomas B.; Kagele, Dominique A.; Runtsch, Marah C.; Bake, Erin; Chaudhuri, Aadel A.; Round, June L.; O’Connell, Ryan M.

    2013-01-01

    Th17 cells are central to the pathogenesis of autoimmune disease, and recently specific noncoding microRNAs (miRNAs) have been shown to regulate their development. However, it remains unclear if miRNAs are also involved in modulating Th17 cell effector functions. Consequently, we examined the role of miR-155 in differentiated Th17 cells during their induction of Experimental Autoimmune Encephalomyelitis (EAE). Using adoptive transfer experiments, we found that highly purified, MOG antigen-specific Th17 cells lacking miR-155 were defective in their capacity to cause EAE. Gene expression profiling of purified miR-155−/− IL-17F+ Th17 cells identified a subset of effector genes that are dependent upon miR-155 for their proper expression through a mechanism involving repression of the transcription factor Ets1. Among the genes reduced in the absence of miR-155 was IL-23R, resulting in miR-155−/− Th17 cells being hypo-responsive to IL-23. Taken together, our study demonstrates a critical role for miR-155 in Th17 cells as they unleash autoimmune inflammation, and finds that this occurs through a signaling network involving miR-155, Ets1 and the clinically relevant IL-23-IL-23R pathway. PMID:23686497

  10. Overexpression of Citrus junos mitochondrial citrate synthase gene in Nicotiana benthamiana confers aluminum tolerance.

    Science.gov (United States)

    Deng, Wei; Luo, Keming; Li, Zhengguo; Yang, Yingwu; Hu, Nan; Wu, Yu

    2009-07-01

    Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study, Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression of mitochondrial CS can be a useful tool to achieve Al tolerance.

  11. Expressing yeast SAMdc gene confers broad changes in gene expression and alters fatty acid composition in tomato fruit.

    Science.gov (United States)

    Kolotilin, Igor; Koltai, Hinanit; Bar-Or, Carmiya; Chen, Lea; Nahon, Sahadia; Shlomo, Haviva; Levin, Ilan; Reuveni, Moshe

    2011-07-01

    Tomato (Solanum lycopersicum) fruits expressing a yeast S-adenosyl methionine decarboxylase (ySAMdc) gene under control of a ripening-induced promoter show altered phytonutrient content and broad changes in gene expression. Genome-wide transcriptional alterations in pericarp tissues of the ySAMdc-expressing fruits are shown. Consistent with the ySAMdc expression pattern from the ripening-induced promoter, very minor transcriptional alterations were detected at the mature green developmental stage. At the breaker and red stages, altered levels of numerous transcripts were observed with a general tendency toward upregulation in the transgenic fruits. Ontological analysis of up- and downregulated transcript groups revealed various affected metabolic processes, mainly carbohydrate and amino acid metabolism, and protein synthesis, which appeared to be intensified in the ripening transgenic fruits. Other functional ontological categories of altered transcripts represented signal transduction, transcription regulation, RNA processing, molecular transport and stress response, as well as metabolism of lipids, glycans, xenobiotics, energy, cofactors and vitamins. In addition, transcript levels of genes encoding structural enzymes for several biosynthetic pathways showed strong correlations to levels of specific metabolites that displayed altered levels in transgenic fruits. Increased transcript levels of fatty acid biosynthesis enzymes were accompanied by a change in the fatty acid profile of transgenic fruits, most notably increasing ω-3 fatty acids at the expense of other lipids. Thus, SAMdc is a prime target in manipulating the nutritional value of tomato fruits. Combined with analyses of selected metabolites in the overripe fruits, a model of enhanced homeostasis of the pericarp tissue in the polyamine-accumulating tomatoes is proposed.

  12. The Seneca Amplification Construction

    Directory of Open Access Journals (Sweden)

    Wallace Chafe

    2012-01-01

    Full Text Available The polysynthetic morphology of the Northern Iroquoian languages presents a challenge to studies of clause combining. The discussion here focuses on a Seneca construction that may appear within a single clause but may also straddle clause boundaries. It amplifies the information provided by a referent, here called the trigger, that is introduced by the pronominal prefix within a verb or occasionally in some other way. The particle neh signals that further information about that referent will follow. This construction is found at four levels of syntactic complexity. At the first level the trigger and its amplification occur within the same prosodic phrase and the amplification is a noun. At the second level the amplification occurs in a separate prosodic phrase but remains a noun. At the third level the amplification exhibits verb morphology but has been lexicalized with a nominal function. At the fourth level the amplification functions as a full clause and neh serves as a marker of clause combining. Several varieties of amplification are discussed, as are cases in which the speaker judges that no amplification is needed. It is suggested that the typologically similar Caddo language illustrates a situation in which this construction could never arise, simply because Caddo verbs lack the pronominal element that triggers the construction in Seneca.

  13. Isolation and characterization of a gene, pmrD, from Salmonella typhimurium that confers resistance to polymyxin when expressed in multiple copies.

    OpenAIRE

    Roland, K L; Esther, C R; Spitznagel, J K

    1994-01-01

    We have isolated from Salmonella typhimurium a gene, designated pmrD, that confers resistance to the membrane-damaging drug, polymyxin B when expressed from the medium-copy-number plasmid pHSG576. The gene maps to 46 min on the standard genetic map, near the menB gene, and is therefore distinct from the previously described pmrA locus. We have mapped the polymyxin resistance activity to a 1.3-kb ClaI-PvuII fragment which contains a small open reading frame that could encode an 85-amino-acid p...

  14. Vat, an Amazing Gene Conferring Resistance to Aphids and Viruses They Carry: From Molecular Structure to Field Effects

    Science.gov (United States)

    Boissot, Nathalie; Schoeny, Alexandra; Vanlerberghe-Masutti, Flavie

    2016-01-01

    We review half a century of research on Cucumis melo resistance to Aphis gossypii from molecular to field levels. The Vat gene is unique in conferring resistance to both A. gossypii and the viruses it transmits. This double phenotype is aphid clone-dependent and has been observed in 25 melon accessions, mostly from Asia. It is controlled by a cluster of genes including CC-NLR, which has been characterized in detail. Copy-number polymorphisms (for the whole gene and for a domain that stands out in the LLR region) and single-nucleotide polymorphisms have been identified in the Vat cluster. The role of these polymorphisms in plant/aphid interactions remains unclear. The Vat gene structure suggests a functioning with separate recognition and response phases. During the recognition phase, the VAT protein is thought to interact (likely indirectly) with an aphid effector introduced during cell puncture by the aphid. A few hours later, several miRNAs are upregulated in Vat plants. Peroxidase activity increases, and callose and lignin are deposited in the walls of the cells adjacent to the stylet path, disturbing aphid behavior. In aphids feeding on Vat plants, Piwi-interacting RNA-like sequences are abundant and the levels of other miRNAs are modified. At the plant level, resistance to aphids is quantitative (aphids escape the plant and display low rates of reproduction). Resistance to viruses is qualitative and local. Durability of NLR genes is highly variable. A. gossypii clones are adapted to Vat resistance, either by introducing a new effector that interferes with the deployment of plant defenses, or by adapting to the defenses it triggered. Viruses transmitted in a non-persistent manner cannot adapt to Vat resistance. At population level, Vat reduces aphid density and genetic diversity. The durability of Vat resistance to A. gossypii populations depends strongly on the agro-ecosystem, including, in particular, the presence of other cucurbit crops serving as

  15. Vat, an amazing gene conferring resistance to aphids and viruses they carry: from molecular structure to field effects

    Directory of Open Access Journals (Sweden)

    Nathalie Boissot

    2016-09-01

    Full Text Available We review half a century of research on Cucumis melo resistance to Aphis gossypii from molecular to field levels. The Vat gene is unique in conferring resistance to both A. gossypii and the viruses it transmits. This double phenotype is aphid clone-dependent and has been observed in 25 melon accessions, mostly from Asia. It is controlled by a cluster of genes including CC-NLR, which has been characterized in detail. Copy-number polymorphisms (for the whole gene and for a domain that stands out in the LLR region and single-nucleotide polymorphisms have been identified in the Vat cluster. The role of these polymorphisms in plant aphid/interactions remains unclear. The Vat gene structure suggests a functioning with separate recognition and response phases. During the recognition phase, the VAT protein is thought to interact (likely indirectly with an aphid effector introduced during cell puncture by the aphid. A few hours later, several miRNAs are upregulated in Vat plants. Peroxidase activity increases, and callose and lignin are deposited in the walls of the cells adjacent to the stylet path, disturbing aphid behavior. In aphids feeding on Vat plants, Piwi-interacting RNA-like sequences are abundant and the levels of other miRNAs are modified. At the plant level, resistance to aphids is quantitative (aphids escape the plant and display low rates of reproduction. Resistance to viruses is qualitative and local.Durability of NLR genes is highly variable. A. gossypii clones are adapted to Vat resistance, either by introducing a new effector that interferes with the deployment of plant defenses, or by adapting to the defenses it triggered. Viruses transmitted in a non-persistent manner cannot adapt to Vat resistance. At population level, Vat reduces aphid density and genetic diversity. The durability of Vat resistance to A. gossypii populations depends strongly on the agro-ecosystem, including, in particular, the presence of other cucurbit crops

  16. Polymorphisms of the ITGAM gene confer higher risk of discoid cutaneous than of systemic lupus erythematosus.

    Directory of Open Access Journals (Sweden)

    Tiina M Järvinen

    Full Text Available BACKGROUND: Lupus erythematosus (LE is a heterogeneous disease ranging from mainly skin-restricted manifestations (discoid LE [DLE] and subacute cutaneous LE to a progressive multisystem disease (systemic LE [SLE]. Genetic association studies have recently identified several strong susceptibility genes for SLE, including integrin alpha M (ITGAM, also known as CD11b, whereas the genetic background of DLE is less clear. PRINCIPAL FINDINGS: To specifically investigate whether ITGAM is a susceptibility gene not only for SLE, but also for cutaneous DLE, we genotyped 177 patients with DLE, 85 patients with sporadic SLE, 190 index cases from SLE families and 395 population control individuals from Finland for nine genetic markers at the ITGAM locus. SLE patients were further subdivided by the presence or absence of discoid rash and renal involvement. In addition, 235 Finnish and Swedish patients positive for Ro/SSA-autoantibodies were included in a subphenotype analysis. Analysis of the ITGAM coding variant rs1143679 showed highly significant association to DLE in patients without signs of systemic disease (P-value  = 4.73×10(-11, OR  = 3.20, 95% CI  = 2.23-4.57. Significant association was also detected to SLE patients (P-value  = 8.29×10(-6, OR  = 2.14, 95% CI  = 1.52-3.00, and even stronger association was found when stratifying SLE patients by presence of discoid rash (P-value  = 3.59×10(-8, OR  = 3.76, 95% CI  = 2.29-6.18. SIGNIFICANCE: We propose ITGAM as a novel susceptibility gene for cutaneous DLE. The risk effect is independent of systemic involvement and has an even stronger genetic influence on the risk of DLE than of SLE.

  17. Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

    Directory of Open Access Journals (Sweden)

    Ann C Smigocki

    Full Text Available Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

  18. Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

    Science.gov (United States)

    Smigocki, Ann C; Ivic-Haymes, Snezana; Li, Haiyan; Savić, Jelena

    2013-01-01

    Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI) was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

  19. Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS using chemogenomics

    Directory of Open Access Journals (Sweden)

    Jaime Maria DLA

    2012-06-01

    Full Text Available Abstract Background Chitosan oligosaccharide (COS, a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS. Results Three different chemogenomic fitness assays, haploinsufficiency (HIP, homozygous deletion (HOP, and multicopy suppression (MSP profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms, membrane functions (e.g. signalling, transport and targeting, membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS. Conclusions Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane

  20. Gene amplification: developments, and applications for medical diagnostics. January 1985-February 1989 (Citations from the Biobusiness data base). Report for January 1985-February 1989

    Energy Technology Data Exchange (ETDEWEB)

    1989-03-01

    This bibliography contains citations concerning genetic amplification, a revolutionary new DNA probe technology. Companies on the cutting edge of this technology are discussed, and mention is made of their licensing agreements. Applications in clinical diagnosis for such diseases as acquired immune deficiency syndrome and leukemia, and applications for prenatal testing, and forensic testing are examined. (Contains 55 citations fully indexed and including a title list.)

  1. The Metallothionein Gene, TaMT3, from Tamarix androssowii Confers Cd2+ Tolerance in Tobacco

    Directory of Open Access Journals (Sweden)

    Boru Zhou

    2014-06-01

    Full Text Available Cadmium (Cd is a nonessential microelement and low concentration Cd2+ has strong toxicity to plant growth. Plant metallothioneins, a class of low molecular, cystein(Cys-rich and heavy-metal binding proteins, play an important role in both metal chaperoning and scavenging of reactive oxygen species (ROS with their large number of cysteine residues and therefore, protect plants from oxidative damage. In this study, a metallothionein gene, TaMT3, isolated from Tamarix androssowii was transformed into tobacco (Nicotiana tobacum through Agrobacterium-mediated leaf disc method, and correctly expressed under the control of 35S promoter. Under Cd2+ stress, the transgenic tobacco showed significant increases of superoxide dismutase (SOD activity and chlorophyll concentration, but decreases of peroxidase (POD activity and malondialdehyde (MDA accumulation when compared to the non-transgenic tobacco. Vigorous growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weight were significantly larger than those of the non-transgenic tobacco under Cd2+ stress. These results demonstrated that the expression of the exogenous TaMT3 gene increased the ability of ROS cleaning-up, indicating a stronger tolerance to Cd2+ stress.

  2. Mutations in the Drosophila pushover gene confer increased neuronal excitability and spontaneous synaptic vesicle fusion

    Energy Technology Data Exchange (ETDEWEB)

    Richards, S.; Hillman, T.; Stern, M. [Rice Univ., Houston, TX (United States)

    1996-04-01

    We describe the identification of a gene called pushover (push), which affects both behavior and synaptic transmission at the neuromuscular junction. Adults carrying either of two mutations in push exhibit sluggishness, uncoordination, a defective escape response, and male sterility. Larvae defective in push exhibit increased release of transmitter at the neuromuscular junction. In particular, the frequency of spontaneous transmitter release and the amount of transmitter release evoked by nerve stimulation are each increased two- to threefold in push mutants at the lowest external [(Ca{sup 2+})] tested (0.15 mM). Furthermore, these mutants are more sensitive than wild type to application of the potassium channel-blocking drug quinidine: following quinidine application, push mutants, but not wild-type, display repetitive firing of the motor axon, leading to repetitive muscle postsynaptic potentials. The push gene thus might affect both neuronal excitability and the transmitter release process. Complementation tests and recombinational mapping suggest that the push mutations are allelic to a previously identified P-element-induced mutation, which also causes behavorial abnormalities and male sterility. 43 refs., 5 figs., 1 tab.

