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Sample records for 53r gene highly

  1. Pathogenicity and immunogenicity of recombinant Tiantan Vaccinia Virus with deleted C12L and A53R genes.

    Science.gov (United States)

    Dai, Kaifan; Liu, Ying; Liu, Mingjie; Xu, Jianqing; Huang, Wei; Huang, Xianggang; Liu, Lianxing; Wan, Yanmin; Hao, Yanling; Shao, Yiming

    2008-09-15

    Interest is increasing regarding replicating poxvirus as HIV vaccine vector. In China, the Tiantan Vaccinia Virus (TV) has been used most extensively in the battle of eradicating smallpox. Recently, TV was developing as vaccine vector to fight against infectious diseases such as human immunodeficiency virus (HIV). However, replicating vaccinia virus sometimes may pose serious post-vaccination complications, especially in immunosuppressed individuals. To develop a safer and more effective TV-based vector, we constructed C12L (vIL-18 binding protein) and A53R (vTNF receptor homolog) gene-deleted mutants which are based on parental TV and VTKgpe (TV expressing HIV gagpol and env gene), respectively. The pathogenicity and immunogenicity were also evaluated. Deleting these two immunomodulatory genes lessened the virulence of the parental virus in both mice and rabbit models. Notably, C12L deletion mutant attenuated the skin virulence of parental virus by as high as approximate 2 logs. Furthermore, VTKgpe with A53R and C12L gene deletion retains the high immunogenicity of the parental virus to elicit strong humoral and cellular responses to the HIV target genes despite the remarkable attenuation. These data suggest that deletion of the cytokine viroceptor gene is feasible to obtain a safer and replication-competent TV vector for vaccination and immunotherapy.

  2. Prevalence of an inherited cancer predisposition syndrome associated with the germ line TP53 R337H mutation in Paraguay.

    Science.gov (United States)

    Legal, Edith Falcon-de; Ascurra, Marta; Custódio, Gislaine; Ayala, Horacio Legal; Monteiro, Magna; Vega, Celeste; Fernández-Nestosa, María José; Vega, Sonia; Sade, Elis R; Coelho, Izabel M M; Ribeiro, Enilze M S F; Cavalli, Iglenir J; Figueiredo, Bonald C

    2015-04-01

    The tumor suppressor gene TP53 is the most frequently mutated gene in human cancer, and the germline TP53 R337H mutation is the most common mutation reported to date. However, this mutation is associated with a lower cumulative lifetime cancer risk than other mutations in the p53 DNA-binding domain. A detailed statistical analysis of 171,500 DNA tests in Brazilian neonates found that 0.27% of the general population is positive for this mutation, and some of the estimated 200,000 Brazilian R337H carriers in southern and southeastern Brazil have already developed cancer. The present study was designed to estimate R337H prevalence in neighboring Paraguay. To address this question, 10,000 dried blood samples stored in Guthrie cards since 2008 were randomly selected from the Paraguayan municipalities located at the border with Brazil. These samples were tested for R337H mutation using the PCR-restriction fragment length polymorphism assay. This germline mutation was detected in five samples (5/10,000), indicating that the total number of R337H carriers in Paraguay may be as high as 3500. Previous studies have shown that other countries (i.e., Portugal, Spain, and Germany) presented one family with this mutation, leading us to conclude that, besides Brazil and Paraguay, other countries may have multiple families carrying this mutation, which is an inherited syndrome that is difficult to control.

  3. Association of the germline TP53 R337H mutation with breast cancer in southern Brazil

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    Srivastava Kumar

    2008-12-01

    Full Text Available Abstract Background The germline TP53-R337H mutation is strongly associated with pediatric adrenocortical tumors (ACT in southern Brazil; it has low penetrance and limited tissue specificity in most families and therefore is not associated with Li-Fraumeni syndrome. However, other tumor types, mainly breast cancer, have been observed in carriers of several unrelated kindreds, raising the possibility that the R337H mutation may also contribute to breast tumorigenesis in a genetic background-specific context. Methods We conducted a case-control study to determine the prevalence of the R337H mutation by sequencing TP53 exon 10 in 123 women with breast cancer and 223 age- and sex-matched control subjects from southern Brazil. Fisher's test was used to compare the prevalence of the R337H. Results The R337H mutation was found in three patients but in none of the controls (p = 0.0442. Among the carriers, two had familial history of cancer meeting the Li-Fraumeni-like criteria. Remarkably, tumors in each of these three cases underwent loss of heterozygosity by eliminating the mutant TP53 allele rather than the wild-type allele. Polymorphisms were identified within the TP53 (R72P and Ins16 and MDM2 (SNP309 genes that may further diminish TP53 tumor suppressor activity. Conclusion These results demonstrate that the R337H mutation can significantly increase the risk of breast cancer in carriers, which likely depends on additional cooperating genetic factors. These findings are also important for understanding how low-penetrant mutant TP53 alleles can differentially influence tumor susceptibility.

  4. Generalist genes and high cognitive abilities.

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    Haworth, Claire M A; Dale, Philip S; Plomin, Robert

    2009-07-01

    The concept of generalist genes operating across diverse domains of cognitive abilities is now widely accepted. Much less is known about the etiology of the high extreme of performance. Is there more specialization at the high extreme? Using a representative sample of 4,000 12-year-old twin pairs from the UK Twins Early Development Study, we investigated the genetic and environmental overlap between web-based tests of general cognitive ability, reading, mathematics and language performance for the top 15% of the distribution using DF extremes analysis. Generalist genes are just as evident at the high extremes of performance as they are for the entire distribution of abilities and for cognitive disabilities. However, a smaller proportion of the phenotypic intercorrelations appears to be explained by genetic influences for high abilities.

  5. Inactivation and inducible oncogenic mutation of p53 in gene targeted pigs.

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    Simon Leuchs

    Full Text Available Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs and live pigs carrying a latent TP53(R167H mutant allele, orthologous to oncogenic human mutant TP53(R175H and mouse Trp53(R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53(R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53(R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53(R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.

  6. Gene Expression in Mammalian Cells After Exposure to 95 MeV Argon Ions

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    Arenz, A.; Hellweg, C. E.; Baumstark-Khan, C.

    Cell response to genotoxic agents is complex and involves the participation of different classes of genes (DNA repair, cell cycle control, signal transduction, apoptosis and oncogenesis). The unique feature of the space radiation environment is the dominance of high-energy charged particles (HZE or high LET radiation) which present a significant hazard to space flight crews, and accelerator-based experiments are underway to quantify the health risks due to unavoidable radiation exposure. High linear energy transfer (LET) radiation has an increased relative biological effectiveness (RBE) as compared to X-rays for cell death induction, gene mutation, genomic instability, and carcinogenesis. The tumour suppressor gene p53 plays a crucial role in maintaining the integrity of the genome. The p53 protein acts as a transcription factor that mediates cell cycle arrest and apoptosis by binding to DNA and activating transcription of specific genes. It is also though to be involved in damage repair by transcriptional activation of the newly identified p53 dependent ribonuclease subunit R2 (p53R2) that is directly involved in the p53 cell cycle checkpoint for repair of damaged DNA. In that case it is responsible for nucleotide delivery for DNA repair synthesis. DNA damages of cultured human cells (e.g. MCF-7, AGS, A549) exposed to accelerated argon ions at the French heavy ion facility GANIL were analysed for expression levels of certain damage- and apoptosis-relevant genes. RNA was extracted from cells exposed to different particle fluences after various recovery times. A real-time QRT-PCR assay was applied, which employs both relative and absolute quantification of a candidate mRNA biomarker. The expressions of different DNA damage inducible genes (e.g. p53R2, GADD45, p21) were analysed. A reproducible up-regulation representing a twofold to fourfold change in p53R2 gene expression level was confirmed for X-irradiated and Ar-ion exposed cells dependent on dose. Kinetics of p

  7. THE GENE EXPRESSION PROFILE OF HIGHLY METASTATIC HUMAN OVARIAN CANCER CELL LINE BY GENE CHIP

    Institute of Scientific and Technical Information of China (English)

    吕桂泉; 许沈华; 牟瀚舟; 朱赤红; 羊正炎; 高永良; 楼洪坤; 刘祥麟; 杨文; 程勇

    2001-01-01

    To study the gene expression of high metastatic human ovarian carcinoma cell line (HO-8910PM) and to screen for novel metastasis- associated genes by cDNA microarray. Methods: The cDNA was retro-transcribed from equal quantity mRNA derived from tissues of highly metastatic ovarian carcinoma cell line and normal ovarian, and was labeled with Cy5 and Cy3 fluorescence as probes. The mixed probes were hybridized with BioDoor 4096 double dot human whole gene chip. The chip was scanned by scanArray 3000 laser scanner. The acquired image was analyzed by ImaGene 3.0 software. Results: By applying the cDNA microarray we found: A total of 323 genes whose expression level were 3 times higher or lower in HO-8910PM cell than normal ovarian epithelium cell were screened out, with 71 higher and 252 lower respectively. Among these 10 were new genes. 67 genes showed expression difference bigger than 6 times between HO-8910PM cell and normal ovarian epithelium cell, among these genes 12 were higher, 55 lower, and two new genes were found. Conclusion: cDNA microarray technique is effective in screening the differentially expressed genes between human ovarian cancer cell line (HO-8910PM) and normal ovarian epithelium cell. Using the cDNA microarray to analyze of human ovarian cancer cell line gene expression profile difference will help the gene diagnosis, treatment and protection.

  8. HIGH EFFICIENCY RETROVIRUS-MEDIATED GENE TRANSFER TO LEUKEMIA CELLS

    Institute of Scientific and Technical Information of China (English)

    FU Jian-xin; CHEN Zi-xing; CEN Jian-nong; WANG Wei; RUAN Chang-geng

    1999-01-01

    Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1.The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR).Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days' culture.Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.

  9. Gene regulation: hacking the network on a sugar high.

    Science.gov (United States)

    Ellis, Tom; Wang, Xiao; Collins, James J

    2008-04-11

    In a recent issue of Molecular Cell, Kaplan et al. (2008) determine the input functions for 19 E. coli sugar-utilization genes by using a two-dimensional high-throughput approach. The resulting input-function map reveals that gene network regulation follows non-Boolean, and often nonmonotonic, logic.

  10. DAVID Knowledgebase: a gene-centered database integrating heterogeneous gene annotation resources to facilitate high-throughput gene functional analysis

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    Baseler Michael W

    2007-11-01

    Full Text Available Abstract Background Due to the complex and distributed nature of biological research, our current biological knowledge is spread over many redundant annotation databases maintained by many independent groups. Analysts usually need to visit many of these bioinformatics databases in order to integrate comprehensive annotation information for their genes, which becomes one of the bottlenecks, particularly for the analytic task associated with a large gene list. Thus, a highly centralized and ready-to-use gene-annotation knowledgebase is in demand for high throughput gene functional analysis. Description The DAVID Knowledgebase is built around the DAVID Gene Concept, a single-linkage method to agglomerate tens of millions of gene/protein identifiers from a variety of public genomic resources into DAVID gene clusters. The grouping of such identifiers improves the cross-reference capability, particularly across NCBI and UniProt systems, enabling more than 40 publicly available functional annotation sources to be comprehensively integrated and centralized by the DAVID gene clusters. The simple, pair-wise, text format files which make up the DAVID Knowledgebase are freely downloadable for various data analysis uses. In addition, a well organized web interface allows users to query different types of heterogeneous annotations in a high-throughput manner. Conclusion The DAVID Knowledgebase is designed to facilitate high throughput gene functional analysis. For a given gene list, it not only provides the quick accessibility to a wide range of heterogeneous annotation data in a centralized location, but also enriches the level of biological information for an individual gene. Moreover, the entire DAVID Knowledgebase is freely downloadable or searchable at http://david.abcc.ncifcrf.gov/knowledgebase/.

  11. High-Diversity Genes in the Arabidopsis Genome

    OpenAIRE

    Cork, Jennifer M.; Purugganan, Michael D.

    2005-01-01

    High-diversity genes represent an important class of loci in organismal genomes. Since elevated levels of nucleotide variation are a key component of the molecular signature for balancing selection or local adaptation, high-diversity genes may represent loci whose alleles are selectively maintained as balanced polymorphisms. Comparison of 4300 random shotgun sequence fragments of the Arabidopsis thaliana Ler ecotype genome with the whole genomic sequence of the Col-0 ecotype identified 60 gen...

  12. The P53R2 Gene involved in a P53-dependent Cell-cycle Checkpoint for DNA Damage%依赖P53的P53R2基因在细胞周期检验点对损伤DNA的修复作用

    Institute of Scientific and Technical Information of China (English)

    张伟; 明镇寰

    2001-01-01

    @@1. p53基因及P53蛋白p53基因最初被认为是一个普通的癌基因,其产物的作用是刺激肿瘤细胞的生长。但后来发现原先研究的p53基因只是野生型p53基因的突变体,只有突变型p53基因的产物才能刺激不正常细胞(如癌细胞)的生长,而野生型p53基因的产物对肿瘤则有抑制作用,正常的p53基因原来是一个抑癌基因。经长期研究发现,突变型p53基因在人

  13. Detecting Key Structural Features within Highly Recombined Genes

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    Wertz, John E; McGregor, Karen F; Bessen, Debra E

    2007-01-01

    Many microorganisms exhibit high levels of intragenic recombination following horizontal gene transfer events. Furthermore, many microbial genes are subject to strong diversifying selection as part of the pathogenic process. A multiple sequence alignment is an essential starting point for many of the tools that provide fundamental insights on gene structure and evolution, such as phylogenetics; however, an accurate alignment is not always possible to attain. In this study, a new analytic approach was developed in order to better quantify the genetic organization of highly diversified genes whose alleles do not align. This BLAST-based method, denoted BLAST Miner, employs an iterative process that places short segments of highly similar sequence into discrete datasets that are designated “modules.” The relative positions of modules along the length of the genes, and their frequency of occurrence, are used to identify sequence duplications, insertions, and rearrangements. Partial alleles of sof from Streptococcus pyogenes, encoding a surface protein under host immune selection, were analyzed for module content. High-frequency Modules 6 and 13 were identified and examined in depth. Nucleotide sequences corresponding to both modules contain numerous duplications and inverted repeats, whereby many codons form palindromic pairs. Combined with evidence for a strong codon usage bias, data suggest that Module 6 and 13 sequences are under selection to preserve their nucleic acid secondary structure. The concentration of overlapping tandem and inverted repeats within a small region of DNA is highly suggestive of a mechanistic role for Module 6 and 13 sequences in promoting aberrant recombination. Analysis of pbp2X alleles from Streptococcus pneumoniae, encoding cell wall enzymes that confer antibiotic resistance, supports the broad applicability of this tool in deciphering the genetic organization of highly recombined genes. BLAST Miner shares with phylogenetics the

  14. High-throughput Binary Vectors for Plant Gene Function Analysis

    Institute of Scientific and Technical Information of China (English)

    Zhi-Yong Lei; Ping Zhao; Min-Jie Cao; Rong Cui; Xi Chen; Li-Zhong Xiong; Qi-Fa Zhang; David J. Oliver; Cheng-Bin Xiang

    2007-01-01

    A series of high-throughput binary cloning vectors were constructed to facilitate gene function analysis in higher plants. This vector series consists of plasmids designed for plant expression, promoter analysis, gene silencing,and green fluorescent protein fusions for protein localization. These vectors provide for high-throughput and efficient cloning utilizing sites for λ phage integrase/excisionase. In addition, unique restriction sites are incorporated in a multiple cloning site and enable promoter replacement. The entire vector series are available with complete sequence information and detailed annotations and are freely distributed to the scientific community for non-commercial uses.

  15. A2 gene of Old World cutaneous Leishmania is a single highly conserved functional gene

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    Derouin Francis

    2005-03-01

    Full Text Available Abstract Background Leishmaniases are among the most proteiform parasitic infections in humans ranging from unapparent to cutaneous, mucocutaneous or visceral diseases. The various clinical issues depend on complex and still poorly understood mechanisms where both host and parasite factors are interacting. Among the candidate factors of parasite virulence are the A2 genes, a family of multiple genes that are developmentally expressed in species of the Leishmania donovani group responsible for visceral diseases (VL. By contrast, in L. major determining cutaneous infections (CL we showed that A2 genes are present in a truncated form only. Furthermore, the A2 genomic sequences of L. major were considered subsequently to represent non-expressed pseudogenes 1. Consequently, it was suggested that the structural and functional properties of A2 genes could play a role in the differential tropism of CL and VL leishmanias. On this basis, it was of importance to determine whether the observed structural/functional particularities of the L. major A2 genes were shared by other CL Leishmania, therefore representing a proper characteristic of CL A2 genes as opposed to those of VL isolates. Methods In the present study we amplified by PCR and sequenced the A2 genes from genomic DNA and from clonal libraries of the four Old World CL species comparatively to a clonal population of L. infantum VL parasites. Using RT-PCR we also amplified and sequenced A2 mRNA transcripts from L. major. Results A unique A2 sequence was identified in Old World cutaneous Leishmania by sequencing. The shared sequence was highly conserved among the various CL strains and species analysed, showing a single polymorphism C/G at position 58. The CL A2 gene was found to be functionally transcribed at both parasite stages. Conclusion The present study shows that cutaneous strains of leishmania share a conserved functional A2 gene. As opposed to the multiple A2 genes described in VL isolates

  16. High pressure-sensitive gene expression in Lactobacillus sanfranciscensis

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    R.F. Vogel

    2005-08-01

    Full Text Available Lactobacillus sanfranciscensis is a Gram-positive lactic acid bacterium used in food biotechnology. It is necessary to investigate many aspects of a model organism to elucidate mechanisms of stress response, to facilitate preparation, application and performance in food fermentation, to understand mechanisms of inactivation, and to identify novel tools for high pressure biotechnology. To investigate the mechanisms of the complex bacterial response to high pressure we have analyzed changes in the proteome and transcriptome by 2-D electrophoresis, and by microarrays and real time PCR, respectively. More than 16 proteins were found to be differentially expressed upon high pressure stress and were compared to those sensitive to other stresses. Except for one apparently high pressure-specific stress protein, no pressure-specific stress proteins were found, and the proteome response to pressure was found to differ from that induced by other stresses. Selected pressure-sensitive proteins were partially sequenced and their genes were identified by reverse genetics. In a transcriptome analysis of a redundancy cleared shot gun library, about 7% of the genes investigated were found to be affected. Most of them appeared to be up-regulated 2- to 4-fold and these results were confirmed by real time PCR. Gene induction was shown for some genes up-regulated at the proteome level (clpL/groEL/rbsK, while the response of others to high hydrostatic pressure at the transcriptome level seemed to differ from that observed at the proteome level. The up-regulation of selected genes supports the view that the cell tries to compensate for pressure-induced impairment of translation and membrane transport.

  17. High-resolution gene mapping using admixture linkage disequilibrium

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    This note reports simulation study on the rate of decay in linkage dis equilibrium (LD) in mixed populations over multiple discrete generations and explores the usefulness of the LD analysis in high-resolution gene mapping. The results indicate that the smaller the recombination fraction and the fewer generati ons since admixtureevent, the higher power of the approach in gene mapping. The expected estimate of recombination fraction would give an estimate that is slig htly biased upwards, if relevant genes are in tight linkage. The estimated recom bination fraction is usually larger than the true value within 2-5 generations. From generations 10-20, the mean estimates are in good agreement with the true value. The method presented here enables estimation of means and corresponding confidence intervals of the recombination fraction at any number of generations.

  18. Identifying the genes of unconventional high temperature superconductors.

    Science.gov (United States)

    Hu, Jiangping

    We elucidate a recently emergent framework in unifying the two families of high temperature (high [Formula: see text]) superconductors, cuprates and iron-based superconductors. The unification suggests that the latter is simply the counterpart of the former to realize robust extended s-wave pairing symmetries in a square lattice. The unification identifies that the key ingredients (gene) of high [Formula: see text] superconductors is a quasi two dimensional electronic environment in which the d-orbitals of cations that participate in strong in-plane couplings to the p-orbitals of anions are isolated near Fermi energy. With this gene, the superexchange magnetic interactions mediated by anions could maximize their contributions to superconductivity. Creating the gene requires special arrangements between local electronic structures and crystal lattice structures. The speciality explains why high [Formula: see text] superconductors are so rare. An explicit prediction is made to realize high [Formula: see text] superconductivity in Co/Ni-based materials with a quasi two dimensional hexagonal lattice structure formed by trigonal bipyramidal complexes.

  19. Galaxy High Throughput Genotyping Pipeline for GeneTitan.

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    Karpenko, Oleksiy; Bahroos, Neil; Chukhman, Morris; Dong, Xiao; Kanabar, Pinal; Arbieva, Zarema; Jackson, Tommie; Hendrickson, William

    2013-01-01

    Latest genotyping solutions allow for rapid testing of more than two million markers in one experiment. Fully automated instruments such as Affymetrix GeneTitan enable processing of large numbers of samples in a truly high-throughput manner. In concert with solutions like Axiom, fully customizable array plates can now utilize automated workflows that can leverage multi-channel instrumentation like the GeneTitan. With the growing size of raw data output, the serial computational architecture of the software, typically distributed by the vendors on turnkey desktop solutions for quality control and genotype calling, becomes legacy rather than an advantage. Advanced software techniques provide power, flexibility, and can be deployed in an HPC environment, but become technically inconvenient for biologists to use. Here we present a pipeline that uses Galaxy as a mechanism to lower the barrier for complex analysis, and increase efficiency by leveraging high-throughput computing.

  20. Immunoinformatics study on highly expressed Mycobacterium tuberculosis genes during infection.

    Science.gov (United States)

    Nguyen Thi, Le Thuy; Sarmiento, Maria Elena; Calero, Romel; Camacho, Frank; Reyes, Fatima; Hossain, Md Murad; Gonzalez, Gustavo Sierra; Norazmi, Mohd Nor; Acosta, Armando

    2014-09-01

    The most important targets for vaccine development are the proteins that are highly expressed by the microorganisms during infection in-vivo. A number of Mycobacterium tuberculosis (Mtb) proteins are also reported to be expressed in-vivo at different phases of infection. In the present study, we analyzed multiple published databases of gene expression profiles of Mtb in-vivo at different phases of infection in animals and humans and selected 38 proteins that are highly expressed in the active, latent and reactivation phases. We predicted T- and B-cell epitopes from the selected proteins using HLAPred for T-cell epitope prediction and BCEPred combined with ABCPred for B-cell epitope prediction. For each selected proteins, regions containing both T- and B-cell epitopes were identified which might be considered as important candidates for vaccine design against tuberculosis.

  1. Sulindac inhibits pancreatic carcinogenesis in LSL-KrasG12D-LSL-Trp53R172H-Pdx-1-Cre mice via suppressing aldo-keto reductase family 1B10 (AKR1B10).

    Science.gov (United States)

    Li, Haonan; Yang, Allison L; Chung, Yeon Tae; Zhang, Wanying; Liao, Jie; Yang, Guang-Yu

    2013-09-01

    Sulindac has been identified as a competitive inhibitor of aldo-keto reductase 1B10 (AKR1B10), an enzyme that plays a key role in carcinogenesis. AKR1B10 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and exhibits lipid substrate specificity, especially for farnesyl and geranylgeranyl. There have been no studies though showing that the inhibition of PDAC by sulindac is via inhibition of AKR1B10, particularly the metabolism of farnesyl/geranylgeranyl and Kras protein prenylation. To determine the chemopreventive effects of sulindac on pancreatic carcinogenesis, 5-week-old LSL-Kras(G12D)-LSL-Trp53(R172H)-Pdx-1-Cre mice (Pan(kras/p53) mice) were fed an AIN93M diet with or without 200 p.p.m. sulindac (n = 20/group). Kaplan-Meier survival analysis showed that average animal survival in Pan(kras/p53) mice was 143.7 ± 8.8 days, and average survival with sulindac was increased to 168.0 ± 8.8 days (P sulindac, the incidence of PDAC was reduced to 56% (P sulindac significantly decreased Ki-67-labeled cell proliferation and markedly reduced the expression of phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Raf and mitogen-activated protein kinase kinase 1 and 2. In in vitro experiments with PDAC cells from Pan(kras/p53) mice, sulindac exhibited dose-dependent inhibition of AKR1B10 activity. By silencing AKR1B10 expression through small interfering RNA or by sulindac treatment, these in vitro models showed a reduction in Kras and human DNA-J homolog 2 protein prenylation, and downregulation of phosphorylated C-raf, ERK1/2 and MEK1/2 expression. Our results demonstrate that sulindac inhibits pancreatic carcinogenesis by the inhibition of Kras protein prenylation by targeting AKR1B10.

  2. High intensity ultrasound transducer used in gene transfection

    Science.gov (United States)

    Morrison, Kyle P.; Keilman, George W.; Noble, Misty L.; Brayman, Andrew A.; Miao, Carol H.

    2012-11-01

    This paper describes a novel therapeutic high intensity non-focused ultrasound (HIU) transducer designed with uniform pressure distribution to aid in accelerated gene transfer in large animal liver tissues in vivo. The underlying HIU transducer was used to initiate homogeneous cavitation throughout the tissue while delivering up to 2.7 MPa at 1.1 MHz across its radiating surface. The HIU transducer was built into a 6 cm diameter x 1.3 cm tall housing ergonomically designed to avoid collateral damage to the surrounding anatomy during dynamic motion. The ultrasound (US) radiation was applied in a 'paintbrush-like' manner to the surface of the liver. The layers and geometry of the transducer were carefully selected to maximize the active diameter (5.74 cm), maximize the electrical to acoustic conversion efficiency (85%) to achieve 2.7 MPa of peak negative pressure, maximize the frequency operating band at the fundamental resonance to within a power transfer delta of 1 dB, and reduce the pressure delta to within 2 dB across the radiating surface. For maximum peak voltage into the transducer, a high performance piezoceramic was chosen and a DC bias circuit was built integral to the system. An apodized two element annular pattern was made from a single piezoceramic element, resulting in significant pressure uniformity enhancement. In addition to using apodization for pressure uniformity, a proprietary multi-layered structure was used to improve efficiency while sustaining an operating band from 900 kHz to 1.3 MHz. The resultant operating band allowed for dithering techniques using frequency modulation. The underlying HIU transducer for use in large animals enhances gene expression up to 6300-fold.

  3. The R package FANet: sparse factor analysis model for high dimensional gene co-expression networks

    OpenAIRE

    Blum, Anne; Houee-Bigot, Magalie; Lagarrigue, Sandrine; Causeur, David

    2014-01-01

    Inference on gene regulatory networks from high-throughput expression data turns out to be one of the main current challenges in systems biology. Such interaction networks are very insightful for the deep understanding of biological relationships between genes. In particular, a functional characterization of gene modules of highly interacting genes enables the identification of biological processes underlying complex traits as diseases. Inference on this dependence structure shall...

  4. Gene expression profile differences in high and low metastatic human ovarian cancer cell lines by gene chip

    Institute of Scientific and Technical Information of China (English)

    许沈华; 牟瀚舟; 吕桂泉; 朱赤红; 羊正炎; 高永良; 楼洪坤; 刘祥麟; 程勇; 杨文

    2002-01-01

    Objectives To study the difference between gene expressions of high (H0-8910PM) and low (HO-8910) metastatic human ovarian carcinoma cell lines and screen novel associated genes by cDNA microarray. Methods cDNA retro-transcribed from equal quantities of mRNA derived from high and low metastatic tumor cells or normal ovarian tissues were labeled with Cy5 and Cy3 fluorescein as probes. The mixed probe was hybridized with two pieces of BioDoor 4096 double dot human whole gene chip and scanned with a ScanArray 3000 laser scanner. The acquired image was analyzed by ImaGene 3.0 software. Results A total of 355 genes with expression levels more than 3 times larger were found by comparing the HO-8910 cell with normal ovarian epithelial cells. A total of 323 genes with expression levels more than 3 times larger in HO-8910PM cells compared to normal ovarian epithelium cells were also detected. A total of 165 genes whose expression levels were more than two times those of HO-8910PM cells compared to their mother cell line (HO-8910) were detected. Twenty-one genes with expression levels >3 times were found from comparison of these two tumor cell lines.Conclusions cDNA microarray techniques are effective in screening differential gene expression between two human ovarian cancer cell lines (H0-8910PM; HO-8910) and normal ovarian epithelial cells. These genes may be related to the genesis and development of ovarian carcinoma. Analysis of the human ovarian cancer gene expression profile with cDNA microarray may help in gene diagnosis, treatment and prevention.

  5. High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta

    Directory of Open Access Journals (Sweden)

    Clark Taane G

    2010-04-01

    Full Text Available Abstract Background Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue. Results Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%. Conclusions Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes

  6. Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish.

    Science.gov (United States)

    Chen, Ailu; Wang, Ruijia; Liu, Shikai; Peatman, Eric; Sun, Luyang; Bao, Lisui; Jiang, Chen; Li, Chao; Li, Yun; Zeng, Qifan; Liu, Zhanjiang

    2016-06-01

    Interspecific hybrids provide a rich source for the analysis of allele-specific expression (ASE). In this work, we analyzed ASE in F1 hybrid catfish using RNA-Seq datasets. While the vast majority of genes were expressed with both alleles, 7-8 % SNPs exhibited significant differences in allele ratios of expression. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5420 (8.2 %) and 13,390 (7.5 %) SNPs were identified as significant ASE-SNPs, respectively. With these SNPs, a total of 1519 and 3075 ASE-genes were identified. Gene Ontology analysis revealed that genes encoding cytoplasmic ribosomal proteins (RP) were highly enriched among ASE genes. Parent-of-origin was determined for 27 and 30 ASE RP genes in the liver and gill, respectively. The results indicated that genes from both channel catfish and blue catfish were involved in ASE. However, each RP gene appeared to be almost exclusively expressed from only one parent, indicating that ribosomes in the hybrid catfish were in the "hybrid" form. Overall representation of RP transcripts among the transcriptome appeared lower in the F1 hybrid catfish than in channel catfish or blue catfish, suggesting that the "hybrid" ribosomes may work more efficiently for translation in the F1 hybrid catfish.

  7. Analysis of Gene Expression in the K562-n High Tumorigenitic Human Leukemia Cell Line

    Institute of Scientific and Technical Information of China (English)

    Shuqing Lü; Xiaoping Xu; Fang Xia; JianMin Wang

    2005-01-01

    OBJECTIVE The human leukemia K562-n cell line displays much higher tumorigenic actively in nude mice compared with its parental K562 cell line. The molecular mechanism of the differences in tumorigenicity between K562-n and K562 in nude mice was examined.METHODS The differences in gene expression between K562 and K562-n cells were analyzed by using cDNA microarrays.RESULTS Among the12,800 genes examined, there was a significant difference in expression of 139 genes between K562-n and K562 cells.Eighty-five of these genes have been registered in the GeneBank and 54are unknown. The genes accessible from the GeneBank include:1)oncogenes and tumor-supressor genes; 2) genes related to transcription regulation, the cell cycle and apoptosis; 3) genes related to the cytoskeleton and cytokinetics; 4) genes related to metabolism and transport; 5) genes related to immune function. There were also some differently expressed genes with mixed functions.CONCLUSION There are many genes differentially expressed between K562-n and K562 cells .The high tumorigenicity of the human leukemia K562-n cell line in nude mice might be related to its specific geneexpression profile.

  8. 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

  9. Measuring the toxic effects of high gene dosage on yeast cells.

    Science.gov (United States)

    Daniel, J

    1996-12-13

    A novel method, which is rapid, reliable and quantitative, is presented for measuring the toxic effects on yeast cells of high dosage of any given gene. It is based on the possibility of monitoring the presence in cells of a plasmid carrying the ADE2 gene from Saccharomyces cerevisiae by direct observation of colonies, the construction of this particular plasmid being easily made by marked homologous recombination in yeast. Four yeast regulatory genes tested were found to result in various degrees of toxicity at high dosage. Possible implications of the measurement of gene toxicity for eukaryotic cell regulatory mechanisms and for the use of novel general approaches to gene selection, such as the gene-gene interference method, are discussed.

  10. Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

    Directory of Open Access Journals (Sweden)

    Senanu M Spring-Pearson

    Full Text Available The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

  11. Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

    Science.gov (United States)

    Spring-Pearson, Senanu M; Stone, Joshua K; Doyle, Adina; Allender, Christopher J; Okinaka, Richard T; Mayo, Mark; Broomall, Stacey M; Hill, Jessica M; Karavis, Mark A; Hubbard, Kyle S; Insalaco, Joseph M; McNew, Lauren A; Rosenzweig, C Nicole; Gibbons, Henry S; Currie, Bart J; Wagner, David M; Keim, Paul; Tuanyok, Apichai

    2015-01-01

    The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

  12. 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.

  13. Substrate-induced gene expression screening: a method for high-throughput screening of metagenome libraries.

    Science.gov (United States)

    Uchiyama, Taku; Miyazaki, Kentaro

    2010-01-01

    The SIGEX (substrate-induced gene expression) method is a novel approach for the screening of gene (genome) libraries. In addition to the commonly used function- and sequence-driven approaches to screening, SIGEX provides a third option; in SIGEX, positives are identified using a reporter gene, and the library is constructed using an "operon-trap" vector. This vector contains the reporter gene immediately downstream of the cloning site for the genomic insert so that the expression of the inserted gene(s) is coupled with that of the reporter gene. This system is especially suitable for screening catabolic genes that are induced in response to metabolically relevant compounds, such as substrates. If expression of the inserted gene(s) is activated in response to the addition of these compounds, then positive clones can be identified based on the reporter signal. The most effective selection is obtained by the use of a FACS (fluorescence-activated cell sorter) in conjunction with a FACS-compatible fluorescent reporter protein, such as GFP (green fluorescent protein). Activity-based screening of metagenomic libraries often suffers from low sensitivity and low throughput. In contrast, the high throughput, high sensitivity, and versatility of SIGEX make it a particularly suitable method for screening metagenomic libraries.

  14. Design of high-performance parallelized gene predictors in MATLAB

    Directory of Open Access Journals (Sweden)

    Rivard Sylvain

    2012-04-01

    Full Text Available Abstract Background This paper proposes a method of implementing parallel gene prediction algorithms in MATLAB. The proposed designs are based on either Goertzel’s algorithm or on FFTs and have been implemented using varying amounts of parallelism on a central processing unit (CPU and on a graphics processing unit (GPU. Findings Results show that an implementation using a straightforward approach can require over 4.5 h to process 15 million base pairs (bps whereas a properly designed one could perform the same task in less than five minutes. In the best case, a GPU implementation can yield these results in 57 s. Conclusions The present work shows how parallelism can be used in MATLAB for gene prediction in very large DNA sequences to produce results that are over 270 times faster than a conventional approach. This is significant as MATLAB is typically overlooked due to its apparent slow processing time even though it offers a convenient environment for bioinformatics. From a practical standpoint, this work proposes two strategies for accelerating genome data processing which rely on different parallelization mechanisms. Using a CPU, the work shows that direct access to the MEX function increases execution speed and that the PARFOR construct should be used in order to take full advantage of the parallelizable Goertzel implementation. When the target is a GPU, the work shows that data needs to be segmented into manageable sizes within the GFOR construct before processing in order to minimize execution time.

  15. The influence of bovine milk high or low in isoflavones on hepatic gene expression in mice

    DEFF Research Database (Denmark)

    Skaanild, Mette Tingleff; Nielsen, Tina Skau

    2012-01-01

    in hepatic gene expression after dietary intake of milk high and low in isoflavones. In addition to pelleted feed female NMRI mice were offered water, water added either 17β-estradiol, equol, Tween 80, and milk high and low in isoflavone content for a week. Gene expression was analyzed using an array q......Isoflavones have generated much attention due to their potential positive effects in various diseases. Phytoestrogens especially equol can be found in bovine milk, as feed ration for dairy cows is comprised of plants containing phytoestrogens. The aim of this study was to analyze the changes......PCR kit. It was revealed that Tween 80 and 17β-estradiol upregulated both phase I and phase II genes to the same extent whereas equol alone, high and low isoflavone milk did not alter the expression of phase I genes but decreased the expression of phase II genes. This study shows that dietary isoflavones...

  16. Isolation and cloning of the gene encoding high affinity phosphate transporter in rice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    High affinity phosphate transporter plays an important role in plant adapting to low phosphorus. Isolation of genes coding this kind of protein has attracted worldwide scholars to accomplish. We aimed to isolate the gene and transfer it to target plants for breeding.

  17. Prediction of highly expressed genes in microbes based on chromatin accessibility

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Ussery, David

    2007-01-01

    BACKGROUND: It is well known that gene expression is dependent on chromatin structure in eukaryotes and it is likely that chromatin can play a role in bacterial gene expression as well. Here, we use a nucleosomal position preference measure of anisotropic DNA flexibility to predict highly expressed...

  18. Physiological effects of high- and low-voltage pulse combinations for gene electrotransfer in muscle

    DEFF Research Database (Denmark)

    Hojman, Pernille; Gissel, Hanne; Andre, Franck M;

    2008-01-01

    Gene transfer by electroporation is gaining momentum now that high-level, long-term expression of transgenes is being obtained. Several different pulse regimens are efficient, yet little information is available about the physiological muscular response to gene electrotransfer. This paper provide...

  19. Recombinant cells that highly express chromosomally-integrated heterologous gene

    Science.gov (United States)

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  20. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EF

  1. High-Resolution Chromosome Ideogram Representation of Currently Recognized Genes for Autism Spectrum Disorders

    Directory of Open Access Journals (Sweden)

    Merlin G. Butler

    2015-03-01

    Full Text Available Recently, autism-related research has focused on the identification of various genes and disturbed pathways causing the genetically heterogeneous group of autism spectrum disorders (ASD. The list of autism-related genes has significantly increased due to better awareness with advances in genetic technology and expanding searchable genomic databases. We compiled a master list of known and clinically relevant autism spectrum disorder genes identified with supporting evidence from peer-reviewed medical literature sources by searching key words related to autism and genetics and from authoritative autism-related public access websites, such as the Simons Foundation Autism Research Institute autism genomic database dedicated to gene discovery and characterization. Our list consists of 792 genes arranged in alphabetical order in tabular form with gene symbols placed on high-resolution human chromosome ideograms, thereby enabling clinical and laboratory geneticists and genetic counsellors to access convenient visual images of the location and distribution of ASD genes. Meaningful correlations of the observed phenotype in patients with suspected/confirmed ASD gene(s at the chromosome region or breakpoint band site can be made to inform diagnosis and gene-based personalized care and provide genetic counselling for families.

  2. Characterization of differentially expressed genes using high-dimensional co-expression networks

    DEFF Research Database (Denmark)

    Coelho Goncalves de Abreu, Gabriel; Labouriau, Rodrigo S.

    2010-01-01

    of spurious information along the network are avoided. The proposed inference procedure is based on the minimization of the Bayesian Information Criterion (BIC) in the class of decomposable graphical models. This class of models can be used to represent complex relationships and has suitable properties...... that allow to make effective inference in problems with high degree of complexity (e.g. several thousands of genes) and small number of observations (e.g. 10-100) as typically occurs in high throughput gene expression studies. Taking advantage of the internal structure of decomposable graphical models, we...... construct a compact representation of the co-expression network that allows to identify the regions with high concentration of differentially expressed genes. It is argued that differentially expressed genes located in highly interconnected regions of the co-expression network are less informative than...

  3. High-performance web services for querying gene and variant annotation.

    Science.gov (United States)

    Xin, Jiwen; Mark, Adam; Afrasiabi, Cyrus; Tsueng, Ginger; Juchler, Moritz; Gopal, Nikhil; Stupp, Gregory S; Putman, Timothy E; Ainscough, Benjamin J; Griffith, Obi L; Torkamani, Ali; Whetzel, Patricia L; Mungall, Christopher J; Mooney, Sean D; Su, Andrew I; Wu, Chunlei

    2016-05-06

    Efficient tools for data management and integration are essential for many aspects of high-throughput biology. In particular, annotations of genes and human genetic variants are commonly used but highly fragmented across many resources. Here, we describe MyGene.info and MyVariant.info, high-performance web services for querying gene and variant annotation information. These web services are currently accessed more than three million times permonth. They also demonstrate a generalizable cloud-based model for organizing and querying biological annotation information. MyGene.info and MyVariant.info are provided as high-performance web services, accessible at http://mygene.info and http://myvariant.info . Both are offered free of charge to the research community.

  4. Identifying the genes of unconventional high temperature superconductors

    OpenAIRE

    Hu, Jiangping

    2016-01-01

    We elucidate a recently emergent framework in unifying the two families of high temperature (high \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$T_{\\rm c}$$\\end{document} T c ) superconductors, cuprates and iron-based superconductors. The unification suggests that the latter is simply the counterpart of the forme...

  5. Screening differentially expressed genes in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Nan Cui; Jian-Wu Tang; Li Hou; Bo Song; Li Li; Ji-Wei Liu

    2005-01-01

    AIM: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis.METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastatic Hca-F and its synogenetic cell line Hca-P with a low metastatic potential was constructed by suppression subtracted hybridization(SSH) method. The screened clones of the subtracted library were sequenced and GeneBank homology search was performed.RESULTS: Fourteen differentially expressed cDNA fragments of Hca-F were obtained with two novel genes.CONCLUSION: SSH is a useful technique to detect differentially expressioned genes and an effective method to clone novel genes.

  6. Transmission of the P250R mutation of the FGFR3 gene in four generations with highly variable phenotype

    DEFF Research Database (Denmark)

    Hove, Hanne Buciek; Dunø, Morten; Daugaard-Jensen, Jette;

    Transmission of the P250R mutation of the FGFR3 gene in four generations with highly variable phenotype.......Transmission of the P250R mutation of the FGFR3 gene in four generations with highly variable phenotype....

  7. Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening

    Directory of Open Access Journals (Sweden)

    Santos Carla S

    2012-11-01

    Full Text Available Abstract Background Pine wilt disease (PWD, caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus, damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant’s molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN and Pinus pinea (less susceptible to PWN. Results Four cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species

  8. Mitogenomic analyses propose positive selection in mitochondrial genes for high-altitude adaptation in galliform birds.

    Science.gov (United States)

    Zhou, Taicheng; Shen, Xuejuan; Irwin, David M; Shen, Yongyi; Zhang, Yaping

    2014-09-01

    Galliform birds inhabit very diverse habitats, including plateaus that are above 3000 m in altitude. At high altitude, lower temperature and hypoxia are two important factors influencing survival. Mitochondria, as the ultimate oxygen transductor, play an important role in aerobic respiration through oxidative phosphorylation (OXPHOS). We analyzed the mitochondrial genomes of six high-altitude phasianidae birds and sixteen low-altitude relatives in an attempt to determine the role of mitochondrial genes in high-altitude adaptation. We reconstructed the phylogenetic relationships of these phasianidae birds and relatives and found at least four lineages that independently occupied this high-altitude habitat. Selective analyses revealed significant evidence for positive selection in the genes ND2, ND4, and ATP6 in three of the high-altitude lineages. This result strongly suggests that adaptive evolution of mitochondrial genes played a critical role during the independent acclimatization to high altitude by galliform birds.

  9. A highly conserved gene island of three genes on chromosome 3B of hexaploid wheat: diverse gene function and genomic structure maintained in a tightly linked block

    Directory of Open Access Journals (Sweden)

    Ma Wujun

    2010-05-01

    Full Text Available Abstract Background The complexity of the wheat genome has resulted from waves of retrotransposable element insertions. Gene deletions and disruptions generated by the fast replacement of repetitive elements in wheat have resulted in disruption of colinearity at a micro (sub-megabase level among the cereals. In view of genomic changes that are possible within a given time span, conservation of genes between species tends to imply an important functional or regional constraint that does not permit a change in genomic structure. The ctg1034 contig completed in this paper was initially studied because it was assigned to the Sr2 resistance locus region, but detailed mapping studies subsequently assigned it to the long arm of 3B and revealed its unusual features. Results BAC shotgun sequencing of the hexaploid wheat (Triticum aestivum cv. Chinese Spring genome has been used to assemble a group of 15 wheat BACs from the chromosome 3B physical map FPC contig ctg1034 into a 783,553 bp genomic sequence. This ctg1034 sequence was annotated for biological features such as genes and transposable elements. A three-gene island was identified among >80% repetitive DNA sequence. Using bioinformatics analysis there were no observable similarity in their gene functions. The ctg1034 gene island also displayed complete conservation of gene order and orientation with syntenic gene islands found in publicly available genome sequences of Brachypodium distachyon, Oryza sativa, Sorghum bicolor and Zea mays, even though the intergenic space and introns were divergent. Conclusion We propose that ctg1034 is located within the heterochromatic C-band region of deletion bin 3BL7 based on the identification of heterochromatic tandem repeats and presence of significant matches to chromodomain-containing gypsy LTR retrotransposable elements. We also speculate that this location, among other highly repetitive sequences, may account for the relative stability in gene order and

  10. Selection for the compactness of highly expressed genes in Gallus gallus

    Directory of Open Access Journals (Sweden)

    Zhou Ming

    2010-05-01

    (n = 1105, and compared the first intron length and the average intron length between highly expressed genes (top 5% expressed genes and weakly expressed genes (bottom 5% expressed genes. We found that the first intron length and the average intron length in highly expressed genes are not different from that in weakly expressed genes. We also made a comparison between ubiquitously expressed genes and narrowly expressed somatic genes with similar expression levels. Our data demonstrated that ubiquitously expressed genes are less compact than narrowly expressed genes with the similar expression levels. Obviously, these observations can not be explained by mutational bias hypotheses either. We also found that the significant trend between genes' compactness and expression level could not be affected by local mutational biases. We argued that the selection of economy model is most likely one to explain the relationship between gene expression and gene characteristics in chicken genome. Conclusion Natural selection appears to favor the compactness of highly expressed genes in chicken genome. This observation can be explained by the selection of economy model. Reviewers This article was reviewed by Dr. Gavin Huttley, Dr. Liran Carmel (nominated by Dr. Eugene V. Koonin and Dr. Araxi Urrutia (nominated by Dr. Laurence D. Hurst.

  11. Highly expressed genes in human high grade gliomas: immunohistochemical analysis of data from the Human Protein Atlas

    Directory of Open Access Journals (Sweden)

    Michael A. Meyer

    2014-06-01

    Full Text Available Gene expression within human glioblastomas were analyzed from data on 20,083 genes entered into the on-line Human Protein Atlas. In selecting genes that are strongly expressed within normal human brain tissue, 58 genes were identified from a search of the 20,083 entries that were rated as showing 90% or greater intensity of expression within normal brain tissues. Of these 58, a subset of 48 genes was identified that not only had expression data for human glioblastomas but also for the human glioblastoma cell line U-251. Four of these 48 selected genes were found to be strongly expressed within the cytoplasm when assessed by both histologic sampling of high grade glioma patient cases as well as U-251 glioblastoma cell line immunofluoresence analysis. These four human genes are: AGBL2 (ATP/GTP binding protein-like 2, BLOC1S6 (biogenesis of lysosomal organelles complex-1, subunit 6, MAP1A (microtubule-associated protein 1A and ZSWIM5 (zinc finger, SWIM-type containing 5, also known as KIAA1511. Further research is advocated to investigate the role of ZSWIM5 and AGBL2 in glioma cell biology.

  12. Global gene analysis of oocytes from early stages in human folliculogenesis shows high expression of novel genes in reproduction.

    Science.gov (United States)

    Markholt, S; Grøndahl, M L; Ernst, E H; Andersen, C Yding; Ernst, E; Lykke-Hartmann, K

    2012-02-01

    The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis. The array data were confirmed by qPCR for selected genes. A total of 6301 unique genes were identified as significantly expressed representing enriched specific functional categories such as 'RNA binding', 'translation initiation' and 'structural molecule activity'. Several genes, some not previously known to be associated with early oocyte development, were identified with exceptionally high expression levels, such as the anti-proliferative transmembrane protein with an epidermal growth factor-like and two follistatin-like domains (TMEFF2), the Rho-GTPase-activating protein oligophrenin 1 (OPHN1) and the mitochondrial-encoded ATPase6 (ATP6). Thus, the present study provides not only a technique to capture and perform transcriptome analysis of the sparse material of human oocytes from the earliest follicle stages but further includes a comprehensive basis for our understanding of the regulatory factors and pathways present during early human folliculogenesis.

  13. Suppression subtractive hybridization reveals differential gene expression in sunflower grown in high P.

    Science.gov (United States)

    Padmanabhan, Priya; Sahi, Shivendra V

    2011-06-01

    Sunflower (Helianthus annuus L.) is a commercially important oilseed crop. Previous studies proved that this crop is a promising plant species for phytoextraction of excess soil phosphorus (P) because of its superior P accumulating characteristics. Suppression subtractive hybridization (SSH) strategy was employed to isolate and characterize genes that are induced in response to high P in this crop. SSH library was prepared using cDNA generated from plants treated with high P as the 'tester'. Based on the results of dot blot analysis, 360 positive cDNA clones were selected from the SSH library for sequencing. A total of 89 non-redundant expressed sequence tags (ESTs) were identified as high P-responsive genes and they were classified into 6 functional groups. Several genes involved in metabolism showed markedly preferential expression in the library. For further confirmation, thirteen of the representative ESTs were selected from all categories for RT-PCR analysis and the results showed up-regulation of these genes in response to high P-treatment. The gene expression data derived from this study suggested that several of the up-regulated genes identified under high P-treatment might be involved in P-accumulation and tolerance in this plant.

  14. High Prevalence and Clinical Relevance of Genes Affected by Chromosomal Breaks in Colorectal Cancer.

    Directory of Open Access Journals (Sweden)

    Evert van den Broek

    Full Text Available Cancer is caused by somatic DNA alterations such as gene point mutations, DNA copy number aberrations (CNA and structural variants (SVs. Genome-wide analyses of SVs in large sample series with well-documented clinical information are still scarce. Consequently, the impact of SVs on carcinogenesis and patient outcome remains poorly understood. This study aimed to perform a systematic analysis of genes that are affected by CNA-associated chromosomal breaks in colorectal cancer (CRC and to determine the clinical relevance of recurrent breakpoint genes.Primary CRC samples of patients with metastatic disease from CAIRO and CAIRO2 clinical trials were previously characterized by array-comparative genomic hybridization. These data were now used to determine the prevalence of CNA-associated chromosomal breaks within genes across 352 CRC samples. In addition, mutation status of the commonly affected APC, TP53, KRAS, PIK3CA, FBXW7, SMAD4, BRAF and NRAS genes was determined for 204 CRC samples by targeted massive parallel sequencing. Clinical relevance was assessed upon stratification of patients based on gene mutations and gene breakpoints that were observed in >3% of CRC cases.In total, 748 genes were identified that were recurrently affected by chromosomal breaks (FDR 3% of cases, indicating that prevalence of gene breakpoints is comparable to the prevalence of well-known gene point mutations. Patient stratification based on gene breakpoints and point mutations revealed one CRC subtype with very poor prognosis.We conclude that CNA-associated chromosomal breaks within genes represent a highly prevalent and clinically relevant subset of SVs in CRC.

  15. High-throughput gene expression profiling of memory differentiation in primary human T cells

    Directory of Open Access Journals (Sweden)

    Russell Kate

    2008-08-01

    Full Text Available Abstract Background The differentiation of naive T and B cells into memory lymphocytes is essential for immunity to pathogens. Therapeutic manipulation of this cellular differentiation program could improve vaccine efficacy and the in vitro expansion of memory cells. However, chemical screens to identify compounds that induce memory differentiation have been limited by 1 the lack of reporter-gene or functional assays that can distinguish naive and memory-phenotype T cells at high throughput and 2 a suitable cell-line representative of naive T cells. Results Here, we describe a method for gene-expression based screening that allows primary naive and memory-phenotype lymphocytes to be discriminated based on complex genes signatures corresponding to these differentiation states. We used ligation-mediated amplification and a fluorescent, bead-based detection system to quantify simultaneously 55 transcripts representing naive and memory-phenotype signatures in purified populations of human T cells. The use of a multi-gene panel allowed better resolution than any constituent single gene. The method was precise, correlated well with Affymetrix microarray data, and could be easily scaled up for high-throughput. Conclusion This method provides a generic solution for high-throughput differentiation screens in primary human T cells where no single-gene or functional assay is available. This screening platform will allow the identification of small molecules, genes or soluble factors that direct memory differentiation in naive human lymphocytes.

  16. A high-throughput gene disruption methodology for the entomopathogenic fungus Metarhizium robertsii.

    Science.gov (United States)

    Xu, Chuan; Zhang, Xing; Qian, Ying; Chen, Xiaoxuan; Liu, Ran; Zeng, Guohong; Zhao, Hong; Fang, Weiguo

    2014-01-01

    Systematic gene disruption is a direct way to interrogate a fungal genome to functionally characterize the full suite of genes involved in various biological processes. Metarhizium robertsii is extraordinarily versatile, and it is a pathogen of arthropods, a saprophyte and a beneficial colonizer of rhizospheres. Thus, M. robertsii can be used as a representative to simultaneously study several major lifestyles that are not shared by the "model" fungi Saccharomyces cerevisiae and Neurospora crassa; a systematic genetic analysis of M. robertsii will benefit studies in other fungi. In order to systematically disrupt genes in M. robertsii, we developed a high-throughput gene disruption methodology, which includes two technologies. One is the modified OSCAR-based, high-throughput construction of gene disruption plasmids. This technology involves two donor plasmids (pA-Bar-OSCAR with the herbicide resistance genes Bar and pA-Sur-OSCAR with another herbicide resistance gene Sur) and a recipient binary plasmid pPK2-OSCAR-GFP that was produced by replacing the Bar cassette in pPK2-bar-GFP with a ccdB cassette and recombination recognition sites. Using this technology, a gene disruption plasmid can be constructed in one cloning step in two days. The other is a highly efficient gene disruption technology based on homologous recombination using a Ku70 deletion mutant (ΔMrKu70) as the recipient strain. The deletion of MrKu70, a gene encoding a key component involved in nonhomologous end-joining DNA repair in fungi, dramatically increases the gene disruption efficiency. The frequency of disrupting the conidiation-associated gene Cag8 in ΔMrKu70 was 93% compared to 7% in the wild-type strain. Since ΔMrKu70 is not different from the wild-type strain in development, pathogenicity and tolerance to various abiotic stresses, it can be used as a recipient strain for a systematic gene disruption project to characterize the whole suite of genes involved in the biological processes of

  17. A high-throughput gene disruption methodology for the entomopathogenic fungus Metarhizium robertsii.

    Directory of Open Access Journals (Sweden)

    Chuan Xu

    Full Text Available Systematic gene disruption is a direct way to interrogate a fungal genome to functionally characterize the full suite of genes involved in various biological processes. Metarhizium robertsii is extraordinarily versatile, and it is a pathogen of arthropods, a saprophyte and a beneficial colonizer of rhizospheres. Thus, M. robertsii can be used as a representative to simultaneously study several major lifestyles that are not shared by the "model" fungi Saccharomyces cerevisiae and Neurospora crassa; a systematic genetic analysis of M. robertsii will benefit studies in other fungi. In order to systematically disrupt genes in M. robertsii, we developed a high-throughput gene disruption methodology, which includes two technologies. One is the modified OSCAR-based, high-throughput construction of gene disruption plasmids. This technology involves two donor plasmids (pA-Bar-OSCAR with the herbicide resistance genes Bar and pA-Sur-OSCAR with another herbicide resistance gene Sur and a recipient binary plasmid pPK2-OSCAR-GFP that was produced by replacing the Bar cassette in pPK2-bar-GFP with a ccdB cassette and recombination recognition sites. Using this technology, a gene disruption plasmid can be constructed in one cloning step in two days. The other is a highly efficient gene disruption technology based on homologous recombination using a Ku70 deletion mutant (ΔMrKu70 as the recipient strain. The deletion of MrKu70, a gene encoding a key component involved in nonhomologous end-joining DNA repair in fungi, dramatically increases the gene disruption efficiency. The frequency of disrupting the conidiation-associated gene Cag8 in ΔMrKu70 was 93% compared to 7% in the wild-type strain. Since ΔMrKu70 is not different from the wild-type strain in development, pathogenicity and tolerance to various abiotic stresses, it can be used as a recipient strain for a systematic gene disruption project to characterize the whole suite of genes involved in the

  18. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Jared Carlson-Stevermer

    2016-01-01

    Full Text Available CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.

  19. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells.

    Science.gov (United States)

    Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

    2016-01-12

    CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.

  20. RELATED GENES IN LUNG CANCER TISSUES ASSOCIATED WITH RESIDENTIAL HIGH RADON EXPOSURE

    Institute of Scientific and Technical Information of China (English)

    夏英; 杨梅英; 张守志; 叶常青

    2002-01-01

    Objective: To investigate the related genes in lung cancer tissues associated with residential high radon exposure. Methods: Differentially expressed gene fragments in lung cancer and normal lung tissues were discovered by differential display and reverse Northern blot hybridization method. The fragments positive in lung cancer and negative in normal lung tissue were determined. Results: Seven differential displayed fragments were sequenced. One of them named NA7 is 95% homologous with AI208667 in EAT of Genbank. Another fragment named NG2 is up to 98% homologous with five fragments. The remained one CA1 may be a new gene fragment. Conclusion: 3 gene fragments were discovered from lung cancer and normal lung tissues of high radon exposure resident.

  1. Pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements.

    Directory of Open Access Journals (Sweden)

    Erik Kristiansson

    Full Text Available The high and sometimes inappropriate use of antibiotics has accelerated the development of antibiotic resistance, creating a major challenge for the sustainable treatment of infections world-wide. Bacterial communities often respond to antibiotic selection pressure by acquiring resistance genes, i.e. mobile genetic elements that can be shared horizontally between species. Environmental microbial communities maintain diverse collections of resistance genes, which can be mobilized into pathogenic bacteria. Recently, exceptional environmental releases of antibiotics have been documented, but the effects on the promotion of resistance genes and the potential for horizontal gene transfer have yet received limited attention. In this study, we have used culture-independent shotgun metagenomics to investigate microbial communities in river sediments exposed to waste water from the production of antibiotics in India. Our analysis identified very high levels of several classes of resistance genes as well as elements for horizontal gene transfer, including integrons, transposons and plasmids. In addition, two abundant previously uncharacterized resistance plasmids were identified. The results suggest that antibiotic contamination plays a role in the promotion of resistance genes and their mobilization from environmental microbes to other species and eventually to human pathogens. The entire life-cycle of antibiotic substances, both before, under and after usage, should therefore be considered to fully evaluate their role in the promotion of resistance.

  2. High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism.

    Directory of Open Access Journals (Sweden)

    Jennifer E Griffin

    2011-09-01

    Full Text Available The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagenesis and deep-sequencing was used to characterize the composition of complex mutant libraries exposed to different conditions. This allowed the unambiguous identification of the genes that are essential for Mtb to grow in vitro, and proved to be a significant improvement over previous approaches. To further explore functions that are required for persistence in the host, we defined the pathways necessary for the utilization of cholesterol, a critical carbon source during infection. Few of the genes we identified had previously been implicated in this adaptation by transcriptional profiling, and only a fraction were encoded in the chromosomal region known to encode sterol catabolic functions. These genes comprise an unexpectedly large percentage of those previously shown to be required for bacterial growth in mouse tissue. Thus, this single nutritional change accounts for a significant fraction of the adaption to the host. This work provides the most comprehensive genetic characterization of a sterol catabolic pathway to date, suggests putative roles for uncharacterized virulence genes, and precisely maps genes encoding potential drug targets.

  3. High-throughput quantification of antibiotic resistance genes from an urban wastewater treatment plant.

    Science.gov (United States)

    Karkman, Antti; Johnson, Timothy A; Lyra, Christina; Stedtfeld, Robert D; Tamminen, Manu; Tiedje, James M; Virta, Marko

    2016-03-01

    Antibiotic resistance among bacteria is a growing problem worldwide, and wastewater treatment plants have been considered as one of the major contributors to the dissemination of antibiotic resistance to the environment. There is a lack of comprehensive quantitative molecular data on extensive numbers of antibiotic resistance genes (ARGs) in different seasons with a sampling strategy that would cover both incoming and outgoing water together with the excess sludge that is removed from the process. In order to fill that gap we present a highly parallel quantitative analysis of ARGs and horizontal gene transfer potential over four seasons at an urban wastewater treatment plant using a high-throughput qPCR array. All analysed transposases and two-thirds of primer sets targeting ARGs were detected in the wastewater. The relative abundance of most of the genes was highest in influent and lower in effluent water and sludge. The resistance profiles of the samples cluster by sample location with a shift from raw influent through the final effluents and dried sludge to the sediments. Wastewater discharge enriched only a few genes, namely Tn25 type transposase gene and clinical class 1 integrons, in the sediment near the discharge pipe, but those enriched genes may indicate a potential for horizontal gene transfer.

  4. High throughput functional genomics: identification of novel genes with tumor suppressor phenotypes.

    Science.gov (United States)

    Koenig-Hoffmann, Kerstin; Bonin-Debs, Angelika L; Boche, Irene; Gawin, Beate; Gnirke, Andrea; Hergersberg, Christoph; Madeo, Frank; Kazinski, Michael; Klein, Matthias; Korherr, Christian; Link, Dieter; Röhrig, Sascha; Schäfer, Rolf; Brinkmann, Ulrich

    2005-01-20

    We have used a combination of high throughput functional genomics, computerized database mining and expression analyses to discover novel human tumor suppressor genes (TSGs). A genome-wide high throughput cDNA phenotype screen was established to identify genes that induce apoptosis or reduce cell viability. TSGs are expressed in normal tissue and frequently act by reduction of growth of transformed cells or induce apoptosis. In agreement with that and thus serving as platform validation, our pro-apoptotic hits included genes for which tumor suppressing activities were known, such as kangai1 and CD81 antigen. Additional genes that so far have been claimed as putative TSGs or associated with tumor inhibitory activities (prostate differentiation factor, hRAS-like suppressor 3, DPH2L1-like and the metastasis inhibitor Kiss1) were confirmed in their proposed TSG-like phenotype by functionally defining their growth inhibitory or pro-apoptotic function towards cancer cells. Finally, novel genes were identified for which neither association with cell growth nor with apoptosis were previously described. A subset of these genes show characteristics of TSGs because they (i) reduce the growth or induce apoptosis in tumor cells; (ii) show reduced expression in tumor vs. normal tissue; and (iii) are located on chromosomal (LOH-) loci for which cancer-associated deletions are described. The pro-apoptotic phenotype and differential expression of these genes in normal and malignant tissue make them promising target candidates for the diagnosis and therapy of various tumors.

  5. Gene therapy trials for the treatment of high-grade gliomas

    OpenAIRE

    2007-01-01

    High-grade gliomas remain relatively resistant to current therapy. Local recurrence is a common feature and the majority of patients progress despite conventional therapy. One modality-gene therapy-has shown a lot of promise in early preclinical and clinical studies aimed at advancing the treatment of this disease. In this review, we provide a comprehensive overview of clinical trials involving gene therapy in the field of neuro-oncology. The use of different delivery vehicles, including lipo...

  6. Comparative analysis of codon usage patterns and identification of predicted highly expressed genes in five Salmonella genomes

    Directory of Open Access Journals (Sweden)

    Mondal U

    2008-01-01

    Full Text Available Purpose: To anlyse codon usage patterns of five complete genomes of Salmonella , predict highly expressed genes, examine horizontally transferred pathogenicity-related genes to detect their presence in the strains, and scrutinize the nature of highly expressed genes to infer upon their lifestyle. Methods: Protein coding genes, ribosomal protein genes, and pathogenicity-related genes were analysed with Codon W and CAI (codon adaptation index Calculator. Results: Translational efficiency plays a role in codon usage variation in Salmonella genes. Low bias was noticed in most of the genes. GC3 (guanine cytosine at third position composition does not influence codon usage variation in the genes of these Salmonella strains. Among the cluster of orthologous groups (COGs, translation, ribosomal structure biogenesis [J], and energy production and conversion [C] contained the highest number of potentially highly expressed (PHX genes. Correspondence analysis reveals the conserved nature of the genes. Highly expressed genes were detected. Conclusions: Selection for translational efficiency is the major source of variation of codon usage in the genes of Salmonella . Evolution of pathogenicity-related genes as a unit suggests their ability to infect and exist as a pathogen. Presence of a lot of PHX genes in the information and storage-processing category of COGs indicated their lifestyle and revealed that they were not subjected to genome reduction.

  7. Hypoxia: adapting to high altitude by mutating EPAS-1, the gene encoding HIF-2α.

    Science.gov (United States)

    van Patot, Martha C Tissot; Gassmann, Max

    2011-01-01

    Living at high altitude is demanding and thus drives adaptational mechanisms. The Tibetan population has had a longer evolutionary period to adapt to high altitude than other mountain populations such as Andeans. As a result, some Tibetans living at high altitudes do not show markedly elevated red blood cell production as compared to South American high altitude natives such as Quechuas or Aymaras, thereby avoiding high blood viscosity creating cardiovascular risk. Unexpectedly, the responsible mutation(s) reducing red blood cell production do not involve either the gene encoding the blood hormone erythropoietin (Epo), or the corresponding regulatory sequences flanking the Epo gene. Similarly, functional mutations in the hypoxia-inducible transcription factor 1α (HIF-1α) gene that represents the oxygen-dependent subunit of the HIF-1 heterodimer, the latter being the main regulator of over 100 hypoxia-inducible genes, have not been described so far. It was not until very recently that three independent groups showed that the gene encoding HIF-2α, EPAS-1 (Wenger et al. 1997), represents a key gene mutated in Tibetan populations adapted to living at high altitudes (Beall et al. 2010 , Yi et al. 2010 , Simonson et al. 2010). Hypoxia-inducible transcription factors were first identified by the description of HIF-1 (Semenza et al. 1991 , 1992), which was subsequently found to enhance transcription of multiple genes that encode proteins necessary for rescuing from hypoxic exposure, including erythropoietic, angiogenic and glycolytic proteins. Then HIF-2 was identified (Ema et al. 1997 ; Flamme et al. 1997 ; Hogenesch et al. 1997 ; and Tian et al. 1997) and although it is highly similar to HIF-1 and has the potential to bind (Camenisch et al. 2001) and mediate (Mole et al. 2009) many of the same genes as HIF-1, its biological actions in response to hypoxia are distinct from those of HIF-1 (reviewed by Loboda et al. 2010). By now, several of these HIF-2 mediated

  8. Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells.

    Science.gov (United States)

    Page, Andrew F; Minocha, Rakesh; Minocha, Subhash C

    2012-01-01

    Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra × maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N (2)-acetylOrn:N-acetyl-Glu acetyltransferase, N (2)-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the

  9. Highly compacted biodegradable DNA nanoparticles capable of overcoming the mucus barrier for inhaled lung gene therapy.

    Science.gov (United States)

    Mastorakos, Panagiotis; da Silva, Adriana L; Chisholm, Jane; Song, Eric; Choi, Won Kyu; Boyle, Michael P; Morales, Marcelo M; Hanes, Justin; Suk, Jung Soo

    2015-07-14

    Gene therapy has emerged as an alternative for the treatment of diseases refractory to conventional therapeutics. Synthetic nanoparticle-based gene delivery systems offer highly tunable platforms for the delivery of therapeutic genes. However, the inability to achieve sustained, high-level transgene expression in vivo presents a significant hurdle. The respiratory system, although readily accessible, remains a challenging target, as effective gene therapy mandates colloidal stability in physiological fluids and the ability to overcome biological barriers found in the lung. We formulated highly stable DNA nanoparticles based on state-of-the-art biodegradable polymers, poly(β-amino esters) (PBAEs), possessing a dense corona of polyethylene glycol. We found that these nanoparticles efficiently penetrated the nanoporous and highly adhesive human mucus gel layer that constitutes a primary barrier to reaching the underlying epithelium. We also discovered that these PBAE-based mucus-penetrating DNA nanoparticles (PBAE-MPPs) provided uniform and high-level transgene expression throughout the mouse lungs, superior to several gold standard gene delivery systems. PBAE-MPPs achieved robust transgene expression over at least 4 mo following a single administration, and their transfection efficiency was not attenuated by repeated administrations, underscoring their clinical relevance. Importantly, PBAE-MPPs demonstrated a favorable safety profile with no signs of toxicity following intratracheal administration.

  10. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth

    DEFF Research Database (Denmark)

    Jakociune, Dziuginta; Herrero-Fresno, Ana; Jelsbak, Lotte;

    2016-01-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality...

  11. Currently recognized genes for schizophrenia: High-resolution chromosome ideogram representation.

    Science.gov (United States)

    Butler, Merlin G; McGuire, Austen B; Masoud, Humaira; Manzardo, Ann M

    2016-03-01

    A large body of genetic data from schizophrenia-related research has identified an assortment of genes and disturbed pathways supporting involvement of complex genetic components for schizophrenia spectrum and other psychotic disorders. Advances in genetic technology and expanding studies with searchable genomic databases have led to multiple published reports, allowing us to compile a master list of known, clinically relevant, or susceptibility genes contributing to schizophrenia. We searched key words related to schizophrenia and genetics from peer-reviewed medical literature sources, authoritative public access psychiatric websites and genomic databases dedicated to gene discovery and characterization of schizophrenia. Our list of 560 genes were arranged in alphabetical order in tabular form with gene symbols placed on high-resolution human chromosome ideograms. Genome wide pathway analysis using GeneAnalytics was carried out on the resulting list of genes to assess the underlying genetic architecture for schizophrenia. Recognized genes of clinical relevance, susceptibility or causation impact a broad range of biological pathways and mechanisms including ion channels (e.g., CACNA1B, CACNA1C, CACNA1H), metabolism (e.g., CYP1A2, CYP2C19, CYP2D6), multiple targets of neurotransmitter pathways impacting dopamine, GABA, glutamate, and serotonin function, brain development (e.g., NRG1, RELN), signaling peptides (e.g., PIK3CA, PIK4CA) and immune function (e.g., HLA-DRB1, HLA-DQA1) and interleukins (e.g., IL1A, IL10, IL6). This summary will enable clinical and laboratory geneticists, genetic counselors, and other clinicians to access convenient pictorial images of the distribution and location of contributing genes to inform diagnosis and gene-based treatment as well as provide risk estimates for genetic counseling of families with affected relatives.

  12. Assembly of Highly Standardized Gene Fragments for High-Level Production of Porphyrins in E. coli

    DEFF Research Database (Denmark)

    Nielsen, Morten Thrane; Madsen, Karina Marie; Seppala, Susanna;

    2015-01-01

    to formulate a molecular cloning pipeline and iteratively assemble and optimize a six-gene pathway for protoporphyrin IX synthesis in Escherichia coli. State of the art production levels were achieved through two simple cycles of engineering and screening. The principles defined here are generally applicable...

  13. Supramolecular Aggregate as a High-Efficiency Gene Carrier Mediated with Optimized Assembly Structure.

    Science.gov (United States)

    Zhang, Yi; Duan, Junkun; Cai, Lingguang; Ma, Dong; Xue, Wei

    2016-11-02

    For cancer gene therapy, a safe and high-efficient gene carrier is a must. To resolve the contradiction between gene transfection efficiency and cytotoxicity, many polymers with complex topological structures have been synthesized, although their synthesis processes and structure control are difficult as well as the high molecular weight also bring high cytotoxicity. We proposed an alternative strategy that uses supramolecular inclusion to construct the aggregate from the small molecules for gene delivery, and to further explore the relationship between the topological assembly structure and their ability to deliver gene. Herein, PEI-1.8k-conjugating β-CD through 6-hydroxyl (PEI-6-CD) and 2-hydroxyl (PEI-2-CD) have been synthesized respectively and then assembled with diferrocene (Fc)-ended polyethylene glycol (PEG-Fc). The obtained aggregates were then used to deliver MMP-9 shRNA plasmid for MCF-7 cancer therapy. It was found that the higher gene transfection efficiency can be obtained by selecting PEI-2-CD as the host and tuning the host/guest molar ratios. With the rational modulation of supramolecular architectures, the aggregate played the functions similar to macromolecules which exhibit higher transfection efficiency than PEI-25k, but show much lower cytotoxicity because of the nature of small/low molecules. In vitro and in vivo assays confirmed that the aggregate could deliver MMP-9 shRNA plasmid effectively into MCF-7 cells and then downregulate MMP-9 expression, which induced the significant MCF-7 cell apoptosis, as well inhibit MCF-7 tumor growth with low toxicity. The supramolecular aggregates maybe become a promising carrier for cancer gene therapy and also provided an alternative strategy for designing new gene carriers.

  14. Parallel Gene Expression Differences between Low and High Latitude Populations of Drosophila melanogaster and D. simulans.

    Science.gov (United States)

    Zhao, Li; Wit, Janneke; Svetec, Nicolas; Begun, David J

    2015-05-01

    Gene expression variation within species is relatively common, however, the role of natural selection in the maintenance of this variation is poorly understood. Here we investigate low and high latitude populations of Drosophila melanogaster and its sister species, D. simulans, to determine whether the two species show similar patterns of population differentiation, consistent with a role for spatially varying selection in maintaining gene expression variation. We compared at two temperatures the whole male transcriptome of D. melanogaster and D. simulans sampled from Panama City (Panama) and Maine (USA). We observed a significant excess of genes exhibiting differential expression in both species, consistent with parallel adaptation to heterogeneous environments. Moreover, the majority of genes showing parallel expression differentiation showed the same direction of differential expression in the two species and the magnitudes of expression differences between high and low latitude populations were correlated across species, further bolstering the conclusion that parallelism for expression phenotypes results from spatially varying selection. However, the species also exhibited important differences in expression phenotypes. For example, the genomic extent of genotype × environment interaction was much more common in D. melanogaster. Highly differentiated SNPs between low and high latitudes were enriched in the 3' UTRs and CDS of the geographically differently expressed genes in both species, consistent with an important role for cis-acting variants in driving local adaptation for expression-related phenotypes.

  15. Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco.

    Science.gov (United States)

    Ye, G N; Hajdukiewicz, P T; Broyles, D; Rodriguez, D; Xu, C W; Nehra, N; Staub, J M

    2001-02-01

    Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.

  16. Regularized logistic regression with adjusted adaptive elastic net for gene selection in high dimensional cancer classification.

    Science.gov (United States)

    Algamal, Zakariya Yahya; Lee, Muhammad Hisyam

    2015-12-01

    Cancer classification and gene selection in high-dimensional data have been popular research topics in genetics and molecular biology. Recently, adaptive regularized logistic regression using the elastic net regularization, which is called the adaptive elastic net, has been successfully applied in high-dimensional cancer classification to tackle both estimating the gene coefficients and performing gene selection simultaneously. The adaptive elastic net originally used elastic net estimates as the initial weight, however, using this weight may not be preferable for certain reasons: First, the elastic net estimator is biased in selecting genes. Second, it does not perform well when the pairwise correlations between variables are not high. Adjusted adaptive regularized logistic regression (AAElastic) is proposed to address these issues and encourage grouping effects simultaneously. The real data results indicate that AAElastic is significantly consistent in selecting genes compared to the other three competitor regularization methods. Additionally, the classification performance of AAElastic is comparable to the adaptive elastic net and better than other regularization methods. Thus, we can conclude that AAElastic is a reliable adaptive regularized logistic regression method in the field of high-dimensional cancer classification.

  17. High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos

    Directory of Open Access Journals (Sweden)

    Ken-ichi T. Suzuki

    2013-03-01

    Recently, gene editing with transcription activator-like effector nucleases (TALENs has been used in the life sciences. TALENs can be easily customized to recognize a specific DNA sequence and efficiently introduce double-strand breaks at the targeted genomic locus. Subsequent non-homologous end-joining repair leads to targeted gene disruption by base insertion, deletion, or both. Here, to readily evaluate the efficacy of TALENs in Xenopus laevis embryos, we performed the targeted gene disruption of tyrosinase (tyr and pax6 genes that are involved in pigmentation and eye formation, respectively. We constructed TALENs targeting tyr and pax6 and injected their mRNAs into fertilized eggs at the one-cell stage. Expectedly, introduction of tyr TALEN mRNA resulted in drastic loss of pigmentation with high efficiency. Similarly, for pax6, TALENs led to deformed eyes in the injected embryos. We confirmed mutations of the target alleles by restriction enzyme digestion and sequence analyses of genomic PCR products. Surprisingly, not only biallelic but also paralogous, gene disruption was observed. Our results demonstrate that targeted gene disruption by TALENs provides a method comparable to antisense morpholinos in analyzing gene function in Xenopus F0 embryos, but also applies beyond embryogenesis to any life stage.

  18. Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity

    Science.gov (United States)

    Corcoran, Martin M.; Phad, Ganesh E.; Bernat, Néstor Vázquez; Stahl-Hennig, Christiane; Sumida, Noriyuki; Persson, Mats A. A.; Martin, Marcel; Hedestam, Gunilla B. Karlsson

    2016-12-01

    Comprehensive knowledge of immunoglobulin genetics is required to advance our understanding of B cell biology. Validated immunoglobulin variable (V) gene databases are close to completion only for human and mouse. We present a novel computational approach, IgDiscover, that identifies germline V genes from expressed repertoires to a specificity of 100%. IgDiscover uses a cluster identification process to produce candidate sequences that, once filtered, results in individualized germline V gene databases. IgDiscover was tested in multiple species, validated by genomic cloning and cross library comparisons and produces comprehensive gene databases even where limited genomic sequence is available. IgDiscover analysis of the allelic content of the Indian and Chinese-origin rhesus macaques reveals high levels of immunoglobulin gene diversity in this species. Further, we describe a novel human IGHV3-21 allele and confirm significant gene differences between Balb/c and C57BL6 mouse strains, demonstrating the power of IgDiscover as a germline V gene discovery tool.

  19. Highly and moderately aggressive mouse ovarian cancer cell lines exhibit differential gene expression

    Science.gov (United States)

    Zhang, Wensheng; Kale, Shubha P.; McFerrin, Harris; Davenport, Ian; Wang, Guangdi; Skripnikova, Elena; Li, Xiao-Lin; Bowen, Nathan J.; McDaniels, Leticia B; Meng, Yuan-Xiang; Polk, Paula; Liu, Yong-Yu; Zhang, Qian-Jin

    2017-01-01

    Patients with advanced epithelial ovarian cancer often experience disease recurrence after standard therapies, a critical factor in determining their five-year survival rate. Recent reports indicated that long-term or short-term survival is associated with varied gene expression of cancer cells. Thus, identification of novel prognostic biomarkers should be considered. Since the mouse genome is similar to the human genome, we explored potential prognostic biomarkers using two groups of mouse ovarian cancer cell lines (group 1: IG-10, IG-10pw, and IG-10pw/agar; group 2: IG-10 clones 2, 3, and 11) which display highly and moderately aggressive phenotypes in vivo. Mice injected with these cell lines have different survival time and rates, capacities of tumor, and ascites formations, reflecting different prognostic potentials. Using an Affymetrix Mouse Genome 430 2.0 Array, a total of 181 genes were differentially expressed (Pdeath. None of the genes from a set of the 72 genes overexpressed in the moderately aggressive cell lines had a function annotation in the David database. Our results suggested that the overexpressed MYC and 109 gene set represented highly aggressive ovarian cancer potential biomarkers while overexpressed AR and 72 gene set represented moderately aggressive ovarian cancer potential biomarkers. Based on our knowledge, the current study is first time to report the potential biomarkers relevant to different aggressive ovarian cancer. These potential biomarkers provide important information for investigating human ovarian cancer prognosis. PMID:26935058

  20. Human cytomegalovirus UL145 gene is highly conserved among clinical strains

    Indian Academy of Sciences (India)

    Zhengrong Sun; Ying Lu; Qiang Ruan; Yaohua Ji; Rong He; Ying Qi; Yanping Ma; Yujing Huang

    2007-09-01

    Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects in newborns. A region (referred to as UL/b′) present in the Toledo strain of HCMV and low-passage clinical isolates) contains 22 additional genes, which are absent in the highly passaged laboratory strain AD169. One of these genes, UL145 open reading frame (ORF), is located between the highly variable genes UL144 and UL146. To assess the structure of the UL145 gene, the UL145 ORF was amplified by PCR and sequenced from 16 low-passage clinical isolates and 15 non-passage strains from suspected congenitally infected infants. Nine UL145 sequences previously published in the GenBank were used for sequence comparison. The identities of the gene and the similarities of its putative protein among all strains were 95.9–100% and 96.6–100%, respectively. The post-translational modification motifs of the UL145 putative protein in clinical strains were conserved, comprising the protein kinase C phosphorylation motif (PKC) and casein kinase II phosphorylation site (CK-II). We conclude that the structure of the UL145 gene and its putative protein are relatively conserved among clinical strains, irrespective of whether the strains come from patients with different manifestations, from different areas of the world, or were passaged or not in human embryonic lung fibroblast (HELF) cells.

  1. Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression.

    Directory of Open Access Journals (Sweden)

    Adam J Book

    2016-06-01

    Full Text Available The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.

  2. Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression.

    Science.gov (United States)

    Book, Adam J; Lewin, Gina R; McDonald, Bradon R; Takasuka, Taichi E; Wendt-Pienkowski, Evelyn; Doering, Drew T; Suh, Steven; Raffa, Kenneth F; Fox, Brian G; Currie, Cameron R

    2016-06-01

    The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.

  3. High-resolution statistical mapping reveals gene territories in live yeast.

    Science.gov (United States)

    Berger, Axel B; Cabal, Ghislain G; Fabre, Emmanuelle; Duong, Tarn; Buc, Henri; Nehrbass, Ulf; Olivo-Marin, Jean-Christophe; Gadal, Olivier; Zimmer, Christophe

    2008-12-01

    The nonrandom positioning of genes inside eukaryotic cell nuclei is implicated in central nuclear functions. However, the spatial organization of the genome remains largely uncharted, owing to limited resolution of optical microscopy, paucity of nuclear landmarks and moderate cell sampling. We developed a computational imaging approach that creates high-resolution probabilistic maps of subnuclear domains occupied by individual loci in budding yeast through automated analysis of thousands of living cells. After validation, we applied the technique to genes involved in galactose metabolism and ribosome biogenesis. We found that genomic loci are confined to 'gene territories' much smaller than the nucleus, which can be remodeled during transcriptional activation, and that the nucleolus is an important landmark for gene positioning. The technique can be used to visualize and quantify territory positions relative to each other and to nuclear landmarks, and should advance studies of nuclear architecture and function.

  4. High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana

    DEFF Research Database (Denmark)

    Bach, Søren Spanner; Bassard, Jean-Étienne André; Andersen-Ranberg, Johan;

    2014-01-01

    To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized...... describe an expression platform for rapid testing of candidate terpenoid biosynthetic genes based on Agrobacterium mediated gene expression in N. benthamiana leaves. Simultaneous expression of multiple genes is facilitated by co-infiltration of leaves with several engineered Agrobacterium strains, possibly...... making this the fastest and most convenient system for the assembly of plant terpenoid biosynthetic routes. Tools for cloning of expression plasmids, N. benthamiana culturing, Agrobacterium preparation, leaf infiltration, metabolite extraction, and automated GC-MS data mining are provided. With all steps...

  5. Highly expressed genes within hippocampal sector CA1: implications for the physiology of memory

    Directory of Open Access Journals (Sweden)

    Michael A. Meyer

    2014-06-01

    Full Text Available As the CA1 sector has been implicated to play a key role in memory formation, a dedicated search for highly expressed genes within this region was made from an on-line atlas of gene expression within the mouse brain (GENSAT. From a data base of 1013 genes, 16 were identified that had selective localization of gene expression within the CA1 region, and included Angpt2, ARHGEF6, CCK, Cntnap1, DRD3, EMP1, Epha2, Itm2b, Lrrtm2, Mdk, PNMT, Ppm1e, Ppp2r2d, RASGRP1, Slitrk5, and Sstr4. Of the 16 identified, the most selective and intense localization for both adult and post-natal day 7 was noted for ARHGEF6, which is known to be linked to non-syndromic mental retardation, and has also been localized to dendritic spines. Further research on the role played by ARHGEF6 in memory formation is strongly advocated.

  6. Plant protein kinase genes induced by drought, high salt and cold stresses

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Drought, high salt and cold are three different kinds of environment stresses that severely influence the growth, development and productivity of crops. They all decrease the water state of plant cells, and consequently result in the harm of plant from water deficit. Several genes encoding protein kinases and induced by drought, high salt and low temperature have been isolated from Arabidopsis. These protein kinases include receptor protein kinase (RPK), MAP kinases, ribosomal-protein kinases and transcription-regulation protein kinase. The expression features of these genes and the regulatory roles of these protein kinases in stress response and signal transduction are discussed.

  7. Transfer RNA gene numbers may not be completely responsible for the codon usage bias in asparagine, isoleucine, phenylalanine, and tyrosine in the high expression genes in bacteria.

    Science.gov (United States)

    Satapathy, Siddhartha Sankar; Dutta, Malay; Buragohain, Alak Kumar; Ray, Suvendra Kumar

    2012-08-01

    It is generally believed that the effect of translational selection on codon usage bias is related to the number of transfer RNA genes in bacteria, which is more with respect to the high expression genes than the whole genome. Keeping this in the background, we analyzed codon usage bias with respect to asparagine, isoleucine, phenylalanine, and tyrosine amino acids. Analysis was done in seventeen bacteria with the available gene expression data and information about the tRNA gene number. In most of the bacteria, it was observed that codon usage bias and tRNA gene number were not in agreement, which was unexpected. We extended the study further to 199 bacteria, limiting to the codon usage bias in the two highly expressed genes rpoB and rpoC which encode the RNA polymerase subunits β and β', respectively. In concordance with the result in the high expression genes, codon usage bias in rpoB and rpoC genes was also found to not be in agreement with tRNA gene number in many of these bacteria. Our study indicates that tRNA gene numbers may not be the sole determining factor for translational selection of codon usage bias in bacterial genomes.

  8. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  9. Cyclen-based cationic lipids for highly efficient gene delivery towards tumor cells.

    Directory of Open Access Journals (Sweden)

    Qing-Dong Huang

    Full Text Available BACKGROUND: Gene therapy has tremendous potential for both inherited and acquired diseases. However, delivery problems limited their clinical application, and new gene delivery vehicles with low cytotoxicity and high transfection efficiency are greatly required. METHODS: In this report, we designed and synthesized three amphiphilic molecules (L1-L3 with the structures involving 1, 4, 7, 10-tetraazacyclododecane (cyclen, imidazolium and a hydrophobic dodecyl chain. Their interactions with plasmid DNA were studied via electrophoretic gel retardation assays, fluorescent quenching experiments, dynamic light scattering and transmission electron microscopy. The in vitro gene transfection assay and cytotoxicity assay were conducted in four cell lines. RESULTS: Results indicated that L1 and L3-formed liposomes could effectively bind to DNA to form well-shaped nanoparticles. Combining with neutral lipid DOPE, L3 was found with high efficiency in gene transfer in three tumor cell lines including A549, HepG2 and H460. The optimized gene transfection efficacy of L3 was nearly 5.5 times more efficient than that of the popular commercially available gene delivery agent Lipofectamine 2000™ in human lung carcinoma cells A549. In addition, since L1 and L3 had nearly no gene transfection performance in normal cells HEK293, these cationic lipids showed tumor cell-targeting property to a certain extent. No significant cytotoxicity was found for the lipoplexes formed by L1-L3, and their cytotoxicities were similar to or slightly lower than the lipoplexes prepared from Lipofectamine 2000™. CONCLUSION: Novel cyclen-based cationic lipids for effective in vitro gene transfection were founded, and these studies here may extend the application areas of macrocyclic polyamines, especially for cyclen.

  10. Dicyema Pax6 and Zic: tool-kit genes in a highly simplified bilaterian

    Directory of Open Access Journals (Sweden)

    Kamiya Akiko

    2007-10-01

    Full Text Available Abstract Background Dicyemid mesozoans (Phylum Dicyemida are simple (8–40-cell cephalopod endoparasites. They have neither body cavities nor differentiated organs, such as nervous and gastrointestinal systems. Whether dicyemids are intermediate between Protozoa and Metazoa (as represented by their "Mesozoa" classification or degenerate species of more complex metazoans is controversial. Recent molecular phylogenetic studies suggested that they are simplified bilaterians belonging to the Lophotrochozoa. We cloned two genes developmentally critical in bilaterian animals (Pax6 and Zic, together with housekeeping genes (actin, fructose-bisphosphate aldolase, and ATP synthase beta subunit from a dicyemid to reveal whether their molecular phylogeny supported the "simplification" hypothesis, and to clarify evolutionary changes in dicyemid gene structure and expression profiles. Results Genomic/cDNA sequence analysis showed that 1 the Pax6 molecular phylogeny and Zic intron positions supported the idea of dicyemids as reduced bilaterians; 2 the aa sequences deduced from the five genes were highly divergent; and 3 Dicyema genes contained very short introns of uniform length. In situ hybridization analyses revealed that Zic genes were expressed in hermaphroditic gonads, and Pax6 was expressed weakly throughout the developmental stages of the 2 types of embryo and in the hermaphroditic gonads. Conclusion The accelerated evolutionary rates and very short and uniform intron may represent a part of Dicyema genomic features. The presence and expression of the two tool-kit genes (Pax6 and Zic in Dicyema suggests that they can be very versatile genes even required for the highly reduced bilaterian like Dicyema. Dicyemids may be useful models of evolutionary body plan simplification.

  11. High-resolution comparative genomic hybridization of inflammatory breast cancer and identification of candidate genes.

    Directory of Open Access Journals (Sweden)

    Ismahane Bekhouche

    Full Text Available BACKGROUND: Inflammatory breast cancer (IBC is an aggressive form of BC poorly defined at the molecular level. We compared the molecular portraits of 63 IBC and 134 non-IBC (nIBC clinical samples. METHODOLOGY/FINDINGS: Genomic imbalances of 49 IBCs and 124 nIBCs were determined using high-resolution array-comparative genomic hybridization, and mRNA expression profiles of 197 samples using whole-genome microarrays. Genomic profiles of IBCs were as heterogeneous as those of nIBCs, and globally relatively close. However, IBCs showed more frequent "complex" patterns and a higher percentage of genes with CNAs per sample. The number of altered regions was similar in both types, although some regions were altered more frequently and/or with higher amplitude in IBCs. Many genes were similarly altered in both types; however, more genes displayed recurrent amplifications in IBCs. The percentage of genes whose mRNA expression correlated with CNAs was similar in both types for the gained genes, but ∼7-fold lower in IBCs for the lost genes. Integrated analysis identified 24 potential candidate IBC-specific genes. Their combined expression accurately distinguished IBCs and nIBCS in an independent validation set, and retained an independent prognostic value in a series of 1,781 nIBCs, reinforcing the hypothesis for a link with IBC aggressiveness. Consistent with the hyperproliferative and invasive phenotype of IBC these genes are notably involved in protein translation, cell cycle, RNA processing and transcription, metabolism, and cell migration. CONCLUSIONS: Our results suggest a higher genomic instability of IBC. We established the first repertory of DNA copy number alterations in this tumor, and provided a list of genes that may contribute to its aggressiveness and represent novel therapeutic targets.

  12. Four genes predict high risk of progression from smoldering to symptomatic multiple myeloma (SWOG S0120).

    Science.gov (United States)

    Khan, Rashid; Dhodapkar, Madhav; Rosenthal, Adam; Heuck, Christoph; Papanikolaou, Xenofon; Qu, Pingping; van Rhee, Frits; Zangari, Maurizio; Jethava, Yogesh; Epstein, Joshua; Yaccoby, Shmuel; Hoering, Antje; Crowley, John; Petty, Nathan; Bailey, Clyde; Morgan, Gareth; Barlogie, Bart

    2015-09-01

    Multiple myeloma is preceded by an asymptomatic phase, comprising monoclonal gammopathy of uncertain significance and smoldering myeloma. Compared to the former, smoldering myeloma has a higher and non-uniform rate of progression to clinical myeloma, reflecting a subset of patients with higher risk. We evaluated the gene expression profile of smoldering myeloma plasma cells among 105 patients enrolled in a prospective observational trial at our institution, with a view to identifying a high-risk signature. Baseline clinical, bone marrow, cytogenetic and radiologic data were evaluated for their potential to predict time to therapy for symptomatic myeloma. A gene signature derived from four genes, at an optimal binary cut-point of 9.28, identified 14 patients (13%) with a 2-year therapy risk of 85.7%. Conversely, a low four-gene score (probe sets showed concordance with indices of chromosome instability. These data demonstrate high discriminatory power of a gene-based assay and suggest a role for dysregulation of mitotic checkpoints in the context of genomic instability as a hallmark of high-risk smoldering myeloma.

  13. Blueprint for a high-performance biomaterial: full-length spider dragline silk genes.

    Directory of Open Access Journals (Sweden)

    Nadia A Ayoub

    Full Text Available Spider dragline (major ampullate silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons, recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers.

  14. The Influence of Bovine Milk High or Low in Isoflavones on Hepatic Gene Expression in Mice

    Directory of Open Access Journals (Sweden)

    Mette T. Skaanild

    2010-01-01

    Full Text Available Isoflavones have generated much attention due to their potential positive effects in various diseases. Phytoestrogens especially equol can be found in bovine milk, as feed ration for dairy cows is comprised of plants containing phytoestrogens. The aim of this study was to analyze the changes in hepatic gene expression after dietary intake of milk high and low in isoflavones. In addition to pelleted feed female NMRI mice were offered water, water added either 17-estradiol, equol, Tween 80, and milk high and low in isoflavone content for a week. Gene expression was analyzed using an array qPCR kit. It was revealed that Tween 80 and 17-estradiol upregulated both phase I and phase II genes to the same extent whereas equol alone, high and low isoflavone milk did not alter the expression of phase I genes but decreased the expression of phase II genes. This study shows that dietary isoflavones can regulate the transcription of especially phase II liver enzymes which potentially could give rise to an increase in reactive oxygen metabolites that may contribute to the development of cancer.

  15. Effects of BST and high energy diet on gene expression in mammary parenchyma of dairy heifers

    Directory of Open Access Journals (Sweden)

    Betina Joyce Lew

    2013-07-01

    Full Text Available The objective of this study was to determine the effects of dietary energy and recombinant bovine somatotropin (bST injection to identify genes that might control mammogenesis. Total RNA was extracted from the parenchymal tissue of 32 heifers randomly assigned to one of four treatments: two diets (a standard diet and a high energy, high protein diet, each with or without bST. To perform microarray experiments, RNA samples were pooled (2 animals/pool before reverse transcription and labeling with Cy3 or Cy5. A 4-node loop design was used to examine the differential gene expression among treatments using a bovine-specific cDNA microarray (National Bovine Functional Genomics Consortium Library, NBFGC containing 18,263 unique expressed sequence tags (EST. Significance levels of differential gene expression among treatments were assessed using a mixed model approach. Injection of bST altered the expression of 12 % of the genes on NBFGC slide related to tissue development, whereas 6% were altered by diet. Administration of bST increases the expression of genes positively related to cell proliferation and mammary parenchyma to a greater extent than a high energy diet.

  16. Prolonged application of high fluid shear to chondrocytes recapitulates gene expression profiles associated with osteoarthritis.