  3. Transgenic banana expressing Pflp gene confers enhanced resistance to Xanthomonas wilt disease.

    Science.gov (United States)

    Namukwaya, B; Tripathi, L; Tripathi, J N; Arinaitwe, G; Mukasa, S B; Tushemereirwe, W K

    2012-08-01

    Banana Xanthomonas wilt (BXW), caused by Xanthomonas campestris pv. musacearum, is one of the most important diseases of banana (Musa sp.) and currently considered as the biggest threat to banana production in Great Lakes region of East and Central Africa. The pathogen is highly contagious and its spread has endangered the livelihood of millions of farmers who rely on banana for food and income. The development of disease resistant banana cultivars remains a high priority since farmers are reluctant to employ labor-intensive disease control measures and there is no host plant resistance among banana cultivars. In this study, we demonstrate that BXW can be efficiently controlled using transgenic technology. Transgenic bananas expressing the plant ferredoxin-like protein (Pflp) gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of banana. These transgenic lines were characterized by molecular analysis. After challenge with X. campestris pv. musacearum transgenic lines showed high resistance. About 67% of transgenic lines evaluated were completely resistant to BXW. These transgenic lines did not show any disease symptoms after artificial inoculation of in vitro plants under laboratory conditions as well as potted plants in the screen-house, whereas non-transgenic control plants showed severe symptoms resulting in complete wilting. This study confirms that expression of the Pflp gene in banana results in enhanced resistance to BXW. This transgenic technology can provide a timely solution to the BXW pandemic.

  4. DNA sequencing conference, 2

    Energy Technology Data Exchange (ETDEWEB)

    Cook-Deegan, R.M. [Georgetown Univ., Kennedy Inst. of Ethics, Washington, DC (United States); Venter, J.C. [National Inst. of Neurological Disorders and Strokes, Bethesda, MD (United States); Gilbert, W. [Harvard Univ., Cambridge, MA (United States); Mulligan, J. [Stanford Univ., CA (United States); Mansfield, B.K. [Oak Ridge National Lab., TN (United States)

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  5. Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaffolder and striped stem borer

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Immature embryos of rice varieties “Xiushui11” and “Chunjiang 11” precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7(containing the spider insecticidal gene).The resistant calli were transferred onto the differentiation medium and plants were regenerated.The transformation frequency reached 56%~72% measured as numbers of Geneticin(G418)-resistant calli produced and 36%~60% measured as numbers of transgenic plants regenerated,respectively.PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome.Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder(Cnaphalocrasis medinalis)after 7d of leaf feeding reached 38%~61% and the corrected mortality of the striped stem borer(Chilo suppressalis)after 7d of leaf feeding reached 16%~75%.The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.

  6. Amplification of NOON States

    CERN Document Server

    Agarwal, G S; Rai, Amit

    2009-01-01

    We examine the behavior of a Non Gaussian state like NOON state under phase insensitive amplification. We derive analytical result for the density matrix of the NOON state for arbitrary gain of the amplifier. We consider cases of both symmetric and antisymmetric amplification of the two modes of the NOON state. We quantitatively evaluate the loss of entanglement by the amplifier in terms of the logarithmic negativity parameter. We find that NOON states are more robust than their Gaussian counterparts.

  7. Amplification of NOON States

    OpenAIRE

    2009-01-01

    We examine the behavior of a Non Gaussian state like NOON state under phase insensitive amplification. We derive analytical result for the density matrix of the NOON state for arbitrary gain of the amplifier. We consider cases of both symmetric and antisymmetric amplification of the two modes of the NOON state. We quantitatively evaluate the loss of entanglement by the amplifier in terms of the logarithmic negativity parameter. We find that NOON states are more robust than their Gaussian coun...

  8. Pathogen-induced expression of a cecropin A-melittin antimicrobial peptide gene confers antifungal resistance in transgenic tobacco.

    Science.gov (United States)

    Yevtushenko, Dmytro P; Romero, Rafael; Forward, Benjamin S; Hancock, Robert E; Kay, William W; Misra, Santosh

    2005-06-01

    Expression of defensive genes from a promoter that is specifically activated in response to pathogen invasion is highly desirable for engineering disease-resistant plants. A plant transformation vector was constructed with transcriptional fusion between the pathogen-responsive win3.12T promoter from poplar and the gene encoding the novel cecropin A-melittin hybrid peptide (CEMA) with strong antimicrobial activity. This promoter-transgene combination was evaluated in transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) for enhanced plant resistance against a highly virulent pathogenic fungus Fusarium solani. Transgene expression in leaves was strongly increased after fungal infection or mechanical wounding, and the accumulation of CEMA transcripts was found to be systemic and positively correlated with the number of transgene insertions. A simple and efficient in vitro regeneration bioassay for preliminary screening of transgenic lines against pathogenic fungi was developed. CEMA had strong antifungal activity in vitro, inhibiting conidia germination at concentrations that were non-toxic to tobacco protoplasts. Most importantly, the expression level of the CEMA peptide in vivo, regulated by the win3.12T promoter, was sufficient to confer resistance against F. solani in transgenic tobacco. The antifungal resistance of plants with high CEMA expression was strong and reproducible. In addition, leaf tissue extracts from transgenic plants significantly reduced the number of fungal colonies arising from germinated conidia. Accumulation of CEMA peptide in transgenic tobacco had no deleterious effect on plant growth and development. This is the first report showing the application of a heterologous pathogen-inducible promoter to direct the expression of an antimicrobial peptide in plants, and the feasibility of this approach to provide disease resistance in tobacco and, possibly, other crops.

  9. Characterization of pbt genes conferring increased Pb2+ and Cd2+ tolerance upon Achromobacter xylosoxidans A8.

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    Hložková, Kateřina; Suman, Jáchym; Strnad, Hynek; Ruml, Tomas; Paces, Vaclav; Kotrba, Pavel

    2013-12-01

    The cluster of pbtTFYRABC genes is carried by plasmid pA81. Its elimination from Achromobacter xylosoxidans A8 resulted in increased sensitivity towards Pb(2+) and Cd(2+). Predicted pbtTRABC products share strong similarities with Pb(2+) uptake transporter PbrT, transcriptional regulator PbrR, metal efflux P1-ATPases PbrA and CadA, undecaprenyl pyrophosphatase PbrB and its signal peptidase PbrC from Cupriavidus metallidurans CH34. Expression of pbtABC or pbtA in a metal-sensitive Escherichia coli GG48 rendered the strain Pb(2+)-, Cd(2+)- and Zn(2+)-tolerant and caused decreased accumulation of the metal ions. Accumulation of Pb(2+), but not of Cd(2+) or Zn(2+), was promoted in E. coli expressing pbtT. Additional genes of the pbt cluster are pbtF and pbtY, which encode the cation diffusion facilitator (CDF)-like transporter and a putative fatty acid hydroxylase of unknown function, respectively. Expression of pbtF did not confer increased metal tolerance upon E. coli GG48, although the protein showed measurable Pb(2+)-efflux activity. Unlike the pbtT promoter, promoters of pbtABC, pbtF and pbtY contain features characteristic of promoters controlled by metal-responsive transcriptional regulators of the MerR family. Upregulation of pbtABC, pbtF and pbtY upon Pb(2+), Cd(2+) and Zn(2+) exposure was confirmed in wild-type Achromobacter xylosoxidans A8. Gel shift assays proved binding of purified PbtR to the respective promoters.

  10. A TagSNP in SIRT1 gene confers susceptibility to myocardial infarction in a Chinese Han population.

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    Jie Cheng

    Full Text Available SIRT1 exerts protective effects against endothelial cells dysfunction, inflammation and atherosclerosis, indicating an important role on myocardial infarction (MI pathogenesis. Nonetheless, the effects of SIRT1 variants on MI risk remain poorly understood. Here we aimed to investigate the influence of SIRT1 polymorphisms on individual susceptibility to MI. Genotyping of three tagSNPs (rs7069102, rs3818292 and rs4746720 in SIRT1 gene was performed in a Chinese Han population, consisting of 287 MI cases and 654 control subjects. In a logistic regression analysis, we found that G allele of rs7069102 had increased MI risk with odds ratio (OR of 1.57 [95% confidence interval (CI = 1.15-2.16, Bonferroni corrected P (Pc = 0.015] after adjustment for conventional risk factors compared to C allele. Similarly, the combined CG/GG genotypes was associated with the increased MI risk (OR = 1.64, 95% CI = 1.14-2.35, Pc = 0.021 compared to the CC genotype. Further stratified analysis revealed a more significant association with MI risk among younger subjects (≤ 55 years old. Consistent with these results, the haplotype rs7069102G-rs3818292A-rs4746720T containing the rs7069102 G allele was also associated with the increased MI risk (OR = 1.41, 95% CI = 1.09-1.84, Pc = 0.040. However, we did not detect any association of rs3818292 and rs4746720 with MI risk. Our study provides the first evidence that the tagSNP rs7069102 and haplotype rs7069102G-rs3818292A-rs4746720T in SIRT1 gene confer susceptibility to MI in the Chinese Han population.

  11. Genetic ablation of the fpr1 gene confers protection from smoking-induced lung emphysema in mice.

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    Cardini, Silvia; Dalli, Jesmond; Fineschi, Silvia; Perretti, Mauro; Lungarella, Giuseppe; Lucattelli, Monica

    2012-09-01

    Cigarette smoke (CS) is the main causative factor of chronic obstructive pulmonary disease (COPD). Current research supports the concept that airway inflammation is central to the development and progression of the disease. Studies have demonstrated that neutrophils are increased in COPD lungs and that neutrophil-associated products correlate with the development and severity of COPD. The peptide FMLP is an active component of CS. FMLP interacts on the neutrophil and macrophage membranes with a high-affinity receptor subtype (FPR1) and with a low-affinity subtype FPRL1, promoting a chemotactic response, superoxide anion production, and degranulation. Bacterial colonization of the lower respiratory tract and lung cell damage may represent further sources of formyl peptides in patients with COPD. We investigated the role of FPR in a mouse model on lung inflammation and emphysema induced by CS. Here, we report the novel observation that genetic ablation of the FPR1 gene (Fpr1) confers protection from smoking-induced lung emphysema in mice. Compared with wild-type mice, Fpr1 knockout mice displayed marked decreases in the lung migration of neutrophils and macrophages after CS exposure. Upon transgenic approach, the changes in cell numbers were accompanied by marked modulation of the expression of genes implicated in the inflammatory response. Administration of the FPR1 antagonist cyclosporine H to wild-type mice attenuated the acute inflammatory response evoked by CS. These findings may have clinical significance because current smokers and subjects with emphysema showed increased FPR expression in bronchoalveolar fluids and on peripheral neutrophils. Modulating the FPR1 signal should be explored as a potential new therapy.

  12. Overexpression of a tea flavanone 3-hydroxylase gene confers tolerance to salt stress and Alternaria solani in transgenic tobacco.

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    Mahajan, Monika; Yadav, Sudesh Kumar

    2014-08-01

    Flavan-3-ols are the major flavonoids present in tea (Camellia sinensis) leaves. These are known to have antioxidant and free radical scavenging properties in vitro. Flavanone 3-hydroxylase is considered to be an important enzyme of flavonoid pathway leading to accumulation of flavan-3-ols in tea. Expression analysis revealed the upregulation in transcript levels of C. sinensis flavanone 3-hydroxylase (CsF3H) encoding gene under salt stress. In this study, the biotechnological potential of CsF3H was evaluated by gene overexpression in tobacco (Nicotiana tabacum cv. Xanthi). Overexpression of CsF3H cDNA increased the content of flavan-3-ols in tobacco and conferred tolerance to salt stress and fungus Alternaria solani infection. Transgenic tobaccos were observed for increase in primary root length, number of lateral roots, chlorophyll content, antioxidant enzyme expression and their activities. Also, they showed lesser malondialdehyde content and electrolyte leakage compared to control tobacco plants. Further, transgenic plants produced higher degree of pectin methyl esterification via decreasing pectin methyl esterase (PME) activity in roots and leaves under unstressed and salt stressed conditions. The effect of flavan-3-ols on pectin methyl esterification under salt stressed conditions was further validated through in vitro experiments in which non-transgenic (wild) tobacco seedlings were exposed to salt stress in presence of flavan-3-ols, epicatechin and epigallocatechin. The in vitro exposed seedlings showed similar trend of increase in pectin methyl esterification through decreasing PME activity as observed in CsF3H transgenic lines. Taken together, overexpression of CsF3H provided tolerance to salt stress and fungus A. solani infection to transgenic tobacco through improved antioxidant system and enhanced pectin methyl esterification.

  13. Common variant rs9939609 in gene FTO confers risk to polycystic ovary syndrome.

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    Tao Li

    Full Text Available BACKGROUND: Fat mass and obesity-associated gene (FTO has been associated with obesity, especially the common variant rs9939609. Polycystic ovary syndrome (PCOS is a complex endocrine-metabolic disorder and over 50% of patients are overweight/obese. Thus FTO is a potential candidate gene for PCOS but their relationship is confusing and remains to be clarified in different population with a large sample size. METHOD: This study was performed adopting a two-stage design by genotyping SNP rs9939609. The first set comprise of 741 PCOS and 704 control subjects, with data from our previous GWAS. The second phase of replication study was performed among another independent group of 2858 PCOS and 2358 control subjects using TaqMan-MGB probe assay. All subjects are from Han Chinese. RESULTS: The less meaningful association of FTO rs9939609 and PCOS discovered in GWAS (P = 2.47E-03, was further confirmed in the replication study (P = 1.86E-09. Using meta-analysis, the P-meta value has reached 6.89E-12, over-exceeding the genome-wide association level of 5.00E-8. By combination, the P value was 1.26E-11 and after BMI adjustment it remained significant(P = 1.82E-06. To further elucidate whether this association is resulted from obesity or PCOS per se, the samples were divided into two groups-obese and non-obese PCOS, and the results were still positive in obese group (P obese = 5.81E-05, OR = 1.55, as well as in non-obese PCOS group (P non-obese = 7.06E-04, OR = 1.28. CONCLUSION: Variant rs9939609 in FTO is associated with PCOS in Chinese women, not only in obese PCOS subjects, but also in non-obese cases.

  14. Salmonella enterica serovar Typhimurium lacking hfq gene confers protective immunity against murine typhoid.

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    Uday Shankar Allam

    Full Text Available Salmonella enterica is an important enteric pathogen and its various serovars are involved in causing both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella leading to increased morbidity and mortality has further complicated its management. Live attenuated vaccines have been proven superior over killed or subunit vaccines due to their ability to induce protective immunity. Of the various strategies used for the generation of live attenuated vaccine strains, focus has gradually shifted towards manipulation of virulence regulator genes. Hfq is a RNA chaperon which mediates the binding of small RNAs to the mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we evaluated the efficacy of the Salmonella Typhimurium Δhfq strain as a candidate for live oral vaccine in murine model of typhoid fever. Salmonella hfq deletion mutant is highly attenuated in cell culture and animal model implying a significant role of Hfq in bacterial virulence. Oral immunization with the Salmonella hfq deletion mutant efficiently protects mice against subsequent oral challenge with virulent strain of Salmonella Typhimurium. Moreover, protection was induced upon both multiple as well as single dose of immunizations. The vaccine strain appears to be safe for use in pregnant mice and the protection is mediated by the increase in the number of CD4(+ T lymphocytes upon vaccination. The levels of serum IgG and secretory-IgA in intestinal washes specific to lipopolysaccharide and outer membrane protein were significantly increased upon vaccination. Furthermore, hfq deletion mutant showed enhanced antigen presentation by dendritic cells compared to the wild type strain. Taken together, the studies in murine immunization model suggest that the Salmonella hfq deletion mutant can be a novel live oral vaccine candidate.

  15. Stable gene transfer of CCR5 and CXCR4 siRNAs by sleeping beauty transposon system to confer HIV-1 resistance

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    Akkina Ramesh

    2008-07-01

    Full Text Available Abstract Background Thus far gene therapy strategies for HIV/AIDS have used either conventional retroviral vectors or lentiviral vectors for gene transfer. Although highly efficient, their use poses a certain degree of risk in terms of viral mediated oncogenesis. Sleeping Beauty (SB transposon system offers a non-viral method of gene transfer to avoid this possible risk. With respect to conferring HIV resistance, stable knock down of HIV-1 coreceptors CCR5 and CXCR4 by the use of lentiviral vector delivered siRNAs has proved to be a promising strategy to protect cells from HIV-1 infection. In the current studies our aim is to evaluate the utility of SB system for stable gene transfer of CCR5 and CXCR4 siRNA genes to derive HIV resistant cells as a first step towards using this system for gene therapy. Results Two well characterized siRNAs against the HIV-1 coreceptors CCR5 and CXCR4 were chosen based on their previous efficacy for the SB transposon gene delivery. The siRNA transgenes were incorporated individually into a modified SB transfer plasmid containing a FACS sortable red fluorescence protein (RFP reporter and a drug selectable neomycin resistance gene. Gene transfer was achieved by co-delivery with a construct expressing a hyperactive transposase (HSB5 into the GHOST-R3/X4/R5 cell line, which expresses the major HIV receptor CD4 and and the co-receptors CCR5 and CXCR4. SB constructs expressing CCR5 or CXCR4 siRNAs were also transfected into MAGI-CCR5 or MAGI-CXCR4 cell lines, respectively. Near complete downregulation of CCR5 and CXCR4 surface expression was observed in transfected cells. During viral challenge with X4-tropic (NL4.3 or R5-tropic (BaL HIV-1 strains, the respective transposed cells showed marked viral resistance. Conclusion SB transposon system can be used to deliver siRNA genes for stable gene transfer. The siRNA genes against HIV-1 coreceptors CCR5 and CXCR4 are able to downregulate the respective cell surface proteins

  16. Hemizygosity of transsulfuration genes confers increased vulnerability against acetaminophen-induced hepatotoxicity in mice

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    Hagiya, Yoshifumi; Kamata, Shotaro; Mitsuoka, Saya; Okada, Norihiko; Yoshida, Saori; Yamamoto, Junya; Ohkubo, Rika [Department of Biochemistry, Keio University School of Pharmaceutical Sciences, Tokyo 105-8512 (Japan); Abiko, Yumi [Environmental Biology Laboratory, School of Medicine, University of Tsukuba, Ibaraki 305-8575 (Japan); Yamada, Hidenori [Jobu Hospital for Respiratory Diseases, Maebashi 371-0048 (Japan); Akahoshi, Noriyuki [Department of Immunology, Akita University Graduate School of Medicine, Akita 010-8543 (Japan); Kasahara, Tadashi [Department of Biochemistry, Keio University School of Pharmaceutical Sciences, Tokyo 105-8512 (Japan); Kumagai, Yoshito [Environmental Biology Laboratory, School of Medicine, University of Tsukuba, Ibaraki 305-8575 (Japan); Ishii, Isao, E-mail: isao-ishii@umin.ac.jp [Department of Biochemistry, Keio University School of Pharmaceutical Sciences, Tokyo 105-8512 (Japan)

    2015-01-15

    The key mechanism for acetaminophen hepatotoxicity is cytochrome P450 (CYP)-dependent formation of N-acetyl-p-benzoquinone imine, a potent electrophile that forms protein adducts. Previous studies revealed the fundamental role of glutathione, which binds to and detoxifies N-acetyl-p-benzoquinone imine. Glutathione is synthesized from cysteine in the liver, and N-acetylcysteine is used as a sole antidote for acetaminophen poisoning. Here, we evaluated the potential roles of transsulfuration enzymes essential for cysteine biosynthesis, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), in acetaminophen hepatotoxicity using hemizygous (Cbs{sup +/−} or Cth{sup +/−}) and homozygous (Cth{sup −/−}) knockout mice. At 4 h after intraperitoneal acetaminophen injection, serum alanine aminotransferase levels were highly elevated in Cth{sup −/−} mice at 150 mg/kg dose, and also in Cbs{sup +/−} or Cth{sup +/−} mice at 250 mg/kg dose, which was associated with characteristic centrilobular hepatocyte oncosis. Hepatic glutathione was depleted while serum malondialdehyde accumulated in acetaminophen-injected Cth{sup −/−} mice but not wild-type mice, although glutamate–cysteine ligase (composed of catalytic [GCLC] and modifier [GCLM] subunits) became more activated in the livers of Cth{sup −/−} mice with lower K{sub m} values for Cys and Glu. Proteome analysis using fluorescent two-dimensional difference gel electrophoresis revealed 47 differentially expressed proteins after injection of 150 mg acetaminophen/kg into Cth{sup −/−} mice; the profiles were similar to 1000 mg acetaminophen/kg-treated wild-type mice. The prevalence of Cbs or Cth hemizygosity is estimated to be 1:200–300 population; therefore, the deletion or polymorphism of either transsulfuration gene may underlie idiosyncratic acetaminophen vulnerability along with the differences in Cyp, Gclc, and Gclm gene activities. - Highlights: • Cbs{sup +/−}, Cth{sup +/−}, and

  17. Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease

    Science.gov (United States)

    2014-01-01

    Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimer's disease (LOAD). These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case-control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer's disease in seven independent case-control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer's disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer's disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer's disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-β precursor protein (APP) and extracellular Aβ42 and Aβ40 (the 42- and 40-residue isoforms of the amyloid-β peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex

  18. Distress of ostracism: oxytocin receptor gene polymorphism confers sensitivity to social exclusion.

    Science.gov (United States)

    McQuaid, Robyn J; McInnis, Opal A; Matheson, Kimberly; Anisman, Hymie

    2015-08-01

    A single-nucleotide polymorphism on the oxytocin receptor gene (OXTR), rs53576, involving a guanine (G) to adenine (A) substitution has been associated with altered prosocial features. Specifically, individuals with the GG genotype (i.e. the absence of the polymorphism) display beneficial traits including enhanced trust, empathy and self-esteem. However, because G carriers might also be more socially sensitive, this may render them more vulnerable to the adverse effects of a negative social stressor. The current investigation, conducted among 128 white female undergraduate students, demonstrated that relative to individuals with AA genotype, G carriers were more emotionally sensitive (lower self-esteem) in response to social ostracism promoted through an on-line ball tossing game (Cyberball). Furthermore, GG individuals also exhibited altered blood pressure and cortisol levels following rejection, effects not apparent among A carriers. The data support the view that the presence of the G allele not only promotes prosocial behaviors but also favors sensitivity to a negative social stressor.

  19. A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum.

    Science.gov (United States)

    Magalhaes, Jurandir V; Liu, Jiping; Guimarães, Claudia T; Lana, Ubiraci G P; Alves, Vera M C; Wang, Yi-Hong; Schaffert, Robert E; Hoekenga, Owen A; Piñeros, Miguel A; Shaff, Jon E; Klein, Patricia E; Carneiro, Newton P; Coelho, Cintia M; Trick, Harold N; Kochian, Leon V

    2007-09-01

    Crop yields are significantly reduced by aluminum toxicity on highly acidic soils, which comprise up to 50% of the world's arable land. Candidate aluminum tolerance proteins include organic acid efflux transporters, with the organic acids forming non-toxic complexes with rhizosphere aluminum. In this study, we used positional cloning to identify the gene encoding a member of the multidrug and toxic compound extrusion (MATE) family, an aluminum-activated citrate transporter, as responsible for the major sorghum (Sorghum bicolor) aluminum tolerance locus, Alt(SB). Polymorphisms in regulatory regions of Alt(SB) are likely to contribute to large allelic effects, acting to increase Alt(SB) expression in the root apex of tolerant genotypes. Furthermore, aluminum-inducible Alt(SB) expression is associated with induction of aluminum tolerance via enhanced root citrate exudation. These findings will allow us to identify superior Alt(SB) haplotypes that can be incorporated via molecular breeding and biotechnology into acid soil breeding programs, thus helping to increase crop yields in developing countries where acidic soils predominate.

  20. Genetic analysis of dilated cardiomyopathy--HLA and immunoglobulin genes may confer susceptibility.

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    Nishi, H; Kimura, A; Fukuta, S; Kusukawa, R; Kawamura, K; Nimura, Y; Nagano, M; Yasuda, H; Kawai, C; Sugimoto, T

    1992-10-01

    To identify genetic factors in the immune system which control the susceptibility to dilated cardiomyopathy (DCM), HLA class II DNA typing was performed in 61 Japanese patients, using PCR/SSO probe analyses. The frequencies of HLA-DQB1*0503 (15% vs 5%; RR = 3.06, chi 2 = 7.19) and DQB1*0604 (21% vs 10%; RR = 2.41, chi 2 = 6.20) were significantly increased and that of HLA-DQB1*0502 (RR = 1.74) was slightly increased in the DCM patients. The frequency of DQB1*0303 (16% vs 31%; RR = 0.44, chi 2 = 5.16) was significantly decreased in the patients. The increased HLA-DQB1 alleles have a histidine residue in common at the 30th codon for the HLA-DQ beta chain. Among the genetic markers studied by Southern blot analyses, IGLV (immunoglobulin lambda light chain, pV3.3) showed a strong association with DCM, i.e. A2/A2 genotype was found in 37.7% of patients whereas it was observed in only 18.9% of the control subjects (RR = 2.6, chi 2 = 7.77). The frequency of this genotype was higher in patients under age 45 years at the time of diagnosis (45.5%, RR = 3.6, chi 2 = 10.02). These results suggest that HLA and immunoglobulin genes are closely linked to susceptibility to DCM.

  1. Expression of a chitinase gene from Metarhizium anisopliae in tobacco plants confers resistance against Rhizoctonia solani.

    Science.gov (United States)

    Kern, Marcelo Fernando; Maraschin, Simone de Faria; Vom Endt, Débora; Schrank, Augusto; Vainstein, Marilene Henning; Pasquali, Giancarlo

    2010-04-01

    The chit1 gene from the entomopathogenic fungus Metarhizium anisopliae, encoding the endochitinase CHIT42, was placed under the control of the CaMV 35S promoter, and the resulting construct was transferred to tobacco. Seventeen kanamycin-resistant transgenic lines were recovered, and the presence of the transgene was confirmed by polymerase chain reactions and Southern blot hybridization. The number of chit1 copies was determined to be varying from one to four. Copy number had observable effects neither on plant growth nor development. Substantial heterogeneity concerning production of the recombinant chitinase, and both general and specific chitinolytic activities were detected in leaf extracts from primary transformants. The highest chitinase activities were found in plants harboring two copies of chit1 inserts at different loci. Progeny derived from self-pollination of the primary transgenics revealed a stable inheritance pattern, with transgene segregation following a mendelian dihybrid ratio. Two selected plants expressing high levels of CHIT42 were consistently resistant to the soilborne pathogen Rhizoctonia solani, suggesting a direct relationship between enzyme activity and reduction of foliar area affected by fungal lesions. To date, this is the first report of resistance to fungal attack in plants mediated by a recombinant chitinase from an entomopathogenic and acaricide fungus.