    Directory of Open Access Journals (Sweden)

    Fei Zhu

    Full Text Available BACKGROUND: Excessive mechanical loading of articular cartilage producing hydrostatic stress, tensile strain and fluid flow leads to irreversible cartilage erosion and osteoarthritic (OA disease. Since application of high fluid shear to chondrocytes recapitulates some of the earmarks of OA, we aimed to screen the gene expression profiles of shear-activated chondrocytes and assess potential similarities with OA chondrocytes. METHODOLOGY/PRINCIPAL FINDINGS: Using a cDNA microarray technology, we screened the differentially-regulated genes in human T/C-28a2 chondrocytes subjected to high fluid shear (20 dyn/cm(2 for 48 h and 72 h relative to static controls. Confirmation of the expression patterns of select genes was obtained by qRT-PCR. Using significance analysis of microarrays with a 5% false discovery rate, 71 and 60 non-redundant transcripts were identified to be ≥2-fold up-regulated and ≤0.6-fold down-regulated, respectively, in sheared chondrocytes. Published data sets indicate that 42 of these genes, which are related to extracellular matrix/degradation, cell proliferation/differentiation, inflammation and cell survival/death, are differentially-regulated in OA chondrocytes. In view of the pivotal role of cyclooxygenase-2 (COX-2 in the pathogenesis and/or progression of OA in vivo and regulation of shear-induced inflammation and apoptosis in vitro, we identified a collection of genes that are either up- or down-regulated by shear-induced COX-2. COX-2 and L-prostaglandin D synthase (L-PGDS induce reactive oxygen species production, and negatively regulate genes of the histone and cell cycle families, which may play a critical role in chondrocyte death. CONCLUSIONS/SIGNIFICANCE: Prolonged application of high fluid shear stress to chondrocytes recapitulates gene expression profiles associated with osteoarthritis. Our data suggest a potential link between exposure of chondrocytes/cartilage to abnormal mechanical loading and the pathogenesis

  17. Mx1 gene protects mice against the highly lethal human H5N1 influenza virus.

    Science.gov (United States)

    Salomon, Rachelle; Staeheli, Peter; Kochs, Georg; Yen, Hui-Ling; Franks, John; Rehg, Jerold E; Webster, Robert G; Hoffmann, Erich

    2007-10-01

    We investigated the importance of the host Mx1 gene in protection against highly pathogenic H5N1 avian influenza virus. Mice expressing the Mx1 gene survived infection with the lethal human H5N1 isolate A/Vietnam/1203/04 and with reassortants combining its genes with those of the non-lethal virus A/chicken/Vietnam/C58/04, while all Mx1-/- mice succumbed. Mx1-expressing mice showed lower organ virus titers, fewer lesions, and less pulmonary inflammation. Our data support the hypothesis that Mx1 expression protects mice against the high pathogenicity of H5N1 virus through inhibition of viral polymerase activity ultimately resulting in reduced viral growth and spread. Drugs that mimic this mechanism may be protective in humans.

  18. High-throughput genomic mapping of vector integration sites in gene therapy studies.

    Science.gov (United States)

    Beard, Brian C; Adair, Jennifer E; Trobridge, Grant D; Kiem, Hans-Peter

    2014-01-01

    Gene therapy has enormous potential to treat a variety of infectious and genetic diseases. To date hundreds of patients worldwide have received hematopoietic cell products that have been gene-modified with retrovirus vectors carrying therapeutic transgenes, and many patients have been cured or demonstrated disease stabilization as a result (Adair et al., Sci Transl Med 4:133ra57, 2012; Biffi et al., Science 341:1233158, 2013; Aiuti et al., Science 341:1233151, 2013; Fischer et al., Gene 525:170-173, 2013). Unfortunately, for some patients the provirus integration dysregulated the expression of nearby genes leading to clonal outgrowth and, in some cases, cancer. Thus, the unwanted side effect of insertional mutagenesis has become a major concern for retrovirus gene therapy. The careful study of retrovirus integration sites (RIS) and the contribution of individual gene-modified clones to hematopoietic repopulating cells is of crucial importance for all gene therapy studies. Supporting this, the US Food and Drug Administration (FDA) has mandated the careful monitoring of RIS in all clinical trials of gene therapy. An invaluable method was developed: linear amplification mediated-polymerase chain reaction (LAM-PCR) capable of analyzing in vitro and complex in vivo samples, capturing valuable genomic information directly flanking the site of provirus integration. Linking this method and similar methods to high-throughput sequencing has now made possible an unprecedented understanding of the integration profile of various retrovirus vectors, and allows for sensitive monitoring of their safety. It also allows for a detailed comparison of improved safety-enhanced gene therapy vectors. An important readout of safety is the relative contribution of individual gene-modified repopulating clones. One limitation of LAM-PCR is that the ability to capture the relative contribution of individual clones is compromised because of the initial linear PCR common to all current methods

  19. Identification of genes preferentially expressed by highly virulent piscine Streptococcus agalactiae upon interaction with macrophages.

    Directory of Open Access Journals (Sweden)

    Chang-Ming Guo

    Full Text Available Streptococcus agalactiae, long recognized as a mammalian pathogen, is an emerging concern with regard to fish. In this study, we used a mouse model and in vitro cell infection to evaluate the pathogenetic characteristics of S. agalactiae GD201008-001, isolated from tilapia in China. This bacterium was found to be highly virulent and capable of inducing brain damage by migrating into the brain by crossing the blood-brain barrier (BBB. The phagocytosis assays indicated that this bacterium could be internalized by murine macrophages and survive intracellularly for more than 24 h, inducing injury to macrophages. Further, selective capture of transcribed sequences (SCOTS was used to investigate microbial gene expression associated with intracellular survival. This positive cDNA selection technique identified 60 distinct genes that could be characterized into 6 functional categories. More than 50% of the differentially expressed genes were involved in metabolic adaptation. Some genes have previously been described as associated with virulence in other bacteria, and four showed no significant similarities to any other previously described genes. This study constitutes the first step in further gene expression analyses that will lead to a better understanding of the molecular mechanisms used by S. agalactiae to survive in macrophages and to cross the BBB.

  20. Photosynthetic Genes and Genes Associated with the C4 Trait in Maize Are Characterized by a Unique Class of Highly Regulated Histone Acetylation Peaks on Upstream Promoters.

    Science.gov (United States)

    Perduns, Renke; Horst-Niessen, Ina; Peterhansel, Christoph

    2015-08-01

    Histone modifications contribute to gene regulation in eukaryotes. We analyzed genome-wide histone H3 Lysine (Lys) 4 trimethylation and histone H3 Lys 9 acetylation (two modifications typically associated with active genes) in meristematic cells at the base and expanded cells in the blade of the maize (Zea mays) leaf. These data were compared with transcript levels of associated genes. For individual genes, regulations (fold changes) of histone modifications and transcript levels were much better correlated than absolute intensities. When focusing on regulated histone modification sites, we identified highly regulated secondary H3 Lys 9 acetylation peaks on upstream promoters (regulated secondary upstream peaks [R-SUPs]) on 10% of all genes. R-SUPs were more often found on genes that were up-regulated toward the blade than on down-regulated genes and specifically, photosynthetic genes. Among those genes, we identified six genes encoding enzymes of the C4 cycle and a significant enrichment of genes associated with the C4 trait derived from transcriptomic studies. On the DNA level, R-SUPs are frequently associated with ethylene-responsive elements. Based on these data, we suggest coevolution of epigenetic promoter elements during the establishment of C4 photosynthesis.

  1. Gene expression profiles in testis of pigs with extreme high and low levels of androstenone

    DEFF Research Database (Denmark)

    Moe, Maren; Meuwissen, Theo; Lien, Sigbjørn;

    2007-01-01

    Boar taint is a major obstacle when using uncastrated male pigs for swine production. One of the main compounds causing this taint is androstenone, a pheromone produced in porcine testis. Here we use microarrays to study the expression of thousands of genes simultaneously in testis of high and low...

  2. High School Students' Understanding of Chromosome/Gene Behavior during Meiosis.

    Science.gov (United States)

    Stewart, Jim; Dale, Michael

    1989-01-01

    Investigates high school students' understanding of the physical relationship of chromosomes and genes as expressed in their conceptual models and in their ability to manipulate the models to explain solutions to dihybrid cross problems. Describes three typical models and three students' reasoning processes. Discusses four implications. (YP)

  3. Genes involved in complex adaptive processes tend to have highly conserved upstream regions in mammalian genomes

    Directory of Open Access Journals (Sweden)

    Kohane Isaac

    2005-11-01

    Full Text Available Abstract Background Recent advances in genome sequencing suggest a remarkable conservation in gene content of mammalian organisms. The similarity in gene repertoire present in different organisms has increased interest in studying regulatory mechanisms of gene expression aimed at elucidating the differences in phenotypes. In particular, a proximal promoter region contains a large number of regulatory elements that control the expression of its downstream gene. Although many studies have focused on identification of these elements, a broader picture on the complexity of transcriptional regulation of different biological processes has not been addressed in mammals. The regulatory complexity may strongly correlate with gene function, as different evolutionary forces must act on the regulatory systems under different biological conditions. We investigate this hypothesis by comparing the conservation of promoters upstream of genes classified in different functional categories. Results By conducting a rank correlation analysis between functional annotation and upstream sequence alignment scores obtained by human-mouse and human-dog comparison, we found a significantly greater conservation of the upstream sequence of genes involved in development, cell communication, neural functions and signaling processes than those involved in more basic processes shared with unicellular organisms such as metabolism and ribosomal function. This observation persists after controlling for G+C content. Considering conservation as a functional signature, we hypothesize a higher density of cis-regulatory elements upstream of genes participating in complex and adaptive processes. Conclusion We identified a class of functions that are associated with either high or low promoter conservation in mammals. We detected a significant tendency that points to complex and adaptive processes were associated with higher promoter conservation, despite the fact that they have emerged

  4. Monitoring yeast physiology during very high gravity wort fermentations by frequent analysis of gene expression.

    Science.gov (United States)

    Rautio, Jari J; Huuskonen, Anne; Vuokko, Heikki; Vidgren, Virve; Londesborough, John

    2007-09-01

    Brewer's yeast experiences constantly changing environmental conditions during wort fermentation. Cells can rapidly adapt to changing surroundings by transcriptional regulation. Changes in genomic expression can indicate the physiological condition of yeast in the brewing process. We monitored, using the transcript analysis with aid of affinity capture (TRAC) method, the expression of some 70 selected genes relevant to wort fermentation at high frequency through 9-10 day fermentations of very high gravity wort (25 degrees P) by an industrial lager strain. Rapid changes in expression occurred during the first hours of fermentations for several genes, e.g. genes involved in maltose metabolism, glycolysis and ergosterol synthesis were strongly upregulated 2-6 h after pitching. By the time yeast growth had stopped (72 h) and total sugars had dropped by about 50%, most selected genes had passed their highest expression levels and total mRNA was less than half the levels during growth. There was an unexpected upregulation of some genes of oxygen-requiring pathways during the final fermentation stages. For five genes, expression of both the Saccharomyces cerevisiae and S. bayanus components of the hybrid lager strain were determined. Expression profiles were either markedly different (ADH1, ERG3) or very similar (MALx1, ILV5, ATF1) between these two components. By frequent analysis of a chosen set of genes, TRAC provided a detailed and dynamic picture of the physiological state of the fermenting yeast. This approach offers a possible way to monitor and optimize the performance of yeast in a complex process environment.

  5. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases.

    Science.gov (United States)

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Wang, Ping; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-06-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.

  6. Average gene length is highly conserved in prokaryotes and eukaryotes and diverges only between the two kingdoms.

    Science.gov (United States)

    Xu, Lin; Chen, Hong; Hu, Xiaohua; Zhang, Rongmei; Zhang, Ze; Luo, Z W

    2006-06-01

    The average length of genes in a eukaryote is larger than in a prokaryote, implying that evolution of complexity is related to change of gene lengths. Here, we show that although the average lengths of genes in prokaryotes and eukaryotes are much different, the average lengths of genes are highly conserved within either of the two kingdoms. This suggests that natural selection has clearly set a strong limitation on gene elongation within the kingdom. Furthermore, the average gene size adds another distinct characteristic for the discrimination between the two kingdoms of organisms.

  7. High occurrence of functional new chimeric genes in survey of rice chromosome 3 short arm genome sequences.

    Science.gov (United States)

    Zhang, Chengjun; Wang, Jun; Marowsky, Nicholas C; Long, Manyuan; Wing, Rod A; Fan, Chuanzhu

    2013-01-01

    In an effort to identify newly evolved genes in rice, we searched the genomes of Asian-cultivated rice Oryza sativa ssp. japonica and its wild progenitors, looking for lineage-specific genes. Using genome pairwise comparison of approximately 20-Mb DNA sequences from the chromosome 3 short arm (Chr3s) in six rice species, O. sativa, O. nivara, O. rufipogon, O. glaberrima, O. barthii, and O. punctata, combined with synonymous substitution rate tests and other evidence, we were able to identify potential recently duplicated genes, which evolved within the last 1 Myr. We identified 28 functional O. sativa genes, which likely originated after O. sativa diverged from O. glaberrima. These genes account for around 1% (28/3,176) of all annotated genes on O. sativa's Chr3s. Among the 28 new genes, two recently duplicated segments contained eight genes. Fourteen of the 28 new genes consist of chimeric gene structure derived from one or multiple parental genes and flanking targeting sequences. Although the majority of these 28 new genes were formed by single or segmental DNA-based gene duplication and recombination, we found two genes that were likely originated partially through exon shuffling. Sequence divergence tests between new genes and their putative progenitors indicated that new genes were most likely evolving under natural selection. We showed all 28 new genes appeared to be functional, as suggested by Ka/Ks analysis and the presence of RNA-seq, cDNA, expressed sequence tag, massively parallel signature sequencing, and/or small RNA data. The high rate of new gene origination and of chimeric gene formation in rice may demonstrate rice's broad diversification, domestication, its environmental adaptation, and the role of new genes in rice speciation.

  8. Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

    Science.gov (United States)

    Stanton, Richard J; McSharry, Brian P; Armstrong, Melanie; Tomasec, Peter; Wilkinson, Gavin W G

    2008-12-01

    With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

  9. 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-...

  10. The complete mitochondrial genome of the cyclopoid copepod Paracyclopina nana: a highly divergent genome with novel gene order and atypical gene numbers.

    Science.gov (United States)

    Ki, Jang-Seu; Park, Heum Gi; Lee, Jae-Seong

    2009-04-15

    In this paper, we describe the complete mitogenome of the cyclopoid copepod Paracyclopina nana with emphasis on the highly rearranged gene order and high divergence against published copepod mitogenomes. The P. nana mtDNA is 15,981 bp in length (70.9% AT) and consists of 37 genes (12 protein-coding genes, 2 rRNAs, 23 tRNAs) that are atypical for metazoan mitogenomes. Unusually, it contains an extra tRNA (tRNA-Ala) but it does not contain the ATPase 8 gene. The P. nana mitogenome has a long putative control region with high AT content (1351 bp, 77.0% AT). The Cyt b was considerably short in length, compared to other crustaceans. Compared to typical mitogenomes of arthropods and copepods, the gene order of the P. nana mitogenome is highly rearranged with a novel gene structure. In addition, P. nana has highly divergent mt genes (mostly less than 50%), judged by amino acid substitution. We present the first complete mitogenome sequence from a cyclopoid copepod, thereby increasing our understanding of copepod and crustacean evolution from the mitochondrial point of view.

  11. Diet-dependent gene expression in honey bees: honey vs. sucrose or high fructose corn syrup.

    Science.gov (United States)

    Wheeler, Marsha M; Robinson, Gene E

    2014-07-17

    Severe declines in honey bee populations have made it imperative to understand key factors impacting honey bee health. Of major concern is nutrition, as malnutrition in honey bees is associated with immune system impairment and increased pesticide susceptibility. Beekeepers often feed high fructose corn syrup (HFCS) or sucrose after harvesting honey or during periods of nectar dearth. We report that, relative to honey, chronic feeding of either of these two alternative carbohydrate sources elicited hundreds of differences in gene expression in the fat body, a peripheral nutrient-sensing tissue analogous to vertebrate liver and adipose tissues. These expression differences included genes involved in protein metabolism and oxidation-reduction, including some involved in tyrosine and phenylalanine metabolism. Differences between HFCS and sucrose diets were much more subtle and included a few genes involved in carbohydrate and lipid metabolism. Our results suggest that bees receive nutritional components from honey that are not provided by alternative food sources widely used in apiculture.

  12. Rapid high resolution genotyping of Francisella tularensis by whole genome sequence comparison of annotated genes ("MLST+".

    Directory of Open Access Journals (Sweden)

    Markus H Antwerpen

    Full Text Available The zoonotic disease tularemia is caused by the bacterium Francisella tularensis. This pathogen is considered as a category A select agent with potential to be misused in bioterrorism. Molecular typing based on DNA-sequence like canSNP-typing or MLVA has become the accepted standard for this organism. Due to the organism's highly clonal nature, the current typing methods have reached their limit of discrimination for classifying closely related subpopulations within the subspecies F. tularensis ssp. holarctica. We introduce a new gene-by-gene approach, MLST+, based on whole genome data of 15 sequenced F. tularensis ssp. holarctica strains and apply this approach to investigate an epidemic of lethal tularemia among non-human primates in two animal facilities in Germany. Due to the high resolution of MLST+ we are able to demonstrate that three independent clones of this highly infectious pathogen were responsible for these spatially and temporally restricted outbreaks.

  13. Analysis on differential expressed genes of ovarian tissue between high- and low-yield laying hen.

    Science.gov (United States)

    Chen, Wei; Song, Ling-Jun; Zeng, Yong-Qing; Yang, Yun; Wang, Hui

    2013-01-01

    In order to elucidate molecular genetic mechanism of laying hen reproduction at the transcriptional level and the structure of significantly differential genes, the mRNA differential display and reverse northern dot-blot were used to detect the differential expression of genes in the ovary tissue of low-yield laying hens and high-yield laying hens in the present study. Sixteen 32-week-old CAU-pink laying hens divided into two groups were used and the laying performance was measured. The results showed that only the egg numbers were significantly different between the two groups; and from 15 primer pairs, a total of 336 bands were displayed of which 59 cDNA bands were found to be differentially expressed in both high-yield and low-yield laying hen. The sequence analysis indicated that the expression of such bands as H-AP5, H-P5, and H-P4 was significantly potentiated in high-yield laying hen using primer pairs AP5/HT11G, P5/HT11G and P4/HT11G and these transcripts had high homology (98%) to HoxDb, HoxCa, and HoxBa, respectively. The differentially expressed gene fragments may be relevant to the progression of the high-yield hens to the egg-laying stage. And further study is required to elucidate the molecular function to improve the productivity of laying hens.

  14. Putative extremely high rate of proteome innovation in lancelets might be explained by high rate of gene prediction errors.

    Science.gov (United States)

    Bányai, László; Patthy, László

    2016-08-01

    A recent analysis of the genomes of Chinese and Florida lancelets has concluded that the rate of creation of novel protein domain combinations is orders of magnitude greater in lancelets than in other metazoa and it was suggested that continuous activity of transposable elements in lancelets is responsible for this increased rate of protein innovation. Since morphologically Chinese and Florida lancelets are highly conserved, this finding would contradict the observation that high rates of protein innovation are usually associated with major evolutionary innovations. Here we show that the conclusion that the rate of proteome innovation is exceptionally high in lancelets may be unjustified: the differences observed in domain architectures of orthologous proteins of different amphioxus species probably reflect high rates of gene prediction errors rather than true innovation.

  15. Cellular Stress Response Gene Expression During Upper and Lower Body High Intensity Exercises

    Science.gov (United States)

    Kochanowicz, Andrzej; Sawczyn, Stanisław; Niespodziński, Bartłomiej; Mieszkowski, Jan; Kochanowicz, Kazimierz

    2017-01-01

    Objectives The aim was to compare the effect of upper and lower body high-intensity exercise on chosen genes expression in athletes and non-athletes. Method Fourteen elite male artistic gymnasts (EAG) aged 20.6 ± 3.3 years and 14 physically active men (PAM) aged 19.9 ± 1.0 years performed lower and upper body 30 s Wingate Tests. Blood samples were collected before, 5 and 30 minutes after each effort to assess gene expression via PCR. Results Significantly higher mechanical parameters after lower body exercise was observed in both groups, for relative power (8.7 ± 1.2 W/kg in gymnasts, 7.2 ± 1.2 W/kg in controls, p = 0.01) and mean power (6.7 ± 0.7 W/kg in gymnasts, 5.4 ± 0.8 W/kg in controls, p = 0.01). No differences in lower versus upper body gene expression were detected for all tested genes as well as between gymnasts and physical active man. For IL-6 m-RNA time-dependent effect was observed. Conclusions Because of no significant differences in expression of genes associated with cellular stress response the similar adaptive effect to exercise may be obtained so by lower and upper body exercise. PMID:28141870

  16. Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

    Directory of Open Access Journals (Sweden)

    Kaul Rajinder

    2009-11-01

    Full Text Available Abstract Background Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia

  17. High rates of gene flow by pollen and seed in oak populations across Europe.

    Directory of Open Access Journals (Sweden)

    Sophie Gerber

    Full Text Available Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea distributed across Europe. Adult trees within a given area in each stand were exhaustively sampled (range [239, 754], mean 423, mapped, and acorns were collected ([17,147], 51 from several mother trees ([3], [47], 23. Seedlings ([65,387], 178 were harvested and geo-referenced in six of the eight stands. Genetic information was obtained from screening distinct molecular markers spread across the genome, genotyping each tree, acorn or seedling. All samples were thus genotyped at 5-8 nuclear microsatellite loci. Fathers/parents were assigned to acorns and seedlings using likelihood methods. Mating success of male and female parents, pollen and seed dispersal curves, and also hybridisation rates were estimated in each stand and compared on a continental scale. On average, the percentage of the wind-borne pollen from outside the stand was 60%, with large variation among stands (21-88%. Mean seed immigration into the stand was 40%, a high value for oaks that are generally considered to have limited seed dispersal. However, this estimate varied greatly among stands (20-66%. Gene flow was mostly intraspecific, with large variation, as some trees and stands showed particularly high rates of hybridisation. Our results show that mating success was unevenly distributed among trees. The high levels of gene flow suggest that geographically remote oak stands are unlikely to be genetically isolated, questioning the static definition of gene reserves and seed stands.

  18. Bacterial translational regulations: high diversity between all mRNAs and major role in gene expression

    Directory of Open Access Journals (Sweden)

    Picard Flora

    2012-10-01

    Full Text Available Abstract Background In bacteria, the weak correlations at the genome scale between mRNA and protein levels suggest that not all mRNAs are translated with the same efficiency. To experimentally explore mRNA translational level regulation at the systemic level, the detailed translational status (translatome of all mRNAs was measured in the model bacterium Lactococcus lactis in exponential phase growth. Results Results demonstrated that only part of the entire population of each mRNA species was engaged in translation. For transcripts involved in translation, the polysome size reached a maximum of 18 ribosomes. The fraction of mRNA engaged in translation (ribosome occupancy and ribosome density were not constant for all genes. This high degree of variability was analyzed by bioinformatics and statistical modeling in order to identify general rules of translational regulation. For most of the genes, the ribosome density was lower than the maximum value revealing major control of translation by initiation. Gene function was a major translational regulatory determinant. Both ribosome occupancy and ribosome density were particularly high for transcriptional regulators, demonstrating the positive role of translational regulation in the coordination of transcriptional networks. mRNA stability was a negative regulatory factor of ribosome occupancy and ribosome density, suggesting antagonistic regulation of translation and mRNA stability. Furthermore, ribosome occupancy was identified as a key component of intracellular protein levels underlining the importance of translational regulation. Conclusions We have determined, for the first time in a bacterium, the detailed translational status for all mRNAs present in the cell. We have demonstrated experimentally the high diversity of translational states allowing individual gene differentiation and the importance of translation-level regulation in the complex process linking gene expression to protein

  19. High amino acid diversity and positive selection at a putative coral immunity gene (tachylectin-2

    Directory of Open Access Journals (Sweden)

    Hellberg Michael E

    2010-05-01

    Full Text Available Abstract Background Genes involved in immune functions, including pathogen recognition and the activation of innate defense pathways, are among the most genetically variable known, and the proteins that they encode are often characterized by high rates of amino acid substitutions, a hallmark of positive selection. The high levels of variation characteristic of immunity genes make them useful tools for conservation genetics. To date, highly variable immunity genes have yet to be found in corals, keystone organisms of the world's most diverse marine ecosystem, the coral reef. Here, we examine variation in and selection on a putative innate immunity gene from Oculina, a coral genus previously used as a model for studies of coral disease and bleaching. Results In a survey of 244 Oculina alleles, we find high nonsynonymous variation and a signature of positive selection, consistent with a putative role in immunity. Using computational protein structure prediction, we generate a structural model of the Oculina protein that closely matches the known structure of tachylectin-2 from the Japanese horseshoe crab (Tachypleus tridentatus, a protein with demonstrated function in microbial recognition and agglutination. We also demonstrate that at least three other genera of anthozoan cnidarians (Acropora, Montastrea and Nematostella possess proteins structurally similar to tachylectin-2. Conclusions Taken together, the evidence of high amino acid diversity, positive selection and structural correspondence to the horseshoe crab tachylectin-2 suggests that this protein is 1 part of Oculina's innate immunity repertoire, and 2 evolving adaptively, possibly under selective pressure from coral-associated microorganisms. Tachylectin-2 may serve as a candidate locus to screen coral populations for their capacity to respond adaptively to future environmental change.

  20. High Dimensional ODEs Coupled with Mixed-Effects Modeling Techniques for Dynamic Gene Regulatory Network Identification.

    Science.gov (United States)

    Lu, Tao; Liang, Hua; Li, Hongzhe; Wu, Hulin

    2011-01-01

    Gene regulation is a complicated process. The interaction of many genes and their products forms an intricate biological network. Identification of this dynamic network will help us understand the biological process in a systematic way. However, the construction of such a dynamic network is very challenging for a high-dimensional system. In this article we propose to use a set of ordinary differential equations (ODE), coupled with dimensional reduction by clustering and mixed-effects modeling techniques, to model the dynamic gene regulatory network (GRN). The ODE models allow us to quantify both positive and negative gene regulations as well as feedback effects of one set of genes in a functional module on the dynamic expression changes of the genes in another functional module, which results in a directed graph network. A five-step procedure, Clustering, Smoothing, regulation Identification, parameter Estimates refining and Function enrichment analysis (CSIEF) is developed to identify the ODE-based dynamic GRN. In the proposed CSIEF procedure, a series of cutting-edge statistical methods and techniques are employed, that include non-parametric mixed-effects models with a mixture distribution for clustering, nonparametric mixed-effects smoothing-based methods for ODE models, the smoothly clipped absolute deviation (SCAD)-based variable selection, and stochastic approximation EM (SAEM) approach for mixed-effects ODE model parameter estimation. The key step, the SCAD-based variable selection of the proposed procedure is justified by investigating its asymptotic properties and validated by Monte Carlo simulations. We apply the proposed method to identify the dynamic GRN for yeast cell cycle progression data. We are able to annotate the identified modules through function enrichment analyses. Some interesting biological findings are discussed. The proposed procedure is a promising tool for constructing a general dynamic GRN and more complicated dynamic networks.

  1. Impact of high predation risk on genome-wide hippocampal gene expression in snowshoe hares.

    Science.gov (United States)

    Lavergne, Sophia G; McGowan, Patrick O; Krebs, Charles J; Boonstra, Rudy

    2014-11-01

    The population dynamics of snowshoe hares (Lepus americanus) are fundamental to the ecosystem dynamics of Canada's boreal forest. During the 8- to 11-year population cycle, hare densities can fluctuate up to 40-fold. Predators in this system (lynx, coyotes, great-horned owls) affect population numbers not only through direct mortality but also through sublethal effects. The chronic stress hypothesis posits that high predation risk during the decline severely stresses hares, leading to greater stress responses, heightened ability to mobilize cortisol and energy, and a poorer body condition. These effects may result in, or be mediated by, differential gene expression. We used an oligonucleotide microarray designed for a closely-related species, the European rabbit (Oryctolagus cuniculus), to characterize differences in genome-wide hippocampal RNA transcript abundance in wild hares from the Yukon during peak and decline phases of a single cycle. A total of 106 genes were differentially regulated between phases. Array results were validated with quantitative real-time PCR, and mammalian protein sequence similarity was used to infer gene function. In comparison to hares from the peak, decline phase hares showed increased expression of genes involved in metabolic processes and hormone response, and decreased expression of immune response and blood cell formation genes. We found evidence for predation risk effects on the expression of genes whose putative functions correspond with physiological impacts known to be induced by predation risk in snowshoe hares. This study shows, for the first time, a link between changes in demography and alterations in neural RNA transcript abundance in a natural population.

  2. The highly recombinogenic bz locus lies in an unusually gene-rich region of the maize genome.

    Science.gov (United States)

    Fu, H; Park, W; Yan, X; Zheng, Z; Shen, B; Dooner, H K

    2001-07-17

    The bronze (bz) locus exhibits the highest rate of recombination of any gene in higher plants. To investigate the possible basis of this high rate of recombination, we have analyzed the physical organization of the region around the bz locus. Two adjacent bacterial artificial chromosome clones, comprising a 240-kb contig centered around the Bz-McC allele, were isolated, and 60 kb of contiguous DNA spanning the two bacterial artificial chromosome clones was sequenced. We find that the bz locus lies in an unusually gene-rich region of the maize genome. Ten genes, at least eight of which are shown to be transcribed, are contained in a 32-kb stretch of DNA that is uninterrupted by retrotransposons. We have isolated nearly full length cDNAs corresponding to the five proximal genes in the cluster. The average intertranscript distance between them is just 1 kb, revealing a surprisingly compact packaging of adjacent genes in this part of the genome. At least 11 small insertions, including several previously described miniature inverted repeat transposable elements, were detected in the introns and 3' untranslated regions of genes and between genes. The gene-rich region is flanked at the proximal and distal ends by retrotransposon blocks. Thus, the maize genome appears to have scattered regions of high gene density similar to those found in other plants. The unusually high rate of intragenic recombination seen in bz may be related to the very high gene density of the region.

  3. Cyclophosphamide alters the gene expression profile in patients treated with high doses prior to stem cell transplantation.

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    Ibrahim El-Serafi

    Full Text Available BACKGROUND: Hematopoietic stem cell transplantation is a curative treatment for several haematological malignancies. However, treatment related morbidity and mortality still is a limiting factor. Cyclophosphamide is widely used in condition regimens either in combination with other chemotherapy or with total body irradiation. METHODS: We present the gene expression profile during cyclophosphamide treatment in 11 patients conditioned with cyclophosphamide for 2 days followed by total body irradiation prior to hematopoietic stem cell transplantation. 299 genes were identified as specific for cyclophosphamide treatment and were arranged into 4 clusters highly down-regulated genes, highly up-regulated genes, early up-regulated but later normalized genes and moderately up-regulated genes. RESULTS: Cyclophosphamide treatment down-regulated expression of several genes mapped to immune/autoimmune activation and graft rejection including CD3, CD28, CTLA4, MHC II, PRF1, GZMB and IL-2R, and up-regulated immune-related receptor genes, e.g. IL1R2, IL18R1, and FLT3. Moreover, a high and significant expression of ANGPTL1 and c-JUN genes was observed independent of cyclophosphamide treatment. CONCLUSION: This is the first investigation to provide significant information about alterations in gene expression following cyclophosphamide treatment that may increase our understanding of the cyclophosphamide mechanism of action and hence, in part, avoid its toxicity. Furthermore, ANGPTL1 remained highly expressed throughout the treatment and, in contrast to several other alkylating agents, cyclophosphamide did not influence c-JUN expression.

  4. Highly expressed genes are associated with inverse antisense transcription in mouse

    Indian Academy of Sciences (India)

    Andras Györffy; Pawel Surowiak; Zsolt Tulassay; Balazs Györffy

    2007-08-01

    There is a growing evidence, that antisense transcription might have a key role in a range of human diseases. Although predefined sense–antisense pairs were extensively studied, the antisense expression of the known sense genes is rarely investigated. We retrieved and correlated the expression of sense and antisense sequences of 1182 mouse transcripts to assess the prevalence and to find the characteristic pattern of antisense transcription. We contrasted three Affymetrix MGU74A version 1 mouse genome chips to six MGU74A version 2 chips. For these 1182 transcripts, the version 1 chips contain the antisense sequences of the transcripts presented on the version 2 chips. The original data was taken from the GEO database (GDS431 and GDS432). As the Affymetrix data are semiquantitative, the relative expression levels of antisense partners were analysed. We detected antisense transcription, although the average antisense expression is shifted towards smaller expression values (MGU74A version 1, 516; version 2, 1688). An inverse direct correlation between sense and antisense expression values could be observed at high expression values. At a very high relative expression—above 40,000—the Pearson correlation coefficient is getting closer to −1. Transcripts with high inverse expression ratio may be correlated to the investigated gene (major histocompatibility complex class II trans activator). The ratio of sense to antisense transcripts varied among different chromosomes; on chromosomes 14 and 1 the level of antisense expression was higher than that of sense. We conclude that antisense transcription is a common phenomenon in the mouse genome. The hypothesis of regulatory role of antisense transcripts is supported by the inverse antisense gene expression of highly expressed genes.

  5. Synthesis of a novel multivalent galactoside with high hepatocyte targeting for gene delivery

    Institute of Scientific and Technical Information of China (English)

    Qing Lin Jiang; Li Hai; Lei Chen; Jiao Lu; Zhi Rong Zhang; Yong Wu

    2008-01-01

    A novel bifunctional glycolipid which carried a cluster of thiogalactosides as the bepatocyte targeting ligand for gene delivery was prepared.Hexa-antennary alcohol 1 was used as the core scaffold to attach a cholesterol molecule by a poly(ethylene glycol)chain,while its remaining branches were linked with five acetylgalactosides,which would be deacetylated later to produce pentaantennary galaetoside.Liposome containing the galactoside showed high affinity and transfection activity in hepatoma cells HepG2.

  6. Peripheral blood mononuclear cell gene expression in healthy adults rapidly transported to high altitude

    Directory of Open Access Journals (Sweden)

    Herman NM

    2014-12-01

    Full Text Available Nicole M Herman,1 Diane E Grill,2 Paul J Anderson,1 Andrew D Miller,1 Jacob B Johnson,1 Kathy A O’Malley,1 Maile L Ceridon Richert,1 Bruce D Johnson1 1Department of Cardiovascular Diseases, 2Department of Biostatistics, Mayo Clinic Rochester, MN, USA Abstract: Although mechanisms of high altitude illness have been studied extensively, the processes behind the development of these conditions are still unclear. Few genome-wide studies on rapid exposure to high altitude have been performed. Each year, scientists and support workers are transferred by plane from McMurdo Station in Antarctica (sea level to the Amundsen-Scott South Pole Station at 2,835 meters. This uniform and rapid transfer to altitude provides a unique opportunity to study the effects of hypobaric hypoxia on gene expression that may help illustrate the body's adaptations to these conditions. We hypothesized that an extensive number of genes would change with rapid exposure to altitude and further expected that these genes would correspond to inflammatory pathways proposed as a mechanism in development of acute mountain sickness. Peripheral venous blood samples were drawn from 98 healthy subjects at sea level and again on day two at altitude. Microarray analysis was performed on these samples. In total, 1,118 probe sets with significant P-values and fold changes (90% upregulated were identified and entered into MetaCore™ software. Several pathways, including oxidative phosphorylation, cytoskeleton remodeling, and platelet aggregation, were significantly represented by the data set and all were upregulated. Many genes changed expression, and the vast majority of these increased. Increased metabolism in peripheral blood mononuclear cells suggests increased inflammatory activity. Keywords: peripheral blood mononuclear cells, microarray, gene expression, acute mountain sickness

  7. Testing candidate genes for attention-deficit/hyperactivity disorder in fruit flies using a high throughput assay for complex behavior

    DEFF Research Database (Denmark)

    Rohde, Palle Duun; Madsen, Lisbeth Strøm; Arvidson, Sandra Marie Neumann

    2016-01-01

    Fruit flies are important model organisms for functional testing of candidate genes in multiple disciplines, including the study of human diseases. Here we use a high-throughput locomotor activity assay to test the response on activity behavior of gene disruption in Drosophila melanogaster. The aim...... was to investigate the impact of disruption of 14 candidate genes for human attention-deficit/hyperactivity disorder (ADHD) on fly behavior. By obtaining a range of correlated measures describing the space of variables for behavioral activity we show, that some mutants display similar phenotypic responses...... in fruit flies. Results provide additional support for the investigated genes being risk candidate genes for ADHD in humans....

  8. Using evolutionary conserved modules in gene networks as a strategy to leverage high throughput gene expression queries.

    Directory of Open Access Journals (Sweden)

    Jeanne M Serb

    Full Text Available BACKGROUND: Large-scale gene expression studies have not yielded the expected insight into genetic networks that control complex processes. These anticipated discoveries have been limited not by technology, but by a lack of effective strategies to investigate the data in a manageable and meaningful way. Previous work suggests that using a pre-determined seed-network of gene relationships to query large-scale expression datasets is an effective way to generate candidate genes for further study and network expansion or enrichment. Based on the evolutionary conservation of gene relationships, we test the hypothesis that a seed network derived from studies of retinal cell determination in the fly, Drosophila melanogaster, will be an effective way to identify novel candidate genes for their role in mouse retinal development. METHODOLOGY/PRINCIPAL FINDINGS: Our results demonstrate that a number of gene relationships regulating retinal cell differentiation in the fly are identifiable as pairwise correlations between genes from developing mouse retina. In addition, we demonstrate that our extracted seed-network of correlated mouse genes is an effective tool for querying datasets and provides a context to generate hypotheses. Our query identified 46 genes correlated with our extracted seed-network members. Approximately 54% of these candidates had been previously linked to the developing brain and 33% had been previously linked to the developing retina. Five of six candidate genes investigated further were validated by experiments examining spatial and temporal protein expression in the developing retina. CONCLUSIONS/SIGNIFICANCE: We present an effective strategy for pursuing a systems biology approach that utilizes an evolutionary comparative framework between two model organisms, fly and mouse. Future implementation of this strategy will be useful to determine the extent of network conservation, not just gene conservation, between species and will

  9. High-throughput gene and SNP discovery in Eucalyptus grandis, an uncharacterized genome

    Directory of Open Access Journals (Sweden)

    Pappas Georgios J

    2008-06-01

    Full Text Available Abstract Background Benefits from high-throughput sequencing using 454 pyrosequencing technology may be most apparent for species with high societal or economic value but few genomic resources. Rapid means of gene sequence and SNP discovery using this novel sequencing technology provide a set of baseline tools for genome-level research. However, it is questionable how effective the sequencing of large numbers of short reads for species with essentially no prior gene sequence information will support contig assemblies and sequence annotation. Results With the purpose of generating the first broad survey of gene sequences in Eucalyptus grandis, the most widely planted hardwood tree species, we used 454 technology to sequence and assemble 148 Mbp of expressed sequences (EST. EST sequences were generated from a normalized cDNA pool comprised of multiple tissues and genotypes, promoting discovery of homologues to almost half of Arabidopsis genes, and a comprehensive survey of allelic variation in the transcriptome. By aligning the sequencing reads from multiple genotypes we detected 23,742 SNPs, 83% of which were validated in a sample. Genome-wide nucleotide diversity was estimated for 2,392 contigs using a modified theta (θ parameter, adapted for measuring genetic diversity from polymorphisms detected by randomly sequencing a multi-genotype cDNA pool. Diversity estimates in non-synonymous nucleotides were on average 4x smaller than in synonymous, suggesting purifying selection. Non-synonymous to synonymous substitutions (Ka/Ks among 2,001 contigs averaged 0.30 and was skewed to the right, further supporting that most genes are under purifying selection. Comparison of these estimates among contigs identified major functional classes of genes under purifying and diversifying selection in agreement with previous researches. Conclusion In providing an abundance of foundational transcript sequences where limited prior genomic information existed, this

  10. Rice Mitochondrial Genes Are Transcribed by Multiple Promoters That Are Highly Diverged

    Institute of Scientific and Technical Information of China (English)

    Qun-Yu Zhang; Yao-Guang Liu

    2006-01-01

    Plant mitochondrial genes are often transcribed into complex sets of mRNA. To characterize the transcription initiation and promoter structure, the transcript termini of four mitochondrial genes, atp1, atp6, cob,rps7, in rice (Oryza sativa L.), were determined by using a modified circularized RNA reverse transcriptionpolymerase chain reaction method. The results revealed that three genes (atp1, atp6, rps7) were transcribed from multiple initiation sites, indicating the presence of multiple promoters. Two transcription termination sites were detected in three genes (atp6, cob, rps7), respectively. Analysis on the promoter architecture showed that the YRTA (Y=T or C, R=A or G) motifs that are widely present in the mitochondrial promoters of other monocot and dicot plant species were detected only in two of the 12 analyzed promoters.Our data suggest that the promoter sequences in the rice mitochondrial genome are highly diverged in comparison to those in other plants, and the YRTA motif is not an essential element for the promoter activity.

  11. Transcript analysis of 1003 novel yeast genes using high-throughput northern hybridizations.

    Science.gov (United States)

    Brown, A J; Planta, R J; Restuhadi, F; Bailey, D A; Butler, P R; Cadahia, J L; Cerdan, M E; De Jonge, M; Gardner, D C; Gent, M E; Hayes, A; Kolen, C P; Lombardia, L J; Murad, A M; Oliver, R A; Sefton, M; Thevelein, J M; Tournu, H; van Delft, Y J; Verbart, D J; Winderickx, J; Oliver, S G

    2001-06-15

    The expression of 1008 open reading frames (ORFs) from the yeast Saccharomyces cerevisiae has been examined under eight different physiological conditions, using classical northern analysis. These northern data have been compared with publicly available data from a microarray analysis of the diauxic transition in S.cerevisiae. The results demonstrate the importance of comparing biologically equivalent situations and of the standardization of data normalization procedures. We have also used our northern data to identify co-regulated gene clusters and define the putative target sites of transcriptional activators responsible for their control. Clusters containing genes of known function identify target sites of known activators. In contrast, clusters comprised solely of genes of unknown function usually define novel putative target sites. Finally, we have examined possible global controls on gene expression. It was discovered that ORFs that are highly expressed following a nutritional upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not. These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs.

  12. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation

    Science.gov (United States)

    Doench, John G.; Hartenian, Ella; Graham, Daniel B.; Tothova, Zuzana; Hegde, Mudra; Smith, Ian; Sullender, Meagan; Ebert, Benjamin L.; Xavier, Ramnik J.; Root, David E.

    2014-01-01

    Components of the prokaryotic clustered regularly interspersed palindromic repeat (CRISPR) loci have recently been repurposed for use in mammalian cells1–6. The Cas9 protein can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system7,8. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the proto-spacer adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest. PMID:25184501

  13. The myostatin gene of Mytilus chilensis evidences a high level of polymorphism and ubiquitous transcript expression.

    Science.gov (United States)

    Núñez-Acuña, Gustavo; Gallardo-Escárate, Cristian

    2014-02-15

    Myostatin (MSTN) is a protein of the Transforming Growth Factor-β (TGF-β) superfamily and plays a crucial role in muscular development for higher vertebrates. However, its biological function in marine invertebrates remains undiscovered. This study characterizes the full-length sequence of the Mytilus chilensis myostatin gene (Mc-MSTN). Furthermore, tissue transcription patterns and putative single nucleotide polymorphisms (SNPs) were also identified. The Mc-MSTN cDNA sequence showed 3528 base pairs (bp), consisting of 161 bp of 5' UTR, 2,110 bp of 3' UTR, and an open reading frame of 1,257 bp encoding for 418 amino acids and with an RXXR proteolytic site and nine cysteine-conserved residues. Gene transcription analysis revealed that the Mc-MSTN has ubiquitous expression among several tissues, with higher expression in the gonads and mantle than in the digestive gland, gills, and hemolymph. Furthermore, high levels of polymorphisms were detected (28 SNPs in 3'-UTR and 9 SNPs in the coding region). Two SNPs were non-synonymous and involved amino acid changes between Glu/Asp and Thr/Ile. Until now, the MSTN gene has been mainly related to muscle growth in marine bivalves. However, the present study suggests a putative biological function not entirely associated to muscle tissue and contributes molecular evidence to the current debate about the function of the MSTN gene in marine invertebrates.

  14. Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes.

    Science.gov (United States)

    De Meyer, Sofie E; Briscoe, Leah; Martínez-Hidalgo, Pilar; Agapakis, Christina M; de-Los Santos, Paulina Estrada; Seshadri, Rekha; Reeve, Wayne; Weinstock, George; O'Hara, Graham; Howieson, John G; Hirsch, Ann M

    2016-08-01

    Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.

  15. Pre-thymic somatic mutation leads to high mutant frequency at hypoxanthine-guanine phosphoribosyltransferase gene

    Energy Technology Data Exchange (ETDEWEB)

    Jett, J. [Lawrence Livermore National Lab., CA (United States)

    1994-12-01

    While characterizing the background mutation spectrum of the Hypoxathine-guanine phosphoribosyltransferase (HPRT) gene in a healthy population, an outlier with a high mutant frequency of thioguanine resistant lymphocytes was found. When studied at the age of 46, this individual had been smoking 60 cigarettes per day for 38 years. His mutant frequency was calculated at 3.6 and 4.2x10{sup {minus}4} for two sampling periods eight months apart. Sequencing analysis of the HPRT gene in his mutant thioguanine resistant T lymphocytes was done to find whether the cells had a high rate of mutation, or if the mutation was due to a single occurrence of mutation and, if so, when in the T lymphocyte development the mutation occurred. By T-cell receptor analysis it has been found that out of 35 thioguanine resistant clones there was no dominant gamma T cell receptor gene rearrangement. During my appointment in the Science & Engineering Research Semester, I found that 34 of those clones have the same base substitution of G{yields}T at cDNA position 197. Due to the consistent mutant frequency from both sampling periods and the varying T cell receptors, the high mutant frequency cannot be due to recent proliferation of a mature mutant T lymphocyte. From the TCR and DNA sequence analysis we conclude that the G{yields}T mutation must have occurred in a T lymphocyte precursor before thymic differentiation so that the thioguanine resistant clones share the same base substitution but not the same gamma T cell receptor gene.

  16. Metagenomic approach reveals variation of microbes with arsenic and antimony metabolism genes from highly contaminated soil.

    Science.gov (United States)

    Luo, Jinming; Bai, Yaohui; Liang, Jinsong; Qu, Jiuhui

    2014-01-01

    Microbes have great potential for arsenic (As) and antimony (Sb) bioremediation in heavily contaminated soil because they have the ability to biotransform As and Sb to species that have less toxicity or are more easily removed. In this study, we integrated a metagenomic method with physicochemical characterization to elucidate the composition of microbial community and functional genes (related to As and Sb) in a high As (range from 34.11 to 821.23 mg kg-1) and Sb (range from 226.67 to 3923.07 mg kg-1) contaminated mine field. Metagenomic analysis revealed that microbes from 18 phyla were present in the 5 samples of soil contaminated with high As and Sb. Moreover, redundancy analysis (RDA) of the relationship between the 18 phyla and the concentration of As and Sb demonstrated that 5 phyla of microbes, i.e. Actinobacteria, Firmicutes, Nitrospirae, Tenericutes and Gemmatimonadetes were positively correlated with As and Sb concentration. The distribution, diversity and abundance of functional genes (including arsC, arrA, aioA, arsB and ACR3) were much higher for the samples containing higher As and Sb concentrations. Based on correlation analysis, the results showed a positive relationship between arsC-like (R2 = 0.871) and aioA-like (R2 = 0.675) gene abundance and As concentration, and indicated that intracellular As(V) reduction and As(III) oxidation could be the dominant As detoxification mechanism enabling the microbes to survive in the environment. This study provides a direct and reliable reference on the diversity of microbial community and functional genes in an extremely high concentration As- and Sb-contaminated environment.

  17. High mutability of the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL in cancer.

    Directory of Open Access Journals (Sweden)

    Vladimir I Kashuba

    Full Text Available BACKGROUND: Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas. METHODOLOGY/PRINCIPAL FINDINGS: We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1-2, 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp and in 98 clones of exons 3-5 we found 146 mutations (MF = 0.29. In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10. The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines. CONCLUSIONS/SIGNIFICANCE: This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread.

  18. GeneChip expression profiling reveals the alterations of energy metabolism related genes in osteocytes under large gradient high magnetic fields.