  2. Genomic amplification patterns of human telomerase RNA gene and C-MYC in liquid-based cytological specimens used for the detection of high-grade cervical intraepithelial neoplasia

    Science.gov (United States)

    2012-01-01

    Background The amplification of oncogenes initiated by high-risk human papillomavirus (HPV) infection is an early event in cervical carcinogenesis and can be used for cervical lesion diagnosis. We measured the genomic amplification rates and the patterns of human telomerase RNA gene (TERC) and C-MYC in the liquid-based cytological specimens to evaluate the diagnostic characteristics for the detection of high-grade cervical lesions. Methods Two hundred and forty-three residual cytological specimens were obtained from outpatients aged 25 to 64 years at Qilu Hospital, Shandong University. The specimens were evaluated by fluorescence in situ hybridization (FISH) using chromosome probes to TERC (3q26) and C-MYC (8q24). All of the patients underwent colposcopic examination and histological evaluation. A Chi-square test was used for categorical data analysis. Results In the normal, cervical intraepithelial neoplasia grade 1 (CIN1), grade 2 (CIN2), grade 3 (CIN3) and squamous cervical cancer (SCC) cases, the TERC positive rates were 9.2%, 17.2%, 76.2%, 100.0% and 100.0%, respectively; the C-MYC positive rates were 20.7%, 31.0%, 71.4%, 81.8% and 100.0%, respectively. The TERC and C-MYC positive rates were higher in the CIN2+ (CIN2, CIN3 and SCC) cases than in the normal and CIN1 cases (p C-MYC test showed lower sensitivity (80.0% vs. 84.0%) and higher specificity (77.7% vs. 64.3%). Using a cut-off value of 5% or more aberrant cells, the TERC test showed the highest combination of sensitivity and specificity. The CIN2+ group showed more high-level TERC gene copy number (GCN) cells than did the normal/CIN1 group (p C-MYC, no significant difference between the two histological categories was detected (p > 0.05). Conclusions The TERC test is highly sensitive and is therefore suitable for cervical cancer screening. The C-MYC test is not suitable for cancer screening because of its lower sensitivity. The amplification patterns of TERC become more diverse and complex as the

  3. Neuropeptide Y gene polymorphisms confer risk of early-onset atherosclerosis.

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    Svati H Shah

    2009-01-01

    Full Text Available Neuropeptide Y (NPY is a strong candidate gene for coronary artery disease (CAD. We have previously identified genetic linkage to familial CAD in the genomic region of NPY. We performed follow-up genetic, biostatistical, and functional analysis of NPY in early-onset CAD. In familial CAD (GENECARD, N = 420 families, we found increased microsatellite linkage to chromosome 7p14 (OSA LOD = 4.2, p = 0.004 in 97 earliest age-of-onset families. Tagged NPY SNPs demonstrated linkage to CAD of a 6-SNP block (LOD = 1.58-2.72, family-based association of this block with CAD (p = 0.02, and stronger linkage to CAD in the earliest age-of-onset families. Association of this 6-SNP block with CAD was validated in: (a 556 non-familial early-onset CAD cases and 256 controls (OR 1.46-1.65, p = 0.01-0.05, showing stronger association in youngest cases (OR 1.84-2.20, p = 0.0004-0.09; and (b GENECARD probands versus non-familial controls (OR 1.79-2.06, p = 0.003-0.02. A promoter SNP (rs16147 within this 6-SNP block was associated with higher plasma NPY levels (p = 0.04. To assess a causal role of NPY in atherosclerosis, we applied the NPY1-receptor-antagonist BIBP-3226 adventitially to endothelium-denuded carotid arteries of apolipoprotein E-deficient mice; treatment reduced atherosclerotic neointimal area by 50% (p = 0.03. Thus, NPY variants associate with atherosclerosis in two independent datasets (with strong age-of-onset effects and show allele-specific expression with NPY levels, while NPY receptor antagonism reduces atherosclerosis in mice. We conclude that NPY contributes to atherosclerosis pathogenesis.

  4. Increased radiosensitivity as an indicator of genes conferring breast cancer susceptibility

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    Varga, D.; Kreienberg, R.; Deissler, H.; Sauer, G. [Dept. of Gynecology and Obstetrics, Univ. of Ulm Medical School (Germany); Vogel, W.; Bender, A.; Surowy, H.; Maier, C. [Dept. of Genetics, Univ. of Ulm Medical School (Germany)

    2007-12-15

    Purpose: This paper briefly summarizes the research on increased radiosensitivity in breast cancer patients measured by the micronucleus test (MNT) and its association to genetic variants in DNA repair genes. More preliminary data are presented on the distribution of chromosomes and chromosome fragments in micronuclei (MN) in order to gain more information on clastogenic and aneugenic effects and better understand the phenotype of increased radiosensitivity. Material and Methods: Reports of relevant studies obtained from a search of PubMed and studies referenced in those reports were reviewed. In four patients with high MN frequency (three cancer patients, one control) and four probands with low MN frequency, the presence of chromosome fragments or whole chromosomes in MN was determined by fluorescence in situ hybridization analysis for chromosomes 1, 7, and 17. Results: An increased MN frequency in breast cancer patients compared to controls has consistently been reported with high significance. Higher MN frequencies were observed in 20-50% of breast cancer patients. Chromosomal fragments of chromosome 17, but not of chromosomes 1 and 7 were more frequent in the probands with high MN frequency than in those with low frequency (p = 0.045). Conclusion: The MNT detects a cellular phenotype common to a portion of sporadic breast cancer patients. This phenotype is very likely to be genetically determined. For the genetic dissection of breast cancer susceptibility this phenotype may turn out to be more efficient than breast cancer itself. Additional parameters which can be measured simultaneously with the MN frequency may be able to further enhance its usefulness. (orig.)

  5. Inheritance and Linkage Map Positions of Genes Conferring Agromorphological Traits in Lens culinaris Medik.

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    Gopesh C. Saha

    2013-01-01

    Full Text Available Agromorphological traits have immense importance in breeding lentils for higher yield and stability. We studied the genetics and identified map positions of some important agro-morphological traits including days to 50% flowering, plant height, seed diameter, 100 seed weight, cotyledon color, and growth habit in Lens culinaris. Earlier developed RILs for stemphylium blight resistance (ILL-5888 × ILL-6002, contrasted for those agro-morphological traits, were used in our study. Three QTLs for days to 50% flowering were detected with additive and epistatic effects. One QTL for days to 50% flowering, QLG483 (QTL at linkage group 4 at 83 cM position, accounted for an estimated 20.2% of the variation, while QLG124 × QLG1352 and QLG484 × QLG138 accounted for 15.6% and 24.2% of the variation, respectively. Epistatic effects accounted for most of the variation in plant height, but the main effect of one QTL, QLG84, accounted for 15.3%. For seed diameter, three QTLs were detected, and one QTL, QLG482, accounted for 32.6% of the variation. For 100 seed weight, five QTLs were identified with significant additive effects and four with significant interaction effects. The main effect of one QTL, QLG482, also accounted for 17.5% of the variation in seed diameter. QLG482-83 which appears to affect days to 50% flowering, seed diameter, and 100 seed weight is flanked by RAPD markers, UBC 34 and UBC1. Growth habit and cotyledon color are controlled by single genes with prostrate dominant to erect and red cotyledon dominant to yellow. The QTL information presented here will assist in the selection of breeding lines for early maturity, upright growth habit, and improved seed quality.

  6. ITIH family genes confer risk to schizophrenia and major depressive disorder in the Han Chinese population.

    Science.gov (United States)

    He, Kuanjun; Wang, Qingzhong; Chen, Jianhua; Li, Tao; Li, Zhiqiang; Li, Wenjin; Wen, Zujia; Qiang, Yu; Wang, Meng; Shen, Jiawei; Song, Zhijian; Ji, Jue; Feng, Guoyin; Qi, Shuguang; Lin, He; Shi, Yongyong; Cheng, Zaohuo

    2014-06-03

    As a major extracellular matrix component, ITIHs played an important role in inflammation and carcinogenesis. Several genome-wide association studies have reported that some positive signals which were derived from the tight linkage disequilibrium region on chromosome 3p21 were associated with both schizophrenia and bipolar disorders in the Caucasian population. To further investigate whether this genomic region is also a susceptibility locus of schizophrenia and major depressive disorder in the Han Chinese population, we conducted this study by recruiting 1235 schizophrenia patients, 1045 major depressive disorder patients and 1235 healthy control subjects in the Han Chinese samples for a case-control study. We genotyped seven SNPs within this region using TaqMan® technology. We found that rs2710322 was significantly associated with schizophrenia (adjusted P(allele) = 0.0018, adjusted P(genotype) = 0.006, OR [95% CI] = 1.278 [1.117-1.462]) while rs1042779 was weakly associated with schizophrenia (adjusted P(allele) = 0.048, OR [95% CI] = 1.164 [1.040-1.303]) and major depressive disorder (adjusted P(allele) = 0.042, OR [95% CI] = 1.178 [1.047-1.326]); it was also our finding that rs3821831 was positively associated with major depressive disorder (adjusted P(allele) = 0.003, adjusted P(genotype) = 0.006, OR [95% CI] = 1.426 [1.156-1.760]). Furthermore, no haplotype was found to be associated with schizophrenia and major depressive disorder. Via the association analysis which combines the schizophrenia and major depressive disorder cases, we also notice that rs1042779 and rs3821831 were significantly associated with combined cases (rs1042779: adjusted P(allele) = 0.012, adjusted P(genotype) = 0.018, OR [95% CI] = 1.171 [1.060-1.292]; rs3821831:adjusted P(genotype) = 0.012, OR [95% CI] = 1.193 [1.010-1.410]). Our results revealed that the shared genetic risk factors of both schizophrenia and major depressive disorder exist in ITIH family genes in the Han Chinese

  7. Alfalfa benefits from Medicago truncatula: the RCT1 gene from M. truncatula confers broad-spectrum resistance to anthracnose in alfalfa.

    Science.gov (United States)

    Yang, Shengming; Gao, Muqiang; Xu, Chenwu; Gao, Jianchang; Deshpande, Shweta; Lin, Shaoping; Roe, Bruce A; Zhu, Hongyan

    2008-08-26

    Alfalfa is economically the most important forage legume worldwide. A recurrent challenge to alfalfa production is the significant yield loss caused by disease. Although knowledge of molecular mechanisms underlying host resistance should facilitate the genetic improvement of alfalfa, the acquisition of such knowledge is hampered by alfalfa's tetrasomic inheritance and outcrossing nature. However, alfalfa is congeneric with the reference legume Medicago truncatula, providing an opportunity to use M. truncatula as a surrogate to clone the counterparts of many agronomically important genes in alfalfa. In particular, the high degree of sequence identity and remarkably conserved genome structure and function between the two species enables M. truncatula genes to be used directly in alfalfa improvement. Here we report the map-based cloning of RCT1, a host resistance (R) gene in M. truncatula that confers resistance to multiple races of Colletotrichum trifolii, a hemibiotrophic fungal pathogen that causes anthracnose disease of alfalfa. RCT1 is a member of the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant R genes and confers broad-spectrum anthracnose resistance when transferred into susceptible alfalfa plants. Thus, RCT1 provides a novel resource to develop anthracnose-resistant alfalfa cultivars and contributes to our understanding of host resistance against the fungal genus Colletotrichum. This work demonstrates the potential of using M. truncatula genes for genetic improvement of alfalfa.

  8. High-resolution mapping reveals linkage between genes in common bean cultivar Ouro Negro conferring resistance to the rust, anthracnose, and angular leaf spot diseases.

    Science.gov (United States)

    Valentini, Giseli; Gonçalves-Vidigal, Maria Celeste; Hurtado-Gonzales, Oscar P; de Lima Castro, Sandra Aparecida; Cregan, Perry B; Song, Qijian; Pastor-Corrales, Marcial A

    2017-08-01

    Co-segregation analysis and high-throughput genotyping using SNP, SSR, and KASP markers demonstrated genetic linkage between Ur-14 and Co-3 (4) /Phg-3 loci conferring resistance to the rust, anthracnose and angular leaf spot diseases of common bean. Rust, anthracnose, and angular leaf spot are major diseases of common bean in the Americas and Africa. The cultivar Ouro Negro has the Ur-14 gene that confers broad spectrum resistance to rust and the gene cluster Co-3 (4) /Phg-3 containing two tightly linked genes conferring resistance to anthracnose and angular leaf spot, respectively. We used co-segregation analysis and high-throughput genotyping of 179 F2:3 families from the Rudá (susceptible) × Ouro Negro (resistant) cross-phenotyped separately with races of the rust and anthracnose pathogens. The results confirmed that Ur-14 and Co-3 (4) /Phg-3 cluster in Ouro Negro conferred resistance to rust and anthracnose, respectively, and that Ur-14 and the Co-3 (4) /Phg-3 cluster were closely linked. Genotyping the F2:3 families, first with 5398 SNPs on the Illumina BeadChip BARCBEAN6K_3 and with 15 SSR, and eight KASP markers, specifically designed for the candidate region containing Ur-14 and Co-3 (4) /Phg-3, permitted the creation of a high-resolution genetic linkage map which revealed that Ur-14 was positioned at 2.2 cM from Co-3 (4) /Phg-3 on the short arm of chromosome Pv04 of the common bean genome. Five flanking SSR markers were tightly linked at 0.1 and 0.2 cM from Ur-14, and two flanking KASP markers were tightly linked at 0.1 and 0.3 cM from Co-3 (4) /Phg-3. Many other SSR, SNP, and KASP markers were also linked to these genes. These markers will be useful for the development of common bean cultivars combining the important Ur-14 and Co-3 (4) /Phg-3 genes conferring resistance to three of the most destructive diseases of common bean.

  9. Virulence Associated Genes-Deleted Salmonella Montevideo Is Attenuated, Highly Immunogenic and Confers Protection against Virulent Challenge in Chickens

    Science.gov (United States)

    Lalsiamthara, Jonathan; Lee, John H.

    2016-01-01

    To construct a novel live vaccine against Salmonella enterica serovar Montevideo (SM) infection in chickens, two important bacterial regulatory genes, lon and cpxR, which are associated with invasion and virulence, were deleted from the wild type SM genome. Attenuated strains, JOL1625 (Δlon), JOL1597 (ΔcpxR), and JOL1599 (ΔlonΔcpxR) were thereby generated. Observations with scanning electron microscopy suggested that JOL1625 and JOL1599 cells showed increased ruffled surface which may be related to abundant extracellular polysaccharide (EPS) production. JOL1597 depicted milder ruffled surface but showed increased surface corrugation. ConA affinity-based fluorometric quantification and fluorescence microscopy revealed significant increases in EPS production in JOL1625 and JOL1599. Four weeks old chickens were used for safety and immunological studies. The mutants were not observed in feces beyond day 3 nor in spleen and cecum beyond day 7, whereas wild type SM was detected for at least 2 weeks in spleen and cecum. JOL1599 was further evaluated as a vaccine candidate. Chickens immunized with JOL1599 showed strong humoral responses, as indicated by systemic IgG and secretory IgA levels, as well as strong cell-mediated immune response, as indicated by increased lymphocyte proliferation. JOL1599-immunized groups also showed significant degree of protection against wild type challenge. Our results indicate that Δlon- and/or ΔcpxR-deleted SM exhibited EPS-enhanced immunogenicity and attenuation via reduced bacterial cell intracellular replication, conferred increased protection, and possess safety qualities favorable for effective vaccine development against virulent SM infections. PMID:27785128

  10. 苔藓植物matK基因PCR反应体系的建立和优化%Establishment and Optimization of matK gene-PCR Amplification System for Bryophytes

    Institute of Scientific and Technical Information of China (English)

    郑红建; 张安世

    2012-01-01

    [Objective] To establish and optimize the matK gene-PCR reaction system for Bryophytes. [ Method ] Using genomic DNA of Bryo-phytes extracted via an improved CTAB method, single factor analysis was performed to investigate the impacts of DNA template concentration, primer concentration, 2 x Taq MasterMix concentration on matK gene-PCR amplification and to optimize this system for Bryophytes. [Result] The matK gene-PCR amplification(10.0 μl) suitable for Bryophytes was determined to be composed of 0.5 μl DNA, 0.2 μl primer and 5.6 μl 2 × Taq MasterMix. [ Conclusion] The study laid basis for analyzing the molecular systematics of Bryophytes.%[目的]建立和优化适合于苔藓植物matK基因的PCR反应体系.[方法]以鳞叶藓为材料,利用改良CTAB法提取了基因组DNA,利用单因素试验分析了DNA模板、引物和2×Taq MasterMix的浓度对苔藓植物matK基因PCR反应的影响,对适合苔藓植物的matK基因PCR反应条件进行了优化.[结果]适合苔藓植物的matK基因PCR反应体系为10.0 μl,其中含DNA模板0.5μl,正反引物各0.2μl,2×Taq MasterMix 5.6 μl.[结论]为苔藓植物的分子系统学等研究奠定了基础.

  11. Efficacy Improvement of PCR Amplification of CAG Trinucleotide Repeats in the Coding Sequence of Spinocerebellar Ataxia Type II Gene%提高SCA2编码区CAG三核苷酸重复的PCR扩增效率

    Institute of Scientific and Technical Information of China (English)

    汤熙翔; 夏家辉

    2000-01-01

    To improve the efficacy of PCR amplification of CAG trinucleotide repeats in the coding sequence of spinocerebellar ataxia type II gene(69.2% G+C), hot-start PCR, base-replacement PCR, and the addition of enhancers(1%~12.5% DMSO , 1%~25% glycerol ,1%~12.5% formamide) were performed and compared with normal PCR . The results showed that hot-start PCR, base-replacement PCR and the addition of enhancers(1%~10% DMSO , 5%~20% glycerol , 1%~10% formamide) improved the amplification efficacy of the GC rich region. Gene diagnosis in 70 SCA pedgrees and 60 spontaneous SCA patients were also conducted.%以遗传性脊髓小脑共济失调II型基因(spinocerebellar ataxia type II gene SCA2)编码区内的CAG三核苷酸重复为研究对象(G+C含量为69.2%),比较了热启动PCR、碱基替代PCR、添加增效剂(1%~12.5%二甲亚砜、1%~25%甘油、1%~12.5%甲酰胺)与常规PCR的扩增效率,发现热启动PCR、碱基替代PCR及添加增效剂(1%~10%二甲亚砜、5%~20%甘油、1%~10%甲酰胺)能提高该GC富集区的扩增效率,并对70个SCA家系及60个散发SCA患者进行了SCA2的基因诊断。

  12. Fine mapping and analysis of a candidate gene in tomato accession PI128216 conferring hypersensitive resistance to bacterial spot race T3.

    Science.gov (United States)

    Pei, Chengcheng; Wang, Hui; Zhang, Jieyun; Wang, Yuanyuan; Francis, David M; Yang, Wencai

    2012-02-01

    Bacterial spot caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans and X. gardneri is one of the most destructive diseases in tomatoes (Solanum lycopersicum L.) growing in tropical and subtropical regions. Exploring resistance genes from diverse germplasm and incorporating them into cultivated varieties are critical for controlling this disease. The S. pimpinellifolium accession PI128216 was reported to carry the Rx4 gene on chromosome 11 conferring hypersensitivity and field resistance to race T3. To facilitate the use of marker-assisted selection in breeding and map-based cloning of the gene, an F(2) population derived from a cross between the susceptible variety OH88119 and the resistant accession PI128216 was created for fine mapping of the Rx4 gene. Using 18 markers developed through various approaches, we mapped the gene to a 45.1-kb region between two markers pcc17 and pcc14 on chromosome 11. A NBS-LRR class of resistance gene was identified as the candidate for the Rx4 gene based on annotation results from the International Tomato Annotation Group. Comparison of the genomic DNA sequences of the Rx4 alleles in PI128216 and OH88119 revealed a 6-bp insertion/deletion (InDel) and eight SNPs. The InDel marker was successfully used to distinguish resistance and susceptibility in 12 tomato lines. These results will facilitate cloning the Rx4 gene and provide a useful tool for marker-assisted selection of this gene in tomato breeding programs.

  13. Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia.

    Science.gov (United States)

    Fallahi, Shirzad; Seyyed Tabaei, Seyyed Javad; Pournia, Yadollah; Zebardast, Nozhat; Kazemi, Bahram

    2014-07-01

    Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this paper, a newly described DNA amplification technique, loop-mediated isothermal amplification (LAMP), and nested-PCR targeting the repeated element (RE) and B1 gene, were compared to each other for the detection of Toxoplasma gondii DNA in blood samples of children with leukaemia. One hundred ten blood samples from these patients were analyzed by LAMP and nested-PCR. Out of 50 seropositive samples (IgM+, IgG+), positive results were obtained with 92% and 86% on RE, B1-LAMP and 82% and 68% on RE, B1-nested PCR analyses, respectively. Of the 50 seronegative samples, three, two and one samples were detected positive by RE-LAMP, B1-LAMP and RE-nested PCR assays, respectively, while none were detected positive by B1-nested PCR. None of the 10 IgM-, IgG+ samples was detected positive after testing LAMP and nested-PCR assays in duplicate. This is the first report of a study in which the LAMP method was applied with high sensitivity and efficacy for the diagnosis of T. gonii in blood samples of children with leukaemia.

  14. Amplification and flagellin typing of pseudomonas aeruginosa by molecular method

    Directory of Open Access Journals (Sweden)

    gholamreza goudarzi

    2011-08-01

    Conclusion: This method could be utilized to determine flagellated (Motile and non-flagellated strains of P. aeruginosa, genotyping, amplification of full coding sequence of fliC gene in order to clone and express recombinant flagellin protein.

  15. 1(st) EMBL/DFG Women in Science Network Conference Heidelberg 2016: From Genes, Cells and the Immune System towards Therapies - Meeting Report.

    Science.gov (United States)

    Stripecke, Renata; Gouttefangeas, Cécile; Förster, Irmgard

    2016-11-01

    The 1(st) EMBL/DFG Women in Science (WiS) Conference "From Genes, Cells and the Immune System towards Therapies" was held on 19(th) - 20(th) September 2016 in Heidelberg, Germany. The WiS conference was funded by nine Collaborative Research Centers (CRCs) of the German Research Council (Deutsche Forschungsgemeinschaft, DFG; Table 1) and benefited from an outstanding hosting environment at the Advanced Training Center of the European Molecular Biology Laboratory (EMBL). Scientific talks focused at genetic, cellular and immunologic mechanisms, and immune therapy, and progress from all stages of development covering basic research to clinical developments was described. The presentations were embedded between structured networking sessions and a round table discussion with representatives of the DFG, EMBL, European Molecular Biology Organisation (EMBO), and the German Society of Immunology (DGfI). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Comparative analysis of the EGFR, HER2, c-MYC, and MET variations in colorectal cancer determined by three different measures: gene copy number gain, amplification status and the 2013 ASCO/CAP guideline criterion for HER2 testing of breast cancer.

    Science.gov (United States)

    Kwak, Yoonjin; Yun, Sumi; Nam, Soo Kyung; Seo, An Na; Lee, Kyu Sang; Shin, Eun; Oh, Heung-Kwon; Kim, Duck Woo; Kang, Sung Bum; Kim, Woo Ho; Lee, Hye Seung

    2017-08-01

    The purpose of this study was to explore gene copy number (GCN) variation of EGFR, HER2, c-MYC, and MET in patients with primary colorectal cancer (CRC). Dual-colour silver-enhanced in situ hybridization was performed in tissue samples of 334 primary CRC patients. The amplification status (GCN ratio ≥2) and GCN gain (average GCN ≥4) data for the EGFR, HER2, c-MYC and MET genes were obtained. GCN variation was also assessed by the criterion of the 2013 ASCO/CAP guidelines for HER2 testing. Amplification of EGFR, HER2, c-MYC and MET was detected in 8 (2.4%), 20 (6.0%), 29 (8.7%), and 14 (4.2%) patients, respectively. Of 66 patients with at least one amplified gene, five exhibited co-amplification of genes studied (HER2-MET co-amplification: two patients; HER2-c-MYC co-amplification: two patients; EGFR-c-MYC co-amplification: one patient). There were 109 patients with GCN gains of one or more genes (EGFR: 11/334, HER2: 29/334, c-MYC; 60/334, MET: 48/334) and 32.1% (35/109) had multiple GCN gains. When each GCN was assessed by the criterion of the ASCO/CAP 2013 guideline for HER2 testing, 116 people showed positive or equivocal results for one or more genes. The cumulative amplification status had no association with patients' outcome. However, the cumulative results of the GCN gain and GCN status determined according to the ASCO/CAP guideline had a significant prognostic correlation in the univariate analysis (P values of 0.006 and 0.022, respectively). In the multivariate analysis, GCN gain and GCN status were independent prognostic factors (P values of 0.010 and 0.017, respectively). In this study, we evaluated GCN variation of four genes in a large sample of Korean CRC patients. The amplification status was not related to patient outcome. However, the GCN gain and GCN status according to the ASCO/CAP 2013 guideline were independent prognostic factors.

  17. Identification of amino acids conferring high-level resistance to expanded-spectrum cephalosporins in the penA gene from Neisseria gonorrhoeae strain H041.

    Science.gov (United States)

    Tomberg, Joshua; Unemo, Magnus; Ohnishi, Makoto; Davies, Christopher; Nicholas, Robert A

    2013-07-01

    The recent identification of a high-level-ceftriaxone-resistant (MIC = 2 to 4 μg/ml) isolate of Neisseria gonorrhoeae from Japan (H041) portends the loss of ceftriaxone as an effective treatment for gonococcal infections. This is of grave concern because ceftriaxone is the last remaining option for first-line empirical antimicrobial monotherapy. The penA gene from H041 (penA41) is a mosaic penA allele similar to mosaic alleles conferring intermediate-level cephalosporin resistance (Ceph(i)) worldwide but has 13 additional mutations compared to the mosaic penA gene from the previously studied Ceph(i) strain 35/02 (penA35). When transformed into the wild-type strain FA19, the penA41 allele confers 300- and 570-fold increases in the MICs for ceftriaxone and cefixime, respectively. In order to understand the mechanisms involved in high-level ceftriaxone resistance and to improve surveillance and epidemiology during the potential emergence of ceftriaxone resistance, we sought to identify the minimum number of amino acid alterations above those in penA35 that confer high-level resistance to ceftriaxone. Using restriction fragment exchange and site-directed mutagenesis, we identified three mutations, A311V, T316P, and T483S, that, when incorporated into the mosaic penA35 allele, confer essentially all of the increased resistance of penA41. A311V and T316P are close to the active-site nucleophile Ser310 that forms the acyl-enzyme complex, while Thr483 is predicted to interact with the carboxylate of the β-lactam antibiotic. These three mutations have thus far been described only for penA41, but dissemination of these mutations in other mosaic alleles would spell the end of ceftriaxone as an effective treatment for gonococcal infections.

  18. Biomaterials in light amplification

    Science.gov (United States)

    Mysliwiec, Jaroslaw; Cyprych, Konrad; Sznitko, Lech; Miniewicz, Andrzej

    2017-03-01

    Biologically produced or inspired materials can serve as optical gain media, i.e. they can exhibit the phenomenon of light amplification. Some of these materials, under suitable dye-doping and optical pumping conditions, show lasing phenomena. The emerging branch of research focused on obtaining lasing action in highly disordered and highly light scattering materials, i.e. research on random lasing, is perfectly suited for biological materials. The use of biomaterials in light amplification has been extensively reported in the literature. In this review we attempt to report on progress in the development of biologically derived systems able to show the phenomena of light amplification and random lasing together with the contribution of our group to this field. The rich world of biopolymers modified with molecular aggregates and nanocrystals, and self-organized at the nanoscale, offers a multitude of possibilities for tailoring luminescent and light scattering properties that are not easily replicated in conventional organic or inorganic materials. Of particular importance and interest are light amplification and lasing, or random lasing studies in biological cells and tissues. In this review we will describe nucleic acids and their complexes employed as gain media due to their favorable optical properties and ease of manipulation. We will report on research conducted on various biomaterials showing structural analogy to nucleic acids such as fluorescent proteins, gelatins in which the first distributed feedback laser was realized, and also amyloids or silks, which, due to their dye-doped fiber-like structure, allow for light amplification. Other materials that were investigated in that respect include polysaccharides, like starch exhibiting favorable photostability in comparison to other biomaterials, and chitosan, which forms photonic crystals or cellulose. Light amplification and random lasing was not only observed in processed biomaterials but also in living

  19. Development of Visual Loop-Mediate Isothermal Amplification Assay for CpTI gene%抗虫转CpTI基因成分现场可视化检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    王永; 兰青阔; 朱珠; 赵新; 陈锐; 刘娜; 李欧静; 郭永泽; 程奕

    2014-01-01

    It is important to detect Cowpea Trypsin Inhibitor (CpTI) gene in food or feed materials in order to prevent the illegal diffusion. A rapid visual loop-mediated isothermal amplification (LAMP) method assay for CpTI gene detection was developed. A set of primers were designed according to the nucleotide sequence of the target CpTI gene, seven genetically modified organisms (GMO) were detected by LAMP for method specificity, and meanwhile the mode of artificially contaminated food was constructed to evaluate the sensitivity of LAMP assay and qualitative PCR method. The results showed that the CpTI gene had specific amplification, but the non- CpTI GMO submitted negative reactions. Sensitivity of LAMP assay for Kefeng No.6 genetically modified rice was 0.005%. In conclusion, the LAMP assay developed in the present study is a specific, sensitive, simple and convenient method for the rapid screening of CpTI gene in contaminated foods.%豇豆胰蛋白酶抑制剂(CpTI)基因是目前仅次于Bt基因的广谱性抗虫基因,常用于转基因水稻和转基因棉花的基因工程研究中。根据CpTI基因特异性碱基序列设计引物,应用可视化环介导等温扩增技术,对转基因水稻科丰6号进行扩增,同时建立转基因成分人工污染的食品模型,比较了 LAMP 法与定性PCR方法检测的敏感性。结果表明:该方法仅对CpTI基因产生特异性扩增,灵敏度达0.005%;所建立的CpTI基因现场可视化筛查方法具有较高的特异性与灵敏性,操作简单、快速,可用于含有CpTI基因转基因成分污染的快速检测。

  20. SpxB is a suicide gene of Streptococcus pneumoniae and confers a selective advantage in an in vivo competitive colonization model.