    Directory of Open Access Journals (Sweden)

    Yang Wang

    Full Text Available The diamagnetic levitation as a novel ground-based model for simulating a reduced gravity environment has recently been applied in life science research. In this study a specially designed superconducting magnet with a large gradient high magnetic field (LG-HMF, which can provide three apparent gravity levels (μ-g, 1-g, and 2-g, was used to simulate a space-like gravity environment. Osteocyte, as the most important mechanosensor in bone, takes a pivotal position in mediating the mechano-induced bone remodeling. In this study, the effects of LG-HMF on gene expression profiling of osteocyte-like cell line MLO-Y4 were investigated by Affymetrix DNA microarray. LG-HMF affected osteocyte gene expression profiling. Differentially expressed genes (DEGs and data mining were further analyzed by using bioinfomatic tools, such as DAVID, iReport. 12 energy metabolism related genes (PFKL, AK4, ALDOC, COX7A1, STC1, ADM, CA9, CA12, P4HA1, APLN, GPR35 and GPR84 were further confirmed by real-time PCR. An integrated gene interaction network of 12 DEGs was constructed. Bio-data mining showed that genes involved in glucose metabolic process and apoptosis changed notablly. Our results demostrated that LG-HMF affected the expression of energy metabolism related genes in osteocyte. The identification of sensitive genes to special environments may provide some potential targets for preventing and treating bone loss or osteoporosis.

  19. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    Science.gov (United States)

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids.

  20. Tumor Classification Using High-Order Gene Expression Profiles Based on Multilinear ICA

    Directory of Open Access Journals (Sweden)

    Ming-gang Du

    2009-01-01

    Full Text Available Motivation. Independent Components Analysis (ICA maximizes the statistical independence of the representational components of a training gene expression profiles (GEP ensemble, but it cannot distinguish relations between the different factors, or different modes, and it is not available to high-order GEP Data Mining. In order to generalize ICA, we introduce Multilinear-ICA and apply it to tumor classification using high order GEP. Firstly, we introduce the basis conceptions and operations of tensor and recommend Support Vector Machine (SVM classifier and Multilinear-ICA. Secondly, the higher score genes of original high order GEP are selected by using t-statistics and tabulate tensors. Thirdly, the tensors are performed by Multilinear-ICA. Finally, the SVM is used to classify the tumor subtypes. Results. To show the validity of the proposed method, we apply it to tumor classification using high order GEP. Though we only use three datasets, the experimental results show that the method is effective and feasible. Through this survey, we hope to gain some insight into the problem of high order GEP tumor classification, in aid of further developing more effective tumor classification algorithms.

  1. The Expression Plasticity of Hypoxia Related Genes in High-Altitude and Plains Nanorana parkeri Populations

    Institute of Scientific and Technical Information of China (English)

    Qiong ZHANG; Xingzhi HAN; Robert H S KRAUS; Le YANG; Liqing FAN; Yinzi YE; Yi TAO

    2016-01-01

    For species that have a broad geographic distribution, adaptive variation may be attributable to gene expression plasticity. Nanorana parkeri is an anuran endemic to the southern Tibetan Plateau where it has an extensive altitudinal range (2850 to 5100 m asl). Low oxygen concentration is one of the main environmental characteristics of the Tibetan Plateau. Hypoxia-inducible factor α subunits (HIF-1α and HIF-2α, encoded by Endothelial PAS domain protein 1 (EPAS1)) and associated genes (e.g., vascular endothelial growth factor (VEGF) and Erythropoietin (EPO)) play crucial roles in maintaining oxygen homeostasis. In this study, we compared the expression of HIF-1A, VEGF, EPAS1 and EPO mRNA between two populations of N. parkeri: one population inhabiting the native high altitudes, and the second living in, and being acclimated to, the lower plains (70 m asl). The expression of HIF-1A, VEGF and EPAS1 mRNA in the high altitude population were significantly higher than in the acclimated population, whereas there was no significant difference for EPO between two groups. Our results indicated that gene expression plasticity may make significant contributions to local adaptation of species that have broad altitudinal distributions. In addition, we deepen our understanding of the adaptive potential of this species by evaluating the experiments in the scope of its evolutionary history.

  2. Different pattern of Galleria mellonella jhbp gene expression in high five and Sf9 cells.

    Science.gov (United States)

    Andruszewska, Grażyna; Ożyhar, Andrzej; Kochman, Marian; Schmidt, Marcin

    2013-03-01

    Juvenile hormone binding protein (JHBP) is the key element of the system that transmits hormone signals to target tissues. Recently, we found that the core promoter of the jhbp gene is strongly under the control of the TATA box and the transcription start site. In this report, we have shown that the jhbp promoter contains distal regulatory elements whose functionality clearly depends on the particular cell environment and that the scope of research from one cell line is insufficient to generalize the conclusions of the analysis. Cf1/Usp (where Usp is ultraspiracle protein previously known as Cf1, chorion factor 1) elements suppressed transcription of the reporter gene in the High Five cell line but not in the Sf9 cell line. However, upstream from all three Cf1/Usp elements there is a DNA sequence, containing the Zeste element, which activates jhbp in both systems. We found that juvenile hormone strongly inhibited the activity of the jhbp promoter in the Sf9 cell line, whereas it did not have an effect in the High Five cell line. A second key hormone that controls insect development--20-hydroxyecdysone, was also found to suppress the transcription of jhbp. This is the first report describing how these two hormones affect jhbp gene expression in different cell lines.

  3. A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene delivery system

    Directory of Open Access Journals (Sweden)

    Eroglu E

    2013-04-01

    Full Text Available Erdal Eroglu,1 Pooja M Tiwari,1 Alain B Waffo,1 Michael E Miller,2 Komal Vig,1 Vida A Dennis,1 Shree R Singh1 1Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL, USA; 2Research Instrumentation Facility, Auburn University, AL, USA Abstract: The transport of DNA into eukaryotic cells is minimal because of the cell membrane barrier, and this limits the application of DNA vaccines, gene silencing, and gene therapy. Several available transfection reagents and techniques have been used to circumvent this problem. Alternatively, nonviral nanoscale vectors have been shown to bypass the eukaryotic cell membrane. In the present work, we developed a unique nanomaterial, pHEMA+chitosan nanospheres (PCNSs, which consisted of poly (2-hydroxyethyl methacrylate nanospheres surrounded by a chitosan cationic shell, and we used this for encapsulation of a respiratory syncytial virus (RSV-F gene construct (a model for a DNA vaccine. The new nanomaterial was capable of transfecting various eukaryotic cell lines without the use of a commercial transfection reagent. Using transmission electron microscopy, (TEM, fluorescence activated cell sorting (FACS, and immunofluorescence, we clearly demonstrated that the positively charged PCNSs were able to bind to the negatively charged cell membrane and were taken up by endocytosis, in Cos-7 cells. Using quantitative polymerase chain reaction (qPCR, we also evaluated the efficiency of transfection achieved with PCNSs and without the use of a liposomal-based transfection mediator, in Cos-7, HEp-2, and Vero cells. To assess the transfection efficiency of the PCNSs in vivo, these novel nanomaterials containing RSV-F gene were injected intramuscularly into BALB/c mice, resulting in high copy number of the transgene. In this study, we report, for the first time, the application of the PCNSs as a nanovehicle for gene delivery in vitro and in vivo. Keywords: pHEMA+chitosan nanoparticles, nonviral vector

  4. A highly specific pathogen-responsive promoter element from the immediate-early activated CMPG1 gene in Petroselinum crispum.

    Science.gov (United States)

    Kirsch, C; Logemann, E; Lippok, B; Schmelzer, E; Hahlbrock, K

    2001-04-01

    Within the complex signalling network from pathogen-derived elicitor perception to defense-related gene activation, some immediate-early responding genes may have pivotal roles in downstream transcriptional regulation. We have identified the parsley (Petroselinum crispum) ELI17 gene as a particularly fast-responding gene possessing a new type of W box-containing, elicitor-responsive promoter element, E17. Highly selective E17-mediated reporter gene expression at pathogen infection sites in transgenic Arabidopsis thaliana plants demonstrated the potential of this promoter element for designing new strategies in resistance breeding as well as for further analysis of the early components of defense-related gene activation mechanisms. The protein encoded by the ELI17 gene exhibits various structural characteristics of established transcription factors and is designated as a CMPG protein according to the first four strictly conserved amino acids defining a newly emerging class of plant-specific proteins.

  5. Tailor-made TALEN system for highly efficient targeted gene replacement in the rice blast fungus.

    Science.gov (United States)

    Arazoe, Takayuki; Ogawa, Tetsuo; Miyoshi, Kennosuke; Yamato, Tohru; Ohsato, Shuichi; Sakuma, Tetsushi; Yamamoto, Takashi; Arie, Tsutomu; Kuwata, Shigeru

    2015-07-01

    Genetic manipulation is key to unraveling gene functions and creating genetically modified strains of microbial organisms. Recently, engineered nucleases that can generate DNA double-strand breaks (DSBs) at a specific site in the desired locus within genome are utilized in a rapidly developing genome editing technology via DSBs repair. However, the use of engineered nucleases in filamentous fungi has not been validated. In this study, we demonstrated that tailor-made transcriptional activator-like effector nucleases (TALENs) system, Platinum-Fungal TALENs (PtFg TALENs), could improve the efficiency of homologous recombination-mediated targeted gene replacement by up to 100% in the rice blast fungus Pyricularia oryzae. This high-efficiency PtFg TALEN has great potential for basic and applied biological applications in filamentous fungi.

  6. Innate-like functions of natural killer T cell subsets result from highly divergent gene programs.

    Science.gov (United States)

    Engel, Isaac; Seumois, Grégory; Chavez, Lukas; Samaniego-Castruita, Daniela; White, Brandie; Chawla, Ashu; Mock, Dennis; Vijayanand, Pandurangan; Kronenberg, Mitchell

    2016-06-01

    Natural killer T cells (NKT cells) have stimulatory or inhibitory effects on the immune response that can be attributed in part to the existence of functional subsets of NKT cells. These subsets have been characterized only on the basis of the differential expression of a few transcription factors and cell-surface molecules. Here we have analyzed purified populations of thymic NKT cell subsets at both the transcriptomic level and epigenomic level and by single-cell RNA sequencing. Our data indicated that despite their similar antigen specificity, the functional NKT cell subsets were highly divergent populations with many gene-expression and epigenetic differences. Therefore, the thymus 'imprints' distinct gene programs on subsets of innate-like NKT cells that probably impart differences in proliferative capacity, homing, and effector functions.

  7. Shortening trinucleotide repeats using highly specific endonucleases: a possible approach to gene therapy?

    Science.gov (United States)

    Richard, Guy-Franck

    2015-04-01

    Trinucleotide repeat expansions are involved in more than two dozen neurological and developmental disorders. Conventional therapeutic approaches aimed at regulating the expression level of affected genes, which rely on drugs, oligonucleotides, and/or transgenes, have met with only limited success so far. An alternative approach is to shorten repeats to non-pathological lengths using highly specific nucleases. Here, I review early experiments using meganucleases, zinc-finger nucleases (ZFN), and transcription-activator like effector nucleases (TALENs) to contract trinucleotide repeats, and discuss the possibility of using CRISPR-Cas nucleases to the same end. Although this is a nascent field, I explore the possibility of designing nucleases and effectively delivering them in the context of gene therapy.

  8. Predicted Highly Expressed Genes in the Genomes of Streptomyces Coelicolor and Streptomyces Avermitilis and the Implications for their Metabolism.

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Gang; Culley, David E.; Zhang, Weiwen

    2005-06-01

    SUMMARY-Highly expressed genes in bacteria often have a stronger codon bias than genes expressed at lower levels. In this study, a comparative analysis of predicted highly expressed (PHX) genes in the Streptomyces coelicolor and S. avermitilis genomes was performed using the codon adaptation index (CAI) as a numerical estimator of gene expression level. Although it has been suggested that there is little heterogeneity in codon usage in G+C rich bacteria, considerable heterogeneity was found among genes in two G+C rich Streptomyces genomes. Using ribosomal protein (RP) genes as references, ~10% of the genes were predicted to be PHX genes using a CAI cutoff value of greater than 0.78 and 0.75 in S. coelicolor and S. avermitilis, respectively. Most of the PHX genes were found to be located within the conserved cores of the Streptomyces linear chromosomes. The predicted PHX genes showed good agreement with the experimental data on expression levels collected by proteomic analysis (Hesketh et al., 2002). Among all PHX genes, 368 were conserved in both genomes. These represented most of the genes essential for cell growth, including those involved in protein and DNA biosynthesis, amino acid metabolism, central intermediary and energy metabolisms. Only a few genes directly involved in biosynthesis of secondary metabolites were predicted to be PHX genes. Correspondence analysis showed that the genes responsible for biosynthesis of secondary metabolites possessed different codon usage patterns from RP genes, suggesting that they were either under strong translational selection that may have driven the codon preference in another direction, or they were acquired by horizontal transfer during their origin and evolution. Nevertheless, several key genes responsible for producing precursors for secondary metabolites, such as crotonyl-CoA reductase and propionyl-CoA carboxylase, and genes necessary for initiation of secondary metabolism, such as adenosylmethionine synthetase were

  9. High-throughput profiling of antibiotic resistance genes in drinking water treatment plants and distribution systems.

    Science.gov (United States)

    Xu, Like; Ouyang, Weiying; Qian, Yanyun; Su, Chao; Su, Jianqiang; Chen, Hong

    2016-06-01

    Antibiotic resistance genes (ARGs) are present in surface water and often cannot be completely eliminated by drinking water treatment plants (DWTPs). Improper elimination of the ARG-harboring microorganisms contaminates the water supply and would lead to animal and human disease. Therefore, it is of utmost importance to determine the most effective ways by which DWTPs can eliminate ARGs. Here, we tested water samples from two DWTPs and distribution systems and detected the presence of 285 ARGs, 8 transposases, and intI-1 by utilizing high-throughput qPCR. The prevalence of ARGs differed in the two DWTPs, one of which employed conventional water treatments while the other had advanced treatment processes. The relative abundance of ARGs increased significantly after the treatment with biological activated carbon (BAC), raising the number of detected ARGs from 76 to 150. Furthermore, the final chlorination step enhanced the relative abundance of ARGs in the finished water generated from both DWTPs. The total enrichment of ARGs varied from 6.4-to 109.2-fold in tap water compared to finished water, among which beta-lactam resistance genes displayed the highest enrichment. Six transposase genes were detected in tap water samples, with the transposase gene TnpA-04 showing the greatest enrichment (up to 124.9-fold). We observed significant positive correlations between ARGs and mobile genetic elements (MGEs) during the distribution systems, indicating that transposases and intI-1 may contribute to antibiotic resistance in drinking water. To our knowledge, this is the first study to investigate the diversity and abundance of ARGs in drinking water treatment systems utilizing high-throughput qPCR techniques in China.

  10. Analysis of KLLN as a high-penetrance breast cancer predisposition gene.

    Science.gov (United States)

    Thompson, Ella R; Gorringe, Kylie L; Choong, David Y H; Eccles, Diana M; Mitchell, Gillian; Campbell, Ian G

    2012-07-01

    KLLN is a p53 target gene with DNA binding function and represents a highly plausible candidate breast cancer predisposition gene. We screened for predisposing variants in 860 high-risk breast cancer families using high resolution melt analysis. A germline c.339_340delAG variant predicted to cause premature termination of the protein after 57 alternative amino acid residues was identified in 3/860 families who tested negative for BRCA1 and BRCA2 mutations and in 1/84 sporadic breast cancer cases. However, the variant was also detected in 2/182 families with known BRCA1 or BRCA2 mutations and in 2/464 non-cancer controls. Furthermore, loss of the mutant allele was detected in 2/2 breast tumors. Our data suggest that pathogenic mutations in KLLN are rare in breast cancer families and the c.339_340delAG variant does not represent a high-penetrance breast cancer risk allele.

  11. Characterization of the Highly Variable Immune Response Gene Family, He185/333, in the Sea Urchin, Heliocidaris erythrogramma

    OpenAIRE

    Roth, Mattias O.; Wilkins, Adam G.; Cooke, Georgina M.; Raftos, David A; Nair, Sham V.

    2014-01-01

    This study characterizes the highly variable He185/333 genes, transcripts and proteins in coelomocytes of the sea urchin, Heliocidaris erythrogramma. Originally discovered in the purple sea urchin, Strongylocentrotus purpuratus, the products of this gene family participate in the anti-pathogen defenses of the host animals. Full-length He185/333 genes and transcripts are identified. Complete open reading frames of He185/333 homologues are analyzed as to their element structure, single nucleoti...

  12. A high efficiency gene disruption strategy using a positive-negative split selection marker and electroporation for Fusarium oxysporum.

    Science.gov (United States)

    Liang, Liqin; Li, Jianqiang; Cheng, Lin; Ling, Jian; Luo, Zhongqin; Bai, Miao; Xie, Bingyan

    2014-11-01

    The Fusarium oxysporum species complex consists of fungal pathogens that cause serial vascular wilt disease on more than 100 cultivated species throughout the world. Gene function analysis is rapidly becoming more and more important as the whole-genome sequences of various F. oxysporum strains are being completed. Gene-disruption techniques are a common molecular tool for studying gene function, yet are often a limiting step in gene function identification. In this study we have developed a F. oxysporum high-efficiency gene-disruption strategy based on split-marker homologous recombination cassettes with dual selection and electroporation transformation. The method was efficiently used to delete three RNA-dependent RNA polymerase (RdRP) genes. The gene-disruption cassettes of three genes can be constructed simultaneously within a short time using this technique. The optimal condition for electroporation is 10μF capacitance, 300Ω resistance, 4kV/cm field strength, with 1μg of DNA (gene-disruption cassettes). Under these optimal conditions, we were able to obtain 95 transformants per μg DNA. And after positive-negative selection, the transformants were efficiently screened by PCR, screening efficiency averaged 85%: 90% (RdRP1), 85% (RdRP2) and 77% (RdRP3). This gene-disruption strategy should pave the way for high throughout genetic analysis in F. oxysporum.

  13. In situ Expression of Functional Genes Reveals Nitrogen Cycling at High Temperatures in Terrestrial Hydrothermal Systems

    Science.gov (United States)

    Loiacono, S. T.; Meyer-Dombard, D. R.

    2011-12-01

    An essential element for life, nitrogen occurs in all living organisms and is critical for the synthesis of amino acids, proteins, nucleic acids, and other forms of biomass. Thus, nitrogen cycling likely plays a vital role in microbial metabolic processes as well as nutrient availability. For microorganisms in "extreme" environments, this means developing adaptations that allow them to survive in harsh conditions and still perform the metabolisms essential to sustain life. Recent studies have screened biofilms and thermal sediments of Yellowstone National Park (YNP) thermal features for the presence of nifH genes, which code for a key enzyme in the nitrogen fixation process [1-4]. Furthermore, analysis of nitrogen isotopes in biofilms across a temperature and chemical gradient revealed that nitrogen fixation likely varies across the chemosynthetic/photosynthetic ecotone [5]. Although research has evaluated and confirmed the presence of nifH genes in various thermophilic microbial communities, the existence of a gene in the DNA of an organism does not verify its use. Instead, other methods, such as culturing, isotope tracer assays, and gene expression studies are required to provide direct evidence of biological nitrogen fixation. Culturing and isotope tracer approaches have successfully revealed high-temperature biological nitrogen fixation in both marine hydrothermal vent microbial communities [6] and in acidic, terrestrial hydrothermal sediment [3]. Transcriptomics-based techniques (using mRNA extracted from samples to confirm in situ expression of targeted genes) have been much more limited in number, and only a few studies have, to date, investigated in situ expression of the nifH gene in thermophilic microbial communities [2, 7]. This study explores the presence and expression of nifH genes in several features of the Lower Geyser Basin (LGB) of YNP. Nucleic acids from chemosynthetic and photosynthetic microbial communities were extracted and then amplified

  14. Two ras genes in Dictyostelium minutum show high sequence homology, but different developmental regulation from Dictyostelium discoideum rasD and rasG genes.

    Science.gov (United States)

    van Es, S; Kooistra, R A; Schaap, P

    1997-03-10

    The social amoeba Dictyostelium discoideum expresses five ras genes at different stages of development. One of them, DdrasD is expressed during postaggregative development and transcription is induced by extracellular cAMP. A homologue of DdrasD, the DdrasG gene, is expressed exclusively during vegetative growth. We cloned two ras homologues Dmras1 and Dmras2 from the primitive species D. minutum, which show high homology to DdrasD and DdrasG and less homology to the other Ddras genes. In contrast to the DdrasD and DdrasG genes, both the Dmras1 and Dmras2 genes are expressed during the entire course of development. The expression levels are low during growth, increase at the onset of starvation and do not decrease until fruiting bodies have formed. Expression of neither Dmras1 or Dmras2 is regulated by cAMP. So even though the high degree of homology between the ras genes of different species suggests conservation of function, this function is apparently not associated with a specific developmental stage.

  15. Heterogeneous High Throughput Scientific Computing with APM X-Gene and Intel Xeon Phi

    CERN Document Server

    Abdurachmanov, David; Elmer, Peter; Eulisse, Giulio; Knight, Robert; Muzaffar, Shahzad

    2014-01-01

    Electrical power requirements will be a constraint on the future growth of Distributed High Throughput Computing (DHTC) as used by High Energy Physics. Performance-per-watt is a critical metric for the evaluation of computer architectures for cost- efficient computing. Additionally, future performance growth will come from heterogeneous, many-core, and high computing density platforms with specialized processors. In this paper, we examine the Intel Xeon Phi Many Integrated Cores (MIC) co-processor and Applied Micro X-Gene ARMv8 64-bit low-power server system-on-a-chip (SoC) solutions for scientific computing applications. We report our experience on software porting, performance and energy efficiency and evaluate the potential for use of such technologies in the context of distributed computing systems such as the Worldwide LHC Computing Grid (WLCG).

  16. Heterogeneous High Throughput Scientific Computing with APM X-Gene and Intel Xeon Phi

    Science.gov (United States)

    Abdurachmanov, David; Bockelman, Brian; Elmer, Peter; Eulisse, Giulio; Knight, Robert; Muzaffar, Shahzad

    2015-05-01

    Electrical power requirements will be a constraint on the future growth of Distributed High Throughput Computing (DHTC) as used by High Energy Physics. Performance-per-watt is a critical metric for the evaluation of computer architectures for cost- efficient computing. Additionally, future performance growth will come from heterogeneous, many-core, and high computing density platforms with specialized processors. In this paper, we examine the Intel Xeon Phi Many Integrated Cores (MIC) co-processor and Applied Micro X-Gene ARMv8 64-bit low-power server system-on-a-chip (SoC) solutions for scientific computing applications. We report our experience on software porting, performance and energy efficiency and evaluate the potential for use of such technologies in the context of distributed computing systems such as the Worldwide LHC Computing Grid (WLCG).

  17. Gene expression profiling in human peripheral blood mononuclear cells using high-density filter-based cDNA microarrays.

    Science.gov (United States)

    Walker, J; Rigley, K

    2000-05-26

    Microarray technology has provided the ability to analyse the expression profiles for thousands of genes in parallel. The need for highly specialised equipment to use certain types of microarrays has restricted the application of this technology to a small number of dedicated laboratories. High-density filter-based cDNA microarrays provide a low-cost option for performing high-throughput gene expression analysis. We have used a model system in which filter-based cDNA microarrays representing over 4000 known human genes were used to monitor the kinetics of gene expression in human peripheral blood mononuclear cells (PBMCs) stimulated with phytohaemagluttinin (PHA). Using software-based cluster analysis, we identified 104 genes that altered in expression levels in response to PHA stimulation of PBMCs and showed that there was a considerable overlap between genes with similar temporal expression profiles and similar functional roles. Comparison of microarray quantitation with quantitative PCR showed almost identical expression profiles for a number of genes. Coupled with the fact that our findings are in agreement with a large number of independent observations, we conclude that the use of filter-based cDNA microarrays is a valid and accurate method for high-throughput gene expression profiling.

  18. Genomic Integration of High-Risk HPV Alters Gene Expression in Oropharyngeal Squamous Cell Carcinoma.

    Science.gov (United States)

    Walline, Heather M; Komarck, Christine M; McHugh, Jonathan B; Bellile, Emily L; Brenner, J Chad; Prince, Mark E; McKean, Erin L; Chepeha, Douglas B; Wolf, Gregory T; Worden, Francis P; Bradford, Carol R; Carey, Thomas E

    2016-10-01

    High-risk HPV (hrHPV) is the leading etiologic factor in oropharyngeal cancer. HPV-positive oropharyngeal tumors generally respond well to therapy, with complete recovery in approximately 80% of patients. However, it remains unclear why some patients are nonresponsive to treatment, with 20% of patients recurring within 5 years. In this study, viral factors were examined for possible clues to differences in tumor behavior. Oropharynx tumors that responded well to therapy were compared with those that persisted and recurred. Viral oncogene alternate transcripts were assessed, and cellular sites of viral integration were mapped and sequenced. Effects of integration on gene expression were assessed by transcript analysis at the integration sites. All of the tumors demonstrated active viral oncogenesis, indicated by expression of HPV E6 and E7 oncogenes and alternate E6 splicing. In the responsive tumors, HPV integration occurred exclusively in intergenic chromosome regions, except for one tumor with viral integration into TP63. Each recurrent tumor exhibited complex HPV integration patterns into cancer-associated genes, including TNFRSF13B, SCN2A, SH2B1, UBE2V2, SMOC1, NFIA, and SEMA6D Disrupted cellular transcripts were identified in the region of integration in four of the seven affected genes.

  19. Highly efficient method for gene delivery in mouse dorsal root ganglia neurons

    Directory of Open Access Journals (Sweden)

    Lingli eYu

    2015-02-01

    Full Text Available The development of gene transfection technologies has greatly advanced our understanding of life sciences. While use of viral vectors has clear efficacy, it requires specific expertise and biological containment conditions. Electroporation has become an effective and commonly used method for introducing DNA into neurons and in intact brain tissue. The present study describes the use of the Neon® electroporation system to transfect genes into dorsal root ganglia neurons isolated from embryonic mouse Day 13.5 to 16. This cell type has been particularly recalcitrant and refractory to physical or chemical methods for introduction of DNA. By optimizing the culture condition and parameters including voltage and duration for this specific electroporation system, high efficiency (60 – 80% and low toxicity (> 60% survival were achieved with robust differentiation in response to Nerve growth factor (NGF. Moreover, 3-50 times fewer cells are needed (6x104 compared with other traditional electroporation methods. This approach underlines the efficacy of this type of electroporation, particularly when only limited amount of cells can be obtained, and is expected to greatly facilitate the study of gene function in dorsal root ganglia neuron cultures.

  20. A p53 drug response signature identifies prognostic genes in high-risk neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Eveline Barbieri

    Full Text Available Chemotherapy induces apoptosis and tumor regression primarily through activation of p53-mediated transcription. Neuroblastoma is a p53 wild type malignancy at diagnosis and repression of p53 signaling plays an important role in its pathogenesis. Recently developed small molecule inhibitors of the MDM2-p53 interaction are able to overcome this repression and potently activate p53 dependent apoptosis in malignancies with intact p53 downstream signaling. We used the small molecule MDM2 inhibitor, Nutlin-3a, to determine the p53 drug response signature in neuroblastoma cells. In addition to p53 mediated apoptotic signatures, GSEA and pathway analysis identified a set of p53-repressed genes that were reciprocally over-expressed in neuroblastoma patients with the worst overall outcome in multiple clinical cohorts. Multifactorial regression analysis identified a subset of four genes (CHAF1A, RRM2, MCM3, and MCM6 whose expression together strongly predicted overall and event-free survival (p<0.0001. The expression of these four genes was then validated by quantitative PCR in a large independent clinical cohort. Our findings further support the concept that oncogene-driven transcriptional networks opposing p53 activation are essential for the aggressive behavior and poor response to therapy of high-risk neuroblastoma.

  1. High Expression of Endogenous Retroviral Envelope Gene in the Equine Fetal Part of the Placenta.

    Directory of Open Access Journals (Sweden)

    Valentina Stefanetti

    Full Text Available Endogenous retroviruses (ERVs are proviral phases of exogenous retroviruses that have co-evolved with vertebrate genomes for millions of years. Previous studies have identified the envelope (env protein genes of retroviral origin preferentially expressed in the placenta which suggests a role in placentation based on their membrane fusogenic capacity and therefore they have been named syncytins. Until now, all the characterized syncytins have been associated with three invasive placentation types: the endotheliochorial (Carnivora, the synepitheliochorial (Ruminantia, and the hemochorial placentation (human, mouse where they play a role in the syncytiotrophoblast formation. The purpose of the present study was to evaluate whether EqERV env RNA is expressed in horse tissues as well and investigate if the horse, possessing an epitheliochorial placenta, has "captured" a common retroviral env gene with syncytin-like properties in placental tissues. Interestingly, although in the equine placenta there is no syncytiotrophoblast layer at the maternal-fetal interface, our results showed that EqERV env RNA is highly expressed at that level, as expected for a candidate syncytin-like gene but with reduced abundance in the other somatic tissues (nearly 30-fold lower thus suggesting a possible role in the placental tissue. Although the horse is one of the few domestic animals with a sequenced genome, few studies have been conducted about the EqERV and their expression in placental tissue has never been investigated.

  2. 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

  3. [Recent progress in gene mapping through high-throughput sequencing technology and forward genetic approaches].

    Science.gov (United States)

    Lu, Cairui; Zou, Changsong; Song, Guoli

    2015-08-01

    Traditional gene mapping using forward genetic approaches is conducted primarily through construction of a genetic linkage map, the process of which is tedious and time-consuming, and often results in low accuracy of mapping and large mapping intervals. With the rapid development of high-throughput sequencing technology and decreasing cost of sequencing, a variety of simple and quick methods of gene mapping through sequencing have been developed, including direct sequencing of the mutant genome, sequencing of selective mutant DNA pooling, genetic map construction through sequencing of individuals in population, as well as sequencing of transcriptome and partial genome. These methods can be used to identify mutations at the nucleotide level and has been applied in complex genetic background. Recent reports have shown that sequencing mapping could be even done without the reference of genome sequence, hybridization, and genetic linkage information, which made it possible to perform forward genetic study in many non-model species. In this review, we summarized these new technologies and their application in gene mapping.

  4. Recharging cationic DNA complexes with highly charged polyanions for in vitro and in vivo gene delivery.

    Science.gov (United States)

    Trubetskoy, V S; Wong, S C; Subbotin, V; Budker, V G; Loomis, A; Hagstrom, J E; Wolff, J A

    2003-02-01

    The intravenous delivery of plasmid DNA complexed with either cationic lipids (CL) or polyethyleneimine (PEI) enables high levels of foreign gene expression in lung. However, these cationic DNA complexes cause substantial toxicity. The present study found that the inclusion of polyacrylic acid (pAA) with DNA/polycation and DNA/CL complexes prevented the serum inhibition of the transfection complexes in cultured cells. The mechanism mediating this increase seems to involve both particle size enlargement due to flocculation and electrostatic shielding from opsonizing serum proteins. The use of pAA also increased the levels of lung expression in mice in vivo substantially above the levels achieved with just binary complexes of DNA and linear PEI (lPEI) or CL and reduced their toxicity. Also, the use of a "chaser" injection of pAA 30 min after injection of the ternary DNA/lPEI/pAA complexes further aided this effort to reduce toxicity while not affecting foreign gene expression. By optimizing the amount of pAA, lPEI, and DNA within the ternary complexes and using the "chaser" injection, substantial levels of lung expression were obtained while avoiding adverse effects in lung or liver. These developments will aid the use of cationic DNA complexes in animals and for eventual human gene therapy.

  5. Automated cleaning and pre-processing of immunoglobulin gene sequences from high-throughput sequencing

    Directory of Open Access Journals (Sweden)

    Miri eMichaeli

    2012-12-01

    Full Text Available High throughput sequencing (HTS yields tens of thousands to millions of sequences that require a large amount of pre-processing work to clean various artifacts. Such cleaning cannot be performed manually. Existing programs are not suitable for immunoglobulin (Ig genes, which are variable and often highly mutated. This paper describes Ig-HTS-Cleaner (Ig High Throughput Sequencing Cleaner, a program containing a simple cleaning procedure that successfully deals with pre-processing of Ig sequences derived from HTS, and Ig-Indel-Identifier (Ig Insertion – Deletion Identifier, a program for identifying legitimate and artifact insertions and/or deletions (indels. Our programs were designed for analyzing Ig gene sequences obtained by 454 sequencing, but they are applicable to all types of sequences and sequencing platforms. Ig-HTS-Cleaner and Ig-Indel-Identifier have been implemented in Java and saved as executable JAR files, supported on Linux and MS Windows. No special requirements are needed in order to run the programs, except for correctly constructing the input files as explained in the text. The programs' performance has been tested and validated on real and simulated data sets.

  6. Evaluation and Application of Two High-Iron Transgenic Rice Lines Expressing a Pea Ferritin Gene

    Institute of Scientific and Technical Information of China (English)

    YE Hong-xai; LI Mei; Guo Ze-jian; Shu Qing-yao; xu Xiao-hui; BAO Jin-song; SHEN Sheng-quan

    2008-01-01

    A totaI of 105 transgenic rice lines independently transformed with a pea ferritin gene (Fer)were previously obtained.After seven generations of selfing and β-glucuronidase(GUS)assisted selection,82 transgenic lines with stable agronomic traits were got.Among the 82 transgenic lines,two high-iron transgenic rice lines Fer34 and Fer65,with the iron contents in the milled rice being 4.82 and 3.46 times of that of the wild type Xiushui 11,respectively were identified.In the two transgenic lines,the exogenous Fer gene was highly expressed,and inherited as a single locus.The transgene had no negative effect on the agronomic traits of rice plant,other mineral nutritional components,appearance quailty and eating quailty of the milled rice,indicating that these two lines were elite high-iron breeding lines.Furthermore,the practical application and further studies facilitating utilization of the two elite breeding lines were discussed.

  7. [Pulmonary. High tech in pulmonary medicine: GeneXpert, coils and bronchial thermoplasty].

    Science.gov (United States)

    Rochat, Thierrry; Chappuis-Gisin, Emilie; Ongaro, Gioele; Janssens, Jean-Paul; Soccal, Paola M

    2014-01-15

    We discuss here three recent applications of high technology in pulmonary medicine. GeneXpert has considerably improved the diagnosis of active tuberculosis. However, the test has also its limits so that clinical assessment, radiology, and sputum microscopy remain fundamental. Treatment of severe emphysema by lung volume reduction is no longer confined to surgical approach, but is now possible by bronchoscopy. Implantation of Nitinol coils in the most diseased parts of the emphysematous lung is a promising technique. Finally, several clinical trials on bronchial thermoplasty have been reported for intractable asthma. The recent publication of the long term effects of this technique is the occasion to update on this matter.

  8. High diversity and no significant selection signal of human ADH1B gene in Tibet

    Directory of Open Access Journals (Sweden)

    Lu Yan

    2012-11-01

    Full Text Available Abstract Background ADH1B is one of the most studied human genes with many polymorphic sites. One of the single nucleotide polymorphism (SNP, rs1229984, coding for the Arg48His substitution, have been associated with many serious diseases including alcoholism and cancers of the digestive system. The derived allele, ADH1B*48His, reaches high frequency only in East Asia and Southwest Asia, and is highly associated with agriculture. Micro-evolutionary study has defined seven haplogroups for ADH1B based on seven SNPs encompassing the gene. Three of those haplogroups, H5, H6, and H7, contain the ADH1B*48His allele. H5 occurs in Southwest Asia and the other two are found in East Asia. H7 is derived from H6 by the derived allele of rs3811801. The H7 haplotype has been shown to have undergone significant positive selection in Han Chinese, Hmong, Koreans, Japanese, Khazak, Mongols, and so on. Methods In the present study, we tested whether Tibetans also showed evidence for selection by typing 23 SNPs in the region covering the ADH1B gene in 1,175 individuals from 12 Tibetan populations representing all districts of the Tibet Autonomous Region. Multiple statistics were estimated to examine the gene diversities and positive selection signals among the Tibetans and other populations in East Asia. Results The larger Tibetan populations (Qamdo, Lhasa, Nagqu, Nyingchi, Shannan, and Shigatse comprised mostly farmers, have around 12% of H7, and 2% of H6. The smaller populations, living on hunting or recently switched to farming, have lower H7 frequencies (Tingri 9%, Gongbo 8%, Monba and Sherpa 6%. Luoba (2% and Deng (0% have even lower frequencies. Long-range haplotype analyses revealed very weak signals of positive selection for H7 among Tibetans. Interestingly, the haplotype diversity of H7 is higher in Tibetans than in any other populations studied, indicating a longer diversification history for that haplogroup in Tibetans. Network analysis on the long

  9. Reference genes for high-throughput quantitative reverse transcription-PCR analysis of gene expression in organs and tissues of Eucalyptus grown in various environmental conditions.

    Science.gov (United States)

    Cassan-Wang, Hua; Soler, Marçal; Yu, Hong; Camargo, Eduardo Leal O; Carocha, Victor; Ladouce, Nathalie; Savelli, Bruno; Paiva, Jorge A P; Leplé, Jean-Charles; Grima-Pettenati, Jacqueline

    2012-12-01

    Interest in the genomics of Eucalyptus has skyrocketed thanks to the recent sequencing of the genome of Eucalyptus grandis and to a growing number of large-scale transcriptomic studies. Quantitative reverse transcription-PCR (RT-PCR) is the method of choice for gene expression analysis and can now also be used as a high-throughput method. The selection of appropriate internal controls is becoming of utmost importance to ensure accurate expression results in Eucalyptus. To this end, we selected 21 candidate reference genes and used high-throughput microfluidic dynamic arrays to assess their expression among a large panel of developmental and environmental conditions with a special focus on wood-forming tissues. We analyzed the expression stability of these genes by using three distinct statistical algorithms (geNorm, NormFinder and ΔCt), and used principal component analysis to compare methods and rankings. We showed that the most stable genes identified depended not only on the panel of biological samples considered but also on the statistical method used. We then developed a comprehensive integration of the rankings generated by the three methods and identified the optimal reference genes for 17 distinct experimental sets covering 13 organs and tissues, as well as various developmental and environmental conditions. The expression patterns of Eucalyptus master genes EgMYB1 and EgMYB2 experimentally validated our selection. Our findings provide an important resource for the selection of appropriate reference genes for accurate and reliable normalization of gene expression data in the organs and tissues of Eucalyptus trees grown in a range of conditions including abiotic stresses.

  10. Long QT interval in Turner syndrome--a high prevalence of LQTS gene mutations.

    Directory of Open Access Journals (Sweden)

    Christian Trolle

    Full Text Available OBJECTIVES: QT-interval prolongation of unknown aetiology is common in Turner syndrome. This study set out to explore the presence of known long QT mutations in Turner syndrome and to examine the corrected QT-interval (QTc over time and relate the findings to the Turner syndrome phenotype. METHODS: Adult women with Turner syndrome (n = 88 were examined thrice and 68 age-matched healthy controls were examined once. QTc was measured by one blinded reader (intra-reader variability: 0.7%, and adjusted for influence of heart rate by Bazett's (bQTc and Hodges's formula (hQTc. The prevalence of mutations in genes related to Long QT syndrome was determined in women with Turner syndrome and a QTc >432.0 milliseconds (ms. Echocardiographic assessment of aortic valve morphology, 24-hour blood pressures and blood samples were done. RESULTS: The mean hQTc in women with Turner syndrome (414.0 ± 25.5 ms compared to controls (390.4 ± 17.8 ms was prolonged (p432 ms, 7 had mutations in major Long QT syndrome genes (SCN5A and KCNH2 and one in a minor Long QT syndrome gene (KCNE2. CONCLUSION: There is a high prevalence of mutations in the major LQTS genes in women with TS and prolonged QTc. It remains to be settled, whether these findings are related to the unexplained excess mortality in Turner women. CLINICAL TRIAL REGISTRATION: NCT00624949. https://register.clinicaltrials.gov/prs/app/action/SelectProtocol/sid/S0001FLI/selectaction/View/ts/3/uid/U000099E.

  11. Evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry.

    Science.gov (United States)

    Shmueli, Ron B; Bhise, Nupura S; Green, Jordan J

    2013-03-01

    Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery(1,3) . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers(4) that are hydrolytically degradable(5,6) and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)(7), mouse mammary epithelial cells(8), human brain cancer cells(9) and macrovascular (human umbilical vein, HUVECs) endothelial cells(10). A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained(11). In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi

  12. Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA mircoarray

    Institute of Scientific and Technical Information of China (English)

    Xiao-Jing Liu; Li Yang; Feng-Ming Luo; Hong-Bin Wu; Qu-Qiang

    2004-01-01

    AIM: To characterize the gene expression profiles associated with activation of mouse hepatic stellate cell (HSC) and provide novel insights into the pathogenesis of hepatic fibrosis.METHODS: Mice HSCs were isolated from BALB/c mice by in situ perfusion of collagenase and pronase and singlestep density Nycodenz gradient. Total RNA and mRNA of quiescent HSC and culture-activated HSC were extracted,quantified and reversely transcripted into cDNA. cDNAs from activated HSC were labeled with Cy5 and cDNAs from the quiescent HSC were labeled with Cy3, which were mixed with equal quantity, then hybridized with cDNA chips containing 4 000 genes. Chips were washed, scanned and analyzed. Increased expression of 4 genes and decreased expression of one gene in activated HSC were confirmed by reverse transcription- polymerase chain reaction (RT-PCR).RESULTS: A total of 835 differentially expressed genes were identified by cDNA chip between activated and quiescent HSC, and 465 genes were highly expressed in activated HSC. The differentially expressed genes included those involved in protein synthesis, cell-cycle regulation,apoptosis, and DNA damage response.CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis and proliferation were up-regulated in activated HSC. cDNA microarray is an effective technique in screening for differentially expressed genes between two different situations of the HSC. Further analysis of the obtained genes will help understand the molecular mechanism of activation of HSC and hepatic fibrosis.

  13. Metagenomic analysis revealed highly diverse microbial arsenic metabolism genes in paddy soils with low-arsenic contents.

    Science.gov (United States)

    Xiao, Ke-Qing; Li, Li-Guan; Ma, Li-Ping; Zhang, Si-Yu; Bao, Peng; Zhang, Tong; Zhu, Yong-Guan

    2016-04-01

    Microbe-mediated arsenic (As) metabolism plays a critical role in global As cycle, and As metabolism involves different types of genes encoding proteins facilitating its biotransformation and transportation processes. Here, we used metagenomic analysis based on high-throughput sequencing and constructed As metabolism protein databases to analyze As metabolism genes in five paddy soils with low-As contents. The results showed that highly diverse As metabolism genes were present in these paddy soils, with varied abundances and distribution for different types and subtypes of these genes. Arsenate reduction genes (ars) dominated in all soil samples, and significant correlation existed between the abundance of arr (arsenate respiration), aio (arsenite oxidation), and arsM (arsenite methylation) genes, indicating the co-existence and close-relation of different As resistance systems of microbes in wetland environments similar to these paddy soils after long-term evolution. Among all soil parameters, pH was an important factor controlling the distribution of As metabolism gene in five paddy soils (p = 0.018). To the best of our knowledge, this is the first study using high-throughput sequencing and metagenomics approach in characterizing As metabolism genes in the five paddy soil, showing their great potential in As biotransformation, and therefore in mitigating arsenic risk to humans.

  14. Gene Structure and Expression of the High-affinity Nitrate Transport System in Rice Roots

    Institute of Scientific and Technical Information of China (English)

    Chao Cai; Jun-Yi Wang; Yong-Guan Zhu; Qi-Rong Shen; Bin Li; Yi-Ping Tong; Zhen-Sheng Li

    2008-01-01

    Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.

  15. Gene-carried hepatoma targeting complex induced high gene transfection efficiency with low toxicity and significant antitumor activity

    Directory of Open Access Journals (Sweden)

    Zhao QQ

    2012-06-01

    Full Text Available Qing-Qing Zhao,1,2 Yu-Lan Hu,1 Yang Zhou,3 Ni Li,1 Min Han,1 Gu-Ping Tang,4 Feng Qiu,2 Yasuhiko Tabata,5 Jian-Qing Gao,11Institute of Pharmaceutics, Zhejiang University, Hangzhou, China; 2Department of Pharmacy, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China; 3Institute of Biochemistry, Iowa State University, Ames, IA, USA; 4Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou, China; 5Institute for Frontier Medical Sciences, Kyoto University, Kyoto, JapanBackground: The success of gene transfection is largely dependent on the development of a vehicle or vector that can efficiently deliver a gene to cells with minimal toxicity.Methods: A liver cancer-targeted specific peptide (FQHPSF sequence was successfully synthesized and linked with chitosan-linked polyethylenimine (CP to form a new targeted gene delivery vector called CPT (CP/peptide. The structure of CPT was confirmed by 1H nuclear magnetic resonance spectroscopy and ultraviolet spectrophotometry. The particle size of CPT/DNA complexes was measured using laser diffraction spectrometry and the cytotoxicity of the copolymer was evaluated by methylthiazol tetrazolium method. The transfection efficiency evaluation of the CP copolymer was performed using luciferase activity assay. Cellular internalization of the CP/DNA complex was observed under confocal laser scanning microscopy. The targeting specificity of the polymer coupled to peptide was measured by competitive inhibition transfection study. The liver targeting specificity of the CPT copolymer in vivo was demonstrated by combining the copolymer with a therapeutic gene, interleukin-12, and assessed by its abilities in suppressing the growth of ascites tumor in mouse model.Results: The results showed that the liver cancer-targeted specific peptide was successfully synthesized and linked with CP to form a new targeted gene delivery vector called CPT. The composition of CPT

  16. Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits.

    Science.gov (United States)

    Ravel, Catherine; Fiquet, Samuel; Boudet, Julie; Dardevet, Mireille; Vincent, Jonathan; Merlino, Marielle; Michard, Robin; Martre, Pierre

    2014-01-01

    The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs) in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS) are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules (CCRMs) including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs) and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator). In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression.

  17. Development of a facile method for high throughput screening with reporter gene assays.

    Science.gov (United States)

    Goetz, A S; Andrews, J L; Littleton, T R; Ignar, D M

    2000-10-01

    This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

  18. High-throughput chemiluminometric genotyping of single nucleotide polymorphisms of histamine, serotonin, and adrenergic receptor genes.

    Science.gov (United States)

    Toubanaki, Dimitra K; Christopoulos, Theodore K; Ioannou, Penelope C; Flordellis, Christodoulos S

    2009-02-01

    Several pharmacogenetic studies are focused on the investigation of the relation between the efficacy of various antipsychotic agents (e.g., clozapine) and the genetic profile of the patient with an emphasis on genes that code for neurotransmitter receptors such as histamine, serotonin, and adrenergic receptors. We report a high-throughput method for genotyping of single nucleotide polymorphisms (SNPs) within the genes of histamine H2 receptor (HRH2), serotonin receptor (HTR2A1 and HTR2A2), and beta(3) adrenergic receptor (ADRB3). The method combines the high specificity of allele discrimination by oligonucleotide ligation reaction (OLR) and the superior sensitivity and simplicity of chemiluminometric detection in a microtiter well assay configuration. The genomic region that spans the locus of interest is first amplified by polymerase chain reaction (PCR). Subsequently, an oligonucleotide ligation reaction is performed using a biotinylated common probe and two allele-specific probes that are labeled at the 3' end with digoxigenin and fluorescein. The ligation products are immobilized in polystyrene wells via biotin-streptavidin interaction, and the hybrids are denatured. Detection is accomplished by the addition of alkaline phosphatase-conjugated anti-digoxigenin or anti-fluorescein antibodies in combination with a chemiluminogenic substrate. The ratio of the luminescence signals obtained from digoxigenin and fluorescein indicates the genotype of the sample. The method was applied successfully to the genotyping of 23 blood samples for all four SNPs. The results were in concordance with both PCR-restriction fragment length polymorphism analysis and sequencing.

  19. A Biomimic Reconstituted High Density Lipoprotein Nanosystem for Enhanced VEGF Gene Therapy of Myocardial Ischemia

    Directory of Open Access Journals (Sweden)

    Xiaotian Sun

    2015-01-01

    Full Text Available A biomimic reconstituted high density lipoprotein (rHDL based system, rHDL/Stearic-PEI/VEGF complexes, was fabricated as an advanced nanovector for delivering VEGF plasmid. Here, Stearic-PEI was utilized to effectively condense VEGF plasmid and to incorporate the plasmid into rHDL. The rHDL/Stearic-PEI/VEGF complexes with diameter under 100 nm and neutral surface charge demonstrated enhanced stability under the presence of bovine serum albumin. Moreover, in vitro cytotoxicity and transfection assays on H9C2 cells further revealed their superiority, as they displayed lower cytotoxicity with much higher transfection efficiency when compared to PEI 10K/VEGF and Lipos/Stearic-PEI/VEGF complexes. In addition, in vivo investigation on ischemia/reperfusion rat model implied that rHDL/Stearic-PEI/VEGF complexes possessed high transgene capacity and strong therapeutic activity. These findings indicated that rHDL/Stearic-PEI/VEGF complexes could be an ideal gene delivery system for enhanced VEGF gene therapy of myocardial ischemia, which might be a new promising strategy for effective myocardial ischemia treatment.