    Science.gov (United States)

    Regev-Yochay, Gili; Trzcinski, Krzysztof; Thompson, Claudette M; Lipsitch, Marc; Malley, Richard

    2007-09-01

    The human bacterial pathogen Streptococcus pneumoniae dies spontaneously upon reaching stationary phase. The extent of S. pneumoniae death at stationary phase is unusual in bacteria and has been conventionally attributed to autolysis by the LytA amidase. In this study, we show that spontaneous pneumococcal death is due to hydrogen peroxide (H(2)O(2)), not LytA, and that the gene responsible for H(2)O(2) production (spxB) also confers a survival advantage in colonization. Survival of S. pneumoniae in stationary phase was significantly prolonged by eliminating H(2)O(2) in any of three ways: chemically by supplementing the media with catalase, metabolically by growing the bacteria under anaerobic conditions, or genetically by constructing DeltaspxB mutants that do not produce H(2)O(2). Likewise, addition of H(2)O(2) to exponentially growing S. pneumoniae resulted in a death rate similar to that of cells in stationary phase. While DeltalytA mutants did not lyse at stationary phase, they died at a rate similar to that of the wild-type strain. Furthermore, we show that the death process induced by H(2)O(2) has features of apoptosis, as evidenced by increased annexin V staining, decreased DNA content, and appearance as assessed by transmission electron microscopy. Finally, in an in vivo rat model of competitive colonization, the presence of spxB conferred a selective advantage over the DeltaspxB mutant, suggesting an explanation for the persistence of this gene. We conclude that a suicide gene of pneumococcus is spxB, which induces an apoptosis-like death in pneumococci and confers a selective advantage in nasopharyngeal cocolonization.

  1. Real-time fluorescence PCR for HER2 gene amplification%实时荧光PCR法检测}HER2基因扩增方法的建立

    Institute of Scientific and Technical Information of China (English)

    邓艳华; 吴荃; 陈华云; 李明

    2011-01-01

    Objective To evaluate the double standard curve of Real-time fluorescence PCR to detect human epidermalgrowth factor reeeptor-2(HER2) gene amplification for breast cancer diagnosis and treatment. Methods Genomie DNA was extracted in 500 eases fresh tissue samples of breast cancer for real-time fluorescence PCR and quantified by double standard curve. Data was analyzed by calculating the concentration ratio of the target gene to the referenced gene detected. Fluorescent in situ hybridization (FISH) was chosen as the control method for HER2 detection. Results 72 cases of positive samples and 419 negative samples were detected by this method. The sensitivity was 85.9%, the specificity was 98.79% and the accuracy is 96.74%. Compared with the FISH test, the results of both methods have good consistency. Conclusion The method of double standard curve of Real-time fluoreacence PCR to detect HER2 gene amplification has a wide application prospect for breast cancer diagnosis and treatment.%目的 探讨应用双标准曲线的实时荧光PCR法检测原癌基因人类表皮生长因子受体2(HER2)基因扩增在临床乳腺癌诊治中的可行性.方法 收集500例乳腺癌术后新鲜组织标本,抽提组织DNA进行实时荧光PCR检测,采用双标准曲线法定量,通过计算目的 基因浓度和内标基因浓度的比值来判断HER2基因的扩增情况.选择灵敏度和特异度均较高的荧光原位杂交方法作为对照方法.结果 检测阳性标本72例,阴性样本419例.该方法检测的灵敏度为85.9%,特异度为98.79%,准确度为96.74.与荧光原位杂交法(FISH)检测结果相比,二者具有较好的一致性.结论 双标准曲线的实时荧光PCR法用于检测HER2基因扩增相对准确可靠,有较好的临床应用前景.

  2. Ectopic expression of Arabidopsis glutaredoxin gene AtGRXS17 in tomato (Solanum lycopersicum) confers tolerance to chilling stress

    Science.gov (United States)

    The monothiol glutaredoxin AtGRXS17 from "Arabidopsis" confers thermotolerance in yeast, "Arabidopsis", and tomato plants. Here, we report that AtGRXS17 also enhances tolerance to chilling stress in tomato and is associated with elevation of antioxidant enzyme activities, which are known to be invol...

  3. Analysis of 525 samples to determine the usefulness of PCR amplification and sequencing of the 16S rRNA gene for diagnosis of bone and joint infections.

    Science.gov (United States)

    Fenollar, Florence; Roux, Véronique; Stein, Andréas; Drancourt, Michel; Raoult, Didier

    2006-03-01

    The 16S rRNA gene PCR in the diagnosis of bone and joint infections has not been systematically tested. Five hundred twenty-five bone and joint samples collected from 525 patients were cultured and submitted to 16S rRNA gene PCR detection of bacteria in parallel. The amplicons with mixed sequences were also cloned. When discordant results were observed, culture and PCR were performed once again. Bacteria were detected in 139 of 525 samples. Culture and 16S rRNA gene PCR yielded identical documentation in 475 samples. Discrepancies were linked to 13 false-positive culture results, 5 false-positive PCR results, 9 false-negative PCR results, 16 false-negative culture results, and 7 mixed infections. Cloning and sequencing of 16S rRNA gene amplicons in 6 of 8 patients with mixed infections identified 2 to 8 bacteria per sample. Rarely described human pathogens such as Alcaligenes faecalis, Comamonas terrigena, and 21 anaerobes were characterized. We also detected, by 16S rRNA gene PCR, four previously identified bacteria never reported in human infection, Alkanindiges illinoisensis, dehydroabietic acid-degrading bacterium DhA-73, unidentified Hailaer soda lake bacterium, and uncultured bacterium clone HuCa4. Seven organisms representing new potential species were also detected. PCR followed by cloning and sequencing may help to identify new pathogens involved in mixed bone infection.

  4. Development of a cost-efficient novel method for rapid, concurrent genotyping of five common single nucleotide polymorphisms of the brain derived neurotrophic factor (BDNF) gene by tetra-primer amplification refractory mutation system.

    Science.gov (United States)

    Wang, Cathy K; Xu, Michael S; Ross, Colin J; Lo, Ryan; Procyshyn, Ric M; Vila-Rodriguez, Fidel; White, Randall F; Honer, William G; Barr, Alasdair M

    2015-09-01

    Brain derived neurotrophic factor (BDNF) is a molecular trophic factor that plays a key role in neuronal survival and plasticity. Single nucleotide polymorphisms (SNPs) of the BDNF gene have been associated with specific phenotypic traits in a large number of neuropsychiatric disorders and the response to psychotherapeutic medications in patient populations. Nevertheless, due to study differences and occasionally contrasting findings, substantial further research is required to understand in better detail the association between specific BDNF SNPs and these psychiatric disorders. While considerable progress has been made recently in developing advanced genotyping platforms of SNPs, many high-throughput probe- or array-based detection methods currently available are limited by high costs, slow processing times or access to advanced instrumentation. The polymerase chain reaction (PCR)-based, tetra-primer amplification refractory mutation system (T-ARMS) method is a potential alternative technique for detecting SNP genotypes efficiently, quickly, easily, and cheaply. As a tool in psychopathology research, T-ARMS was shown to be capable of detecting five common SNPs in the BDNF gene (rs6265, rs988748, rs11030104, 11757G/C and rs7103411), which are all SNPs with previously demonstrated clinical relevance to schizophrenia and depression. The present technique therefore represents a suitable protocol for many research laboratories to study the genetic correlates of BDNF in psychiatric disorders. Copyright Copyright © 2015 John Wiley & Sons, Ltd.

  5. Overexpression of pigeonpea stress-induced cold and drought regulatory gene (CcCDR) confers drought, salt, and cold tolerance in Arabidopsis.

    Science.gov (United States)

    Tamirisa, Srinath; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

    2014-09-01

    A potent cold and drought regulatory protein-encoding gene (CcCDR) was isolated from the subtractive cDNA library of pigeonpea plants subjected to drought stress. CcCDR was induced by different abiotic stress conditions in pigeonpea. Overexpression of CcCDR in Arabidopsis thaliana imparted enhanced tolerance against major abiotic stresses, namely drought, salinity, and low temperature, as evidenced by increased biomass, root length, and chlorophyll content. Transgenic plants also showed increased levels of antioxidant enzymes, proline, and reducing sugars under stress conditions. Furthermore, CcCDR-transgenic plants showed enhanced relative water content, osmotic potential, and cell membrane stability, as well as hypersensitivity to abscisic acid (ABA) as compared with control plants. Localization studies confirmed that CcCDR could enter the nucleus, as revealed by intense fluorescence, indicating its possible interaction with various nuclear proteins. Microarray analysis revealed that 1780 genes were up-regulated in CcCDR-transgenics compared with wild-type plants. Real-time PCR analysis on selected stress-responsive genes, involved in ABA-dependent and -independent signalling networks, revealed higher expression levels in transgenic plants, suggesting that CcCDR acts upstream of these genes. The overall results demonstrate the explicit role of CcCDR in conferring multiple abiotic stress tolerance at the whole-plant level. The multifunctional CcCDR seems promising as a prime candidate gene for enhancing abiotic stress tolerance in diverse plants.

  6. Zinc finger protein genes from Cucurbita pepo are promising tools for conferring non-Cucurbitaceae plants with ability to accumulate persistent organic pollutants.

    Science.gov (United States)

    Inui, Hideyuki; Hirota, Matashi; Goto, Junya; Yoshihara, Ryouhei; Kodama, Noriko; Matsui, Tomomi; Yamazaki, Kiyoshi; Eun, Heesoo

    2015-03-01

    Some cultivars of cucumbers, melons, pumpkins, and zucchini, which are members of the Cucurbitaceae family, are uniquely subject to contamination by hydrophobic pollutants such as the organohalogen insecticides DDT. However, the molecular mechanisms for the accumulation of these pollutants in cucurbits have not been determined. Here, cDNA subtraction analysis of Cucurbita pepo cultivars that are low and high accumulators of hydrophobic contaminants revealed that a gene for zinc finger proteins (ZFPs) are preferentially expressed in high accumulators. The cloned CpZFP genes were classified into 2 types: (1) the PBG type, which were expressed in C. pepo cultivars Patty Green, Black Beauty, and Gold Rush, and (2) the BG type, which were expressed in Black Beauty and Gold Rush. Expression of these CpZFP genes in transgenic tobacco plants carrying an aryl hydrocarbon receptor-based inducible gene expression system significantly induced β-glucuronidase activity when the plants were treated with a polychlorinated biphenyl (PCB) compound, indicating that highly hydrophobic PCBs accumulated in the plants. In transgenic tobacco plants carrying CpZFPs, accumulation of dioxins and dioxin-like compounds increased in their aerial parts when they were cultivated in the dioxin-contaminated soil. In summary, we propose that addition of CpZFP genes is a promising tool for conferring noncucurbits with the ability to accumulate hydrophobic contaminants.

  7. Comparison between NuGEN's WT-Ovation Pico and one-direct amplification systems.

    Science.gov (United States)

    Morse, Alison M; Carballo, Valentina; Baldwin, Donald A; Taylor, Christopher G; McIntyre, Lauren M

    2010-09-01

    Differential gene expression between groups of homogenous cell types is a biological question whose time has come. RNA can be extracted from small numbers of cells, such as those isolated by laser-capture microdissection, but the small amounts obtained often require amplification to enable whole genome transcriptome profiling by technologies such as microarray analysis and RNA-seq. Recently, advances in amplification procedures make amplification directly from whole cell lysates possible. The aim of this study was to compare two amplification systems for variations in observed RNA abundance attributable to the amplification procedure for use with small quantities of cells isolated by laser-capture microdissection. Arabidopsis root cells undergoing giant cell formation as a result of nematode infestation and uninfested control root cells were laser-captured and used to evaluate two amplification systems. One, NuGEN's WT-Ovation Pico (Pico) amplification system, uses total RNA as starting material, and the other, NuGEN's WT-One-Direct (One-Direct) amplification system, uses lysate containing the captured cells. The reproducibility of whole genome transcript profiling and correlations of both systems were investigated after microarray analysis. The One-Direct system was less reproducible and more variable than the Pico system. The Pico amplification kit resulted in the detection of thousands of differentially expressed genes between giant cells and control cells. This is in marked contrast to the relatively few genes detected after amplification with the One-Direct amplification kit.

  8. High copy number variation of cancer-related microRNA genes and frequent amplification of DICER1 and DROSHA in lung cancer

    Science.gov (United States)

    Czubak, Karol; Lewandowska, Marzena Anna; Klonowska, Katarzyna; Roszkowski, Krzysztof; Kowalewski, Janusz; Figlerowicz, Marek; Kozlowski, Piotr

    2015-01-01

    A growing body of evidence indicates that miRNAs may be a class of genetic elements that can either drive or suppress oncogenesis. In this study we analyzed the somatic copy number variation of 14 miRNA genes frequently found to be either over- or underexpressed in lung cancer, as well as two miRNA biogenesis genes, DICER1 and DROSHA, in non-small-cell lung cancer (NSCLC). Our analysis showed that most analyzed miRNA genes undergo substantial copy number alteration in lung cancer. The most frequently amplified miRNA genes include the following: miR-30d, miR-21, miR-17 and miR-155. We also showed that both DICER1 and DROSHA are frequently amplified in NSCLC. The copy number variation of DICER1 and DROSHA correlates well with their expression and survival of NSCLC and other cancer patients. The increased expression of DROSHA and DICER1 decreases and increases the survival, respectively. In conclusion, our results show that copy number variation may be an important mechanism of upregulation/downregulation of miRNAs in cancer and suggest an oncogenic role for DROSHA. PMID:26156018

  9. Identification of a novel, invasive, not-yet-cultivated Treponema sp in the large intestine of pigs by PCR amplification of the 16S rRNA gene

    DEFF Research Database (Denmark)

    Mølbak, Lars; Schou, Kirstine Klitgaard; Jensen, Tim Kåre;

    2006-01-01

    Laser capture microdissection in combination with fluorescent in situ hybridization was used to identify an unknown species of spirochetes from the pig colonic mucosa. The 16S rRNA gene was PCR amplified, and the closest related type strain was Treponema bryantii(T) (90.1%). The spirochete, here...

  10. Spurious Amplification of a Plasmodium vivax Small-Subunit RNA Gene by Use of Primers Currently Used To Detect P. knowlesi▿

    Science.gov (United States)

    Imwong, Mallika; Tanomsing, Naowarat; Pukrittayakamee, Sasithon; Day, Nicholas P. J.; White, Nicholas J.; Snounou, Georges

    2009-01-01

    The PCR primers commonly used to detect Plasmodium knowlesi infections in humans were found to cross-react stochastically with P. vivax genomic DNA. A nested primer set that targets one of the P. knowlesi small-subunit rRNA genes was validated for specificity and for sensitivity of detection of <10 parasite genomes. PMID:19812279

  11. A wheat homologue of PHYTOCLOCK 1 is a candidate gene conferring the early heading phenotype to einkorn wheat.

    Science.gov (United States)

    Mizuno, Nobuyuki; Nitta, Miyuki; Sato, Kazuhiro; Nasuda, Shuhei

    2012-01-01

    An X-ray mutant showing an early flowering phenotype has been identified in einkorn wheat (Triticum monococcum L.), for which a major QTL for heading time was previously mapped in the telomeric region on the long arm of chromosome 3A. Recent advances in Triticeae genomics revealed that the gene order in this region is highly conserved between wheat and barley. Thus, we adopted a hypothetical gene order in barley, the so-called GenomeZipper, to develop DNA markers for fine mapping the target gene in wheat. We identified three genes tightly linked to the early heading phenotype. PCR analysis revealed that early-flowering is associated with the deletion of two genes in the mutant. Of the two deleted genes, one is an ortholog of the LUX ARRHYTHMO (LUX)/PHYTOCLOCK 1 (PCL1) gene found in Arabidopsis, which regulates the circadian clock and flowering time. We found distorted expression patterns of two clock genes (TOC1 and LHY) in the einkorn pcl1 deletion mutant as was reported for the Arabidopsis lux mutant. Transcript accumulation levels of photoperiod-response related genes, a photoperiod sensitivity gene (Ppd-1) and two wheat CONSTANS-like genes (WCO1 and TaHd1), were significantly higher in the einkorn wheat mutant. In addition, transcripts of the wheat florigen gene (WFT) accumulated temporally under short-day conditions in the einkorn wheat mutant. These results suggest that deletion of WPCL1 leads to abnormally higher expression of Ppd-1, resulting in the accumulation of WFT transcripts that triggers flowering even under short-day conditions. Our observations from gene mapping, gene deletions, and expression levels of flowering related genes strongly suggest that WPCL1 is the most likely candidate gene for controlling the early flowering phenotype in the einkorn wheat mutant.

  12. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean

    OpenAIRE

    Bingfu eGuo; Yong eGuo; Huilong eHong; Longguo eJin; Lijuan eZhang; Ru-Zhen eChang; Wei eLu; Min eLin; Li-Juan eQiu

    2015-01-01

    Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-...

  13. Amplification of Surface Antigen P43 Gene and Its Application in Detection of Toxoplasma Gondii in Allogeneic Hematopoietic Stem Cell Transplantation

    Institute of Scientific and Technical Information of China (English)

    ZHOUYongan; YUXinbing; 等

    2002-01-01

    Objective:To establish a rapid,specific and sensitive diagnostic technique for the human Toxoplasma gondii infection in the recipi-ents with allogeneic hematopoietic stem cell transplantation and discuss its clinical significance.Methods:30 patients undergoing allogeneic hematopoietic stem cell transplantation were detected by using ELISA and PCR.Results:Among 30 recipients undergiong allogeneic hematopoietic stem cell transplantation,3 were positive for Toxoplasma gondiii antigen and 5 for surface antigen p43 gene with the positive rate being 13.3% and 16.67% respectively.20 healthy people(negative for anti-Tox antibody)were also tested by using ELISA and PCR.Conclusion:PCR is an accurate,relatively rapid,sensitive and specific method for detecting P43 gene of Toxoplasma gondii.Be-canuse PCR can be applied to a variety of different clinical samples,it can be considered as a valuable additional tool for identification of Toxoplasma gondii infections.

  14. Evaluation of PCR primer selectivity and phylogenetic specificity by using amplification of 16S rRNA genes from betaproteobacterial ammonia-oxidizing bacteria in environmental samples.

    Science.gov (United States)

    Junier, Pilar; Kim, Ok-Sun; Hadas, Ora; Imhoff, Johannes F; Witzel, Karl-Paul

    2008-08-01

    The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (betaAOB) was evaluated. betaAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the betaAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations betaAMOf/betaAMOr, betaAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on betaAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.

  15. Technical note: Occurrence in fecal microbiota of genes conferring resistance to both macrolide-lincosamide-streptogramin B and tetracyclines concomitant with feeding of beef cattle with tylosin.

    Science.gov (United States)

    Chen, J; Fluharty, F L; St-Pierre, N; Morrison, M; Yu, Z

    2008-09-01

    Development of antimicrobial resistance in food animals receiving antimicrobials has been well documented among bacterial isolates, especially pathogens, but information on development of antimicrobial resistance at the microbial community level during long-term feeding of antimicrobials is lacking. The objective of this study was to examine the association between inclusion of tylosin in feed and occurrence of resistance to macrolide-lincosamide-streptogramin B (MLS(B)) in the entire fecal microbial communities of beef cattle over a feeding study of 168 d. A completely randomized design included 6 pens housed together in 1 barn, with each pen housing 10 to 11 steers. The control and tylosin groups each had 3 pens, with the former receiving no antimicrobial whereas the latter received both tylosin and monensin (11 and 29.9 mg/ kg of feed, respectively, DM) in feed. The abundance of genes conferring resistance to MLS(B) (erm genes) and tetracyclines (tet genes) were quantified using class-specific, real-time PCR assays. The abundances of erm and tet genes were analyzed with pens as experimental units using the MIXED procedure of SAS. Correlations between abundance of different resistance genes were calculated using the CORR procedure of SAS. We identified 4 classes (B, F, T, and X) of erm genes in fresh fecal samples collected at wk 2, 17, and 21 of feeding. From wk 2 to 17, the abundance of erm(T) and erm(X) increased (P tylosin feeding. Such co-selection of multiresistance at community level by one antimicrobial drug used in animals has the important implication that future studies should examine resistance to not only the antimicrobials used in animals, but also other antimicrobials, especially those used in human medicine, to fully assess the potential risk associated with antimicrobial use in animals. Both the erm and tet genes appeared to be disseminated among the microbial populations in all steers housed together.

  16. Hardness amplification in nondeterministic logspace

    OpenAIRE

    Gupta, Sushmita

    2007-01-01

    A hard problem is one which cannot be easily computed by efficient algorithms. Hardness amplification is a procedure which takes as input a problem of mild hardness and returns a problem of higher hardness. This is closely related to the task of decoding certain error-correcting codes. We show amplification from mild average case hardness to higher average case hardness for nondeterministic logspace and worst-to-average amplification for nondeterministic linspace. Finally we explore possible ...

  17. The ERI-6/7 helicase acts at the first stage of an siRNA amplification pathway that targets recent gene duplications.

    Directory of Open Access Journals (Sweden)

    Sylvia E J Fischer

    2011-11-01

    Full Text Available Endogenous small interfering RNAs (siRNAs are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7-dependent siRNAs require Dicer. We identify that the eri-6/7-dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3-4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7-dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA-associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation.

  18. Targeting MET Amplification as a New Oncogenic Driver

    Energy Technology Data Exchange (ETDEWEB)

    Kawakami, Hisato [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Okamoto, Isamu, E-mail: okamotoi@kokyu.med.kyushu-u.ac.jp [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Center for Clinical and Translational Research, Kyushu University Hospital, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582 (Japan); Okamoto, Wataru [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Division of Transrlational Research, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577 (Japan); Tanizaki, Junko [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, HIM223, 450 Brookline Avenue, Boston, MA 02215 (United States); Nakagawa, Kazuhiko [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Nishio, Kazuto [Department of Genome Biology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan)

    2014-07-22

    Certain genetically defined cancers are dependent on a single overactive oncogene for their proliferation and survival, a phenomenon known as “oncogene addiction”. A new generation of drugs that selectively target such “driver oncogenes” manifests a clinical efficacy greater than that of conventional chemotherapy in appropriate genetically defined patients. MET is a proto-oncogene that encodes a receptor tyrosine kinase, and aberrant activation of MET signaling occurs in a subset of advanced cancers as result of various genetic alterations including gene amplification, polysomy, and gene mutation. Our preclinical studies have shown that inhibition of MET signaling either with the small-molecule MET inhibitor crizotinib or by RNA interference targeted to MET mRNA resulted in marked antitumor effects in cancer cell lines with MET amplification both in vitro and in vivo. Furthermore, patients with non-small cell lung cancer or gastric cancer positive for MET amplification have shown a pronounced clinical response to crizotinib. Accumulating preclinical and clinical evidence thus suggests that MET amplification is an “oncogenic driver” and therefore a valid target for treatment. However, the prevalence of MET amplification has not been fully determined, possibly in part because of the difficulty in evaluating gene amplification. In this review, we provide a rationale for targeting this genetic alteration in cancer therapy.

  19. Targeting MET Amplification as a New Oncogenic Driver

    Directory of Open Access Journals (Sweden)

    Hisato Kawakami

    2014-07-01

    Full Text Available Certain genetically defined cancers are dependent on a single overactive oncogene for their proliferation and survival, a phenomenon known as “oncogene addiction”. A new generation of drugs that selectively target such “driver oncogenes” manifests a clinical efficacy greater than that of conventional chemotherapy in appropriate genetically defined patients. MET is a proto-oncogene that encodes a receptor tyrosine kinase, and aberrant activation of MET signaling occurs in a subset of advanced cancers as result of various genetic alterations including gene amplification, polysomy, and gene mutation. Our preclinical studies have shown that inhibition of MET signaling either with the small-molecule MET inhibitor crizotinib or by RNA interference targeted to MET mRNA resulted in marked antitumor effects in cancer cell lines with MET amplification both in vitro and in vivo. Furthermore, patients with non-small cell lung cancer or gastric cancer positive for MET amplification have shown a pronounced clinical response to crizotinib. Accumulating preclinical and clinical evidence thus suggests that MET amplification is an “oncogenic driver” and therefore a valid target for treatment. However, the prevalence of MET amplification has not been fully determined, possibly in part because of the difficulty in evaluating gene amplification. In this review, we provide a rationale for targeting this genetic alteration in cancer therapy.

  20. Plasma HER2 amplification in cell-free DNA during neoadjuvant chemotherapy in breast cancer

    DEFF Research Database (Denmark)

    Bechmann, Troels; Andersen, Rikke Fredslund; Pallisgaard, Niels

    2013-01-01

    Measurement of human epidermal growth factor receptor 2 (HER2) gene amplification in cell-free DNA (cfDNA) is an evolving technique in breast cancer, enabling liquid biopsies and treatment monitoring. The present study investigated the dynamics of plasma HER2 gene copy number and amplification in...... in cfDNA during neoadjuvant chemotherapy....

  1. Nostradamus conference

    CERN Document Server

    Rössler, Otto; Snášel, Václav; Abraham, Ajith; Corchado, Emilio; Nostradamus: Modern Methods of Prediction, Modeling and Analysis of Nonlinear Systems

    2013-01-01

    This proceeding book of Nostradamus conference (http://nostradamus-conference.org) contains accepted papers presented at this event in 2012. Nostradamus conference was held in the one of the biggest and historic city of Ostrava (the Czech Republic, http://www.ostrava.cz/en), in September 2012. Conference topics are focused on classical as well as modern methods for prediction of dynamical systems with applications in science, engineering and economy. Topics are (but not limited to): prediction by classical and novel methods, predictive control, deterministic chaos and its control, complex systems, modelling and prediction of its dynamics and much more.

  2. Overexpression of MpCYS4, A Phytocystatin Gene from Malus prunifolia (Willd.) Borkh., Enhances Stomatal Closure to Confer Drought Tolerance in Transgenic Arabidopsis and Apple.

    Science.gov (United States)

    Tan, Yanxiao; Li, Mingjun; Yang, Yingli; Sun, Xun; Wang, Na; Liang, Bowen; Ma, Fengwang

    2017-01-01

    Phytocystatins (PhyCys) comprise a group of inhibitors for cysteine proteinases in plants. They play a wide range of important roles in regulating endogenous processes and protecting plants against various environmental stresses, but the underlying mechanisms remain largely unknown. Here, we detailed the biological functions of MpCYS4, a member of cystatin genes isolated from Malus prunifolia. This gene was activated under water deficit, heat (40°C), exogenous abscisic acid (ABA), or methyl viologen (MV) (Tan et al., 2014a). At cellular level, MpCYS4 protein was found to be localized in the nucleus, cytoplasm, and plasma membrane of onion epidermal cells. Recombinant MpCYS4 cystatin expressed in Escherichia coli was purified and it exhibited cysteine protease inhibitor activity. Transgenic overexpression of MpCYS4 in Arabidopsis (Arabidopsis thaliana) and apple (Malus domestica) led to ABA hypersensitivity and series of ABA-associated phenotypes, such as enhanced ABA-induced stomatal closing, altered expression of many ABA/stress-responsive genes, and enhanced drought tolerance. Taken together, our results demonstrate that MpCYS4 is involved in ABA-mediated stress signal transduction and confers drought tolerance at least in part by enhancing stomatal closure and up-regulating the transcriptional levels of ABA- and drought-related genes. These findings provide new insights into the molecular mechanisms by which phytocystatins influence plant growth, development, and tolerance to stress.