  20. Expression Changes of Early Response Genes in Lung Due to High Volume Ventilation

    Institute of Scientific and Technical Information of China (English)

    WANG Yuelan; YAO Shanglong; XIONG Ping

    2005-01-01

    Summary: The expression changes of early response genes due to ventilation with high volume in adult rats in vivo were observed. Forty SD male rats were randomly divided into control and 30, 60, 90 and 120 min ventilation groups, respectively (n=8 in each group). The animals were ventilated with tidal volume of 42 ml/kg and a PEEP level of 0 cmH2O at a rate of 40 breaths per minute in room air with a ventilator was given to the small animals. The expression of Egr-1, C-jun and IL-1β mRNA and proteins was detected by RT-PCR and immunohistochemical technique, respectively. The pathological changes in lung tissues were examined by HE staining. The results indicated that the expression of Egr-1, C-jun and IL-1β mRNA was detectable at 30th min after overventilation, but there was no significant difference in comparison with that in control group until overventilation for 60 min. However, at 90 and 120 min there was a significent increase as compared with 30 min or control group (P<0.05). The expression of Egr-1, C-jun and IL-1β deteced by immunohistochemical assay also showed a similar tendency of the gradual increase. In the 120 min ventilation group, the expression intensity of Egr-1, C-jun and IL-1β proteins in lung cells was the strongest and the nuclear translocation was increased markedly in comparison with any other groups (P<0.05). HE staining suggested that the degree of lung injury was aggravated gradually with the ventialtion going on and had a similar tendency to the expression of these early response genes and proteins. The current data suggested that overventilation activated and upregulated the expression of early response genes and the expression of these genes may be taken as the early signal to predict the onset and degree of lung injury. These results may demonstrated partially that the expression of early response genes induced by the mechanical stretch is associated with biochamic lung injury.

  1. GeneLab for High Schools: Data Mining for the Next Generation

    Science.gov (United States)

    Blaber, Elizabeth A.; Ly, Diana; Sato, Kevin Y.; Taylor, Elizabeth

    2016-01-01

    Modern biological sciences have become increasingly based on molecular biology and high-throughput molecular techniques, such as genomics, transcriptomics, and proteomics. NASA Scientists and the NASA Space Biology Program have aimed to examine the fundamental building blocks of life (RNA, DNA and protein) in order to understand the response of living organisms to space and aid in fundamental research discoveries on Earth. In an effort to enable NASA funded science to be available to everyone, NASA has collected the data from omics studies and curated them in a data system called GeneLab. Whilst most college-level interns, academics and other scientists have had some interaction with omics data sets and analysis tools, high school students often have not. Therefore, the Space Biology Program is implementing a new Summer Program for high-school students that aims to inspire the next generation of scientists to learn about and get involved in space research using GeneLabs Data System. The program consists of three main components core learning modules, focused on developing students knowledge on the Space Biology Program and Space Biology research, Genelab and the data system, and previous research conducted on model organisms in space; networking and team work, enabling students to interact with guest lecturers from local universities and their fellow peers, and also enabling them to visit local universities and genomics centers around the Bay area; and finally an independent learning project, whereby students will be required to form small groups, analyze a dataset on the Genelab platform, generate a hypothesis and develop a research plan to test their hypothesis. This program will not only help inspire high-school students to become involved in space-based research but will also help them develop key critical thinking and bioinformatics skills required for most college degrees and furthermore, will enable them to establish networks with their peers and connections

  2. High-throughput mapping of the promoters of the mouse olfactory receptor genes reveals a new type of mammalian promoter and provides insight into olfactory receptor gene regulation.

    Science.gov (United States)

    Clowney, E Josephine; Magklara, Angeliki; Colquitt, Bradley M; Pathak, Nidhi; Lane, Robert P; Lomvardas, Stavros

    2011-08-01

    The olfactory receptor (OR) genes are the largest mammalian gene family and are expressed in a monogenic and monoallelic fashion in olfactory neurons. Using a high-throughput approach, we mapped the transcription start sites of 1085 of the 1400 murine OR genes and performed computational analysis that revealed potential transcription factor binding sites shared by the majority of these promoters. Our analysis produced a hierarchical model for OR promoter recognition in which unusually high AT content, a unique epigenetic signature, and a stereotypically positioned O/E site distinguish OR promoters from the rest of the murine promoters. Our computations revealed an intriguing correlation between promoter AT content and evolutionary plasticity, as the most AT-rich promoters regulate rapidly evolving gene families. Within the AT-rich promoter category the position of the TATA-box does not correlate with the transcription start site. Instead, a spike in GC composition might define the exact location of the TSS, introducing the concept of "genomic contrast" in transcriptional regulation. Finally, our experiments show that genomic neighborhood rather than promoter sequence correlates with the probability of different OR genes to be expressed in the same olfactory cell.

  3. Gene expression identifies heterogeneity of metastatic propensity in high-grade soft tissue sarcomas

    DEFF Research Database (Denmark)

    Skubitz, Keith M; Francis, Princy; Skubitz, Amy P N;

    2012-01-01

    Metastatic propensity of soft tissue sarcoma (STS) is heterogeneous and may be determined by gene expression patterns that do not correlate well with morphology. The authors have reported gene expression patterns that distinguish 2 broad classes of clear cell renal carcinoma (ccRCC-gene set......), and other patterns that can distinguish heterogeneity of serous ovarian carcinoma (OVCA-gene set) and aggressive fibromatosis (AF-gene set); however, clinical follow-up data were not available for these samples....

  4. Cellular effect of high doses of silica-coated quantum dot profiled with high throughput gene expression analysis and high content cellomics measurements.

    Science.gov (United States)

    Zhang, Tingting; Stilwell, Jackie L; Gerion, Daniele; Ding, Lianghao; Elboudwarej, Omeed; Cooke, Patrick A; Gray, Joe W; Alivisatos, A Paul; Chen, Fanqing Frank

    2006-04-01

    Quantum dots (Qdots) are now used extensively for labeling in biomedical research, and this use is predicted to grow because of their many advantages over alternative labeling methods. Uncoated Qdots made of core/shell CdSe/ZnS are toxic to cells because of the release of Cd2+ ions into the cellular environment. This problem has been partially overcome by coating Qdots with polymers, poly(ethylene glycol) (PEG), or other inert molecules. The most promising coating to date, for reducing toxicity, appears to be PEG. When PEG-coated silanized Qdots (PEG-silane-Qdots) are used to treat cells, toxicity is not observed, even at dosages above 10-20 nM, a concentration inducing death when cells are treated with polymer or mercaptoacid coated Qdots. Because of the importance of Qdots in current and future biomedical and clinical applications, we believe it is essential to more completely understand and verify this negative global response from cells treated with PEG-silane-Qdots. Consequently, we examined the molecular and cellular response of cells treated with two different dosages of PEG-silane-Qdots. Human fibroblasts were exposed to 8 and 80 nM of these Qdots, and both phenotypic as well as whole genome expression measurements were made. PEG-silane-Qdots did not induce any statistically significant cell cycle changes and minimal apoptosis/necrosis in lung fibroblasts (IMR-90) as measured by high content image analysis, regardless of the treatment dosage. A slight increase in apoptosis/necrosis was observed in treated human skin fibroblasts (HSF-42) at both the low and the high dosages. We performed genome-wide expression array analysis of HSF-42 exposed to doses 8 and 80 nM to link the global cell response to a molecular and genetic phenotype. We used a gene array containing approximately 22,000 total probe sets, containing 18,400 probe sets from known genes. Only approximately 50 genes (approximately 0.2% of all the genes tested) exhibited a statistically significant

  5. Bifunctional chimeric SuperCD suicide gene -YCD: YUPRT fusion is highly effective in a rat hepatoma model

    Institute of Scientific and Technical Information of China (English)

    Florian Graepler; Ulrike A Lauer; Reinhard Vonthein; Michael Gregor; Sorin Armeanu; Michael Bitzer; Ulrich M. Lauer; Marie-Luise Lemken; Wolfgang A Wybranietz; Ulrike Schmidt; Irina Smirnow; Christine D Groβ; Martin Spiegel; Andrea Schenk; Hansj(o)rg Graf

    2005-01-01

    AIM: To investigate the effects of catalytically superior gene-directed enzyme prodrug therapy systems on a rat hepatoma model.METHODS: To increase hepatoma cell chemosensitivity for the prodrug 5-fluorocytosine (5-FC), we generated a chimeric bifunctional SuperCD suicide gene, a fusion of the yeast cytosine deaminase (YCD) and the yeast uracil phosphoribosyltransferase (YUPRT) gene.RESULTS: In vitro stably transduced Morris rat hepatoma cells (MH) expressing the bifunctional SuperCD suicide gene (MH SuperCD) showed a clearly marked enhancement in cell killing when incubated with 5-FC as compared with MH ceils stably expressing YCD solely (MH YCD) or the cytosine deaminase gene of bacterial origin(MH BCD), respectively. In vivo, MH SuperCD tumors implanted both subcutaneously as well as orthotopically into the livers of syngeneic ACI rats demonstrated significant tumor regressions (P<0.01) under both high dose as well as low dose systemic 5-FC application,whereas MH tumors without transgene expression (MH naive) showed rapid progression. For the first time, an order of in vivo suicide gene effectiveness (SuperCD>>YCD > > BCD > > > negative control) was defi ned as a result of a directin vivo comparison of all three suicide genes.CONCLUSION: Bifunctional SuperCD suicide gene expression is highly effective in a rat hepatoma model,thereby significantly improving both the therapeutic index and the efficacy of hepatocellular carcinoma killing by fluorocytosine.

  6. Expression of Selenoprotein Genes Is Affected by Obesity of Pigs Fed a High-Fat Diet123

    Science.gov (United States)

    Zhao, Hua; Li, Ke; Tang, Jia-Yong; Zhou, Ji-Chang; Wang, Kang-Ning; Xia, Xin-Jie; Lei, Xin Gen

    2015-01-01

    Background: Relations of the 25 mammalian selenoprotein genes with obesity and the associated inflammation remain unclear. Objective: This study explored impacts of high-fat diet-induced obesity on inflammation and expressions of selenoprotein and obesity-related genes in 10 tissues of pigs. Methods: Plasma and 10 tissues were collected from pigs (n = 10) fed a corn-soy–based control diet or that diet containing 3–7% lard from weanling to finishing (180 d). Plasma concentrations (n = 8) of cytokines and thyroid hormones and tissue mRNA abundance (n = 4) of 25 selenoprotein genes and 16 obesity-related genes were compared between the pigs fed the control and high-fat diets. Stepwise regression was applied to analyze correlations among all these measures, including the previously reported body physical and plasma biochemical variables. Results: The high-fat diet elevated (P < 0.05) plasma concentrations of tumor necrosis factor α, interleukin-6, leptin, and leptin receptor by 29–42% and affected (P < 0.05–0.1) tissue mRNA levels of the selenoprotein and obesity-related genes in 3 patterns. Specifically, the high-fat diet up-regulated 12 selenoprotein genes in 6 tissues, down-regulated 13 selenoprotein genes in 7 tissues, and exerted no effect on 5 genes in any tissue. Body weights and plasma triglyceride concentrations of pigs showed the strongest regressions to tissue mRNA abundances of selenoprotein and obesity-related genes. Among the selenoprotein genes, selenoprotein V and I were ranked as the strongest independent variables for the regression of phenotypic and plasma measures. Meanwhile, agouti signaling protein, adiponectin, and resistin genes represented the strongest independent variables of the obesity-related genes for the regression of tissue selenoprotein mRNA. Conclusions: The high-fat diet induced inflammation in pigs and affected their gene expression of selenoproteins associated with thioredoxin and oxidoreductase systems, local tissue

  7. Comparison of the △12 fatty acid desaturase gene between high-oleic and normal-oleic peanut genotypes

    Institute of Scientific and Technical Information of China (English)

    ShanlinYu; Lijuan Pan; Qingli Yang; Ping Min; Zengkai Ren; Hongsheng Zhang

    2008-01-01

    △12fatty acid desaturase gene has been targeted as a logical candidate controlling the high oleate trait in peanut seeds. By RT-PCR method, the full-length cDNAs of △12 fatty acid desaturase gene were isolated from peanut (Arachis hypogaea L.) genotypes with normal and high ratio of oleic to linoleic acid, which were designated AhFAD2B and AhFAD2B', respectively. Sequence alignment of their coding regions revealed that an extra A was inserted at the position +442 bp of AhFAD2B' sequence of high oleic acid genotypes, which resulted in the shift of open reading frame and a truncated protein AhFAD2B', with the loss of one histidine box involved in metal ion complex required for the reduction of oxygen. Analysis of transcript level showed that the expression of △12 fatty acid desaturase gene in high oleic acid genotype was slightly lower than that in normal genotype. The enzyme activity experiment of yeast (Saccharomyces cerevisiae) cell transformed with AhFAD2B or AhFAD2B' proved that only AhFAD2B gene product showed significant A12 fatty acid desaturase activity,but AhFAD2B' gene product did not. These results suggested that the change of AhFAD2B' gene sequence resulted in lower activity or deactivation of △12 fatty acid desaturase in high oleic acid genotype.

  8. Study of gene function based on spatial co-expression in a high-resolution mouse brain atlas

    Directory of Open Access Journals (Sweden)

    Jiang Tao

    2007-04-01

    Full Text Available Abstract Background The Allen Brain Atlas (ABA project systematically profiles three-dimensional high-resolution gene expression in postnatal mouse brains for thousands of genes. By unveiling gene behaviors at both the cellular and molecular levels, ABA is becoming a unique and comprehensive neuroscience data source for decoding enigmatic biological processes in the brain. Given the unprecedented volume and complexity of the in situ hybridization image data, data mining in this area is extremely challenging. Currently, the ABA database mainly serves as an online reference for visual inspection of individual genes; the underlying rich information of this large data set is yet to be explored by novel computational tools. In this proof-of-concept study, we studied the hypothesis that genes sharing similar three-dimensional expression profiles in the mouse brain are likely to share similar biological functions. Results In order to address the pattern comparison challenge when analyzing the ABA database, we developed a robust image filtering method, dubbed histogram-row-column (HRC algorithm. We demonstrated how the HRC algorithm offers the sensitivity of identifying a manageable number of gene pairs based on automatic pattern searching from an original large brain image collection. This tool enables us to quickly identify genes of similar in situ hybridization patterns in a semi-automatic fashion and consequently allows us to discover several gene expression patterns with expression neighborhoods containing genes of similar functional categories. Conclusion Given a query brain image, HRC is a fully automated algorithm that is able to quickly mine vast number of brain images and identify a manageable subset of genes that potentially shares similar spatial co-distribution patterns for further visual inspection. A three-dimensional in situ hybridization pattern, if statistically significant, could serve as a fingerprint of certain gene function

  9. Whole-exome sequencing and high throughput genotyping identified KCNJ11 as the thirteenth MODY gene.

    Directory of Open Access Journals (Sweden)

    Amélie Bonnefond

    Full Text Available BACKGROUND: Maturity-onset of the young (MODY is a clinically heterogeneous form of diabetes characterized by an autosomal-dominant mode of inheritance, an onset before the age of 25 years, and a primary defect in the pancreatic beta-cell function. Approximately 30% of MODY families remain genetically unexplained (MODY-X. Here, we aimed to use whole-exome sequencing (WES in a four-generation MODY-X family to identify a new susceptibility gene for MODY. METHODOLOGY: WES (Agilent-SureSelect capture/Illumina-GAIIx sequencing was performed in three affected and one non-affected relatives in the MODY-X family. We then performed a high-throughput multiplex genotyping (Illumina-GoldenGate assay of the putative causal mutations in the whole family and in 406 controls. A linkage analysis was also carried out. PRINCIPAL FINDINGS: By focusing on variants of interest (i.e. gains of stop codon, frameshift, non-synonymous and splice-site variants not reported in dbSNP130 present in the three affected relatives and not present in the control, we found 69 mutations. However, as WES was not uniform between samples, a total of 324 mutations had to be assessed in the whole family and in controls. Only one mutation (p.Glu227Lys in KCNJ11 co-segregated with diabetes in the family (with a LOD-score of 3.68. No KCNJ11 mutation was found in 25 other MODY-X unrelated subjects. CONCLUSIONS/SIGNIFICANCE: Beyond neonatal diabetes mellitus (NDM, KCNJ11 is also a MODY gene ('MODY13', confirming the wide spectrum of diabetes related phenotypes due to mutations in NDM genes (i.e. KCNJ11, ABCC8 and INS. Therefore, the molecular diagnosis of MODY should include KCNJ11 as affected carriers can be ideally treated with oral sulfonylureas.

  10. The Novel Gene CRNDE Encodes a Nuclear Peptide (CRNDEP Which Is Overexpressed in Highly Proliferating Tissues.

    Directory of Open Access Journals (Sweden)

    Lukasz Michal Szafron

    Full Text Available CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene.

  11. Down-regulation of EPHX2 gene transcription by Sp1 under high-glucose conditions.

    Science.gov (United States)

    Oguro, Ami; Oida, Shoko; Imaoka, Susumu

    2015-09-15

    sEH (soluble epoxide hydrolase), which is encoded by the EPHX2 gene, regulates the actions of bioactive lipids, EETs (epoxyeicosatrienoic acids). Previously, we found that high-glucose-induced oxidative stress suppressed sEH levels in a hepatocarcinoma cell line (Hep3B) and sEH was decreased in streptozotocin-induced diabetic mice in vivo. In the present study, we investigated the regulatory mechanisms underlying EPHX2 transcriptional suppression under high-glucose conditions. The decrease in sEH was prevented by an Sp1 (specificity protein 1) inhibitor, mithramycin A, and overexpression or knockdown of Sp1 revealed that Sp1 suppressively regulated sEH expression, in contrast with the general role of Sp1 on transcriptional activation. In addition, we found that AP2α (activating protein 2α) promoted EPHX2 transcription. The nuclear transport of Sp1, but not that of AP2α, was increased under high glucose concomitantly with the decrease in sEH. Within the EPHX2 promoter -56/+32, five Sp1-binding sites were identified, and the mutation of each of these sites showed that the first one (SP1_1) was important in both suppression by Sp1 and activation by AP2α. Furthermore, overexpression of Sp1 diminished the binding of AP2α by DNA-affinity precipitation assay and ChIP, suggesting competition between Sp1 and AP2α on the EPHX2 promoter. These findings provide novel insights into the role of Sp1 in transcriptional suppression, which may be applicable to the transcriptional regulation of other genes.

  12. 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....

  13. Locus heterogeneity disease genes encode proteins with high interconnectivity in the human protein interaction network.

    Science.gov (United States)

    Keith, Benjamin P; Robertson, David L; Hentges, Kathryn E

    2014-01-01

    Mutations in genes potentially lead to a number of genetic diseases with differing severity. These disease genes have been the focus of research in recent years showing that the disease gene population as a whole is not homogeneous, and can be categorized according to their interactions. Locus heterogeneity describes a single disorder caused by mutations in different genes each acting individually to cause the same disease. Using datasets of experimentally derived human disease genes and protein interactions, we created a protein interaction network to investigate the relationships between the products of genes associated with a disease displaying locus heterogeneity, and use network parameters to suggest properties that distinguish these disease genes from the overall disease gene population. Through the manual curation of known causative genes of 100 diseases displaying locus heterogeneity and 397 single-gene Mendelian disorders, we use network parameters to show that our locus heterogeneity network displays distinct properties from the global disease network and a Mendelian network. Using the global human proteome, through random simulation of the network we show that heterogeneous genes display significant interconnectivity. Further topological analysis of this network revealed clustering of locus heterogeneity genes that cause identical disorders, indicating that these disease genes are involved in similar biological processes. We then use this information to suggest additional genes that may contribute to diseases with locus heterogeneity.

  14. Post genome-wide association studies of novel genes associated with type 2 diabetes show gene-gene interaction and high predictive value.

    Directory of Open Access Journals (Sweden)

    Stéphane Cauchi

    Full Text Available BACKGROUND: Recently, several Genome Wide Association (GWA studies in populations of European descent have identified and validated novel single nucleotide polymorphisms (SNPs, highly associated with type 2 diabetes (T2D. Our aims were to validate these markers in other European and non-European populations, then to assess their combined effect in a large French study comparing T2D and normal glucose tolerant (NGT individuals. METHODOLOGY/PRINCIPAL FINDINGS: In the same French population analyzed in our previous GWA study (3,295 T2D and 3,595 NGT, strong associations with T2D were found for CDKAL1 (OR(rs7756992 = 1.30[1.19-1.42], P = 2.3x10(-9, CDKN2A/2B (OR(rs10811661 = 0.74[0.66-0.82], P = 3.5x10(-8 and more modestly for IGFBP2 (OR(rs1470579 = 1.17[1.07-1.27], P = 0.0003 SNPs. These results were replicated in both Israeli Ashkenazi (577 T2D and 552 NGT and Austrian (504 T2D and 753 NGT populations (except for CDKAL1 but not in the Moroccan population (521 T2D and 423 NGT. In the overall group of French subjects (4,232 T2D and 4,595 NGT, IGFBP2 and CXCR4 synergistically interacted with (LOC38776, SLC30A8, HHEX and (NGN3, CDKN2A/2B, respectively, encoding for proteins presumably regulating pancreatic endocrine cell development and function. The T2D risk increased strongly when risk alleles, including the previously discovered T2D-associated TCF7L2 rs7903146 SNP, were combined (8.68-fold for the 14% of French individuals carrying 18 to 30 risk alleles with an allelic OR of 1.24. With an area under the ROC curve of 0.86, only 15 novel loci were necessary to discriminate French individuals susceptible to develop T2D. CONCLUSIONS/SIGNIFICANCE: In addition to TCF7L2, SLC30A8 and HHEX, initially identified by the French GWA scan, CDKAL1, IGFBP2 and CDKN2A/2B strongly associate with T2D in French individuals, and mostly in populations of Central European descent but not in Moroccan subjects. Genes expressed in the pancreas interact together and their

  15. Nonviral gene-delivery by highly fluorinated gemini bispyridinium surfactant-based DNA nanoparticles.

    Science.gov (United States)

    Fisicaro, Emilia; Compari, Carlotta; Bacciottini, Franco; Contardi, Laura; Pongiluppi, Erika; Barbero, Nadia; Viscardi, Guido; Quagliotto, Pierluigi; Donofrio, Gaetano; Krafft, Marie Pierre

    2017-02-01

    Biological and thermodynamic properties of a new homologous series of highly fluorinated bispyridinium cationic gemini surfactants, differing in the length of the spacer bridging the pyridinium polar heads in 1,1' position, are reported for the first time. Interestingly, gene delivery ability is closely associated with the spacer length due to a structural change of the molecule in solution. This conformation change is allowed when the spacer reaches the right length, and it is suggested by the trends of the apparent and partial molar enthalpies vs molality. To assess the compounds' biological activity, they were tested with an agarose gel electrophoresis mobility shift assay (EMSA), MTT proliferation assay and Transient Transfection assays on a human rhabdomyosarcoma cell line. Data from atomic force microscopy (AFM) allow for morphological characterization of DNA nanoparticles. Dilution enthalpies, measured at 298K, enabled the determination of apparent and partial molar enthalpies vs molality. All tested compounds (except that with the longest spacer), at different levels, can deliver the plasmid when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE). The compound with a spacer formed by eight carbon atoms gives rise to a gene delivery ability that is comparable to that of the commercial reagent. The compound with the longest spacer compacts DNA in loosely condensed structures by forming bows, which are not suitable for transfection. Regarding the compounds' hydrogenated counterparts, the tight relationship between the solution thermodynamics data and their biological performance is amazing, making "old" methods the foundation to deeply understanding "new" applications.

  16. Single cell visualization of transcription kinetics variance of highly mobile identical genes using 3D nanoimaging.

    Science.gov (United States)

    Annibale, Paolo; Gratton, Enrico

    2015-03-19

    Multi-cell biochemical assays and single cell fluorescence measurements revealed that the elongation rate of Polymerase II (PolII) in eukaryotes varies largely across different cell types and genes. However, there is not yet a consensus whether intrinsic factors such as the position, local mobility or the engagement by an active molecular mechanism of a genetic locus could be the determinants of the observed heterogeneity. Here by employing high-speed 3D fluorescence nanoimaging techniques we resolve and track at the single cell level multiple, distinct regions of mRNA synthesis within the model system of a large transgene array. We demonstrate that these regions are active transcription sites that release mRNA molecules in the nucleoplasm. Using fluctuation spectroscopy and the phasor analysis approach we were able to extract the local PolII elongation rate at each site as a function of time. We measured a four-fold variation in the average elongation between identical copies of the same gene measured simultaneously within the same cell, demonstrating a correlation between local transcription kinetics and the movement of the transcription site. Together these observations demonstrate that local factors, such as chromatin local mobility and the microenvironment of the transcription site, are an important source of transcription kinetics variability.

  17. Zebrafish Cx35: cloning and characterization of a gap junction gene highly expressed in the retina.

    Science.gov (United States)

    McLachlan, Elizabeth; White, Thomas W; Ugonabo, Chioma; Olson, Carl; Nagy, James I; Valdimarsson, Gunnar

    2003-09-15

    The vertebrate connexin gene family encodes protein subunits of gap junction channels, which provide a route for direct intercellular communication. Consequently, gap junctions play a vital role in many developmental and homeostatic processes. Aberrant functioning of gap junctions is implicated in many human diseases. Zebrafish are an ideal vertebrate model to study development of the visual system as they produce transparent embryos that develop rapidly, thereby facilitating morphological and behavioral testing. In this study, zebrafish connexin35 has been cloned from a P1 artificial chromosome (PAC) library. Sequence analysis shows a high degree of similarity to the Cx35/36 orthologous group, which are expressed primarily in nervous tissue, including the retina. The gene encodes a 304-amino acid protein with a predicted molecular weight of approximately 35 kDa. Injection of zebrafish Cx35 RNA into paired Xenopus oocytes elicited intercellular electrical coupling with weak voltage sensitivity. In development, Cx35 is first detectable by Northern analysis and RT-PCR, at 2 days post-fertilization (2 dpf), and in the adult it is expressed in the brain and retina. Immunohistochemical analysis revealed that the Cx35 protein is expressed in two sublaminae of the inner plexiform layer of the adult retina. A similar pattern was seen in the 4 and 5 dpf retina, but no labeling was detected in the retina of earlier embryos.

  18. Low and high expressing alleles of the LMNA gene: implications for laminopathy disease development.

    Directory of Open Access Journals (Sweden)

    Sofía Rodríguez

    Full Text Available Today, there are at least a dozen different genetic disorders caused by mutations within the LMNA gene, and collectively, they are named laminopathies. Interestingly, the same mutation can cause phenotypes with different severities or even different disorders and might, in some cases, be asymptomatic. We hypothesized that one possible contributing mechanism for this phenotypic variability could be the existence of high and low expressing alleles in the LMNA locus. To investigate this hypothesis, we developed an allele-specific absolute quantification method for lamin A and lamin C transcripts using the polymorphic rs4641(C/TLMNA coding SNP. The contribution of each allele to the total transcript level was investigated in nine informative human primary dermal fibroblast cultures from Hutchinson-Gilford progeria syndrome (HGPS and unaffected controls. Our results show differential expression of the two alleles. The C allele is more frequently expressed and accounts for ∼70% of the lamin A and lamin C transcripts. Analysis of samples from six patients with Hutchinson-Gilford progeria syndrome showed that the c.1824C>T, p.G608G mutation is located in both the C and the T allele, which might account for the variability in phenotype seen among HGPS patients. Our method should be useful for further studies of human samples with mutations in the LMNA gene and to increase the understanding of the link between genotype and phenotype in laminopathies.

  19. Total beta-globin gene deletion has high frequency in Filipinos

    Energy Technology Data Exchange (ETDEWEB)

    Patrick, N.; Miyakawa, F.; Hunt, J.A. [Univ. of Hawaii, Honolulu, HI (United States)] [and others

    1994-09-01

    The distribution of {beta}-thalassemia [{beta}{sup Th}] mutations is unique to each ethnic group. Most mutations affect one or a few bases; large deletions have been rare. Among families screened in Hawaii, [{beta}{sup Th}] heterozygotes were diagnosed by microcytosis, absence of abnormal hemoglobins on isoelectric focusing, and raised Hb A{sub 2} by chromatography. Gene frequency for {beta}{sup Th} was 0.02 in Filipinos. In Filipinos, polymerase chain reaction [PCR] with denaturing gradient gel electrophoresis for {beta}{sup Th} mutations detected a mutation in only 6 of 42 {beta}{sup Th} heterozygotes; an IVS2-666 C/T polymorphism showed non-heterozygosity in 37 and heterozygosity in only 5 of these {beta}{sup Th} heterozygotes. One {beta}{sup Th}/{beta}{sup Th} major patient and his mother had no mutation detected by allele-specific oligomer hybridization; PCR failed to amplify any DNA from his {beta}-globin gene. After a total {beta}-globin gene deletion [{beta}{sup Del}] was found in a Filipino family in Ontario, specific PCR amplification for {beta}{sup Del} detected this in 43 of 53 {beta}{sup Th} Filipino samples tested; the above {beta}{sup Th}/{beta}{sup Th} patient was a ({beta}{sup Del}/{beta}{sup Del}) homozygote. The {beta}{sup Del} may account for over 60% of all {beta}{sup Th} alleles in Filipinos; this is the highest proportion of a deletion {beta}{sup Th} mutation reported from any population. Most but not all {beta}{sup Del} heterozygotes had high Hb F [5.13 {plus_minus} 3.94 mean {plus_minus} 1 s.d.] compared to the codon 41/42 four base deletion common in Chinese [2.30 {plus_minus} 0.86], or to {beta}{sup Th} heterozygotes with normal {alpha}-globin genes [2.23 {plus_minus} 0.80].

  20. The active gene that encodes human High Mobility Group 1 protein (HMG1) contains introns and maps to chromosome 13

    Energy Technology Data Exchange (ETDEWEB)

    Ferrari, S. [Dipartimento di Genetica e di Biologia dei Microrganismi, Milan (Italy); Finelli, P.; Rocchi, M. [Istituto di Genetica, Bari (Italy)] [and others

    1996-07-15

    The human genome contains a large number of sequences related to the cDNA for High Mobility Group 1 protein (HMG1), which so far has hampered the cloning and mapping of the active HMG1 gene. We show that the human HMG1 gene contains introns, while the HMG1-related sequences do not and most likely are retrotransposed pseudogenes. We identified eight YACs from the ICI and CEPH libraries that contain the human HMG1 gene. The HMG1 gene is similar in structure to the previously characterized murine homologue and maps to human chromosome 13 and q12, as determined by in situ hybridization. The mouse Hmg1 gene maps to the telomeric region of murine Chromosome 5, which is syntenic to the human 13q12 band. 18 refs., 3 figs.

  1. Detection of the esp gene in high-level gentamicin resistant Enterococcus faecalis strains from pet animals in Japan.

    Science.gov (United States)

    Harada, Tetsuya; Tsuji, Noboru; Otsuki, Koichi; Murase, Toshiyuki

    2005-03-20

    We investigated the prevalence of the esp gene and the susceptibility to gentamicin in Enterococcus faecalis and E. faecium strains obtained from pet animals. Nine of 30 E. faecalis and 2 of 38 E. faecium strains from the pet animals had the esp gene. Three esp-positive E. faecalis strains, which were isolated from two dogs and a cat, showed gentamicin MICs of > or =256 microg/ml and harbored the high-level gentamicin resistance (HLGR) gene, aac(6')-Ie-aph(2'')-Ia. Of the nine esp-positive E. faecalis strains, five, including the three strains with the HLGR gene, were closely related by numerical analysis of PFGE patterns. Longitudinal investigation needs to elucidate whether the HLGR gene was incorporated into a subpopulation of the esp-positive E. faecalis.

  2. High frequency of virulence factor genes tdh, trh, and tlh in Vibrio parahaemolyticus strains isolated from a pristine estuary.

    Science.gov (United States)

    Gutierrez West, Casandra K; Klein, Savannah L; Lovell, Charles R

    2013-04-01

    Virulence factor genes encoding the thermostable direct hemolysin (tdh) and the thermostable direct hemolysin-related hemolysin (trh) are strongly correlated with virulence of the emergent human pathogen Vibrio parahaemolyticus. The gene encoding the thermolabile hemolysin (tlh) is also considered a signature molecular marker for the species. These genes are typically reported in very low percentages (1 to 2%) of nonclinical strains. V. parahaemolyticus strains were isolated from various niches within a pristine estuary (North Inlet, SC) and were screened for these genes using both newly designed PCR primers and more commonly used primers. DNA sequences of tdh and trh were recovered from 48% and 8.3%, respectively, of these North Inlet strains. The recovery of pathogenic V. parahaemolyticus strains in such high proportions from an estuarine ecosystem that is virtually free of anthropogenic influences indicates the potential for additional, perhaps environmental roles of the tdh and trh genes.

  3. Validation of a prognostic multi-gene signature in high-risk neuroblastoma using the high throughput digital NanoString nCounter™ system.

    Science.gov (United States)

    Stricker, Thomas P; Morales La Madrid, Andres; Chlenski, Alexandre; Guerrero, Lisa; Salwen, Helen R; Gosiengfiao, Yasmin; Perlman, Elizabeth J; Furman, Wayne; Bahrami, Armita; Shohet, Jason M; Zage, Peter E; Hicks, M John; Shimada, Hiroyuki; Suganuma, Rie; Park, Julie R; So, Sara; London, Wendy B; Pytel, Peter; Maclean, Kirsteen H; Cohn, Susan L

    2014-05-01

    Microarray-based molecular signatures have not been widely integrated into neuroblastoma diagnostic classification systems due to the complexities of the assay and requirement for high-quality RNA. New digital technologies that accurately quantify gene expression using RNA isolated from formalin-fixed paraffin embedded (FFPE) tissues are now available. In this study, we describe the first use of a high-throughput digital system to assay the expression of genes in an "ultra-high risk" microarray classifier in FFPE high-risk neuroblastoma tumors. Customized probes corresponding to the 42 genes in a published multi-gene neuroblastoma signature were hybridized to RNA isolated from 107 FFPE high-risk neuroblastoma samples using the NanoString nCounter™ Analysis System. For classification of each patient, the Pearson's correlation coefficient was calculated between the standardized nCounter™ data and the molecular signature from the microarray data. We demonstrate that the nCounter™ 42-gene panel sub-stratified the high-risk cohort into two subsets with statistically significantly different overall survival (p = 0.0027) and event-free survival (p = 0.028). In contrast, none of the established prognostic risk markers (age, stage, tumor histology, MYCN status, and ploidy) were significantly associated with survival. We conclude that the nCounter™ System can reproducibly quantify expression levels of signature genes in FFPE tumor samples. Validation of this microarray signature in our high-risk patient cohort using a completely different technology emphasizes the prognostic relevance of this classifier. Prospective studies testing the prognostic value of molecular signatures in high-risk neuroblastoma patients using FFPE tumor samples and the nCounter™ System are warranted.

  4. A highly efficient molecular cloning platform that utilises a small bacterial toxin gene.

    Science.gov (United States)

    Mok, Wendy W K; Li, Yingfu

    2013-04-15

    Molecular cloning technologies that have emerged in recent years are more efficient and simpler to use than traditional strategies, but many have the disadvantages of requiring multiple steps and expensive proprietary enzymes. We have engineered cloning vectors containing variants of IbsC, a 19-residue toxin from Escherichia coli K-12. These toxic peptides offer selectivity to minimise the background, labour, and cost associated with conventional molecular cloning. As demonstrated with the cloning of reporter genes, this "detox cloning" system consistently produced over 95 % positive clones. Purification steps between digestion and ligation are not necessary, and the total time between digestion and plating of transformants can be as little as three hours. Thus, these IbsC-based cloning vectors are as reliable and amenable to high-throughput cloning as commercially available systems, and have the advantage of being more time-efficient and cost-effective.

  5. Introgression of High Yield Genes from Lycopersicon hirsutum acc. LA1777 Using CAPS Marker

    Institute of Scientific and Technical Information of China (English)

    LI Hong; WANG Xiao-xuan; SONG Ming; GAO Jian-chang; GUO Yan-mei; ZHU De-wei; DAI Shan-shu; DU Yong-chen

    2007-01-01

    The idea behind this study is to show that using high yield genes from a wild tomato can enrich tomato breeding resources and accelerate tomato breeding programs. In this study, the near-isogenic line TA1229 containing a 24-cM introgression at the bottom of chromosome 1 from Lycopersicon acc. LA1777, affects several higher yield traits. The TA1229 × 9706 BC1population was analyzed by marker-assisted selection and the traits of the population were evaluated. Twenty-three recombinant individuals that carried a shorter segment than TA1229 were obtained. Among them, 16 lines with the chromosome 1 recombinant segment can increase tomato yield and a QTL affecting yield was found between TG53 and TG158. Sixteen recombinant lines are useful to improve the tomato variety.

  6. Identification of Abiotic Stress Responsive Genes from Indian High Altitude Lepidium latifolium L. (Short Communication

    Directory of Open Access Journals (Sweden)

    Sanjay Mohan Gupta

    2012-09-01

    Full Text Available Abiotic stresses are major environmental factors that periodically account for significant loss in crop productivity. In order to improve the abiotic stress tolerance in vegetable crops through transgenic approaches, authors isolated and cloned six up-regulated, LlaDREB1b (JN214345, LlaGPAT (JN398166, LlaNAC (FJ423495, LlaCIPK (FJ423496, LlaPR5 (GQ853409 and LlaIPK (FJ487575 and two down-regulated LlaRan (JN214347 and LlaDRT (JN214346 abiotic stress responsive genes from Indian high altitude Lepidium latifolium L. plant that that may be used for abiotic stress-tolerance engineering upon functional validation.Defence Science Journal, 2012, 62(5, pp.315-318, DOI:http://dx.doi.org/10.14429/dsj.62.1495

  7. Homeostasis of Retinol in Lecithin:retinol Acyltransferase Gene Knockout Mice Fed a High Retinol Diet

    OpenAIRE

    Liu, Limin; Tang, Xiao-Han; Gudas, Lorraine J.

    2008-01-01

    We analyzed the retinoid levels and gene expression in various tissues after wild type (Wt) and lecithin:retinol acyltransferase knockout (LRAT−/−) mice were fed a high retinol diet (250 IU/gram). As compared to Wt, LRAT−/− mice exhibited a greater and faster increase in serum retinol concentration (mean±S.D., Wt, 1.3±0.2 µM to 1.5±0.3 µM in 48 hours, p>0.05; LRAT−/−, 1.3±0.2 µM to 2.2±0.3 µM in 48 hours, p

  8. Mapping the transcription repressive domain in the highly conserved human gene hnulp1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    HNULP1,a new member of the basic helixloop-helix transcription factors,contains a DUF654 domain in its C-terminus and is highly conserved from Drosophilae,yeast,zebrafish to mouse.The function of this motif,however,is currently unknown.In this research,we fused five deletion fragments of the DUF654 domain to the GAL4 DNA-binding domain and then co-transfected with plasmids L8G5-Luc and VP-16.The analysis of the GAL4 luciferase reporter gene indicated that fragments from 228 to 407 amino acids in the DUF654 domain had a strong transcription repression activity.Therefore,this study lays a solid foundation for research on the mechanism of hnulp1 transcriptional regulation and the function of the DUF654 domain.

  9. Generation of a high-titer retroviral vector capable of expressing high levels of the human β-globin gene

    NARCIS (Netherlands)

    M. Sadelain (Michel); C.H.J. Wang (Jason); M. Antoniou (Michael); F.G. Grosveld (Frank); R.C. Mulligan

    1995-01-01

    textabstractRetrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and

  10. Cryptochrome genes are highly expressed in the ovary of the African clawed frog, Xenopus tropicalis.

    Directory of Open Access Journals (Sweden)

    Yoko Kubo

    Full Text Available Cryptochromes (CRYs are flavoproteins sharing high homology with photolyases. Some of them have function(s including transcription regulation in the circadian clock oscillation, blue-light photoreception for resetting the clock phase, and light-dependent magnetoreception. Vertebrates retain multiple sets of CRY or CRY-related genes, but their functions are yet unclear especially in the lower vertebrates. Although CRYs and the other circadian clock components have been extensively studied in the higher vertebrates such as mice, only a few model species have been studied in the lower vertebrates. In this study, we identified two CRYs, XtCRY1 and XtCRY2 in Xenopus tropicalis, an excellent experimental model species. Examination of tissue specificity of their mRNA expression by real-time PCR analysis revealed that both the XtCRYs showed extremely high mRNA expression levels in the ovary. The mRNA levels in the ovary were about 28-fold (XtCry1 and 48-fold (XtCry2 higher than levels in the next abundant tissues, the retina and kidney, respectively. For the functional analysis of the XtCRYs, we cloned circadian positive regulator XtCLOCK and XtBMAL1, and found circadian enhancer E-box in the upstream of XtPer1 gene. XtCLOCK and XtBMAL1 exhibited strong transactivation from the XtPer1 E-box element, and both the XtCRYs inhibited the XtCLOCK:XtBMAL1-mediated transactivation, thereby suggesting this element to drive the circadian transcription. These results revealed a conserved main feedback loop in the X. tropicalis circadian clockwork and imply a possible physiological importance of CRYs in the ovarian functions such as synthesis of steroid hormones and/or control of estrus cycles via the transcription regulation.

  11. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi.

    Science.gov (United States)

    Gantz, Valentino M; Jasinskiene, Nijole; Tatarenkova, Olga; Fazekas, Aniko; Macias, Vanessa M; Bier, Ethan; James, Anthony A

    2015-12-08

    Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.

  12. The SKP1-like gene family of Arabidopsis exhibits a high degree of differential gene expression and gene product interaction during development.

    Science.gov (United States)

    Dezfulian, Mohammad H; Soulliere, Danielle M; Dhaliwal, Rajdeep K; Sareen, Madhulika; Crosby, William L

    2012-01-01

    The Arabidopsis thaliana genome encodes several families of polypeptides that are known or predicted to participate in the formation of the SCF-class of E3-ubiquitin ligase complexes. One such gene family encodes the Skp1-like class of polypeptide subunits, where 21 genes have been identified and are known to be expressed in Arabidopsis. Phylogenetic analysis based on deduced polypeptide sequence organizes the family of ASK proteins into 7 clades. The complexity of the ASK gene family, together with the close structural similarity among its members raises the prospect of significant functional redundancy among select paralogs. We have assessed the potential for functional redundancy within the ASK gene family by analyzing an expanded set of criteria that define redundancy with higher resolution. The criteria used include quantitative expression of locus-specific transcripts using qRT-PCR, assessment of the sub-cellular localization of individual ASK:YFP auto-fluorescent fusion proteins expressed in vivo as well as the in planta assessment of individual ASK-F-Box protein interactions using bimolecular fluorescent complementation techniques in combination with confocal imagery in live cells. The results indicate significant functional divergence of steady state transcript abundance and protein-protein interaction specificity involving ASK proteins in a pattern that is poorly predicted by sequence-based phylogeny. The information emerging from this and related studies will prove important for defining the functional intersection of expression, localization and gene product interaction that better predicts the formation of discrete SCF complexes, as a prelude to investigating their molecular mode of action.

  13. The SKP1-like gene family of Arabidopsis exhibits a high degree of differential gene expression and gene product interaction during development.

    Directory of Open Access Journals (Sweden)

    Mohammad H Dezfulian

    Full Text Available The Arabidopsis thaliana genome encodes several families of polypeptides that are known or predicted to participate in the formation of the SCF-class of E3-ubiquitin ligase complexes. One such gene family encodes the Skp1-like class of polypeptide subunits, where 21 genes have been identified and are known to be expressed in Arabidopsis. Phylogenetic analysis based on deduced polypeptide sequence organizes the family of ASK proteins into 7 clades. The complexity of the ASK gene family, together with the close structural similarity among its members raises the prospect of significant functional redundancy among select paralogs. We have assessed the potential for functional redundancy within the ASK gene family by analyzing an expanded set of criteria that define redundancy with higher resolution. The criteria used include quantitative expression of locus-specific transcripts using qRT-PCR, assessment of the sub-cellular localization of individual ASK:YFP auto-fluorescent fusion proteins expressed in vivo as well as the in planta assessment of individual ASK-F-Box protein interactions using bimolecular fluorescent complementation techniques in combination with confocal imagery in live cells. The results indicate significant functional divergence of steady state transcript abundance and protein-protein interaction specificity involving ASK proteins in a pattern that is poorly predicted by sequence-based phylogeny. The information emerging from this and related studies will prove important for defining the functional intersection of expression, localization and gene product interaction that better predicts the formation of discrete SCF complexes, as a prelude to investigating their molecular mode of action.

  14. Identification and characterization of the lamprey high-mobility group box 1 gene.

    Directory of Open Access Journals (Sweden)

    Yue Pang

    Full Text Available High-mobility group box 1 (HMGB1, a highly conserved DNA-binding protein, plays an important role in maintaining nucleosome structures, transcription, and inflammation. We identified a homolog of HMGB1 in the Japanese lamprey (Lampetra japonica. The Lampetra japonica HMGB1 gene (Lj-HMGB1 has over 70% sequence identity with its homologs in jawed vertebrates. Despite the reasonably high sequence identity with other HMGB1 proteins, Lj-HMGB1 did not group together with these proteins in a phylogenetic analysis. We examined Lj-HMGB1 expression in lymphocyte-like cells, and the kidneys, heart, gills, and intestines of lampreys before and after the animals were challenged with lipopolysaccharide (LPS and concanavalin A (ConA. Lj-HMGB1 was initially expressed at a higher level in the heart, but after treatment with LPS and ConA only the gills demonstrated a significant up-regulation of expression. The recombinant Lj-HMGB1 (rLj-HMGB1 protein bound double-stranded DNA and induced the proliferation of human adenocarcinoma cells to a similar extent as human HMGB1. We further revealed that Lj-HMGB1 was able to induce the production of tumor necrosis factor-α (TNF-α, a pro-inflammatory mediator, in activated human acute monocytic leukemia cells. These results suggest that lampreys use HMGB1 to activate their innate immunity for the purpose of pathogen defense.

  15. Hepatic Gene Expression Profiling in Nrf2 Knockout Mice after Long-Term High-Fat Diet-Induced Obesity

    Directory of Open Access Journals (Sweden)

    Dionysios V. Chartoumpekis

    2013-01-01

    Full Text Available Introduction. The transcription factor NFE2-related factor 2 (Nrf2 is a central regulator of antioxidant and detoxification gene expression in response to electrophilic or oxidative stress. Nrf2 has recently been shown to cross-talk with metabolic pathways, and its gene deletion protected mice from high-fat-diet-(HFD- induced obesity and insulin resistance. This study aimed to identify potential Nrf2-regulated genes of metabolic interest by comparing gene expression profiles of livers of wild-type (WT versus Nrf2 knockout (Nrf2-KO mice after a long-term HFD. Methods. WT and Nrf2-KO mice were fed an HFD for 180 days; total RNA was prepared from liver and used for microarray analysis and quantitative real-time RT-PCR (qRT-PCR. Results. The microarray analysis identified 601 genes that were differentially expressed between WT and Nrf2-KO mice after long-term HFD. Selected genes, including ones known to be involved in metabolic regulation, were prioritized for verification by qRT-PCR: Cyp7a1 and Fabp5 were significantly overexpressed in Nrf2-KO mice; in contrast, Car, Cyp2b10, Lipocalin 13, Aquaporin 8, Cbr3, Me1, and Nqo1 were significantly underexpressed in Nrf2-KO mice. Conclusion. Transcriptome profiling after HFD-induced obesity confirms that Nrf2 is implicated in liver metabolic gene networks. The specific genes identified here may provide insights into Nrf2-dependent mechanisms of metabolic regulation.

  16. Gene expression profiles of adipose tissue of high-fat diet-induced obese rats by cDNA microarrays.

    Science.gov (United States)

    Qiu, Jie; Cheng, Rui; Zhou, Xiao-yu; Zhu, Jin-gai; Zhu, Chun; Qin, Da-ni; Kou, Chun-zhao; Guo, Xi-rong

    2010-12-01

    To better understand the molecular basis of dietary obesity, we examined adipose tissue genes differentially expressed in a well-characterized rat model of high-fat diet (HFD)-induced obesity using cDNA microarrays. Male Sprague-Dawley rats were fed either the HFD or the normal diet. Seven weeks later, the weights of obese models (362.92 ± 39.65 g) were significantly higher than those of normal control rats (315.22 ± 42.30 g, P obese models. cDNA microarrays containing 9 216 genes/Ests were used to investigate gene expression of adipose tissue. Autoradiographic analysis showed that 532, 154, and 22 genes were differently expressed over 2-, 3-, and 5-fold, respectively. The analysis of gene expression profiles indicated that 276 genes were up-regulated and 432 genes were down-regulated in response to HFD-induced obesity. Different clusters of genes associated with lipid metabolism, extracellular matrix, signal transduction, cytoskeleton, cell apoptosis, etc., such as VLCS-H2, DGAT, ACADVL, PHYH, SCD, ACACA, ACS, MMP-2, MMP-15, CD38, CAMK2D, CACNA1F, CAPZA2, TMOD3, ARPC2, KNS2, TPM1, MAPK8, GADD45B, DAXX, TOK-1, PRKACA, STAT6, were concerned.

  17. Novel genes associated with colorectal cancer are revealed by high resolution cytogenetic analysis in a patient specific manner.

    Directory of Open Access Journals (Sweden)

    Hisham Eldai

    Full Text Available Genomic abnormalities leading to colorectal cancer (CRC include somatic events causing copy number aberrations (CNAs as well as copy neutral manifestations such as loss of heterozygosity (LOH and uniparental disomy (UPD. We studied the causal effect of these events by analyzing high resolution cytogenetic microarray data of 15 tumor-normal paired samples. We detected 144 genes affected by CNAs. A subset of 91 genes are known to be CRC related yet high GISTIC scores indicate 24 genes on chromosomes 7, 8, 18 and 20 to be strongly relevant. Combining GISTIC ranking with functional analyses and degree of loss/gain we identify three genes in regions of significant loss (ATP8B1, NARS, and ATP5A1 and eight in regions of gain (CTCFL, SPO11, ZNF217, PLEKHA8, HOXA3, GPNMB, IGF2BP3 and PCAT1 as novel in their association with CRC. Pathway and target prediction analysis of CNA affected genes and microRNAs, respectively indicates TGF-β signaling pathway to be involved in causing CRC. Finally, LOH and UPD collectively affected nine cancer related genes. Transcription factor binding sites on regions of >35% copy number loss/gain influenced 16 CRC genes. Our analysis shows patient specific CRC manifestations at the genomic level and that these different events affect individual CRC patients differently.

  18. Screening the dystrophin gene suggests a high rate of polymorphism in general but no exonic deletions in schizophrenics

    Energy Technology Data Exchange (ETDEWEB)

    Lindor, N.M.; Sobell, J.L.; Thibodeau, S.N. [Mayo Clinic/Foundation, Rochester, MN (United States)] [and others

    1994-03-15

    The dystrophin gene, located at chromosome Xp21, was evaluated as a candidate gene in chronic schizophrenia in response to the report of a large family in which schizophrenia cosegregated with Becker muscular dystrophy. Genomic DNA from 94 men with chronic schizophrenia was evaluated by Southern blot analysis using cDNA probes that span exons 1-59. No exonic deletions were identified. An unexpectedly high rate of polymorphism was calculated in this study and two novel polymorphisms were found, demonstrating the usefulness of the candidate gene approach even when results of the original study are negative. 41 refs., 1 fig., 4 tabs.

  19. Building Next-Generation Technologies for Low-Cost Gene Synthesis and High-Accuracy Genome Engineering

    OpenAIRE

    2014-01-01

    The technologies that enable writing and editing of DNA form the foundation of modern molecular biology and biotechnology. However, a number of methodological barriers have limited the widespread adoption of both high-throughput de novo gene synthesis and large-scale genome alteration. Increasingly, work in the fields of synthetic biology, protein design, and gene therapy has been hindered by shortcomings in current DNA writing and editing technologies. The goal of this dissertation has be...

  20. Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bacillus thuringiensis collection.

    Science.gov (United States)

    Beard, Cheryl E; Court, Leon; Boets, Annemie; Mourant, Roslyn; Van Rie, Jeroen; Akhurst, Raymond J

    2008-09-01

    Of 188 Australian Bacillus thuringiensis strains screened for genes encoding soluble insecticidal proteins by polymerase chain reaction/restriction-length fragment polymorphism (RFLP) analysis, 87% showed the presence of such genes. Although 135 isolates (72%) produced an RFLP pattern identical to that expected for vip3A genes, 29 isolates possessed a novel vip-like gene. The novel vip-like gene was cloned from B. thuringiensis isolate C81, and sequence analysis demonstrated that it was 94% identical to the vip3Ba1 gene. The new gene was designated vip3Bb2. Cell-free supernatants from both the B. thuringiensis strain C81 and from Escherichia coli expressing the Vip3Bb2 protein were toxic for the cotton bollworm, Helicoverpa armigera.

  1. Knowledge-driven analysis identifies a gene-gene interaction affecting high-density lipoprotein cholesterol levels in multi-ethnic populations.

    Science.gov (United States)

    Ma, Li; Brautbar, Ariel; Boerwinkle, Eric; Sing, Charles F; Clark, Andrew G; Keinan, Alon

    2012-01-01

    Total cholesterol, low-density lipoprotein cholesterol, triglyceride, and high-density lipoprotein cholesterol (HDL-C) levels are among the most important risk factors for coronary artery disease. We tested for gene-gene interactions affecting the level of these four lipids based on prior knowledge of established genome-wide association study (GWAS) hits, protein-protein interactions, and pathway information. Using genotype data from 9,713 European Americans from the Atherosclerosis Risk in Communities (ARIC) study, we identified an interaction between HMGCR and a locus near LIPC in their effect on HDL-C levels (Bonferroni corrected P(c) = 0.002). Using an adaptive locus-based validation procedure, we successfully validated this gene-gene interaction in the European American cohorts from the Framingham Heart Study (P(c) = 0.002) and the Multi-Ethnic Study of Atherosclerosis (MESA; P(c) = 0.006). The interaction between these two loci is also significant in the African American sample from ARIC (P(c) = 0.004) and in the Hispanic American sample from MESA (P(c) = 0.04). Both HMGCR and LIPC are involved in the metabolism of lipids, and genome-wide association studies have previously identified LIPC as associated with levels of HDL-C. However, the effect on HDL-C of the novel gene-gene interaction reported here is twice as pronounced as that predicted by the sum of the marginal effects of the two loci. In conclusion, based on a knowledge-driven analysis of epistasis, together with a new locus-based validation method, we successfully identified and validated an interaction affecting a complex trait in multi-ethnic populations.

  2. Identification of genes and genomic islands correlated with high pathogenicity in Streptococcus suis using whole genome tiling microarrays.

    Directory of Open Access Journals (Sweden)

    Xiao Zheng

    Full Text Available Streptococcus suis is an important zoonotic pathogen that can cause meningitis and sepsis in both pigs and humans. Infections in humans have been sporadic worldwide but two severe outbreaks occurred in China in recent years, while infections in pigs are a major problem in the swine industry. Some S. suis strains are more pathogenic than others with 2 sequence types (ST, ST1 and ST7, being well recognized as highly pathogenic. We analyzed 31 isolates from 23 serotypes and 25 STs by NimbleGen tiling microarray using the genome of a high pathogenicity (HP ST1 strain, GZ1, as reference and a new algorithm to detect gene content difference. The number of genes absent in a strain ranged from 49 to 225 with a total of 632 genes absent in at least one strain, while 1346 genes were found to be invariably present in all strains as the core genome of S. suis, accounting for 68% of the GZ1 genome. The majority of genes are located in chromosomal blocks with two or more contiguous genes. Sixty two blocks are absent in two or more strains and defined as regions of difference (RDs, among which 26 are putative genomic islands (GIs. Clustering and statistical analyses revealed that 8 RDs including 6 putative GIs and 21 genes within these RDs are significantly associated with HP. Three RDs encode known virulence related factors including the extracellular factor, the capsular polysaccharide and a SrtF pilus. The strains were divided into 5 groups based on population genetic analysis of multilocus sequence typing data and the distribution of the RDs among the groups revealed gain and loss of RDs in different groups. Our study elucidated the gene content diversity of S. suis and identified genes that potentially promote HP.

  3. Global gene analysis of oocytes from early stages in human folliculogenesis shows high expression of novel genes in reproduction

    DEFF Research Database (Denmark)

    Markholt, Sara; Grøndahl, M L; Ernst, Erik

    2012-01-01

    The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood...... but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis......) and the mitochondrial-encoded ATPase6 (ATP6). Thus, the present study provides not only a technique to capture and perform transcriptome analysis of the sparse material of human oocytes from the earliest follicle stages but further includes a comprehensive basis for our understanding of the regulatory factors...

  4. The phenotypic patterns of essential hypertension are the key to identifying "high blood pressure" genes.

    Science.gov (United States)

    Korner, P I

    2010-01-01

    hypertension provide targets for identifying high BP genes. Reading the genome from the phenotype will require new approaches, such as those used in developmental genetics. In addition, transgenic technology may help verify hypotheses and examine whether an observed effect is through single or multiple mechanisms. To obtain answers will require substantial collaborative efforts between physiologists and geneticists.

  5. [Clinical significance and distribution of BRCA genes mutation in sporadic high grade serous ovarian cancer].

    Science.gov (United States)

    Liu, W L; Wang, Z Z; Zhao, J Z; Hou, Y Y; Wu, X X; Li, W; Dong, B; Tong, T T; Guo, Y J

    2017-01-25

    Objective: To investigate the mutations of BRCA genes in sporadic high grade serous ovarian cancer (HGSOC) and study its clinical significance. Methods: Sixty-eight patients between January 2015 and January 2016 from the Affiliated Cancer Hospital of Zhengzhou University were collected who were based on pathological diagnosis of ovarian cancer and had no reported family history, and all patients firstly hospitalized were untreated in other hospitals before. (1) The BRCA genes were detected by next-generation sequencing (NGS) method. (2) The serum tumor markers included carcinoembryonic antigen (CEA), CA(125), CA(199), and human epididymis protein 4 (HE4) were detected by the chemiluminescence methods, and their correlation was analyzed by Pearson linear correlation. Descriptive statistics and comparisons were performed using two-tailed t-tests, Pearson's chi square test, Fisher's exact tests or logistic regression analysis as appropriate to research the clinicopathologic features associated with BRCA mutations, including age, International Federation of Gynecology and Obstetrics (FIGO) stage, platinum-based chemotherapy sensitivity, distant metastases, serum tumor markers (STM) . Results: (1) Fifteen cases (22%, 15/68) BRCA mutations were identified (BRCA1: 11 cases; BRCA2: 4 cases), and four novel mutations were observed. (2) The levels of CEA, CA(199), and HE4 were lower in BRCA mutations compared to that in control group, while no significant differences were found (P>0.05), but the level of CA(125) was much higher in BRCA mutation group than that in controls (t=-3.536, P=0.003). Further linear regression analysis found that there was a significant linear correlation between CA(125) and HE4 group (r=0.494, PBRCA(+) and BRCA(-) group (P>0.05), while significant differences were found in CA(125) and sensitivity to platinum-based chemotherapy between the patients with BRCA mutation and wild type (PBRCA mutations in sporadic HGSOC (P=0.007). Conclusion: The

  6. Application of high-resolution, massively parallel pyrosequencing for estimation of haplotypes and gene expression levels of swine leukocyte antigen (SLA) class I genes.

    Science.gov (United States)

    Kita, Yuki F; Ando, Asako; Tanaka, Keiko; Suzuki, Shingo; Ozaki, Yuki; Uenishi, Hirohide; Inoko, Hidetoshi; Kulski, Jerzy K; Shiina, Takashi

    2012-03-01

    The swine is an important animal model for allo- and xeno-transplantation donor studies, which necessitates an extensive characterization of the expression and sequence variations within the highly polygenic and polymorphic swine leukocyte antigen (SLA) region. Massively parallel pyrosequencing is potentially an effective new 2ndGen method for simultaneous high-throughput genotyping and detection of SLA class I gene expression levels. In this study, we compared the 2ndGen method using the Roche Genome Sequencer 454 FLX with the conventional method using sub-cloning and Sanger sequencing to genotype SLA class I genes in five pigs of the Clawn breed and four pigs of the Landrace breed. We obtained an average of 10.4 SLA class I sequences per pig by the 2ndGen method, consistent with the inheritance data, and an average of only 6.0 sequences by the conventional method. We also performed a correlation analysis between the sequence read numbers obtained by the 2ndGen method and the relative expression values obtained by quantitative real-time PCR analysis at the allele level. A significant correlation coefficient (r = 0.899, P SLA class I genes SLA-1, SLA-2, and SLA-3, suggesting that the sequence read numbers closely reflect the gene expression levels in white blood cells. Overall, five novel class I sequences, different haplotype-specific expression patterns and a splice variant for one of the SLA class I genes were identified by the 2ndGen method at greater efficiency and sensitivity than the conventional method.

  7. Multiplex high-throughput gene mutation analysis in acute myeloid leukemia.

    Science.gov (United States)

    Dunlap, Jennifer; Beadling, Carol; Warrick, Andrea; Neff, Tanaya; Fleming, William H; Loriaux, Marc; Heinrich, Michael C; Kovacsovics, Tibor; Kelemen, Katalin; Leeborg, Nicky; Gatter, Ken; Braziel, Rita M; Press, Richard; Corless, Christopher L; Fan, Guang

    2012-12-01

    Classification of acute myeloid leukemia increasingly depends on genetic analysis. However, the number of known mutations in acute myeloid leukemia is expanding rapidly. Therefore, we tested a high-throughput screening method for acute myeloid leukemia mutation analysis using a multiplex mass spectrometry-based approach. To our knowledge, this is the first reported application of this approach to genotype leukemias in a clinical setting. One hundred seven acute myeloid leukemia cases were screened for mutations using a panel that covers 344 point mutations across 31 genes known to be associated with leukemia. The analysis was performed by multiplex polymerase chain reaction for mutations in genes of interest followed by primer extension reactions. Products were analyzed on a Sequenom MassARRAY system (San Diego, CA). The multiplex panel yielded mutations in 58% of acute myeloid leukemia cases with normal cytogenetics and 21% of cases with abnormal cytogenetics. Cytogenetics and routine polymerase chain reaction-based screening of NPM1, CEBPA, FLT3-ITD, and KIT was also performed on a subset of cases. When combined with the results of these standard polymerase chain reaction-based tests, the mutation frequency reached 78% in cases with normal cytogenetics. Of these, 42% harbored multiple mutations primarily involving NPM1 with NRAS, KRAS, CEBPA, PTPN11, IDH1, or FLT3. In contrast, cases with abnormal cytogenetics rarely harbored more than 1 mutation (1.5%), suggesting different underlying biology. This study demonstrates the feasibility and utility of broad-based mutation profiling of acute myeloid leukemia in a clinical setting. This approach will be helpful in defining prognostic subgroups of acute myeloid leukemia and contribute to the selection of patients for enrollment into trials with novel inhibitors.

  8. 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.

  9. Population divergence and gene flow in an endangered and highly mobile seabird

    Science.gov (United States)

    Welch, A. J.; Fleischer, R. C.; James, H. F.; Wiley, A. E.; Ostrom, P. H.; Adams, J.; Duvall, F.; Holmes, N.; Hu, D.; Penniman, J.; Swindle, K. A.

    2012-01-01

    Seabirds are highly vagile and can disperse up to thousands of kilometers, making it difficult to identify the factors that promote isolation between populations. The endemic Hawaiian petrel (Pterodroma sandwichensis) is one such species. Today it is endangered, and known to breed only on the islands of Hawaii, Maui, Lanai and Kauai. Historical records indicate that a large population formerly bred on Molokai as well, but this population has recently been extirpated. Given the great dispersal potential of these petrels, it remains unclear if populations are genetically distinct and which factors may contribute to isolation between them. We sampled petrels from across their range, including individuals from the presumably extirpated Molokai population. We sequenced 524 bp of mitochondrial DNA, 741 bp from three nuclear introns, and genotyped 18 microsatellite loci in order to examine the patterns of divergence in this species and to investigate the potential underlying mechanisms. Both mitochondrial and nuclear data sets indicated significant genetic differentiation among all modern populations, but no differentiation was found between historic samples from Molokai and modern birds from Lanai. Population-specific nonbreeding distribution and strong natal philopatry may reduce gene flow between populations. However, the lack of population structure between extirpated Molokai birds and modern birds on Lanai indicates that there was substantial gene flow between these populations and that petrels may be able to overcome barriers to dispersal prior to complete extirpation. Hawaiian petrel populations could be considered distinct management units, however, the dwindling population on Hawaii may require translocation to prevent extirpation in the near future.

  10. Whole cell strategies based on lux genes for high throughput applications toward new antimicrobials.

    Science.gov (United States)

    Galluzzi, Lorenzo; Karp, Matti

    2006-08-01

    The discovery/development of novel drug candidates has witnessed dramatic changes over the last two decades. Old methods to identify lead compounds are not suitable to screen wide libraries generated by combinatorial chemistry techniques. High throughput screening (HTS) has become irreplaceable and hundreds of different approaches have been described. Assays based on purified components are flanked by whole cell-based assays, in which reporter genes are used to monitor, directly or indirectly, the influence of a chemical over the metabolism of living cells. The most convenient and widely used reporters for real-time measurements are luciferases, light emitting enzymes from evolutionarily distant organisms. Autofluorescent proteins have been also extensively employed, but proved to be more suitable for end-point measurements, in situ applications - such as the localization of fusion proteins in specific subcellular compartments - or environmental studies on microbial populations. The trend toward miniaturization and the technical advances in detection and liquid handling systems will allow to reach an ultra high throughput screening (uHTS), with 100,000 of compounds routinely screened each day. Here we show how similar approaches may be applied also to the search for new and potent antimicrobial agents.

  11. Expression of HIF-1α and Its Target Genes in the Nanorana parkeri Heart:Implications for High Altitude Adaptation

    Institute of Scientific and Technical Information of China (English)

    Qiong ZHANG; Xingzhi HAN; Yinzi YE; Robert H S KRAUS; Liqing FAN; Le YANG; Yi TAO

    2016-01-01

    Hypoxia-inducible factor 1 alpha (HIF-1α) and its target genes vascular endothelial growth factor (VEGF) and transferrins (TF) play an important role in native endothermic animals’ adaptation to the high altitude environments. For ectothermic animals – especially frogs – it remains undetermined whether HIF-1α and its target genes (VEGF and TF) play an important role in high altitude adaptation, too. In this study, we compared the gene sequences and expression of HIF-1α and its target genes (VEGF and TF) between three Nanorana parkeri populations from different altitudes (3008 m a.s.l., 3440 m a.s.l. and 4312 m a.s.l.). We observed that the cDNA sequences of HIF-1A exhibited high sequence similarity (99.38%) among the three altitudinally separated populations; but with increasing altitude, the expression of HIF-1A and its target genes (VEGF and TF) increased significantly. These results indicate that HIF-1αplays an important role in N. parkeri adaptation to the high altitude, similar to its role in endothermic animals.

  12. Automation of gene assignments to metabolic pathways using high-throughput expression data

    Directory of Open Access Journals (Sweden)

    Yona Golan

    2005-08-01

    Full Text Available Abstract Background Accurate assignment of genes to pathways is essential in order to understand the functional role of genes and to map the existing pathways in a given genome. Existing algorithms predict pathways by extrapolating experimental data in one organism to other organisms for which this data is not available. However, current systems classify all genes that belong to a specific EC family to all the pathways that contain the corresponding enzymatic reaction, and thus introduce ambiguity. Results Here we describe an algorithm for assignment of genes to cellular pathways that addresses this problem by selectively assigning specific genes to pathways. Our algorithm uses the set of experimentally elucidated metabolic pathways from MetaCyc, together with statistical models of enzyme families and expression data to assign genes to enzyme families and pathways by optimizing correlated co-expression, while minimizing conflicts due to shared assignments among pathways. Our algorithm also identifies alternative ("backup" genes and addresses the multi-domain nature of proteins. We apply our model to assign genes to pathways in the Yeast genome and compare the results for genes that were assigned experimentally. Our assignments are consistent with the experimentally verified assignments and reflect characteristic properties of cellular pathways. Conclusion We present an algorithm for automatic assignment of genes to metabolic pathways. The algorithm utilizes expression data and reduces the ambiguity that characterizes assignments that are based only on EC numbers.

  13. Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates.

    Science.gov (United States)

    Ikebe, Tadayoshi; Ato, Manabu; Matsumura, Takayuki; Hasegawa, Hideki; Sata, Tetsutaro; Kobayashi, Kazuo; Watanabe, Haruo

    2010-04-01

    Streptococcal toxic shock syndrome (STSS) is a severe invasive infection characterized by the sudden onset of shock and multiorgan failure; it has a high mortality rate. Although a number of studies have attempted to determine the crucial factors behind the onset of STSS, the responsible genes in group A Streptococcus have not been clarified. We previously reported that mutations of csrS/csrR genes, a two-component negative regulator system for multiple virulence genes of Streptococcus pyogenes, are found among the isolates from STSS patients. In the present study, mutations of another negative regulator, rgg, were also found in clinical isolates of STSS patients. The rgg mutants from STSS clinical isolates enhanced lethality and impaired various organs in the mouse models, similar to the csrS mutants, and precluded their being killed by human neutrophils, mainly due to an overproduction of SLO. When we assessed the mutation frequency of csrS, csrR, and rgg genes among S. pyogenes isolates from STSS (164 isolates) and non-invasive infections (59 isolates), 57.3% of the STSS isolates had mutations of one or more genes among three genes, while isolates from patients with non-invasive disease had significantly fewer mutations in these genes (1.7%). The results of the present study suggest that mutations in the negative regulators csrS/csrR and rgg of S. pyogenes are crucial factors in the pathogenesis of STSS, as they lead to the overproduction of multiple virulence factors.

  14. Risk Factors in Normal-Tension Glaucoma and High-Tension Glaucoma in relation to Polymorphisms of Endothelin-1 Gene and Endothelin-1 Receptor Type A Gene

    Directory of Open Access Journals (Sweden)

    Dominika Wróbel-Dudzińska

    2015-01-01

    Full Text Available The aim of the research is to analyse the influence of polymorphisms of endothelin-1 gene and endothelin-1 receptor type A gene on the clinical condition of patients with primary open angle glaucoma. Methods. 285 Polish patients took part in the research (160 normal-tension glaucoma and 125 high-tension glaucoma. DNA was isolated by standard methods and genotype distributions of four polymorphisms in genes encoding endothelin-1 (K198N and endothelin-1 receptor type A polymorphisms (C1222T, C70G, and G231A were determined. Genotype distributions were compared between NTG and HTG groups. The clinical condition of participants was examined for association with polymorphisms. Results. A similar frequency of occurrence of the polymorphic varieties of the studied genes was observed in patients with NTG and HTG. There is no relation between NTG risk factors and examined polymorphisms. NTG patients with TT genotype of K198N polymorphism presented with the lowest intraocular pressure in comparison to GG + GT genotype (p=0.03. In NTG patients with CC genotype of C1222T polymorphism (p=0.028 and GG of C70G polymorphism (p=0.03 the lowest values of mean blood pressure were observed. Conclusions. The studied polymorphic varieties (K198N, C1222T do have an influence on intraocular pressure as well as arterial blood pressure in NTG patients.

  15. Identification of genes that promote or antagonize somatic homolog pairing using a high-throughput FISH-based screen.

    Directory of Open Access Journals (Sweden)

    Eric F Joyce

    Full Text Available The pairing of homologous chromosomes is a fundamental feature of the meiotic cell. In addition, a number of species exhibit homolog pairing in nonmeiotic, somatic cells as well, with evidence for its impact on both gene regulation and double-strand break (DSB repair. An extreme example of somatic pairing can be observed in Drosophila melanogaster, where homologous chromosomes remain aligned throughout most of development. However, our understanding of the mechanism of somatic homolog pairing remains unclear, as only a few genes have been implicated in this process. In this study, we introduce a novel high-throughput fluorescent in situ hybridization (FISH technology that enabled us to conduct a genome-wide RNAi screen for factors involved in the robust somatic pairing observed in Drosophila. We identified both candidate "pairing promoting genes" and candidate "anti-pairing genes," providing evidence that pairing is a dynamic process that can be both enhanced and antagonized. Many of the genes found to be important for promoting pairing are highly enriched for functions associated with mitotic cell division, suggesting a genetic framework for a long-standing link between chromosome dynamics during mitosis and nuclear organization during interphase. In contrast, several of the candidate anti-pairing genes have known interphase functions associated with S-phase progression, DNA replication, and chromatin compaction, including several components of the condensin II complex. In combination with a variety of secondary assays, these results provide insights into the mechanism and dynamics of somatic pairing.

  16. Identification of differentially expressed genes in esophageal cancer through SSH in com- bination with high throughput reverse Northern screening

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To understand the molecular mechanisms of carcinogenesis of esophagus and to isolate genes with different expression levels in esophageal cancer, suppression subtractive hybridization (SSH) was combined with PCR-based cDNA synthesis and reverse Northern on the cancer tissues and matched almost normal mucosa using 5 microgram of total RNA as starting marterial. Eight genes were found expressed differentially in esophageal cancer, in which 5 were known genes and 3 were novel ones; and 6 were down-regulated in cancer tissues, while 2 were up-regulated; 6 were of mid-high abundance and 2 were of low abundance in esophagus. The results revealed that alteration in expression level of multiple genes underlied the initiation and development of esophageal cancer. The differentially expressed genes identified in this study such as liporcotinⅠ, cystatin A, cystatin B, cytokeratin 13 may play roles in dedifferentiation, transformation and malignant proliferation of esophageal cancer. The combination of SSH with PCR-based double- strand cDNA synthesis and high throughput reverse Northern screening is an efficient way to isolate differentially expressed genes from microgram of total RNA.

  17. High Level Aminoglycoside Resistance and Distribution of Aminoglycoside Resistant Genes among Clinical Isolates of Enterococcus Species in Chennai, India

    Directory of Open Access Journals (Sweden)

    Elango Padmasini

    2014-01-01

    Full Text Available Enterococci are nosocomial pathogen with multiple-drug resistance by intrinsic and extrinsic mechanisms. Aminoglycosides along with cell wall inhibitors are given clinically for treating enterococcal infections. 178 enterococcal isolates were analyzed in this study. E. faecalis is identified to be the predominant Enterococcus species, along with E. faecium, E. avium, E. hirae, E. durans, E. dispar and E. gallinarum. High level aminoglycoside resistance (HLAR by MIC for gentamicin (GM, streptomycin (SM and both (GM + SM antibiotics was found to be 42.7%, 29.8%, and 21.9%, respectively. Detection of aminoglycoside modifying enzyme encoding genes (AME in enterococci was identified by multiplex PCR for aac(6′-Ie-aph(2′′-Ia; aph(2′′-Ib; aph(2′′-Ic; aph(2′′-Id and aph(3′-IIIa genes. 38.2% isolates carried aac(6′-Ie-aph(2′′-Ia gene and 40.4% isolates carried aph(3′-IIIa gene. aph(2′′-Ib; aph(2′′-Ic; aph(2′′-Id were not detected among our study isolates. aac(6′-Ie-aph(2′′-Ia and aph(3′-IIIa genes were also observed in HLAR E. durans, E. avium, E. hirae, and E. gallinarum isolates. This indicates that high level aminoglycoside resistance genes are widely disseminated among isolates of enterococci from Chennai.

  18. High level aminoglycoside resistance and distribution of aminoglycoside resistant genes among clinical isolates of Enterococcus species in Chennai, India.

    Science.gov (United States)

    Padmasini, Elango; Padmaraj, R; Ramesh, S Srivani

    2014-01-01

    Enterococci are nosocomial pathogen with multiple-drug resistance by intrinsic and extrinsic mechanisms. Aminoglycosides along with cell wall inhibitors are given clinically for treating enterococcal infections. 178 enterococcal isolates were analyzed in this study. E. faecalis is identified to be the predominant Enterococcus species, along with E. faecium, E. avium, E. hirae, E. durans, E. dispar and E. gallinarum. High level aminoglycoside resistance (HLAR) by MIC for gentamicin (GM), streptomycin (SM) and both (GM + SM) antibiotics was found to be 42.7%, 29.8%, and 21.9%, respectively. Detection of aminoglycoside modifying enzyme encoding genes (AME) in enterococci was identified by multiplex PCR for aac(6')-Ie-aph(2'')-Ia; aph(2'')-Ib; aph(2'')-Ic; aph(2'')-Id and aph(3')-IIIa genes. 38.2% isolates carried aac(6')-Ie-aph(2'')-Ia gene and 40.4% isolates carried aph(3')-IIIa gene. aph(2'')-Ib; aph(2'')-Ic; aph(2'')-Id were not detected among our study isolates. aac(6')-Ie-aph(2'')-Ia and aph(3')-IIIa genes were also observed in HLAR E. durans, E. avium, E. hirae, and E. gallinarum isolates. This indicates that high level aminoglycoside resistance genes are widely disseminated among isolates of enterococci from Chennai.

  19. BABELOMICS: a suite of web tools for functional annotation and analysis of groups of genes in high-throughput experiments.

    Science.gov (United States)

    Al-Shahrour, Fátima; Minguez, Pablo; Vaquerizas, Juan M; Conde, Lucía; Dopazo, Joaquín

    2005-07-01

    We present Babelomics, a complete suite of web tools for the functional analysis of groups of genes in high-throughput experiments, which includes the use of information on Gene Ontology terms, interpro motifs, KEGG pathways, Swiss-Prot keywords, analysis of predicted transcription factor binding sites, chromosomal positions and presence in tissues with determined histological characteristics, through five integrated modules: FatiGO (fast assignment and transference of information), FatiWise, transcription factor association test, GenomeGO and tissues mining tool, respectively. Additionally, another module, FatiScan, provides a new procedure that integrates biological information in combination with experimental results in order to find groups of genes with modest but coordinate significant differential behaviour. FatiScan is highly sensitive and is capable of finding significant asymmetries in the distribution of genes of common function across a list of ordered genes even if these asymmetries were not extreme. The strong multiple-testing nature of the contrasts made by the tools is taken into account. All the tools are integrated in the gene expression analysis package GEPAS. Babelomics is the natural evolution of our tool FatiGO (which analysed almost 22,000 experiments during the last year) to include more sources on information and new modes of using it. Babelomics can be found at http://www.babelomics.org.

  20. Gene Expression of Stress Proteins and Identification of Molecular Markers of Plant Resistance to High Temperatures and Drought

    Directory of Open Access Journals (Sweden)

    L.P. Khokhlova

    2016-06-01

    Full Text Available Molecular biomarkers of plant resistance to both individual and combined action of high tempera-tures (42 °C and drought have been identified. For this purpose, correlation between gene expression of four stress proteins (non-photosynthetic malic enzyme (TaNADP-ME2, serine-threonine kinase (W55a, dehydrin (DHN14, and lipocalin (TaTIL and resistance of eight spring wheat cultivars has been determined for the first time. Gene expression has been studied using the RT-PCR method based on the content of transcripts on electrophoregrams. The absence of species-specific responses of two genes, TaNADP-ME2 and W55a, the gene activity of which did not depend on the resistance of cultivars to heat shock and water deficit, has been shown. However, gene expression of two other genes, DHN14 and TaTIL, was genotypically determined and positively correlated with the high resistance of particular cultivars. It has been concluded that the activities of DHN14 and TaTIL are potential molecular markers of heat and drought resistance in spring wheat and, therefore, can be used in transgenic selection technologies to create new phenotypes of agricultural crops that would be better adapted to the environmental conditions.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor®

    Directory of Open Access Journals (Sweden)

    Vincent Kate

    2009-12-01

    Full Text Available Abstract Background TILLING (Targeting Induced Local Lesions IN Genomes is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor® software, aimed at simultaneous detection of mutations in three homoeologous genes. Results We demonstrate that High Resolution Melting (HRM analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor® is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation. Conclusion Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence

  3. Characterization of new bacterial catabolic genes and mobile genetic elements by high throughput genetic screening of a soil metagenomic library.

    Science.gov (United States)

    Jacquiod, Samuel; Demanèche, Sandrine; Franqueville, Laure; Ausec, Luka; Xu, Zhuofei; Delmont, Tom O; Dunon, Vincent; Cagnon, Christine; Mandic-Mulec, Ines; Vogel, Timothy M; Simonet, Pascal

    2014-11-20

    A mix of oligonucleotide probes was used to hybridize soil metagenomic DNA from a fosmid clone library spotted on high density membranes. The pooled radio-labeled probes were designed to target genes encoding glycoside hydrolases GH18, dehalogenases, bacterial laccases and mobile genetic elements (integrases from integrons and insertion sequences). Positive hybridizing spots were affiliated to the corresponding clones in the library and the metagenomic inserts were sequenced. After assembly and annotation, new coding DNA sequences related to genes of interest were identified with low protein similarity against the closest hits in databases. This work highlights the sensitivity of DNA/DNA hybridization techniques as an effective and complementary way to recover novel genes from large metagenomic clone libraries. This study also supports that some of the identified catabolic genes might be associated with horizontal transfer events.

  4. Gene polymorphisms of micrornas in Helicobacter pylori-induced high risk atrophic gastritis and gastric cancer.

    Directory of Open Access Journals (Sweden)

    Juozas Kupcinskas

    Full Text Available BACKGROUND AND AIMS: MicroRNAs (miRNAs are known for their function as translational regulators of tumor suppressor or oncogenes. Single nucleotide polymorphisms (SNPs in miRNAs related genes have been shown to affect the regulatory capacity of miRNAs and were linked with gastric cancer (GC and premalignant gastric conditions. The purpose of this study was to evaluate potential associations between miRNA-related gene polymorphisms (miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608 and the presence of GC or high risk atrophic gastritis (HRAG in European population. METHODS: Gene polymorphisms were analyzed in 995 subjects (controls: n = 351; GC: n = 363; HRAG: n = 281 of European descent. MiR-27a T>C (rs895819, miR-146a G>C (rs2910164, miR-196a-2 C>T (rs11614913, miR-492 G>C (rs2289030 and miR-608 C>G (rs4919510 SNPs were genotyped by RT-PCR. RESULTS: Overall, SNPs of miRNAs were not associated with the presence of GC or HRAG. We observed a tendency for miR-196a-2 CT genotype to be associated with higher risk of GC when compared to CC genotype, however, the difference did not reach the adjusted P-value (odds ratio (OR - 1.46, 95% confidence interval (CI 1.03-2.07, P = 0.032. MiR-608 GG genotype was more frequent in GC when compared to controls (OR -2.34, 95% CI 1.08-5.04, but significance remained marginal (P = 0.029. A similar tendency was observed in a recessive model for miR-608, where CC + CG vs GG genotype comparison showed a tendency for increased risk of GC with OR of 2.44 (95% CI 1.14-5.22, P = 0.021. The genotypes and alleles of miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608 SNPs had similar distribution between histological subtypes of GC and were not linked with the presence of diffuse or intestinal-type GC. CONCLUSIONS: Gene polymorphisms of miR-27a, miR-146a, miR-196a-2, miR-492, miR-492a and miR-608 were not associated with the presence of HRAG, GC or different histological subtypes of GC in European

  5. Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions.

    Science.gov (United States)

    Butaye, Katleen M J; Goderis, Inge J W M; Wouters, Piet F J; Pues, Jonathan M-T G; Delauré, Stijn L; Broekaert, Willem F; Depicker, Ann; Cammue, Bruno P A; De Bolle, Miguel F C

    2004-08-01

    Basic and applied research involving transgenic plants often requires consistent high-level expression of transgenes. However, high inter-transformant variability of transgene expression caused by various phenomena, including gene silencing, is frequently observed. Here, we show that stable, high-level transgene expression is obtained using Arabidopsis thaliana post-transcriptional gene silencing (PTGS) sgs2 and sgs3 mutants. In populations of first generation (T1) A. thaliana plants transformed with a beta-glucuronidase (GUS) gene (uidA) driven by the 35S cauliflower mosaic virus promoter (p35S), the incidence of highly expressing transformants shifted from 20% in wild type background to 100% in sgs2 and sgs3 backgrounds. Likewise, when sgs2 mutants were transformed with a cyclin-dependent kinase inhibitor 6 gene under control of p35S, all transformants showed a clear phenotype typified by serrated leaves, whereas such phenotype was only observed in about one of five wild type transformants. p35S-driven uidA expression remained high and steady in T2 sgs2 and sgs3 transformants, in marked contrast to the variable expression patterns observed in wild type T2 populations. We further show that T-DNA constructs flanked by matrix attachment regions of the chicken lysozyme gene (chiMARs) cause a boost in GUS activity by fivefold in sgs2 and 12-fold in sgs3 plants, reaching up to 10% of the total soluble proteins, whereas no such boost is observed in the wild type background. MAR-based plant transformation vectors used in a PTGS mutant background might be of high value for efficient high-throughput screening of transgene-based phenotypes as well as for obtaining extremely high transgene expression in plants.

  6. Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle

    Institute of Scientific and Technical Information of China (English)

    Shengli Yu; Junjie Luo; Zhiyuan Song; Fangrong Ding; Yunping Dai; Ning Li

    2011-01-01

    Dear Editor,Gene targeting is in widespread use as a gold standard for determining the function of genes in mice and human embryonic stem cells [1].However,the poor efficiency of this technology has hindered its application to domestic animals,for which embryonic stem cells are not available.Although gene-targeted large domestic animals have been produced successfully by combination of homologous recombination-based targeting strategy and cloning [2-4],the efficiency is very low and,more importantly,the disruption of the targeted gene is usually mono-allelic.It thus takes a long time to obtain a null mutant.

  7. High Genetic Diversity of Microbial Cellulase and Hemicellulase Genes in the Hindgut of Holotrichia parallela Larvae

    Directory of Open Access Journals (Sweden)

    Ping Sheng

    2015-07-01

    Full Text Available In this study, we used a culture-independent method based on library construction and sequencing to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the hindgut of Holotrichia parallela larvae. The results indicate that there is a large, diverse set of bacterial genes encoding lignocellulose hydrolysis enzymes in the hindgut of H. parallela. The total of 101 distinct gene fragments (similarity <95% of glycosyl hydrolase families including GH2 (24 genes, GH8 (27 genes, GH10 (19 genes, GH11 (14 genes and GH36 (17 genes families was retrieved, and certain sequences of GH2 (10.61%, GH8 (3.33%, and GH11 (18.42% families had <60% identities with known sequences in GenBank, indicating their novelty. Based on phylogenetic analysis, sequences from hemicellulase families were related to enzymes from Bacteroidetes and Firmicutes. Fragments from cellulase family were most associated with the phylum of Proteobacteria. Furthermore, a full-length endo-xylanase gene was obtained, and the enzyme exhibited activity over a broad range of pH levels. Our results indicate that there are large number of cellulolytic and xylanolytic bacteria in the hindgut of H. parallela larvae, and these symbiotic bacteria play an important role in the degradation of roots and other organic matter for the host insect.

  8. Analysis of genetic interaction networks shows that alternatively spliced genes are highly versatile.

    Science.gov (United States)

    Talavera, David; Sheoran, Ritika; Lovell, Simon C

    2013-01-01

    Alternative splicing has the potential to increase the diversity of the transcriptome and proteome. Where more than one transcript arises from a gene they are often so different that they are quite unlikely to have the same function. However, it remains unclear if alternative splicing generally leads to a gene being involved in multiple biological processes or whether it alters the function within a single process. Knowing that genetic interactions occur between functionally related genes, we have used them as a proxy for functional versatility, and have analysed the sets of genes of two well-characterised model organisms: Caenorhabditis elegans and Drosophila melanogaster. Using network analyses we find that few genes are functionally homogenous (only involved in a few functionally-related biological processes). Moreover, there are differences between alternatively spliced genes and genes with a single transcript; specifically, genes with alternatively splicing are, on average, involved in more biological processes. Finally, we suggest that factors other than specific functional classes determine whether a gene is alternatively spliced.

  9. Isolation and characterization of "GmScream" promoters that regulate highly expressing soybean (Glycine max Merr.) genes.

    Science.gov (United States)

    Zhang, Ning; McHale, Leah K; Finer, John J

    2015-12-01

    To increase our understanding of the regulatory components that control gene expression, it is important to identify, isolate and characterize new promoters. In this study, a group of highly expressed soybean (Glycine max Merr.) genes, which we have named "GmScream", were first identified from RNA-Seq data. The promoter regions were then identified, cloned and fused with the coding region of the green fluorescent protein (gfp) gene, for introduction and analysis in different tissues using 3 tools for validation. Approximately half of the GmScream promoters identified showed levels of GFP expression comparable to or higher than the Cauliflower Mosaic Virus 35S (35S) promoter. Using transient expression in lima bean cotyledonary tissues, the strongest GmScream promoters gave over 6-fold higher expression than the 35S promoter while several other GmScream promoters showed 2- to 3-fold higher expression. The two highest expressing promoters, GmScreamM4 and GmScreamM8, regulated two different elongation factor 1A genes in soybean. In stably transformed soybean tissues, GFP driven by the GmScreamM4 or GmScreamM8 promoter exhibited constitutive high expression in most tissues with preferentially higher expression in proliferative embryogenic tissues, procambium, vascular tissues, root tips and young embryos. Using deletion analysis of the promoter, two proximal regions of the GmScreamM8 promoter were identified as contributing significantly to high levels of gene expression.

  10. Cloning and characterization of the gene encoding the highly expressed ribosomal protein l3 of the ciliated protozoan Tetrahymena thermophila. Evidence for differential codon usage in highly expressed genes

    DEFF Research Database (Denmark)

    Larsen, L K; Andreasen, P H; Dreisig, H

    1999-01-01

    We have cloned and characterized the cDNA and the macronuclear genomic copy of the highly conserved ribosomal protein (r-protein) L3 of Tetrahymena thermophila. The r-protein L3 is encoded by a single copy gene interrupted by one intron. The organization of the promoter region exhibits features...

  11. Global transcript structure resolution of high gene density genomes through multi-platform data integration.

    Science.gov (United States)

    O'Grady, Tina; Wang, Xia; Höner Zu Bentrup, Kerstin; Baddoo, Melody; Concha, Monica; Flemington, Erik K

    2016-10-14

    Annotation of herpesvirus genomes has traditionally been undertaken through the detection of open reading frames and other genomic motifs, supplemented with sequencing of individual cDNAs. Second generation sequencing and high-density microarray studies have revealed vastly greater herpesvirus transcriptome complexity than is captured by existing annotation. The pervasive nature of overlapping transcription throughout herpesvirus genomes, however, poses substantial problems in resolving transcript structures using these methods alone. We present an approach that combines the unique attributes of Pacific Biosciences Iso-Seq long-read, Illumina short-read and deepCAGE (Cap Analysis of Gene Expression) sequencing to globally resolve polyadenylated isoform structures in replicating Epstein-Barr virus (EBV). Our method, Transcriptome Resolution through Integration of Multi-platform Data (TRIMD), identifies nearly 300 novel EBV transcripts, quadrupling the size of the annotated viral transcriptome. These findings illustrate an array of mechanisms through which EBV achieves functional diversity in its relatively small, compact genome including programmed alternative splicing (e.g. across the IR1 repeats), alternative promoter usage by LMP2 and other latency-associated transcripts, intergenic splicing at the BZLF2 locus, and antisense transcription and pervasive readthrough transcription throughout the genome.

  12. Classification and Clinical Management of Variants of Uncertain Significance in High Penetrance Cancer Predisposition Genes.

    Science.gov (United States)

    Moghadasi, Setareh; Eccles, Diana M; Devilee, Peter; Vreeswijk, Maaike P G; van Asperen, Christi J

    2016-04-01

    In 2008, the International Agency for Research on Cancer (IARC) proposed a system for classifying sequence variants in highly penetrant breast and colon cancer susceptibility genes, linked to clinical actions. This system uses a multifactorial likelihood model to calculate the posterior probability that an altered DNA sequence is pathogenic. Variants between 5%-94.9% (class 3) are categorized as variants of uncertain significance (VUS). This interval is wide and might include variants with a substantial difference in pathogenicity at either end of the spectrum. We think that carriers of class 3 variants would benefit from a fine-tuning of this classification. Classification of VUS to a category with a defined clinical significance is very important because for carriers of a pathogenic mutation full surveillance and risk-reducing surgery can reduce cancer incidence. Counselees who are not carriers of a pathogenic mutation can be discharged from intensive follow-up and avoid unnecessary risk-reducing surgery. By means of examples, we show how, in selected cases, additional data can lead to reclassification of some variants to a different class with different recommendations for surveillance and therapy. To improve the clinical utility of this classification system, we suggest a pragmatic adaptation to clinical practice.

  13. Angiotensin II receptor 1 gene variants are associated with high-altitude pulmonary edema risk.

    Science.gov (United States)

    Jin, Tianbo; Ren, Yongchao; Zhu, Xikai; Li, Xun; Ouyang, Yongri; He, Xue; Zhang, Zhiying; Zhang, Yuan; Kang, Longli; Yuan, Dongya

    2016-11-22

    Previous studies demonstrated that Angiotensin II Receptor 1 (AGTR1) may play an important role in the development of high-altitude pulmonary edema. We envisaged a role for AGTR1 gene variants in the pathogenesis of HAPE and investigated their potential associations with HAPE in a Han Chinese population. We genotyped seven AGTR1 polymorphisms in 267 patients with diagnosed HAPE and 304 controls and evaluated their association with risk of HAPE. Statistically significant associations were found for the single nucleotide polymorphisms (SNPs) rs275651 (p = 0.017; odds ratio [OR] = 0.65) and rs275652 (p = 0.016; OR = 0.64). Another SNP rs10941679 showed a marginally significant association after adjusting for age and sex in the additive genetic model (adjusted OR = 1.44, 95% CI = 1.01-2.04, p = 0.040). Haplotype analysis confirmed that the haplotype "AG" was associated with a 35% reduction in the risk of developing HAPE, while the haplotype "AA" increased the risk of developing HAPE by 44%. These results provide the first evidence linking genetic variations in AGTR1 with HAPE risk in Han Chinese individuals.

  14. Molecular Analysis of Hemagglutinin Gene of a Goose Origin Highly Pathogenic Avian Influenza Virus

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The hemagglutinin (HA) of avian influenza virus (AIV) plays a key role in determining the pathogenicity, cell receptor-binding property and host range of the virus. A goose origin AIV A/Goose/Guangdong/1/96(H5N1) (GD/96) was confirmed as a highly pathogenic AIV (HPAIV) by the tests of intravenous pathogenic index (IVPI) and the assay of plaque formation. The sequence results of the HA gene cDNA of the isolate reveal that there is an insertion of 6 basic amino acids ( R-R-R-K-K-R-) in the cleavage site between the HA1 and HA2, which is the characterization of the H5 subtype HPAIV. When compared with the lethal A/Hongkong/156/97 (H5N1) (HK/97), there is a homology of 98% at the nucleotide level and 98. 2% at the amino acid level. Furthermore, no difference of nucleotides related to all of the 6 potential glycosylation sites, the 2 receptor-binding sites and the basic amino acid insert within the HA existed between GD/96 and HK/97. These results imply that the GD/96 and HK/97 have a closely related common ancestor and share the same biological properties decided by the HA.