  3. Expression of a codon-optimized dsdA gene in tobacco plastids and rice nuclear confers D-serine tolerance

    Directory of Open Access Journals (Sweden)

    Yanmei eLi

    2016-05-01

    Full Text Available D-serine is toxic to plants. D-serine ammonia lyase, which is encoded by the dsdA gene, can attenuate this toxicity with high specificity. In the present study, we explored the function of codon-optimized dsdA with tobacco plastids and rice nuclear transformation system. It was shown that the dsdA gene was site-specifically integrated into the tobacco chloroplast genome and displayed a high level of expression. Genetic analysis of the progenies showed that the dsdA gene is maternally inherited and confers sufficient D-serine resistance in tobacco. The effective screening concentrations of D-serine for seed germination, callus regeneration and foliar spray were 10 mM, 30 mM and 75 mM, respectively. In addition, calluses from homozygous transgenic rice lines also showed significant tolerance to D-serine (up to 75 mM. Our study proves the feasibility of using dsdA gene as a selectable marker in both chloroplast and nuclear transformation systems.

  4. Expression of a Codon-Optimized dsdA Gene in Tobacco Plastids and Rice Nucleus Confers D-Serine Tolerance.

    Science.gov (United States)

    Li, Yanmei; Wang, Rui; Hu, Zongliang; Li, Hongcai; Lu, Shizhan; Zhang, Juanjuan; Lin, Yongjun; Zhou, Fei

    2016-01-01

    D-serine is toxic to plants. D-serine ammonia lyase, which is encoded by the dsdA gene, can attenuate this toxicity with high specificity. In the present study, we explored the function of codon-optimized dsdA with tobacco plastids and rice nuclear transformation system. It was shown that dsdA gene was site-specifically integrated into the tobacco plastid genome and displayed a high level of expression. Genetic analysis of the progenies showed that dsdA gene is maternally inherited and confers sufficient D-serine resistance in tobacco. The effective screening concentrations of D-serine for seed germination, callus regeneration and foliar spray were 10, 30, and 75 mM, respectively. In addition, calluses from homozygous transgenic rice lines also showed significant tolerance to D-serine (up to 75 mM). Our study proves the feasibility of using dsdA gene as a selectable marker in both plastid and nuclear transformation systems.

  5. Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.

    Science.gov (United States)

    Wendlandt, Sarah; Kadlec, Kristina; Feßler, Andrea T; Schwarz, Stefan

    2015-06-12

    The aim of this study was to investigate the genetic basis of combined pleuromutilin-lincosamide-streptogramin A resistance in 26 unrelated methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) from dairy cows suffering from mastitis. The 26 pleuromutilin-resistant staphylococcal isolates were screened for the presence of the genes vga(A), vga(B), vga(C), vga(E), vga(E) variant, sal(A), vmlR, cfr, lsa(A), lsa(B), lsa(C), and lsa(E) by PCR. None of the 26 isolates carried the genes vga(B), vga(C), vga(E), vga(E) variant, vmlR, cfr, lsa(A), lsa(B), or lsa(C). Two Staphylococcus haemolyticus and single Staphylococcus xylosus, Staphylococcus lentus, and Staphylococcus hominis were vga(A)-positive. Twelve S. aureus, two Staphylococcus warneri, as well as single S. lentus and S. xylosus carried the lsa(E) gene. Moreover, single S. aureus, S. haemolyticus, S. xylosus, and Staphylococcus epidermidis were positive for both genes, vga(A) and lsa(E). The sal(A) gene was found in a single Staphylococcus sciuri. All ABC transporter genes were located in the chromosomal DNA, except for a plasmid-borne vga(A) gene in the S. epidermidis isolate. The genetic environment of the lsa(E)-positive isolates was analyzed using previously described PCR assays. Except for the S. warneri and S. xylosus, all lsa(E)-positive isolates harbored a part of the previously described enterococcal multiresistance gene cluster. This is the first report of the novel lsa(E) gene in the aforementioned bovine CoNS species. This is also the first identification of the sal(A) gene in a S. sciuri from a case of bovine mastitis. Moreover, the sal(A) gene was shown to also confer pleuromutilin resistance. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Expression of the grape VqSTS21 gene in Arabidopsis confers resistance to osmotic stress and biotrophic pathogens but not Botrytis cinerea

    Directory of Open Access Journals (Sweden)

    Li Huang

    2016-09-01

    Full Text Available Stilbene synthase (STS is a key gene in the biosynthesis of various stilbenoids, including resveratrol and its derivative glucosides (such as piceid, that has been shown to contribute to disease resistance in plants. However, the mechanism behind such a role has yet to be elucidated. Furthermore, the function of STS genes in osmotic stress tolerance remains unclear. As such, we sought to elucidate the role of STS genes in the defense against biotic and abiotic stress in the model plant Arabidopsis thaliana. Expression profiling of 31 VqSTS genes from Vitis quinquangularis revealed that VqSTS21 was up-regulated in response to powdery mildew (PM infection. To provide a deeper understanding of the function of this gene, we cloned the full-length coding sequence of VqSTS21 and overexpressed it in Arabidopsis thaliana via Agrobacterium-mediated transformation. The resulting VqSTS21 Arabidopsis lines produced trans-piceid rather than resveratrol as their main stilbenoid product and exhibited improved disease resistance to PM and Pseudomonas syringae pv. tomato DC3000, but displayed increased susceptibility to Botrytis cinerea. In addition, transgenic Arabidopsis lines were found to confer tolerance to salt and drought stress from seed germination through plant maturity. Intriguingly, qPCR assays of defense-related genes involved in salicylic acid, jasmonic acid, and abscisic acid-induced signaling pathways in these transgenic lines suggested that VqSTS21 plays a role in various phytohormone-related pathways, providing insight into the mechanism behind VqSTS21-mediated resistance to biotic and abiotic stress.

  7. Expression of the Grape VqSTS21 Gene in Arabidopsis Confers Resistance to Osmotic Stress and Biotrophic Pathogens but Not Botrytis cinerea

    Science.gov (United States)

    Huang, Li; Zhang, Songlin; Singer, Stacy D.; Yin, Xiangjing; Yang, Jinhua; Wang, Yuejin; Wang, Xiping

    2016-01-01

    Stilbene synthase (STS) is a key gene in the biosynthesis of various stilbenoids, including resveratrol and its derivative glucosides (such as piceid), that has been shown to contribute to disease resistance in plants. However, the mechanism behind such a role has yet to be elucidated. Furthermore, the function of STS genes in osmotic stress tolerance remains unclear. As such, we sought to elucidate the role of STS genes in the defense against biotic and abiotic stress in the model plant Arabidopsis thaliana. Expression profiling of 31 VqSTS genes from Vitis quinquangularis revealed that VqSTS21 was up-regulated in response to powdery mildew (PM) infection. To provide a deeper understanding of the function of this gene, we cloned the full-length coding sequence of VqSTS21 and overexpressed it in Arabidopsis thaliana via Agrobacterium-mediated transformation. The resulting VqSTS21 Arabidopsis lines produced trans-piceid rather than resveratrol as their main stilbenoid product and exhibited improved disease resistance to PM and Pseudomonas syringae pv. tomato DC3000, but displayed increased susceptibility to Botrytis cinerea. In addition, transgenic Arabidopsis lines were found to confer tolerance to salt and drought stress from seed germination through plant maturity. Intriguingly, qPCR assays of defense-related genes involved in salicylic acid, jasmonic acid, and abscisic acid-induced signaling pathways in these transgenic lines suggested that VqSTS21 plays a role in various phytohormone-related pathways, providing insight into the mechanism behind VqSTS21-mediated resistance to biotic and abiotic stress. PMID:27695466

  8. Accurately assessing the risk of schizophrenia conferred by rare copy-number variation affecting genes with brain function.

    Science.gov (United States)

    Raychaudhuri, Soumya; Korn, Joshua M; McCarroll, Steven A; Altshuler, David; Sklar, Pamela; Purcell, Shaun; Daly, Mark J

    2010-09-09

    Investigators have linked rare copy number variation (CNVs) to neuropsychiatric diseases, such as schizophrenia. One hypothesis is that CNV events cause disease by affecting genes with specific brain functions. Under these circumstances, we expect that CNV events in cases should impact brain-function genes more frequently than those events in controls. Previous publications have applied "pathway" analyses to genes within neuropsychiatric case CNVs to show enrichment for brain-functions. While such analyses have been suggestive, they often have not rigorously compared the rates of CNVs impacting genes with brain function in cases to controls, and therefore do not address important confounders such as the large size of brain genes and overall differences in rates and sizes of CNVs. To demonstrate the potential impact of confounders, we genotyped rare CNV events in 2,415 unaffected controls with Affymetrix 6.0; we then applied standard pathway analyses using four sets of brain-function genes and observed an apparently highly significant enrichment for each set. The enrichment is simply driven by the large size of brain-function genes. Instead, we propose a case-control statistical test, cnv-enrichment-test, to compare the rate of CNVs impacting specific gene sets in cases versus controls. With simulations, we demonstrate that cnv-enrichment-test is robust to case-control differences in CNV size, CNV rate, and systematic differences in gene size. Finally, we apply cnv-enrichment-test to rare CNV events published by the International Schizophrenia Consortium (ISC). This approach reveals nominal evidence of case-association in neuronal-activity and the learning gene sets, but not the other two examined gene sets. The neuronal-activity genes have been associated in a separate set of schizophrenia cases and controls; however, testing in independent samples is necessary to definitively confirm this association. Our method is implemented in the PLINK software package.

  9. Accurately Assessing the Risk of Schizophrenia Conferred by Rare Copy-Number Variation Affecting Genes with Brain Function

    Science.gov (United States)

    Raychaudhuri, Soumya; Korn, Joshua M.; McCarroll, Steven A.; Altshuler, David; Sklar, Pamela; Purcell, Shaun; Daly, Mark J.

    2010-01-01

    Investigators have linked rare copy number variation (CNVs) to neuropsychiatric diseases, such as schizophrenia. One hypothesis is that CNV events cause disease by affecting genes with specific brain functions. Under these circumstances, we expect that CNV events in cases should impact brain-function genes more frequently than those events in controls. Previous publications have applied “pathway” analyses to genes within neuropsychiatric case CNVs to show enrichment for brain-functions. While such analyses have been suggestive, they often have not rigorously compared the rates of CNVs impacting genes with brain function in cases to controls, and therefore do not address important confounders such as the large size of brain genes and overall differences in rates and sizes of CNVs. To demonstrate the potential impact of confounders, we genotyped rare CNV events in 2,415 unaffected controls with Affymetrix 6.0; we then applied standard pathway analyses using four sets of brain-function genes and observed an apparently highly significant enrichment for each set. The enrichment is simply driven by the large size of brain-function genes. Instead, we propose a case-control statistical test, cnv-enrichment-test, to compare the rate of CNVs impacting specific gene sets in cases versus controls. With simulations, we demonstrate that cnv-enrichment-test is robust to case-control differences in CNV size, CNV rate, and systematic differences in gene size. Finally, we apply cnv-enrichment-test to rare CNV events published by the International Schizophrenia Consortium (ISC). This approach reveals nominal evidence of case-association in neuronal-activity and the learning gene sets, but not the other two examined gene sets. The neuronal-activity genes have been associated in a separate set of schizophrenia cases and controls; however, testing in independent samples is necessary to definitively confirm this association. Our method is implemented in the PLINK software package

  10. Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study

    Science.gov (United States)

    Yi, Xiaoyan; Zhang, Huan; Liu, Guangxing

    2014-05-01

    Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene (18S rDNA) libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18s rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.

  11. Amplification, cloning and expression of Brucella melitensis bp26 gene (OMP28 isolated from Markazi province (Iran and purification of Bp26 Protein

    Directory of Open Access Journals (Sweden)

    Hosseini, S.D.

    2013-12-01

    Full Text Available Brucellosis is a debilitative disease that imposes costs on both economy and society. It is shown that although the vaccine can prevent abortion, it does not provide complete protection against infection. In Iran, Brucella melitensis is a common causative agent for brucellosis and BP26 protein of this bacterium having a good antigenesity and an important vaccine candidate. In this study B. melitensis bp26 gene was cloned first in to PTZ57R/T vector and accessed on the PET28a vector and sequenced. Recombinant vector transformed and expressed in to E. coli BL21 (DE3 and then recombinant protein was purified with Ni-NTA column of chromatography against His tag. Obtained rOmp28 could be used as a research experimental tool to find its potential as a detection kit and vaccine candidate.

  12. A New Method for Sex Determination Based on Detection of SRY, STS and Amelogenin Gene Regions with Simultaneous Amplification of Their Homologous Sequences by a Multiplex PCR

    Directory of Open Access Journals (Sweden)

    Morikawa,Toshio

    2011-04-01

    Full Text Available We have developed a new method for sex determination based on simultaneous detection of the SRY (sex-determining region Y, STS (steroid sulfatase and amelogenin (AMELX and AMELY gene regions and their homologous sequences. The sex of 246 blood samples was correctly determined by this method. An AMELY-deleted male sample, which would have been erroneously considered female based solely on analysis of the amelogenin locus, was successfully identified as male by the present method. The detection limit of this method was 63 pg of genomic DNA, and the male DNA component could be detected from mixed samples having a male:female ratio as low as 1:10. This method was useful for degraded DNA and possessed the human specificity. Practical application to 35 autopsy cases is described.

  13. A new method for sex determination based on detection of SRY, STS and amelogenin gene regions with simultaneous amplification of their homologous sequences by a multiplex PCR.

    Science.gov (United States)

    Morikawa, Toshio; Yamamoto, Yuji; Miyaishi, Satoru

    2011-04-01

    We have developed a new method for sex determination based on simultaneous detection of the SRY (sex-determining region Y), STS (steroid sulfatase) and amelogenin (AMELX and AMELY) gene regions and their homologous sequences. The sex of 246 blood samples was correctly determined by this method. An AMELY-deleted male sample, which would have been erroneously considered female based solely on analysis of the amelogenin locus, was successfully identified as male by the present method. The detection limit of this method was 63 pg of genomic DNA, and the male DNA component could be detected from mixed samples having a male:female ratio as low as 1:10. This method was useful for degraded DNA and possessed the human specificity. Practical a