  15. A locus on chromosome 20 encompassing genes that are highly expressed in the epididymis

    Institute of Scientific and Technical Information of China (English)

    (A)ke Lundwall

    2007-01-01

    During liquefaction of the ejaculate, the semen coagulum proteins semenogelin Ⅰ (SEMG1) and semenogelin Ⅱ (SEMG2) are degraded to low molecular mass fragments by kallikrein-related peptidase 3 (KLK3), also known as prostate-specific antigen. Semenogelin molecules initiate their own destruction by chelating Zn2+ that normally would completely inhibit the proteolytic activity of KLK3. In a similar way, semenogelins might regulate the activity of kallikrein-related peptidases in the epididymis, something that might be of importance for the maturation of spermatozoa or generation of anti-bacterial peptides. Studies on the evolution of semen coagulum proteins have revealed that most of them carry an exon that displays a rapid and unusual evolution. As a consequence, homologous proteins in rodents and primates show almost no conservation in primary structure. Further studies on their evolution suggest that the progenitor of the semen coagulum proteins probably was a protease inhibitor that might have displayed antimicrobial activity. The semenogelin locus on chromosome 20 contains at least 17 homologous genes encoding probable protease inhibitors with homology to semen coagulum proteins. All of these are highly expressed in the epididymis where they, similar to the semenogelins, could affect the maturation of spermatozoa or display antibacterial properties.

  16. Identifying the 'inorganic gene' for high-temperature piezoelectric perovskites through statistical learning.

    Science.gov (United States)

    Balachandran, Prasanna V; Broderick, Scott R; Rajan, Krishna

    2011-08-01

    This paper develops a statistical learning approach to identify potentially new high-temperature ferroelectric piezoelectric perovskite compounds. Unlike most computational studies on crystal chemistry, where the starting point is some form of electronic structure calculation, we use a data-driven approach to initiate our search. This is accomplished by identifying patterns of behaviour between discrete scalar descriptors associated with crystal and electronic structure and the reported Curie temperature (TC) of known compounds; extracting design rules that govern critical structure-property relationships; and discovering in a quantitative fashion the exact role of these materials descriptors. Our approach applies linear manifold methods for data dimensionality reduction to discover the dominant descriptors governing structure-property correlations (the 'genes') and Shannon entropy metrics coupled to recursive partitioning methods to quantitatively assess the specific combination of descriptors that govern the link between crystal chemistry and TC (their 'sequencing'). We use this information to develop predictive models that can suggest new structure/chemistries and/or properties. In this manner, BiTmO3-PbTiO3 and BiLuO3-PbTiO3 are predicted to have a TC of 730(°)C and 705(°)C, respectively. A quantitative structure-property relationship model similar to those used in biology and drug discovery not only predicts our new chemistries but also validates published reports.

  17. High prevalence of an anti-hypertriglyceridemic variant of the MLXIPL gene in Central Asia.

    Science.gov (United States)

    Nakayama, Kazuhiro; Yanagisawa, Yoshiko; Ogawa, Ayumi; Ishizuka, Yuumi; Munkhtulga, Lkhagvasuren; Charupoonphol, Phitaya; Supannnatas, Somjit; Kuartei, Stevenson; Chimedregzen, Ulziiburen; Koda, Yoshiro; Ishida, Takafumi; Kagawa, Yasuo; Iwamoto, Sadahiko

    2011-12-01

    MLXIPL is a transcription factor integral to the regulation of glycolysis and lipogenesis in the liver. Common variants of the MLXIPL gene (MLXIPL) are known to influence plasma triglyceride levels in people of European descent. As MLXIPL has a key role in energy storage, genetic variations of the MLXIPL may be relevant to physiological adaptations to nutritional stresses that have occurred during the evolution of modern humans. In the present study, we assessed the phenotypic consequences of the Q241H variant of MLXIPL in populations of Asian and Oceanian origin and also surveyed the prevalence of Q241H variant in populations worldwide. Multiple linear regression models based on 2373 individuals of Asian origin showed that the H allele was significantly associated with decreased concentrations of plasma triglycerides (P=0.0003). Direct genotyping of 1455 individuals from Africa, Asia and Oceania showed that the triglyceride-lowering H allele was found at quite low frequencies (0.00-0.16) in most of the populations examined. The exceptions were some Central Asian populations, including Mongolians, Tibetans and Uyghurs, which exhibited much higher frequencies of the H allele (0.21-0.26). The high prevalence of the H allele in Central Asia implies that the Q241H variant of MLXIPL might have been significant for utilization of carbohydrates and fats in the common ancestors of these populations, who successfully adapted to the environment of Central Asia by relying on nomadic livestock herding.

  18. Mutations in the WTX - gene are found in some high-grade microsatellite instable (MSI-H colorectal cancers

    Directory of Open Access Journals (Sweden)

    Scheel Silvio K

    2010-08-01

    Full Text Available Abstract Background Genetically, colorectal cancers (CRCs can be subdivided into tumors with chromosomal instability (CIN or microsatellite instability (MSI. In both types of CRCs genes that are involved in the degradation of β-CATENIN are frequently mutated. Whereas in CIN CRCs APC (Adenomatous Polyposis Coli is affected in most cases, high grade MSI (MSI-H CRCs frequently display mutations in various genes, like the APC-, AXIN2- or CTNNBI (β-CATENIN gene itself. Recently in Wilms tumors, WTX (Wilms tumor gene on the X-chromosome was discovered as another gene involved in the destruction of β-CATENIN. As the WTX-gene harbors a short T6-microsatellite in its N-terminal coding region, we hypothesized that frameshift-mutations might occur in MSI-H CRCs in the WTX gene, thus additionally contributing to the stabilization of β-CATENIN in human CRCs. Methods DNA was extracted from 632 formalin-fixed, paraffin-embedded metastatic CRCs (UICCIV and analyzed for MSI-H by investigating the stability of the highly sensitive microsatellite markers BAT25 and BAT26 applying fluorescence capillary electrophoresis (FCE. Then, in the MSI-H cases, well described mutational hot spot regions from the APC-, AXIN2- and CTNNBI genes were analyzed for genomic alterations by didesoxy-sequencing while the WTX T6-microsatellite was analyzed by fragment analysis. Additionally, the PCR products of T5-repeats were subcloned and mutations were validated using didesoxy-sequencing. Furthermore, the KRAS and the BRAF proto-oncogenes were analyzed for the most common activating mutations applying pyro-sequencing. mRNA expression of WTX from MSI-H and MSS cases and a panel of colorectal cancer cell lines was investigated using reverse transcription (RT- PCR and FCE. Results In our cohort of 632 metastatic CRCs (UICCIV we identified 41 MSI-H cases (6.5%. Two of the 41 MSI-H cases (4.8% displayed a frameshift mutation in the T6-repeat resulting in a T5 sequence. Only one case, a

  19. Environmental selection on transcriptome-derived SNPs in a high gene flow marine fish, the Atlantic herring (Clupea harengus)

    DEFF Research Database (Denmark)

    Limborg, Morten; Helyar, S.J.; de Bruyn, M.;

    2012-01-01

    High gene flow is considered the norm for most marine organisms and is expected to limit their ability to adapt to local environments. Few studies have directly compared the patterns of differentiation at neutral and selected gene loci in marine organisms. We analysed a transcriptome-derived panel...... of 281 SNPs in Atlantic herring (Clupea harengus), a highly migratory small pelagic fish, for elucidating neutral and selected genetic variation among populations and to identify candidate genes for environmental adaptation. We analysed 607 individuals from 18 spawning locations in the northeast Atlantic......, including two temperature clines (5–12 C) and two salinity clines (5–35&). By combining genome scan and landscape genetic analyses, four genetically distinct groups of herring were identified: Baltic Sea, Baltic–North Sea transition area, North Sea ⁄ British Isles and North Atlantic; notably, samples...

  20. Rasch-based high-dimensionality data reduction and class prediction with applications to microarray gene expression data

    CERN Document Server

    Kastrin, Andrej

    2010-01-01

    Class prediction is an important application of microarray gene expression data analysis. The high-dimensionality of microarray data, where number of genes (variables) is very large compared to the number of samples (obser- vations), makes the application of many prediction techniques (e.g., logistic regression, discriminant analysis) difficult. An efficient way to solve this prob- lem is by using dimension reduction statistical techniques. Increasingly used in psychology-related applications, Rasch model (RM) provides an appealing framework for handling high-dimensional microarray data. In this paper, we study the potential of RM-based modeling in dimensionality reduction with binarized microarray gene expression data and investigate its prediction ac- curacy in the context of class prediction using linear discriminant analysis. Two different publicly available microarray data sets are used to illustrate a general framework of the approach. Performance of the proposed method is assessed by re-randomization s...

  1. No allelic variation in genes with high gliadin homology in patients with celiac disease and type 1 diabetes

    DEFF Research Database (Denmark)

    Nielsen, Christian; Hansen, Dorte; Husby, Steffen

    2004-01-01

    Celiac disease (CD) is a complex inflammatory disorder of the small intestine, induced by dietary gluten in genetically susceptible individuals. CD is strongly associated with HLA-DQ2 and it has recently been established that gut-derived DQ2-restricted T cells from patients with CD predominantly...... recognize gluten-derived peptides in which specific glutamine residues are deamidated to glutamic acid by tissue transglutaminase. Recently, intestinally expressed human genes with high homology to DQ2-gliadin celiac T-cell epitopes have been identified. Single or double point mutations which would increase...... the celiac T-cell epitope homology, and mutation in these genes, leading to the expression of glutamic acid at particular positions, could hypothetically be involved in the initiation of CD in HLA-DQ2-positive children. Six gene regions with high celiac T-cell epitope homology were investigated for single...

  2. Identification of rare high-risk copy number variants affecting the dopamine transporter gene in mental disorders

    DEFF Research Database (Denmark)

    Hoeffding, Louise K; Duong, Linh T T; Ingason, Andrés;

    2015-01-01

    BACKGROUND: The dopamine transporter, also known as solute carrier 6A3 (SLC6A3), plays an important role in synaptic transmission by regulating the reuptake of dopamine in the synapses. In line with this, variations in the gene encoding this transporter have been linked to both schizophrenia...... rare high-risk variants of psychiatric disorders. METHODS: We performed a systematic screening for CNVs affecting SLC6A3 in 761 healthy controls, 672 schizophrenia patients, and 194 patients with bipolar disorder in addition to 253 family members from six large pedigrees affected by mental disorders...... sizes and two affected several genes in addition to SLC6A3. CONCLUSION: Our findings suggest that rare high-risk CNVs affecting the gene encoding the dopamine transporter contribute to the pathogenesis of schizophrenia and affective disorders....

  3. High rates of gene flow by pollen and seed in oak populations across Europe

    NARCIS (Netherlands)

    Gerber, S.; Chadoeuf, J.; Gugerli, F.; Lascoux, M.; Buiteveld, J.; Cottrell, J.; Dounavi, A.; Fineschi, S.; Forrest, L.; Fogelqvist, J.; Goicoechea, P.G.; Jensen, J.S.; Salvini, D.; Vendramin, G.G.; Kremer, A.

    2014-01-01

    Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea) distributed across Europe. Adult tree

  4. Detecting robust gene signature through integrated analysis of multiple types of high-throughput data in liver cancer

    Institute of Scientific and Technical Information of China (English)

    Xin-yu ZHANG; Tian-tian LI; Xiang-jun LIU

    2007-01-01

    Aim: To investigate the robust gene signature in liver cancer, we applied an integrated approach to perform a joint analysis of a highly diverse collection of liver cancer genome-wide datasets, including genomic alterations and transcrip- tion profiles. Methods: 1-class Significance Analysis of Microarrays coupled with ranking score method were used to identify the robust gene signature in liver tumor tissue. Results: In total, 1 625 051 gene expression measurements from 16 public microarrays, 2 pairs of serial analyses of gene expression experiments, and 252 loss of heterozygosity reports obtained from 568 publications were used in this integrated study. The resulting robust gene signatures included 90 genes, which may be of great importance to liver cancer research. A system assessment analysis revealed that our integrative method had an accuracy of 92% and a correlation coefficient value of 0.88. Conclusion: The system assessment results indicated that our method had the ability of integrating the datasets from various types of sources, and eliciting more accurate results, as can be very useful in the study of liver cancer.

  5. Cross-linked Polyethylenimine as Potential DNA Vector for Gene Delivery with High Efficiency and Low Cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Wei DONG; Guang-Hui JIN; Shu-Feng LI; Qi-Ming SUN; Ding-Yuan MA; Zi-Chun HUA

    2006-01-01

    Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on its molecular weight. To enhance its gene delivery efficiency and minimize cytotoxicity, we have synthesized small cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro. In this study, branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate [ 1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h. The efficiencies of the cross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein (EGFP) reporter gene were assessed in melanoma B 16F10 cell line and other cell lines. Flow cytometry was used to quantify the cellular entry efficiency of plasmid and the transgene expression level. The cytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay. EGDMA-PEI 800-4h, a typical cross-linked PEI reported here, mediated a more efficient expression of reporter gene than the commercially available 25-kDa branched PEI control, and resulted in a 9-fold increase in gene delivery in B16F10 cells and a 16-fold increase in 293T cells, while no cytotoxicity was found at the optimized condition for gene delivery. Furthermore, the transfection activity of polyplexes was preserved in the presence of serum proteins.

  6. A cholesterol-free, high-fat diet suppresses gene expression of cholesterol transporters in murine small intestine

    NARCIS (Netherlands)

    den Bosch, Heleen M. de Vogel-van; de Wit, Nicole J. W.; Hooiveld, Guido J. E. J.; Vermeulen, Hanneke; van der Veen, Jelske N.; Houten, Sander M.; Kuipers, Folkert; Mueller, Michael; van der Meer, Roelof

    2008-01-01

    A cholesterol-free, high-fat diet suppresses gene expression of cholesterol transporters in murine small intestine. Am J Physiol Gastrointest Liver Physiol 294: G1171-G1180, 2008. First published March 20, 2008; doi:10.1152/ajpgi.00360.2007.-Transporters present in the epithelium of the small intest

  7. Replication fitness determines high virulence of influenza A virus in mice carrying functional Mx1 resistance gene.

    Science.gov (United States)

    Grimm, Daniel; Staeheli, Peter; Hufbauer, Martin; Koerner, Iris; Martínez-Sobrido, Luis; Solórzano, Alicia; García-Sastre, Adolfo; Haller, Otto; Kochs, Georg

    2007-04-17

    The IFN-induced resistance factor Mx1 is a critical component of innate immunity against influenza A viruses (FLUAV) in mice. Animals carrying a wild-type Mx1 gene (Mx1(+/+)) differ from regular laboratory mice (Mx1(-/-)) in that they are highly resistant to infection with standard FLUAV strains. We identified an extraordinary variant of the FLUAV strain, A/PR/8/34 (H1N1) (designated hvPR8), which is unusually virulent in Mx1(+/+) mice. hvPR8 was well controlled in Mx1(+/+) but not Mx1(-/-) mice provided that the animals were treated with IFN before infection, indicating that hvPR8 exhibits normal sensitivity to growth restriction by Mx1. hvPR8 multiplied much faster than standard PR8 early in infection because of highly efficient viral gene expression in infected cells. Studies with reassortant viruses containing defined genome segments of both hvPR8 and standard PR8 demonstrated that the HA, neuraminidase, and polymerase genes of hvPR8 all contributed to virulence, indicating that efficient host cell entry and early gene expression renders hvPR8 highly pathogenic. These results reveal a surprisingly simple concept of how influenza viruses may gain virulence and illustrate that high speed of virus growth can outcompete the antiviral response of the infected host.

  8. Cis-eQTL analysis and functional validation of candidate susceptibility genes for high-grade serous ovarian cancer

    NARCIS (Netherlands)

    Lawrenson, K.; Li, Q.; Kar, S.; Seo, J.H.; Tyrer, J.; Spindler, T.J.; Lee, J. van der; Chen, Y; Karst, A.; Drapkin, R.; Aben, K.K.H.; Anton-Culver, H.; Antonenkova, N.; Baker, H.; Bandera, E.V.; Bean, Y.; Beckmann, M.W.; Berchuck, A.; Bisogna, M.; Bjorge, L.; Bogdanova, N.; Brinton, L.A.; Brooks-Wilson, A.; Bruinsma, F.; Butzow, R.; Campbell, I.G.; Carty, K.; Chang-Claude, J.; Chenevix-Trench, G.; Chen, A; Chen, Z.; Cook, L.S.; Cramer, D.W; Cunningham, J.M.; Cybulski, C.; Dansonka-Mieszkowska, A.; Dennis, J.; Dicks, E.; Doherty, J.A.; Dork, T.; Bois, A. du; Durst, M.; Eccles, D.; Easton, D.T.; Edwards, R.P.; Eilber, U.; Ekici, A.B.; Fasching, P.A.; Fridley, B.L.; Gao, Y.T.; Gentry-Maharaj, A.; Giles, G.G.; Glasspool, R.; Goode, E.L.; Goodman, M.T.; Grownwald, J.; Harrington, P.; Harter, P.; Hasmad, H.N.; Hein, A.; Heitz, F.; Hildebrandt, M.A.; Hillemanns, P.; Hogdall, E.; Hogdall, C.; Hosono, S.; Iversen, E.S.; Jakubowska, A.; James, P.; Jensen, A.; Ji, B.T.; Karlan, B.Y.; Kjaer, S. Kruger; Kelemen, L.E.; Kellar, M.; Kelley, J.L.; Kiemeney, L.A.; Krakstad, C.; Kupryjanczyk, J.; Lambrechts, D.; Lambrechts, S.; Le, N.D.; Lee, A.W.; Lele, S.; Leminen, A.; Lester, J.; Levine, D.A.; Liang, D.; Lissowska, J.; Lu, K.; Lubinski, J.; Lundvall, L.; Massuger, L.F.; Matsuo, K.; McGuire, V.; McLaughlin, J.R.; Nevanlinna, H.; McNeish, I.; Menon, U.; Modugno, F.

    2015-01-01

    Genome-wide association studies have reported 11 regions conferring risk of high-grade serous epithelial ovarian cancer (HGSOC). Expression quantitative trait locus (eQTL) analyses can identify candidate susceptibility genes at risk loci. Here we evaluate cis-eQTL associations at 47 regions associat

  9. Effect of metformin on proliferation and related genes expression of human osteoblast MG63 under high glucose

    Institute of Scientific and Technical Information of China (English)

    曹小俊

    2013-01-01

    Objective To study the effect of metformin on proliferation and related genes expression of human osteoblast.Methods The proliferation of MG63 cells under high glucose intervened with metformin was measured by CCK-8 assay. The activity of intracellular alkaline phosphatase

  10. An emerging avian influenza A virus H5N7 is a genetic reassortant of highly pathogenic genes

    DEFF Research Database (Denmark)

    Bragstad, K.; Jørgensen, Poul Henrik; Handberg, Kurt;

    2006-01-01

    We full genome characterised the newly discovered avian influenza virus H5N7 subtype combination isolated from a stock of Danish game ducks to investigate the composition of the genome and possible features of high pathogenicity. It was found that the haemagglutinin and the acidic polymerase genes...... low pathogenic avian influenza A viruses. (c) 2006 Elsevier Ltd. All rights reserved....

  11. The AFT1 transcriptional factor is differentially required for expression of high-affinity iron uptake genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Casas, C; Aldea, M; Espinet, C; Gallego, C; Gil, R; Herrero, E

    1997-06-15

    High-affinity iron uptake in Saccharomyces cerevisiae involves the extracytoplasmic reduction of ferric ions by FRE1 and FRE2 reductases. Ferrous ions are then transported across the plasma membrane through the FET3 oxidase-FTR1 permease complex. Expression of the high-affinity iron uptake genes is induced upon iron deprivation. We demonstrate that AFT1 is differentially involved in such regulation. Aft1 protein is required for maintaining detectable non-induced level of FET3 expression and for induction of FRE2 in iron starvation conditions. On the contrary, FRE1 mRNA induction is normal in the absence of Aft1, although the existence of AFT1 point mutations causing constitutive expression of FRE1 (Yamaguchi-Iwai et al., EMBO J. 14: 1231-1239, 1995) indicates that Aft1 may also participate in FRE1 expression in a dispensable way. The alterations in the basal levels of expression of the high-affinity iron uptake genes may explain why the AFT1 mutant is unable to grow on respirable carbon sources. Overexpression of AFT1 leads to growth arrest of the G1 stage of the cell cycle. Aft1 is a transcriptional activator that would be part of the different transcriptional complexes interacting with the promoter of the high-affinity iron uptake genes. Aft1 displays phosphorylation modifications depending on the growth stage of the cells, and it might link induction of genes for iron uptake to other metabolically dominant requirement for cell growth.

  12. cDNA microarray reveals the alterations of cytoskeleton-related genes in osteoblast under high magneto-gravitational environment

    Institute of Scientific and Technical Information of China (English)

    Airong Qian; Shengmeng Di; Xiang Gao; Wei Zhang; Zongcheng Tian; Jingbao Li; Lifang Hu; Pengfei Yang; Dachuan Yin; Peng Shang

    2009-01-01

    The diamagnetic levitation as a novel ground-based model for simulating a reduced gravity environment has been widely applied in many fields.In this study,a special designed superconducting magnet,which can produce three apparent gravity levels (0,1,and 2 g),namely high magneto-gravitational environment (HMGE),was used to simulate space gravity environment.The effects of HMGE on osteoblast gene expression profile were investigated by microarray.Genes sensitive to diamagnetic levitation environment (0 g),gravity changes,and high magnetic field changes were sorted on the basis of typical cell func-tions.Cytoskeleton,as an intracellular load-bearing struc-ture,plays an important role in gravity perception.Therefore,13 cytoskeleton-related genes were chosen according to the results of microarray analysis,and the expressions of these genes were found to be altered under HMGE by real-time PCR.Based on the PCR results,the expressions of WASF2 (WAS protein family,member 2),WIPFI (WAS/WASL interacting protein family,member 1),paxillin:and talin 1 were further identified by western blot assay.Results indicated that WASF2 and WIPF1 were more sensitive to altered gravity levels,and talin 1 and paxillin were sensitive to both magnetic field and gravity changes.Our findings demonstrated that HMGE can affect osteoblast gene expression profile and cytoskele-ton-related genes expression.The identification of mechanosensitive genes may enhance our understandings to the mechanism of bone loss induced by microgravity and may provide some potential targets for preventing and treating bone loss or osteoporosis.

  13. Integrated analyses of microRNAs demonstrate their widespread influence on gene expression in high-grade serous ovarian carcinoma.

    Directory of Open Access Journals (Sweden)

    Chad J Creighton

    Full Text Available BACKGROUND: The Cancer Genome Atlas (TCGA Network recently comprehensively catalogued the molecular aberrations in 487 high-grade serous ovarian cancers, with much remaining to be elucidated regarding the microRNAs (miRNAs. Here, using TCGA ovarian data, we surveyed the miRNAs, in the context of their predicted gene targets. METHODS AND RESULTS: Integration of miRNA and gene patterns yielded evidence that proximal pairs of miRNAs are processed from polycistronic primary transcripts, and that intronic miRNAs and their host gene mRNAs derive from common transcripts. Patterns of miRNA expression revealed multiple tumor subtypes and a set of 34 miRNAs predictive of overall patient survival. In a global analysis, miRNA:mRNA pairs anti-correlated in expression across tumors showed a higher frequency of in silico predicted target sites in the mRNA 3'-untranslated region (with less frequency observed for coding sequence and 5'-untranslated regions. The miR-29 family and predicted target genes were among the most strongly anti-correlated miRNA:mRNA pairs; over-expression of miR-29a in vitro repressed several anti-correlated genes (including DNMT3A and DNMT3B and substantially decreased ovarian cancer cell viability. CONCLUSIONS: This study establishes miRNAs as having a widespread impact on gene expression programs in ovarian cancer, further strengthening our understanding of miRNA biology as it applies to human cancer. As with gene transcripts, miRNAs exhibit high diversity reflecting the genomic heterogeneity within a clinically homogeneous disease population. Putative miRNA:mRNA interactions, as identified using integrative analysis, can be validated. TCGA data are a valuable resource for the identification of novel tumor suppressive miRNAs in ovarian as well as other cancers.

  14. RGD peptide-modified dendrimer-entrapped gold nanoparticles enable highly efficient and specific gene delivery to stem cells.

    Science.gov (United States)

    Kong, Lingdan; Alves, Carla S; Hou, Wenxiu; Qiu, Jieru; Möhwald, Helmuth; Tomás, Helena; Shi, Xiangyang

    2015-03-04

    We report the use of arginine-glycine-aspartic (Arg-Gly-Asp, RGD) peptide-modified dendrimer-entrapped gold nanoparticles (Au DENPs) for highly efficient and specific gene delivery to stem cells. In this study, generation 5 poly(amidoamine) dendrimers modified with RGD via a poly(ethylene glycol) (PEG) spacer and with PEG monomethyl ether were used as templates to entrap gold nanoparticles (AuNPs). The native and the RGD-modified PEGylated dendrimers and the respective well characterized Au DENPs were used as vectors to transfect human mesenchymal stem cells (hMSCs) with plasmid DNA (pDNA) carrying both the enhanced green fluorescent protein and the luciferase (pEGFPLuc) reporter genes, as well as pDNA encoding the human bone morphogenetic protein-2 (hBMP-2) gene. We show that all vectors are capable of transfecting the hMSCs with both pDNAs. Gene transfection using pEGFPLuc was demonstrated by quantitative Luc activity assay and qualitative evaluation by fluorescence microscopy. For the transfection with hBMP-2, the gene delivery efficiency was evaluated by monitoring the hBMP-2 concentration and the level of osteogenic differentiation of the hMSCs via alkaline phosphatase activity, osteocalcin secretion, calcium deposition, and von Kossa staining assays. Our results reveal that the stem cell gene delivery efficiency is largely dependent on the composition and the surface functionality of the dendrimer-based vectors. The coexistence of RGD and AuNPs rendered the designed dendrimeric vector with specific stem cell binding ability likely via binding of integrin receptor on the cell surface and improved three-dimensional conformation of dendrimers, which is beneficial for highly efficient and specific stem cell gene delivery applications.

  15. Expression Characterization of Stress Genes Under High and Low Temperature Stresses in the Pacific Oyster, Crassostrea gigas.

    Science.gov (United States)

    Zhu, Qihui; Zhang, Linlin; Li, Li; Que, Huayong; Zhang, Guofan

    2016-04-01

    As a characteristic sessile inhabitant of the intertidal zone, the Pacific oyster Crassostrea gigas occupies one of the most physically stressful environments on earth. With high exposure to terrestrial conditions, oysters must tolerate broad fluctuations in temperature range. However, oysters' cellular and molecular responses to temperature stresses have not been fully characterized. Here, we analyzed oyster transcriptome data under high and low temperatures. We also identified over 30 key temperature stress-responsive candidate genes, which encoded stress proteins such as heat shock proteins and apoptosis-associated proteins. The expression characterization of these genes under short-term cold and hot environments (5 and 35 °C) and long-term cold environments (5 °C) was detected by quantitative real-time PCR. Most of these genes reached expression peaks during the recovery stage after 24 h of heat stress, and these genes were greatly induced around day 3 in long-term cold stress while responded little to short-term cold stress. In addition, in the second heat stress after 2 days of recovery, oysters showed milder expression in these genes and a lower mortality rate, which indicated the existence of plasticity in the oyster's response to heat stress. We confirmed that homeostatic flexibility and anti-apoptosis might be crucial centers of temperature stress responses in oysters. Furthermore, we analyzed stress gene families in 11 different species and found that the linage-specific expansion of stress genes might be implicated in adaptive evolution. These results indicated that both plasticity and evolution played an important role in the stress response adaptation of oysters.

  16. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Keisuke Nakajima

    2015-01-01

    Full Text Available Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3′UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3′UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf. In contrast, TALEN mRNAs without this 3′UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  17. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

    Science.gov (United States)

    Nakajima, Keisuke; Yaoita, Yoshio

    2015-01-16

    Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3'UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3'UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf). In contrast, TALEN mRNAs without this 3'UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT) stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  18. Characterization of a bZIP gene highly expressed during ripening of the peach fruit.

    Science.gov (United States)

    Lovisetto, Alessandro; Guzzo, Flavia; Tadiello, Alice; Confortin, Enrico; Pavanello, Anna; Botton, Alessandro; Casadoro, Giorgio

    2013-09-01

    A ripening specific bZIP gene of peach was studied by ectopically expressing it in tomato. Two lines, with either a mild or a strong phenotype, respectively, were analyzed in detail. Transgenic fruit morphology was normal, yet the time spent to proceed through the various ripening stages was longer compared to wild type. In agreement with this finding the transgenic berries produced less ethylene, and also had a modified expression of some ripening-related genes that was particularly evident in berries with a strong phenotype. In particular, in the latter fruits polygalacturonase and lipoxygenase genes, but also genes coding for transcription factors (TFs) important for tomato ripening (i.e. TAGL1, CNR, APETALA2a, NOR) did not show the expected decreased expression in the red berries. As regards the RIN gene, its expression continued to increase in both mild and strong lines, and this is in agreement with the dilated ripening times. Interestingly, a metabolomic analysis of berries at various stages of ripening showed that the longer time spent by the transgenic berries to proceed from a stage to another was not due to a slackened metabolism. In fact, the differences in amount of stage-specific marker metabolites indicated that the transgenic berries had a very active metabolism. Therefore, the dilated ripening and the enhanced metabolism of the berries over-expressing the bZIP gene suggest that such gene might regulate ripening by acting as a pacemaker for some of the ripening metabolic pathways.

  19. Characters of homogentisate oxygenase gene mutation and high clonality of the natural pigment-producing Vibrio cholerae strains

    Directory of Open Access Journals (Sweden)

    Diao Baowei

    2011-05-01

    Full Text Available Abstract Background Some microorganisms can produce pigments such as melanin, which has been associated with virulence in the host and with a survival advantage in the environment. In Vibrio cholerae, studies have shown that pigment-producing mutants are more virulent than the parental strain in terms of increased UV resistance, production of major virulence factors, and colonization. To date, almost all of the pigmented V. cholerae strains investigated have been induced by chemicals, culture stress, or transposon mutagenesis. However, during our cholera surveillance, some nontoxigenic serogroup O139 strains and one toxigenic O1 strain, which can produce pigment steadily under the commonly used experimental growth conditions, were obtained in different years and from different areas. The genes VC1344 to VC1347, which correspond to the El Tor strain N16961 genome and which comprise an operon in the tyrosine catabolic pathway, have been confirmed to be associated with a pigmented phenotype. In the present study, we investigated the mechanism of pigment production in these strains. Results Sequencing of the VC1344, VC1345, VC1346, and VC1347 genes in these pigmented strains suggested that a deletion mutation in the homogentisate oxygenase gene (VC1345 may be associated with the pigmented phenotype, and gene complementation confirmed the role of this gene in pigment production. An identical 15-bp deletion was found in the VC1345 gene of all six O139 pigment-producing strains examined, and a 10-bp deletion was found in the VC1345 gene of the O1 strain. Strict sequence conservation in the VC1344 gene but higher variance in the other three genes of this operon were observed, indicating the different stress response functions of these genes in environmental adaption and selection. On the basis of pulsed-field gel electrophoresis typing, the pigment-producing O139 strains showed high clonality, even though they were isolated in different years and from

  20. Local adaptation with high gene flow: temperature parameters drive adaptation to altitude in the common frog (Rana temporaria)

    OpenAIRE

    2014-01-01

    Both environmental and genetic influences can result in phenotypic variation. Quantifying the relative contributions of local adaptation and phenotypic plasticity to phenotypes is key to understanding the effect of environmental variation on populations. Identifying the selective pressures that drive divergence is an important, but often lacking, next step. High gene flow between high- and low-altitude common frog (Rana temporaria) breeding sites has previously been demonstrated in Scotland. ...

  1. Effect of high temperature on the expressions of genes encoding starch synthesis enzymes in developing rice endosperms

    Institute of Scientific and Technical Information of China (English)

    CAO Zhen-zhen; PAN Gang; WANG Fu-biao; WEI Ke-su; LI Zhao-wei; SHI Chun-hai; GENG Wei; CHENG Fang-min

    2015-01-01

    High temperature is the major environmental factor affecting grain starch properties of cooking rice cultivars. In this study, two non-waxy indica rice genotypes, cv. 9311 and its mutant with extremely high amylose phenotype (9311eha) were used to study the differential expressions of genes in starch synthesis and their responses to high temperature (HT). Signiifcant increase in apparent amylose content and hot-water-soluble starch content in mutant 9311eha were genetical y caused by a substitution from AGTTATA to AGGTATA at the leader intron 5´ splice site in Wx gene. This mutation resulted in different mRNA transcript levels, mRNA splicing efifciencies and protein levels of Wx between the two rice genotypes, which also lead to the genotype-dependent alteration in the temporal pattern of Wx transcription and granule-bound starch synthase (GBSS) activity in response to HT. However, changes in the activities of other starch synthesizing enzymes and their expressions of distinct isoform genes were not signiifcant with the Wx gene mutation, thus only minor difference in the particle size of starch granule, chain-length distribution and gelatinization enthalpy were found between the two genotypes. The tempo-ral-speciifc expression of multiple isoform genes responsive to different temperature regiments indicated that the reduction of GBSS transcript expression under HT was general y accompanied by the decreased expressions of SSSIIa, SSSIIIa and SBEIIb. Consequently, high temperature-ripened grains in 9311eha showed high proportion of intermediate and long B chains and somewhat lower level of short A chain compared to the wildtype. The temperature-dependent alteration of amylose content was not only attributed to the reduced expression of GBSS, but also associated with the complimentary effect of SSSIIa and SBEIIb.

  2. Indica rice cultivar IRGA 424, transformed with cry genes of B. thuringiensis, provided high resistance against Spodoptera frugiperda (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Pinto, Laura Massochin Nunes; Fiuza, Lidia Mariana; Ziegler, Denize; De Oliveira, Jaime Vargas; Menezes, Valmir Gaedke; Bourrié, Isabelle; Meynard, Donaldo; Guiderdoni, Emmanuel; Breitler, Jean-Christophe; Altosaar, Illimar; Gantet, Pascal

    2013-12-01

    Plant expression of the entomopathogenic bacteria Bacillus thuringiensis cry gene has reduced the damage created by insect pests in several economically important cultures. For this study, we have conducted genetic transformation of the indica rice "IRGA 424", via Agrobacterium tumefaciens, using the B. thuringiensis cry1Aa and cry1B genes, with the objective of obtaining rice plants resistant to the insect pests from this culture. The gene constructions harbor the promoters maize proteinase inhibitor and ubiquitin. The results showed that high concentration of the hormone 2,4-dichlorophenoxyacetic acid and agarose as the gelling agent helped the production of embryogenic calli for the analyzed cultivar. More than 80% of the obtained transformed plants revealed the integration, using polymerase chain reaction, of the cry1Aa and cry1B genes. Analysis of the expression of the heterologous protein by Western blotting revealed the expression of the Cry1B delta-endotoxin in IRGA 424 plants transformed with the ubiquitin promoter. Data showed the production and dissemination of a high number of embryogenic calli in addition to obtaining plants transformed with the cry1Aa and cry1B genes until the reproductive phase. The feed bioassays with the transformed plants and Spodoptera frugiperda (JE Smith) larvae indicated high rates of mortality to the insect target. The highest corrected mortality rate achieved under laboratory conditions with Bt-rice plants transformed with the cry1B and cry1Aa genes was 94 and 84%, respectively. Thus, our results demonstrated the great potential of transformed Bt-rice plants in controlling the damage caused by these insect pests in rice paddy fields.

  3. Local adaptation with high gene flow: temperature parameters drive adaptation to altitude in the common frog (Rana temporaria).

    Science.gov (United States)

    Muir, A P; Biek, R; Thomas, R; Mable, B K

    2014-02-01

    Both environmental and genetic influences can result in phenotypic variation. Quantifying the relative contributions of local adaptation and phenotypic plasticity to phenotypes is key to understanding the effect of environmental variation on populations. Identifying the selective pressures that drive divergence is an important, but often lacking, next step. High gene flow between high- and low-altitude common frog (Rana temporaria) breeding sites has previously been demonstrated in Scotland. The aim of this study was to assess whether local adaptation occurs in the face of high gene flow and to identify potential environmental selection pressures that drive adaptation. Phenotypic variation in larval traits was quantified in R. temporaria from paired high- and low-altitude sites using three common temperature treatments. Local adaptation was assessed using Q(ST)-F(ST) analyses, and quantitative phenotypic divergence was related to environmental parameters using Mantel tests. Although evidence of local adaptation was found for all traits measured, only variation in larval period and growth rate was consistent with adaptation to altitude. Moreover, this was only evident in the three mountains with the highest high-altitude sites. This variation was correlated with mean summer and winter temperatures, suggesting that temperature parameters are potentially strong selective pressures maintaining local adaptation, despite high gene flow.

  4. Using the synergism strategy for highly sensitive and specific electrochemical sensing of Streptococcus pneumoniae Lyt-1 gene sequence.

    Science.gov (United States)

    Li, Fengqin; Yu, Zhigang; Xu, Yanmei; Ma, Huiyuan; Zhang, Guiling; Song, Yongbin; Yan, Hong; He, Xunjun

    2015-07-30

    With the help of the interaction mode of capture probe-target-signal probe (CP-T-SP), an electrochemical sensing method based on the synergism strategy of dual-hybridized signaling probes modified with 6 MB (methylene blue), background suppression and large surface area Au electrode is developed for the detection of Streptococcus pneumoniae (S. pneumoniae) Lyt-1 gene sequence. The proposed sensor features a very low detection limit (LOD) of ∼0.5 fM for the target. This method also exhibits highly versatility and can apply to the construction of other sensors for the analysis of similar designated pathogenic bacteria gene sequence (PBGS).

  5. Gene Expression of Stress Proteins and Identification of Molecular Markers of Plant Resistance to High Temperatures and Drought

    OpenAIRE

    L.P. Khokhlova

    2016-01-01

    Molecular biomarkers of plant resistance to both individual and combined action of high tempera-tures (42 °C) and drought have been identified. For this purpose, correlation between gene expression of four stress proteins (non-photosynthetic malic enzyme (TaNADP-ME2), serine-threonine kinase (W55a), dehydrin (DHN14), and lipocalin (TaTIL)) and resistance of eight spring wheat cultivars has been determined for the first time. Gene expression has been studied using the RT-PCR method based on th...

  6. piggyBac as a high-capacity transgenesis and gene-therapy vector in human cells and mice

    Directory of Open Access Journals (Sweden)

    Rongbo Li

    2013-05-01

    The stable genomic integration and expression of a large transgene is a major hurdle in gene therapy. We show that the modified piggyBac (PB transposon system can be used to introduce a 207 kb genomic DNA fragment containing the RORγ/γt locus into human cells and mice. PB-mediated transgenesis results in a single copy of a stably inherited and expressed transgene. These results indicate that PB could serve as an effective high-capacity vector for functional analysis of the mammalian genome and for gene therapy in human cells.

  7. A Highly Efficient Gene Expression Programming (GEP Model for Auxiliary Diagnosis of Small Cell Lung Cancer.

    Directory of Open Access Journals (Sweden)

    Zhuang Yu

    Full Text Available Lung cancer is an important and common cancer that constitutes a major public health problem, but early detection of small cell lung cancer can significantly improve the survival rate of cancer patients. A number of serum biomarkers have been used in the diagnosis of lung cancers; however, they exhibit low sensitivity and specificity.We used biochemical methods to measure blood levels of lactate dehydrogenase (LDH, C-reactive protein (CRP, Na+, Cl-, carcino-embryonic antigen (CEA, and neuron specific enolase (NSE in 145 small cell lung cancer (SCLC patients and 155 non-small cell lung cancer and 155 normal controls. A gene expression programming (GEP model and Receiver Operating Characteristic (ROC curves incorporating these biomarkers was developed for the auxiliary diagnosis of SCLC.After appropriate modification of the parameters, the GEP model was initially set up based on a training set of 115 SCLC patients and 125 normal controls for GEP model generation. Then the GEP was applied to the remaining 60 subjects (the test set for model validation. GEP successfully discriminated 281 out of 300 cases, showing a correct classification rate for lung cancer patients of 93.75% (225/240 and 93.33% (56/60 for the training and test sets, respectively. Another GEP model incorporating four biomarkers, including CEA, NSE, LDH, and CRP, exhibited slightly lower detection sensitivity than the GEP model, including six biomarkers. We repeat the models on artificial neural network (ANN, and our results showed that the accuracy of GEP models were higher than that in ANN. GEP model incorporating six serum biomarkers performed by NSCLC patients and normal controls showed low accuracy than SCLC patients and was enough to prove that the GEP model is suitable for the SCLC patients.We have developed a GEP model with high sensitivity and specificity for the auxiliary diagnosis of SCLC. This GEP model has the potential for the wide use for detection of SCLC in less

  8. Acquisition of useful and high ability genes for acidophilic bacteria; Kosansei saikin ni takai noryoku wo fuyosuru idenshi no kakutoku

    Energy Technology Data Exchange (ETDEWEB)

    Senda, T.; Inoue, C.; Shinbori, Y. [Tohoku University, Sendai (Japan)

    1997-02-01

    This effort aims at the development of high-performance bacteria usable in bio-leaching in metal smelting by acquiring genes capable of realizing such. A method is used of choosing some isolated strains exhibiting high-performance traits and acquiring target genes therefrom by use of genetic engineering. Approximately 200 kinds in the aggregate of acidophilic bacteria are currently available for the study, including isolated iron-oxidizing and sulfur-oxidizing bacteria, standard species acquired for the study, and strains previously isolated by the laboratory. The bacteria are tested with respect to their Fe{sup 2+}-oxidizing rates, sulfur-oxidizing capabilities, and strength to withstand inhibiting substances (Ag{sup +}, Cl{sup -}, Mo{sup 6+}, etc.), which results in the nomination of 8 strains. The study planned to follow includes processes involving the extraction of chromosome DNAs from the 8 strains and their refinement, gene cloning by the Southern hybridization method, determination of their base sequences, determination of the difference between the strains in point of gene expression, and investigations of the relations that the results of these processes bear toward the said high-performance traits. Also under way is a study about the infuence-exerting factors revealed during the evaluation of the abilities of acidphlic bacteria. 2 refs., 2 tabs.

  9. Targeted high-throughput growth hormone 1 gene sequencing reveals high within-breed genetic diversity in South African goats.

    Science.gov (United States)

    Ncube, K T; Mdladla, K; Dzomba, E F; Muchadeyi, F C

    2016-06-01

    This study assessed the genetic diversity in the growth hormone 1 gene (GH1) within and between South African goat breeds. Polymerase chain reaction-targeted gene amplification together with Illumina MiSeq next-generation sequencing (NGS) was used to generate the full length (2.54 kb) of the growth hormone 1 gene and screen for SNPs in the South African Boer (SAB) (n = 17), Tankwa (n = 15) and South African village (n = 35) goat populations. A range of 27-58 SNPs per population were observed. Mutations resulting in amino acid changes were observed at exons 2 and 5. Higher within-breed diversity of 97.37% was observed within the population category consisting of SA village ecotypes and the Tankwa goats. Highest pairwise FST values ranging from 0.148 to 0.356 were observed between the SAB and both the South African village and Tankwa feral goat populations. Phylogenetic analysis indicated nine genetic clusters, which reflected close relationships between the South African populations and the other international breeds with the exception of the Italian Sarda breeds. Results imply greater potential for within-population selection programs, particularly with SA village goats.

  10. Detecting differential allelic expression using high-resolution melting curve analysis: application to the breast cancer susceptibility gene CHEK2

    Directory of Open Access Journals (Sweden)

    Sinilnikova Olga

    2011-05-01

    Full Text Available Abstract Background The gene CHEK2 encodes a checkpoint kinase playing a key role in the DNA damage pathway. Though CHEK2 has been identified as an intermediate breast cancer susceptibility gene, only a small proportion of high-risk families have been explained by genetic variants located in its coding region. Alteration in gene expression regulation provides a potential mechanism for generating disease susceptibility. The detection of differential allelic expression (DAE represents a sensitive assay to direct the search for a functional sequence variant within the transcriptional regulatory elements of a candidate gene. We aimed to assess whether CHEK2 was subject to DAE in lymphoblastoid cell lines (LCLs from high-risk breast cancer patients for whom no mutation in BRCA1 or BRCA2 had been identified. Methods We implemented an assay based on high-resolution melting (HRM curve analysis and developed an analysis tool for DAE assessment. Results We observed allelic expression imbalance in 4 of the 41 LCLs examined. All four were carriers of the truncating mutation 1100delC. We confirmed previous findings that this mutation induces non-sense mediated mRNA decay. In our series, we ruled out the possibility of a functional sequence variant located in the promoter region or in a regulatory element of CHEK2 that would lead to DAE in the transcriptional regulatory milieu of freely proliferating LCLs. Conclusions Our results support that HRM is a sensitive and accurate method for DAE assessment. This approach would be of great interest for high-throughput mutation screening projects aiming to identify genes carrying functional regulatory polymorphisms.

  11. Efficiency of high- and low-voltage pulse combinations for gene electrotransfer in muscle, liver, tumor, and skin

    DEFF Research Database (Denmark)

    André, F M; Gehl, J; Sersa, G;

    2008-01-01

    Gene electrotransfer is gaining momentum as an efficient methodology for nonviral gene transfer. In skeletal muscle, data suggest that electric pulses play two roles: structurally permeabilizing the muscle fibers and electrophoretically supporting the migration of DNA toward or across......, but not to liver; and (4) efficient gene electrotransfer was achieved with HV field strengths below the detectability thresholds for permeabilization; and (5) the lag time interval between the HV and LV pulses decreased sensitivity to the HV pulses, enabling a wider HV amplitude range. In conclusion, HV plus LV...... the permeabilized membrane. To investigate this further, combinations of permeabilizing short high-voltage pulses (HV; hundreds of V/cm) and mainly electrophoretic long low-voltage pulses (LV; tens of V/cm) were investigated in muscle, liver, tumor, and skin in rodent models. The following observations were made...

  12. Common variant in myocilin gene is associated with high myopia in isolated population of Korcula Island, Croatia.

    LENUS (Irish Health Repository)

    Vatavuk, Zoran

    2012-01-31

    AIM: To study the association between genetic variants in myocilin and collagen type I alpha 1 genes and high myopia in an isolated island population. METHODS: A total of 944 examinees from the genetic epidemiology study conducted on the island of Korcula, Croatia, were included in the study. We selected 2 short nucleotide polymorphisms (SNP) available in our genome-wide scan set of SNPs that were previously associated with high myopia and used them to replicate previous claims of possible association. RESULTS: Nineteen cases of high myopia, defined as the refraction of <\\/=-6.00 diopters, were identified and included in the analysis. We showed that rs2075555 in the COL1A1 gene was not associated with high myopia. In contrast, rs2421853 in the myocilin gene was significantly associated in both bivariate (P=0.006) and age- and sex-adjusted analysis (P=0.049). CONCLUSION: Myocilin seems to be a very strong candidate for explaining some of the pathophysiological pathways leading to the development of both glaucoma and high myopia. As our finding was obtained in a relatively under-powered sample, further research and replication of these results is needed.

  13. Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates.

    Directory of Open Access Journals (Sweden)

    Tadayoshi Ikebe

    2010-04-01

    Full Text Available Streptococcal toxic shock syndrome (STSS is a severe invasive infection characterized by the sudden onset of shock and multiorgan failure; it has a high mortality rate. Although a number of studies have attempted to determine the crucial factors behind the onset of STSS, the responsible genes in group A Streptococcus have not been clarified. We previously reported that mutations of csrS/csrR genes, a two-component negative regulator system for multiple virulence genes of Streptococcus pyogenes, are found among the isolates from STSS patients. In the present study, mutations of another negative regulator, rgg, were also found in clinical isolates of STSS patients. The rgg mutants from STSS clinical isolates enhanced lethality and impaired various organs in the mouse models, similar to the csrS mutants, and precluded their being killed by human neutrophils, mainly due to an overproduction of SLO. When we assessed the mutation frequency of csrS, csrR, and rgg genes among S. pyogenes isolates from STSS (164 isolates and non-invasive infections (59 isolates, 57.3% of the STSS isolates had mutations of one or more genes among three genes, while isolates from patients with non-invasive disease had significantly fewer mutations in these genes (1.7%. The results of the present study suggest that mutations in the negative regulators csrS/csrR and rgg of S. pyogenes are crucial factors in the pathogenesis of STSS, as they lead to the overproduction of multiple virulence factors.

  14. Hypermethylated SFRP2 gene in fecal DNA is a high potential biomarker for colorectal cancer noninvasive screening

    Institute of Scientific and Technical Information of China (English)

    Dao-Rong Wang; Dong Tang

    2008-01-01

    AIM: To investigate the feasibility of detecting hypermethylated secreted frizzled-related protein 2 (SFRP2) gene in fecal DNA as a non-invasive screening tool for colorectal cancer (CRC).METHODS: Fluorescence-based real-time PCR assay (MethyLight) was performed to analyze SFRP2 gene promoter methylation status in a blinded fashion in tumor tissues and in stool samples taken from 69 CRC patients preoperatively and at the 9th postoperative day, 34 patients with adenoma ≥ 1 cm, 26 with hyperplastic polyp, and 30 endoscopically normal subjects. Simultaneously the relationship between hypermethylation of SFRP2 gene and clinicopathological features was analyzed.RESULTS: SFRP2 gene was hypermethylated in 91.3% (63/69) CRC, 79.4% (27/34) and 53.8% (14/26) adenoma and hyperplastic polyp tissues, and in 87.0% (60/69), 61.8% (21/34) and 42.3% (11/26) of corresponding fecal samples, respectively. In contrast, no methylated SFRP2 gene was detected in mucosal tissues of normal controls, while two cases of matched fecal samples from normal controls were detected with hypermethylated SFRP2. A significant decrease (P < 0.001) in the rate of hypermethylated SFRP2 gene was detected in the postoperative (8.7%, 6/69) fecal samples as compared with the preoperative fecal samples (87%, 60/69) of CRC patients. Moreover, no significant associations were observed between SFRP2 hypermethylation and clinicopathological features including sex, age, tumor stage, site, lymph node status and histological grade, etc.CONCLUSION: Hypermethylation of SFRP2 gene infecal DNA is a novel molecular biomarker of CRC and carries a high potential for the remote detection of CRC and premalignant lesions as noninvasive screening method.

  15. Human IL-12 p40 as a reporter gene for high-throughput screening of engineered mouse embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Shaffer Benjamin

    2008-06-01

    Full Text Available Abstract Background Establishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12 p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells. Results A series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii the utility of IL-12 p40 as a reporter gene for analyzing the activity of cis-acting genetic elements; (iv expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi demonstration of pluripotency of IL-12 p40 expressing ES cells in vitro and in vivo; and (vii germline transmission of the IL-12 p40 reporter gene. Conclusion IL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using

  16. Differential Expression of Non-Shelterin Genes Associated with High Telomerase Levels and Telomere Shortening in Plasma Cell Disorders

    Science.gov (United States)

    Panero, Julieta; Stella, Flavia; Schutz, Natalia; Fantl, Dorotea Beatriz; Slavutsky, Irma

    2015-01-01

    Telomerase, shelterin proteins and various interacting factors, named non-shelterin proteins, are involved in the regulation of telomere length (TL). Altered expression of any of these telomere-associated genes can lead to telomere dysfunction, causing genomic instability and disease development. In this study, we investigated the expression profile of a set of non-shelterin genes involved in essential processes such as replication (RPA1), DNA damage repair pathways (MRE11-RAD50-NBS1) and stabilization of telomerase complex (DKC1), in 35 patients with monoclonal gammopathy of undetermined significance (MGUS) and 40 cases with multiple myeloma (MM). Results were correlated with hTERT expression, TL and clinical parameters. Overall, a significant increase in DKC1, RAD50, MRE11, NBS1 and RPA1 expression along with an upregulation of hTERT in MM compared with MGUS was observed (p≤0.032). Interestingly, in both entities high mRNA levels of non-shelterin genes were associated with short TLs and increased hTERT expression. Significant differences were observed for DKC1 in MM (p ≤0.026), suggesting an important role for this gene in the maintenance of short telomeres by telomerase in myeloma plasma cells. With regard to clinical associations, we observed a significant increase in DKC1, RAD50, MRE11 and RPA1 expression in MM cases with high bone marrow infiltration (p≤0.03) and a tendency towards cases with advanced ISS stage, providing the first evidence of non-shelterin genes associated to risk factors in MM. Taken together, our findings bring new insights into the intricate mechanisms by which telomere-associated proteins collaborate in the maintenance of plasma cells immortalization and suggest a role for the upregulation of these genes in the progression of the disease. PMID:26366868

  17. Differential Expression of Non-Shelterin Genes Associated with High Telomerase Levels and Telomere Shortening in Plasma Cell Disorders.

    Directory of Open Access Journals (Sweden)

    Julieta Panero

    Full Text Available Telomerase, shelterin proteins and various interacting factors, named non-shelterin proteins, are involved in the regulation of telomere length (TL. Altered expression of any of these telomere-associated genes can lead to telomere dysfunction, causing genomic instability and disease development. In this study, we investigated the expression profile of a set of non-shelterin genes involved in essential processes such as replication (RPA1, DNA damage repair pathways (MRE11-RAD50-NBS1 and stabilization of telomerase complex (DKC1, in 35 patients with monoclonal gammopathy of undetermined significance (MGUS and 40 cases with multiple myeloma (MM. Results were correlated with hTERT expression, TL and clinical parameters. Overall, a significant increase in DKC1, RAD50, MRE11, NBS1 and RPA1 expression along with an upregulation of hTERT in MM compared with MGUS was observed (p≤0.032. Interestingly, in both entities high mRNA levels of non-shelterin genes were associated with short TLs and increased hTERT expression. Significant differences were observed for DKC1 in MM (p ≤0.026, suggesting an important role for this gene in the maintenance of short telomeres by telomerase in myeloma plasma cells. With regard to clinical associations, we observed a significant increase in DKC1, RAD50, MRE11 and RPA1 expression in MM cases with high bone marrow infiltration (p≤0.03 and a tendency towards cases with advanced ISS stage, providing the first evidence of non-shelterin genes associated to risk factors in MM. Taken together, our findings bring new insights into the intricate mechanisms by which telomere-associated proteins collaborate in the maintenance of plasma cells immortalization and suggest a role for the upregulation of these genes in the progression of the disease.

  18. Gene network activity in cultivated primary hepatocytes is highly similar to diseased mammalian liver tissue.

    Science.gov (United States)

    Godoy, Patricio; Widera, Agata; Schmidt-Heck, Wolfgang; Campos, Gisela; Meyer, Christoph; Cadenas, Cristina; Reif, Raymond; Stöber, Regina; Hammad, Seddik; Pütter, Larissa; Gianmoena, Kathrin; Marchan, Rosemarie; Ghallab, Ahmed; Edlund, Karolina; Nüssler, Andreas; Thasler, Wolfgang E; Damm, Georg; Seehofer, Daniel; Weiss, Thomas S; Dirsch, Olaf; Dahmen, Uta; Gebhardt, Rolf; Chaudhari, Umesh; Meganathan, Kesavan; Sachinidis, Agapios; Kelm, Jens; Hofmann, Ute; Zahedi, René P; Guthke, Reinhard; Blüthgen, Nils; Dooley, Steven; Hengstler, Jan G

    2016-10-01

    It is well known that isolation and cultivation of primary hepatocytes cause major gene expression alterations. In the present genome-wide, time-resolved study of cultivated human and mouse hepatocytes, we made the observation that expression changes in culture strongly resemble alterations in liver diseases. Hepatocytes of both species were cultivated in collagen sandwich and in monolayer conditions. Genome-wide data were also obtained from human NAFLD, cirrhosis, HCC and hepatitis B virus-infected tissue as well as mouse livers after partial hepatectomy, CCl4 intoxication, obesity, HCC and LPS. A strong similarity between cultivation and disease-induced expression alterations was observed. For example, expression changes in hepatocytes induced by 1-day cultivation and 1-day CCl4 exposure in vivo correlated with R = 0.615 (p < 0.001). Interspecies comparison identified predominantly similar responses in human and mouse hepatocytes but also a set of genes that responded differently. Unsupervised clustering of altered genes identified three main clusters: (1) downregulated genes corresponding to mature liver functions, (2) upregulation of an inflammation/RNA processing cluster and (3) upregulated migration/cell cycle-associated genes. Gene regulatory network analysis highlights overrepresented and deregulated HNF4 and CAR (Cluster 1), Krüppel-like factors MafF and ELK1 (Cluster 2) as well as ETF (Cluster 3) among the interspecies conserved key regulators of expression changes. Interventions ameliorating but not abrogating cultivation-induced responses include removal of non-parenchymal cells, generation of the hepatocytes' own matrix in spheroids, supplementation with bile salts and siRNA-mediated suppression of key transcription factors. In conclusion, this study shows that gene regulatory network alterations of cultivated hepatocytes resemble those of inflammatory liver diseases and should therefore be considered and exploited as disease models.

  19. High-throughput sequencing-based gene profiling on multi-staged fruit development of date palm (Phoenix dactylifera, L.).

    Science.gov (United States)

    Yin, Yuxin; Zhang, Xiaowei; Fang, Yongjun; Pan, Linlin; Sun, Gaoyuan; Xin, Chengqi; Ba Abdullah, Mohammed M; Yu, Xiaoguang; Hu, Songnian; Al-Mssallem, Ibrahim S; Yu, Jun

    2012-04-01

    Date palm provides both staple food and gardening for the Middle East and North African countries for thousands of years. Its fruits have diversified significantly, such as nutritional content, size, length, weight color, and ripping process. Dates palm represent an excellent model system for the study of fruit development and diversity of fruit-bearing palm species that produce the most versatile fruit types as compared to other plant families. Using Roche/454 GS FLX instrument, we acquired 7.6 million sequence tags from seven fruiting stages (F1-F7). Over 99% of the raw reads are assembled, and the numbers of isotigs (equivalent to transcription units or unigenes) range from 30,684 to 40,378 during different fruiting stages. We annotated isotigs using BLASTX and BLASTN, and mapped 74% of the isotigs to known functional sequences or genes. Based on gene ontology categorization and pathway analysis, we have identified 10 core cell division genes, 18 ripening related genes, and 7 starch metabolic enzymes, which are involved as nutrition storage and sugar/starch metabolisms. We noticed that many metabolic pathways vary significantly during fruit development, and carbohydrate metabolism (especially sugar synthesis) is particularly prominent during fruit ripening. Transcriptomics study on various fruiting stages of date palm shows complicated metabolic activities during fruit development, ripening, synthesis and accumulation of starch enzymes and other related sugars. Most Genes are highly expressed in early stages of development, while late developmental stages are critical for fruit ripening including most of the metabolism associated ones.

  20. Global gene expression analysis of Saccharomyces cerevisiae grown under redox potential-controlled very-high-gravity conditions.

    Science.gov (United States)

    Liu, Chen-Guang; Lin, Yen-Han; Bai, Feng-Wu

    2013-11-01

    Redox potential (ORP) plays a pivotal role in yeast viability and ethanol production during very-high-gravity (VHG) ethanol fermentation. In order to identify the correlation between redox potential profiles and gene expression patterns, global gene expression of Saccharomyces cerevisiae was investigated. Results indicated that significant changes in gene expression occurred at the periods of 0 - 6 h and 30 - 36 h, respectively. Changes noted in the period of 0 - 6 h were mainly related to carbohydrate metabolism. In contrast, gene expression variation at 30 - 36 h could be attributed primarily to stress response. Although CDC19 was down-regulated, expression of PYK2, PDC6 and ADH2 correlated inversely with ORP. Meanwhile, expression of GPD1 decreased due to the depletion of dissolved oxygen in the fermentation broth, but expression of GPD2 correlated with ORP. Transcription of genes encoding heat shock proteins was characterized by uphill, downhill, valley and plateau expression profiles, accordingly to specific function in stress response. These results highlight the role of ORP in modulating yeast physiology and metabolism under VHG conditions.

  1. High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water.

    Directory of Open Access Journals (Sweden)

    David W Verner-Jeffreys

    Full Text Available BACKGROUND: Antimicrobials are used to directly control bacterial infections in pet (ornamental fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport. METHODOLOGY/PRINCIPAL FINDINGS: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to > or =15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR. Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, bla(TEM-1, tet(A, tet(D, tet(E, qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. CONCLUSIONS: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.

  2. Pathway-Based Factor Analysis of Gene Expression Data Produces Highly Heritable Phenotypes That Associate with Age

    Science.gov (United States)

    Anand Brown, Andrew; Ding, Zhihao; Viñuela, Ana; Glass, Dan; Parts, Leopold; Spector, Tim; Winn, John; Durbin, Richard

    2015-01-01

    Statistical factor analysis methods have previously been used to remove noise components from high-dimensional data prior to genetic association mapping and, in a guided fashion, to summarize biologically relevant sources of variation. Here, we show how the derived factors summarizing pathway expression can be used to analyze the relationships between expression, heritability, and aging. We used skin gene expression data from 647 twins from the MuTHER Consortium and applied factor analysis to concisely summarize patterns of gene expression to remove broad confounding influences and to produce concise pathway-level phenotypes. We derived 930 “pathway phenotypes” that summarized patterns of variation across 186 KEGG pathways (five phenotypes per pathway). We identified 69 significant associations of age with phenotype from 57 distinct KEGG pathways at a stringent Bonferroni threshold (P<5.38×10−5). These phenotypes are more heritable (h2=0.32) than gene expression levels. On average, expression levels of 16% of genes within these pathways are associated with age. Several significant pathways relate to metabolizing sugars and fatty acids; others relate to insulin signaling. We have demonstrated that factor analysis methods combined with biological knowledge can produce more reliable phenotypes with less stochastic noise than the individual gene expression levels, which increases our power to discover biologically relevant associations. These phenotypes could also be applied to discover associations with other environmental factors. PMID:25758824

  3. Effect of high-fructose corn syrup on Streptococcus mutans virulence gene expression and on tooth demineralization.

    Science.gov (United States)

    Sun, Minmin; Kang, Qiongyi; Li, Tingting; Huang, Lili; Jiang, Yuntao; Xia, Wenwei

    2014-06-01

    High-fructose corn syrup-55 (HFCS-55) has been widely welcomed in recent years as a substitute for sucrose on the basis of its favourable properties and price. The objective of this study was to determine the influence of HFCS-55 on the expression of Streptococcus mutans UA159 virulence genes and on tooth demineralization. Real-time reverse-transcription PCR (real-time RT-PCR) and microhardness evaluations were performed to examine gene expression and enamel demineralization, respectively, after treatment with HFCS-55 and/or sucrose. Significant up-regulation of glucosyltransferase B (gtfB) by HFCS-55 was found. A mixture of HFCS-55 and sucrose could positively enhance expression of glucan-binding protein (gbp) genes. Regarding acidogenicity, expression of the lactate dehydrogenase (ldh) gene was unaffected by HFCS-55. A notable finding in this study was that 5% HFCS-55 significantly enhanced expression of the intracellular response gene of the two-component VicRK signal transduction system (vicR). Demineralization testing showed that the microhardness of teeth decreased by a greater extent in response to HFCS-55 than in response to sucrose. The results indicate that HFCS-55 can enhance S. mutans biofilm formation indirectly in the presence of sucrose and that HFCS-55 has a more acidogenic potential than does sucrose. Summing up the real-time PCR and demineralization results, HFCS-55 appears to be no less cariogenic than sucrose in vitro - at least, not under the conditions of our experiments.

  4. The Max-Min High-Order Dynamic Bayesian Network for Learning Gene Regulatory Networks with Time-Delayed Regulations.

    Science.gov (United States)

    Li, Yifeng; Chen, Haifen; Zheng, Jie; Ngom, Alioune

    2016-01-01

    Accurately reconstructing gene regulatory network (GRN) from gene expression data is a challenging task in systems biology. Although some progresses have been made, the performance of GRN reconstruction still has much room for improvement. Because many regulatory events are asynchronous, learning gene interactions with multiple time delays is an effective way to improve the accuracy of GRN reconstruction. Here, we propose a new approach, called Max-Min high-order dynamic Bayesian network (MMHO-DBN) by extending the Max-Min hill-climbing Bayesian network technique originally devised for learning a Bayesian network's structure from static data. Our MMHO-DBN can explicitly model the time lags between regulators and targets in an efficient manner. It first uses constraint-based ideas to limit the space of potential structures, and then applies search-and-score ideas to search for an optimal HO-DBN structure. The performance of MMHO-DBN to GRN reconstruction was evaluated using both synthetic and real gene expression time-series data. Results show that MMHO-DBN is more accurate than current time-delayed GRN learning methods, and has an intermediate computing performance. Furthermore, it is able to learn long time-delayed relationships between genes. We applied sensitivity analysis on our model to study the performance variation along different parameter settings. The result provides hints on the setting of parameters of MMHO-DBN.

  5. mRNA Noise Reveals that Activators Induce a Biphasic Response in the Promoter Kinetics of Highly Regulated Genes

    Science.gov (United States)

    Quinn, Katie; To, Tsz-Leung; Maheshri, Narendra

    2012-02-01

    A dominant source of fluctuations in gene expression is thought to be the process of transcription. The statistics of these fluctuations arise from the kinetics of transcription. Multiple studies suggest the bulk of fluctuations can be understood by a simple process where genes are inactive for exponentially distributed times punctuated by geometric bursts of mRNA. Yet it's largely unknown how cis and trans factors affect the two lumped kinetic parameters, burst size and burst frequency, that describe this process. Importantly, how these parameters are regulated in a single gene can qualitatively affect the dynamical behavior of the network it is embedded within. Here, we ask whether transcriptional activators increase gene expression by increasing the burst size or burst frequency. We do so by deducing these parameters from steady-state mRNA distributions measured in individual yeast cells using single molecule mRNA FISH. We find that for both a synthetic and natural promoter, activators appear to first increase burst size, then burst frequency. We suggest this biphasic response may be common to all highly regulated genes and was previously unappreciated because of measurement techniques. Furthermore, its origins appear to relate to cis events at the promoter, and may arise from combinations of basal and activator-dependent bursts. Our measurements shed new light on transcriptional mechanisms and should assist in building synthetic promoters with tunable statistics.

  6. W::Neo: a novel dual-selection marker for high efficiency gene targeting in Drosophila.

    Directory of Open Access Journals (Sweden)

    Wenke Zhou

    Full Text Available We have recently developed a so-called genomic engineering approach that allows for directed, efficient and versatile modifications of Drosophila genome by combining the homologous recombination (HR-based gene targeting with site-specific DNA integration. In genomic engineering and several similar approaches, a "founder" knock-out line must be generated first through HR-based gene targeting, which can still be a potentially time and resource intensive process. To significantly improve the efficiency and success rate of HR-based gene targeting in Drosophila, we have generated a new dual-selection marker termed W::Neo, which is a direct fusion between proteins of eye color marker White (W and neomycin resistance (Neo. In HR-based gene targeting experiments, mutants carrying W::Neo as the selection marker can be enriched as much as fifty times by taking advantage of the antibiotic selection in Drosophila larvae. We have successfully carried out three independent gene targeting experiments using the W::Neo to generate genomic engineering founder knock-out lines in Drosophila.

  7. Genomic characterisation, chromosomal assignment and in vivo localisation of the canine High Mobility Group A1 (HMGA1 gene

    Directory of Open Access Journals (Sweden)

    Reimann-Berg Nicola

    2008-07-01

    Full Text Available Abstract Background The high mobility group A1 proteins (HMGA1a/HMGA1b are highly conserved between mammalian species and widely described as participating in various cellular processes. By inducing DNA conformation changes the HMGA1 proteins indirectly influence the binding of various transcription factors and therefore effect the transcription regulation. In humans chromosomal aberrations affecting the HMGA1 gene locus on HSA 6p21 were described to be the cause for various benign mesenchymal tumours while high titres of HMGA1 proteins were shown to be associated with the neoplastic potential of various types of cancer. Interestingly, the absence of HMGA1 proteins was shown to cause insulin resistance and diabetes in humans and mice. Due to the various similarities in biology and presentation of human and canine cancers the dog has joined the common rodent animal model for therapeutic and preclinical studies. Accordingly, the canine genome was sequenced completely twice but unfortunately this could not solve the structure of canine HMGA1 gene. Results Herein we report the characterisation of the genomic structure of the canine HMGA1 gene consisting of 7 exons and 6 introns spanning in total 9524 bp, the in vivo localisation of the HMGA1 protein to the nucleus, and a chromosomal assignment of the gene by FISH to CFA12q11. Additionally, we evaluated a described canine HMGA1 exon 6 SNP in 55 Dachshunds. Conclusion The performed characterisations will make comparative analyses of aberrations affecting the human and canine gene and proteins possible, thereby providing a basis for revealing mechanisms involved in HMGA1 related pathogenesis in both species.

  8. Several different lactase persistence associated alleles and high diversity of the lactase gene in the admixed Brazilian population.

    Science.gov (United States)

    Friedrich, Deise C; Santos, Sidney E B; Ribeiro-dos-Santos, Ândrea K C; Hutz, Mara H

    2012-01-01

    Adult-type hypolactasia is a common phenotype caused by the lactase enzyme deficiency. The -13910 C>T polymorphism, located 14 Kb upstream of the lactase gene (LCT) in the MCM6 gene was associated with lactase persistence (LP) in Europeans. This polymorphism is rare in Africa but several other variants associated with lactase persistence were observed in Africans. The aims of this study were to identify polymorphisms in the MCM6 region associated with the lactase persistence phenotype and to determine the distribution of LCT gene haplotypes in 981 individuals from North, Northeast and South Brazil. These polymorphisms were genotyped by PCR based methods and sequencing. The -13779*C,-13910*T, -13937*A, -14010*C, -14011*T LP alleles previously described in the MCM6 gene region that acts as an enhancer for the LCT gene were identified in Brazilians. The most common LP allele was -13910*T. Its frequency was highly correlated with European ancestry in the Brazilian populations investigated. The -13910*T was higher (0.295) in southern Brazilians of European ancestry and lower (0.175) in the Northern admixed population. LCT haplotypes were derived from the 10 LCT SNPs genotyped. Overall twenty six haplotypes previously described were identified in the four Brazilian populations studied. The Multidimensional Scaling analysis showed that Belém, in the north, was closer to Amerindians. Northeastern and southern Afro-descendants were more related with Bantu-speaking South Africans whereas the Southern population with European ancestry grouped with Southern and Northern Europeans. This study shows a high variability considering the number of LCT haplotypes observed. Due to the highly admixed nature of the Brazilian populations, the diagnosis of hypolactasia in Brazil, based only in the investigation of the -13910*T allele is an oversimplification.

  9. Several different lactase persistence associated alleles and high diversity of the lactase gene in the admixed Brazilian population.

    Directory of Open Access Journals (Sweden)

    Deise C Friedrich

    Full Text Available Adult-type hypolactasia is a common phenotype caused by the lactase enzyme deficiency. The -13910 C>T polymorphism, located 14 Kb upstream of the lactase gene (LCT in the MCM6 gene was associated with lactase persistence (LP in Europeans. This polymorphism is rare in Africa but several other variants associated with lactase persistence were observed in Africans. The aims of this study were to identify polymorphisms in the MCM6 region associated with the lactase persistence phenotype and to determine the distribution of LCT gene haplotypes in 981 individuals from North, Northeast and South Brazil. These polymorphisms were genotyped by PCR based methods and sequencing. The -13779*C,-13910*T, -13937*A, -14010*C, -14011*T LP alleles previously described in the MCM6 gene region that acts as an enhancer for the LCT gene were identified in Brazilians. The most common LP allele was -13910*T. Its frequency was highly correlated with European ancestry in the Brazilian populations investigated. The -13910*T was higher (0.295 in southern Brazilians of European ancestry and lower (0.175 in the Northern admixed population. LCT haplotypes were derived from the 10 LCT SNPs genotyped. Overall twenty six haplotypes previously described were identified in the four Brazilian populations studied. The Multidimensional Scaling analysis showed that Belém, in the north, was closer to Amerindians. Northeastern and southern Afro-descendants were more related with Bantu-speaking South Africans whereas the Southern population with European ancestry grouped with Southern and Northern Europeans. This study shows a high variability considering the number of LCT haplotypes observed. Due to the highly admixed nature of the Brazilian populations, the diagnosis of hypolactasia in Brazil, based only in the investigation of the -13910*T allele is an oversimplification.

  10. High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous.

    Science.gov (United States)

    Verwaal, René; Wang, Jing; Meijnen, Jean-Paul; Visser, Hans; Sandmann, Gerhard; van den Berg, Johan A; van Ooyen, Albert J J

    2007-07-01

    To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially beta-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product beta-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of beta-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of beta-carotene by S. cerevisiae.

  11. Identification of Differentially Expressed Genes during Bacillus subtilis Spore Outgrowth in High-Salinity Environments Using RNA Sequencing

    Science.gov (United States)

    Nagler, Katja; Krawczyk, Antonina O.; De Jong, Anne; Madela, Kazimierz; Hoffmann, Tamara; Laue, Michael; Kuipers, Oscar P.; Bremer, Erhard; Moeller, Ralf

    2016-01-01

    In its natural habitat, the soil bacterium Bacillus subtilis often has to cope with fluctuating osmolality and nutrient availability. Upon nutrient depletion it can form dormant spores, which can revive to form vegetative cells when nutrients become available again. While the effects of salt stress on spore germination have been analyzed previously, detailed knowledge on the salt stress response during the subsequent outgrowth phase is lacking. In this study, we investigated the changes in gene expression during B. subtilis outgrowth in the presence of 1.2 M NaCl using RNA sequencing. In total, 402 different genes were upregulated and 632 genes were downregulated during 90 min of outgrowth in the presence of salt. The salt stress response of outgrowing spores largely resembled the osmospecific response of vegetative cells exposed to sustained high salinity and included strong upregulation of genes involved in osmoprotectant uptake and compatible solute synthesis. The σB-dependent general stress response typically triggered by salt shocks was not induced, whereas the σW regulon appears to play an important role for osmoadaptation of outgrowing spores. Furthermore, high salinity induced many changes in the membrane protein and transporter transcriptome. Overall, salt stress seemed to slow down the complex molecular reorganization processes (“ripening”) of outgrowing spores by exerting detrimental effects on vegetative functions such as amino acid metabolism. PMID:27766092

  12. High frequencies of antibiotic resistance genes in infants’ meconium and early fecal samples

    DEFF Research Database (Denmark)

    Gosalbes, M. J.; Vallès, Y.; Jiménez-Hernández, N.

    2016-01-01

    The gastrointestinal tract (GIT) microbiota has been identified as an important reservoir of antibiotic resistance genes (ARGs) that can be horizontally transferred to pathogenic species. Maternal GIT microbes can be transmitted to the offspring, and recent work indicates that such transfer starts......-resistant Staphylococcus aureus, and although its detection does not imply the presence of this pathogen, it does implicate the young infant’s GIT as a noteworthy reservoir of this gene....... for a variety of genes conferring each. To evaluate whether ARGs could have been inherited by maternal transmission, we have screened perinatal fecal samples of the 1-week-old babies’ mothers, as well as a mother–infant series including meconium, fecal samples collected through the infant’s 1st year, maternal...

  13. High-throughput in vivo analysis of gene expression in Caenorhabditis elegans.

    Science.gov (United States)

    Hunt-Newbury, Rebecca; Viveiros, Ryan; Johnsen, Robert; Mah, Allan; Anastas, Dina; Fang, Lily; Halfnight, Erin; Lee, David; Lin, John; Lorch, Adam; McKay, Sheldon; Okada, H Mark; Pan, Jie; Schulz, Ana K; Tu, Domena; Wong, Kim; Zhao, Z; Alexeyenko, Andrey; Burglin, Thomas; Sonnhammer, Eric; Schnabel, Ralf; Jones, Steven J; Marra, Marco A; Baillie, David L; Moerman, Donald G

    2007-09-01

    Using DNA sequences 5' to open reading frames, we have constructed green fluorescent protein (GFP) fusions and generated spatial and temporal tissue expression profiles for 1,886 specific genes in the nematode Caenorhabditis elegans. This effort encompasses about 10% of all genes identified in this organism. GFP-expressing wild-type animals were analyzed at each stage of development from embryo to adult. We have identified 5' DNA regions regulating expression at all developmental stages and in 38 different cell and tissue types in this organism. Among the regulatory regions identified are sequences that regulate expression in all cells, in specific tissues, in combinations of tissues, and in single cells. Most of the genes we have examined in C. elegans have human orthologs. All the images and expression pattern data generated by this project are available at WormAtlas (http://gfpweb.aecom.yu.edu/index) and through WormBase (http://www.wormbase.org).

  14. High-throughput in vivo analysis of gene expression in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Rebecca Hunt-Newbury

    2007-09-01

    Full Text Available Using DNA sequences 5' to open reading frames, we have constructed green fluorescent protein (GFP fusions and generated spatial and temporal tissue expression profiles for 1,886 specific genes in the nematode Caenorhabditis elegans. This effort encompasses about 10% of all genes identified in this organism. GFP-expressing wild-type animals were analyzed at each stage of development from embryo to adult. We have identified 5' DNA regions regulating expression at all developmental stages and in 38 different cell and tissue types in this organism. Among the regulatory regions identified are sequences that regulate expression in all cells, in specific tissues, in combinations of tissues, and in single cells. Most of the genes we have examined in C. elegans have human orthologs. All the images and expression pattern data generated by this project are available at WormAtlas (http://gfpweb.aecom.yu.edu/index and through WormBase (http://www.wormbase.org.

  15. Identification of highly methylated genes across various types of B-cell non-hodgkin lymphoma.

    Directory of Open Access Journals (Sweden)

    Nicole Bethge

    Full Text Available Epigenetic alterations of gene expression are important in the development of cancer. In this study, we identified genes which are epigenetically altered in major lymphoma types. We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified 233 genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples (n = 480 when compared to normal B cells (n = 5. The top 30 genes were further analyzed by methylation specific PCR (MSP in 18 lymphoma cell lines. Seven of the genes were methylated in more than 70% of the cell lines and were further subjected to quantitative MSP in 37 B-cell lymphoma patient samples (diffuse large B-cell lymphoma (activated B-cell like and germinal center B-cell like subtypes, follicular lymphoma and Burkitt`s lymphoma and normal B lymphocytes from 10 healthy donors. The promoters of DSP, FZD8, KCNH2, and PPP1R14A were methylated in 28%, 67%, 22%, and 78% of the 36 tumor samples, respectively, but not in control samples. Validation using a second series of healthy donor controls (n = 42; normal B cells, peripheral blood mononuclear cells, bone marrow, tonsils and follicular hyperplasia and fresh-frozen lymphoma biopsies (n = 25, confirmed the results. The DNA methylation biomarker panel consisting of DSP, FZD8, KCNH2, and PPP1R14A was positive in 89% (54/61 of all lymphomas. Receiver operating characteristic analysis to determine the discriminative power between lymphoma and healthy control samples showed a c-statistic of 0.96, indicating a possible role for the biomarker panel in monitoring of lymphoma patients.

  16. Gene expression profile of Jurkat cells exposed to high power terahertz radiation

    Science.gov (United States)

    Grundt, Jessica E.; Roth, Caleb C.; Rivest, Benjamin D.; Doroski, Michael L.; Payne, Jason; Ibey, Bennett L.; Wilmink, Gerald J.

    2011-03-01

    Terahertz (THz) radiation sources are now being used in a host of military, defense, and medical applications. Widespread employment of these applications has prompted concerns regarding the health effects associated with THz radiation. In this study, we examined the gene expression profile of mammalian cells exposed to THz radiation. We hypothesized that if THz radiation couples directly to cellular constituents, then exposed cells may express a specific gene expression profile indicative of ensuing damage. To test this hypothesis, Jurkat cells were irradiated with a molecular gas THz laser (2.52 THz, 636 mWcm-2, durations: 5, 10, 20, 30, 40, or 50 minutes). Viability was assessed 24 h post-exposure using MTT assays, and gene expression profiles were evaluated 4 h post-exposure using mRNA microarrays. Comparable analyses were also performed for hyperthermic positive controls (44°C for 40 minutes). We found that cellular temperatures increased by ~6 °C during THz exposures. We also found that cell death increased with exposure duration, and the median lethal dose (LD50) was calculated to be ~44 minutes. The microarray data showed that THz radiation induced the transcriptional activation of genes associated with cellular proliferation, differentiation, transcriptional activation, chaperone protein stabilization, and apoptosis. For most genes, we found that the magnitude of differential expression was comparable for both the THz and thermal exposure groups; however, several genes were specifically activated by the THz exposure. These results suggest that THz radiation may elicit effects that are not exclusively due to the temperature rise created during THz exposures (i.e. thermal effects). In future work, we plan to verify the results of our microarray experiments using qPCR techniques.

  17. Live imaging of muscles in Drosophila metamorphosis: Towards high-throughput gene identification and function analysis.

    Science.gov (United States)

    Puah, Wee Choo; Wasser, Martin

    2016-03-01

    Time-lapse microscopy in developmental biology is an emerging tool for functional genomics. Phenotypic effects of gene perturbations can be studied non-invasively at multiple time points in chronological order. During metamorphosis of Drosophila melanogaster, time-lapse microscopy using fluorescent reporters allows visualization of alternative fates of larval muscles, which are a model for the study of genes related to muscle wasting. While doomed muscles enter hormone-induced programmed cell death, a smaller population of persistent muscles survives to adulthood and undergoes morphological remodeling that involves atrophy in early, and hypertrophy in late pupation. We developed a method that combines in vivo imaging, targeted gene perturbation and image analysis to identify and characterize genes involved in muscle development. Macrozoom microscopy helps to screen for interesting muscle phenotypes, while confocal microscopy in multiple locations over 4-5 days produces time-lapse images that are used to quantify changes in cell morphology. Performing a similar investigation using fixed pupal tissues would be too time-consuming and therefore impractical. We describe three applications of our pipeline. First, we show how quantitative microscopy can track and measure morphological changes of muscle throughout metamorphosis and analyze genes involved in atrophy. Second, our assay can help to identify genes that either promote or prevent histolysis of abdominal muscles. Third, we apply our approach to test new fluorescent proteins as live markers for muscle development. We describe mKO2 tagged Cysteine proteinase 1 (Cp1) and Troponin-I (TnI) as examples of proteins showing developmental changes in subcellular localization. Finally, we discuss strategies to improve throughput of our pipeline to permit genome-wide screens in the future.

  18. A high soy diet enhances neurotropin receptor and Bcl-XL gene expression in the brains of ovariectomized female rats.

    Science.gov (United States)

    Lovekamp-Swan, Tara; Glendenning, Michele L; Schreihofer, Derek A

    2007-07-23

    Estrogen is a powerful neuroprotective agent with the ability to induce trophic and antiapoptotic genes. However, concerns about negative overall health consequences of estrogen replacement after menopause have led to the adoption of other strategies to obtain estrogen's benefits in the brain, including the use of selective estrogen receptor modulators, high soy diets, or isoflavone supplements. This study sought to determine the ability of a high soy diet to induce neuroprotective gene expression in the female rat brain and compare the actions of soy with estrogen. Adult ovariectomized female rats were treated with 3 days of high dose estrogen or 2 weeks of a soy-free diet, a high soy diet, or chronic low dose estrogen. Different brain regions were microdissected and subjected to real time RT-PCR for neuroprotective genes previously shown to be estrogen-regulated. The principle findings are that a high soy diet led to the widespread increase in the mRNA for neurotropin receptors TrkA and p75-NTR, and the antiapoptotic Bcl-2 family member Bcl-X(L). Immunohistochemistry confirmed increases in both TrkA and Bcl-X(L). Chronic low dose estrogen mimicked some of these effects, but acute high dose estrogen did not. The effects of a high soy diet were particularly evident in the parietal cortex and hippocampus, two regions protected by estrogen in animal models of neurological disease and injury. These results suggest that a high soy diet may provide beneficial effects to the brain similar to low dose chronic estrogen treatment such as that used for postmenopausal hormone replacement.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. High-throughput identification of antigen-specific TCRs by TCR gene capture

    DEFF Research Database (Denmark)

    Linnemann, Carsten; Heemskerk, Bianca; Kvistborg, Pia;

    2013-01-01

    The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We...... the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge...

  1. Fasciola hepatica demonstrates high levels of genetic diversity, a lack of population structure and high gene flow: possible implications for drug resistance.

    Science.gov (United States)

    Beesley, Nicola J; Williams, Diana J L; Paterson, Steve; Hodgkinson, Jane

    2017-01-01

    Fasciola hepatica, the liver fluke, is a trematode parasite of considerable economic importance to the livestock industry and is a re-emerging zoonosis that poses a risk to human health in F. hepatica-endemic areas worldwide. Drug resistance is a substantial threat to the current and future control of F. hepatica, yet little is known about how the biology of the parasite influences the development and spread of resistance. Given that F. hepatica can self-fertilise and therefore inbreed, there is the potential for greater population differentiation and an increased likelihood of recessive alleles, such as drug resistance genes, coming together. This could be compounded by clonal expansion within the snail intermediate host and aggregation of parasites of the same genotype on pasture. Alternatively, widespread movement of animals that typically occurs in the UK could promote high levels of gene flow and prevent population differentiation. We identified clonal parasites with identical multilocus genotypes in 61% of hosts. Despite this, 84% of 1579 adult parasites had unique multilocus genotypes, which supports high levels of genotypic diversity within F. hepatica populations. Our analyses indicate a selfing rate no greater than 2%, suggesting that this diversity is in part due to the propensity for F. hepatica to cross-fertilise. Finally, although we identified high genetic diversity within a given host, there was little evidence for differentiation between populations from different hosts, indicating a single panmictic population. This implies that, once those emerge, anthelmintic resistance genes have the potential to spread rapidly through liver fluke populations.

  2. Detection and identification of vegetative insecticidal proteins vip3 genes of Bacillus thuringiensis strains using polymerase chain reaction-high resolution melt analysis.

    Science.gov (United States)

    Li, Haitao; Shu, Changlong; He, Xiaoming; Gao, JiGuo; Liu, Rongmei; Huang, Dafang

    2012-05-01

    In this study, vegetative insecticidal proteins vip3 genes from Bacillus thuringiensis strains were detected based on polymerase chain reaction-high resolution melt (PCR-HRM) analysis. A pair of primers was designed according to the conservative sequences in 150 bp region of the known vip3 subfamily. The 150 bp regions of difference vip3 genes have only a few nucleotide difference vip3 genes were detected in 8 of 11 standard B. thuringiensis strains, and vip3Aa genes, vip3Af genes and vip3Ba gene can be distinguished as different melting curves by this method. The results demonstrate the utility of the HRM assay for mutant screening using vip3 gene. The PCR-HRM method may be a valuable and reliable tool for specific detection and identification of vip3 genes.

  3. Cloning and selection of carotenoid ketolase genes for the engineering of high-yield astaxanthin in plants.

    Science.gov (United States)

    Huang, Junchao; Zhong, Yujuan; Sandmann, Gerhard; Liu, Jin; Chen, Feng

    2012-08-01

    β-Carotene ketolase (BKT) catalyzes the rate-limiting steps for the biosynthesis of astaxanthin. Several bkt genes have been isolated and explored to modify plant carotenoids to astaxanthin with limited success. In this study, five algal BKT cDNAs were isolated and characterized for the engineering of high-yield astaxanthin in plants. The products of the cDNAs showed high similarity in sequence and enzymatic activity of converting β-carotene into canthaxanthin. However, the enzymes exhibited extremely different activities in converting zeaxanthin into astaxanthin. Chlamydomonas reinhardtii BKT showed the highest conversion rate (ca 85%), whereas, Neochloris wimmeri BKT exhibited very poor activity of ketolating zeaxanthin. Expression of C. reinhardtii BKT in tobacco led to a twofold increase of total carotenoids in the leaves with astaxanthin being the predominant. The bkt genes described here provide a valuable resource for metabolic engineering of plants as cell factories for astaxanthin production.

  4. High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1.

    Science.gov (United States)

    Bovy, Arnaud; de Vos, Ric; Kemper, Mark; Schijlen, Elio; Almenar Pertejo, Maria; Muir, Shelagh; Collins, Geoff; Robinson, Sue; Verhoeyen, Martine; Hughes, Steve; Santos-Buelga, Celestino; van Tunen, Arjen

    2002-10-01

    Flavonoids are a group of polyphenolic plant secondary metabolites important for plant biology and human nutrition. In particular flavonols are potent antioxidants, and their dietary intake is correlated with a reduced risk of cardiovascular diseases. Tomato fruit contain only in their peel small amounts of flavonoids, mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside. To increase flavonoid levels in tomato, we expressed the maize transcription factor genes LC and C1 in the fruit of genetically modified tomato plants. Expression of both genes was required and sufficient to upregulate the flavonoid pathway in tomato fruit flesh, a tissue that normally does not produce any flavonoids. These fruit accumulated high levels of the flavonol kaempferol and, to a lesser extent, the flavanone naringenin in their flesh. All flavonoids detected were present as glycosides. Anthocyanins, previously reported to accumulate upon LC expression in several plant species, were present in LC/C1 tomato leaves but could not be detected in ripe LC/C1 fruit. RNA expression analysis of ripening fruit revealed that, with the exception of chalcone isomerase, all of the structural genes required for the production of kaempferol-type flavonols and pelargonidin-type anthocyanins were induced strongly by the LC/C1 transcription factors. Expression of the genes encoding flavanone-3'-hydroxylase and flavanone-3'5'-hydroxylase, which are required for the modification of B-ring hydroxylation patterns, was not affected by LC/C1. Comparison of flavonoid profiles and gene expression data between tomato leaves and fruit indicates that the absence of anthocyanins in LC/C1 fruit is attributable primarily to an insufficient expression of the gene encoding flavanone-3'5'-hydroxylase, in combination with a strong preference of the tomato dihydroflavonol reductase enzyme to use the flavanone-3'5'-hydroxylase reaction product dihydromyricetin as a substrate.

  5. High Rate of Chimeric Gene Origination by Retroposition in Plant Genomes

    DEFF Research Database (Denmark)

    Wang, Wen; Zheng, Hongkung; Fan, Chuanzhu;

    2006-01-01

    and a reported long terminal repeat (LTR) retrotransposon-mediated mechanism of retroposing cellular genes in maize (Zea mays). We show extensive retropositions in the rice (Oryza sativa) genome, with 1235 identified primary retrogenes. We identified 27 of these primary retrogenes within LTR retrotransposons...

  6. Obtaining High Pest-resistant Tobacco Plants Carrying B.t. insecticidal Gene

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To increase the expression level of CryIA(c) gene in transgenic plants, a plant expression vector pBinMoBc carrying the CryIA(c) gene under control of chimeric OM promoter and Ω factor was constructed. As a control, pBinoBc carrying the CryIA(c) gene with the CaMV 35S promoter was also constructed. The vectors were transferred into tobacco plants respectively via Agrobacterium-mediated transformation. ELISA assay showed that the expression level of the CryIA(c) gene in pBinMoBc transgenic tobacco plants was 2.44-times that in pBinoBc transgenic tobacco plants, and it could be up to 0.255% of total soluble proteins. Bioassay showed that pBinMoBc transgenic tobacco plants had more notable insecticidal effect than pBinoBc transgenic tobacco plants. The above results showed that the chimeric OM promoter was a stronger promoter than CaMV 35S promoter that was widely used in plant genetic engineering, and this is very useful in pest-resistant plant genetic engineering.

  7. Gene expression responses to highly pathogenic avian influenza H5N1 virus infections in ducks

    Science.gov (United States)

    Differences in host response to infection with avian influenza (AI) viruses were investigated by identifying genes differentially expressed in tissues of infected ducks. Clear differences in pathogenicity were observed among ducks inoculated with five H5N1 HPAI viruses. Virus titers in tissues cor...

  8. NEC1, a novel gene, highly expressed in the nectary tissue of Petunia hybrida

    NARCIS (Netherlands)

    Ge, X.Y.; Angenent, G.C.; Wittich, P.E.; Peters, J.; Franken, J.; Busscher, M.; Zhang Lanying,; Dahlhaus, E.; Kater, M.M.; Wullems, G.J.; Creemers-Molenaar, T.

    2000-01-01

    To study the molecular regulation of nectary development, we cloned NEC1, a gene predominantly expressed in the nectaries of Petunia hybrida, by using the differential display RTPCR technique. The secondary structure of the putative NEC1 protein is reminiscent of a transmembrane protein, indicating

  9. High diversity of genes for nonhost resistance of barley to heterologous rust fungi

    NARCIS (Netherlands)

    Jafary, H.; Albertazzi, G.; Marcel, T.C.; Niks, R.E.

    2008-01-01

    Inheritance studies on the nonhost resistance of plants would normally require interspecific crosses that suffer from sterility and abnormal segregation. Therefore, we developed the barley¿Puccinia rust model system to study, using forward genetics, the specificity, number, and diversity of genes in

  10. Reduced Variance of Gene Expression at Numerous Loci in a Population of Chickens Selected for High Feather Pecking

    DEFF Research Database (Denmark)

    Hughes, A L; Buitenhuis, A J

    2010-01-01

    Changes in gene expression in response to selection were studied by comparing microarray expression profiles among a population of domestic chickens selected for high feather pecking (FP) with a control population and a population selected for low FP. No transcripts showed significant differences...... and gentle FP were distinct, suggesting that very distinct underlying neural mechanisms underlie these 2 behaviors, with SFP showing more signs of an association with synaptic plasticity and with an immunosuppressive stress response...

  11. High-level expression of a chemically synthesized gene for human interferon-gamma using a prokaryotic expression vector.

    OpenAIRE

    1984-01-01

    A chemically synthesized gene for human interferon-gamma has been cloned into a prokaryotic expression vector under the regulation of a synthetic constitutive transcriptional-translational control unit that contains a strong bacteriophage T5 early promoter and a strong ribosome-binding site. Cells harboring the recombinant plasmid express high levels (4 X 10(9) units per liter of culture) of antiviral activity specific for interferon-gamma. Analysis of total cell lysates on NaDodSO4/polyacryl...

  12. Pathway-based factor analysis of gene expression data produces highly heritable phenotypes that associate with age

    OpenAIRE

    Brown, Andrew Anand; Ding, Zhihao; Viñuela, Ana; Glass, Dan; Parts, Leopold; Spector, Tim; Winn, John; Durbin, Richard

    2015-01-01

    Statistical factor analysis methods have previously been used to remove noise components from high-dimensional data prior to genetic association mapping and, in a guided fashion, to summarize biologically relevant sources of variation. Here, we show how the derived factors summarizing pathway expression can be used to analyze the relationships between expression, heritability, and aging. We used skin gene expression data from 647 twins from the MuTHER Consortium and applied factor analysis to...

  13. The use of high-frequency ultrasound imaging and biofluorescence for in vivo evaluation of gene therapy vectors

    OpenAIRE

    Ingram, N.; Macnab, SA; Marston, G; Scott, N.; Carr, IM; Markham, AF; Whitehouse, A; Coletta, PL

    2013-01-01

    Background: Non-invasive imaging of the biodistribution of novel therapeutics including gene therapy vectors in animal models is essential. Methods: This study assessed the utility of high-frequency ultrasound (HF-US) combined with biofluoresence imaging (BFI) to determine the longitudinal impact of a Herpesvirus saimiri amplicon on human colorectal cancer xenograft growth. Results: HF-US imaging of xenografts resulted in an accurate and informative xenograft volume in a longitudinal study. T...

  14. 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.

  15. Microarray analysis of gene expression patterns of high lycopene tomato generated from seeds after long-term space flight

    Science.gov (United States)

    Lu, Jinying; Ren, Chunxiao; Pan, Yi; Nechitailo, Galina S.; Liu, Min

    Lycopene content is a most vital trait of tomatoes due to the role of lycopene in reducing the risk of some kinds of cancers. In this experiment, we gained a high lycopene (hl) tomato (named HY-2), after seven generations of self-cross selection, from seeds Russian MNP-1 carried in Russia MIR space station for six years. HPLC result showed that the lycopene content was 1.6 times more than that in Russian MNP-1 (the wild type). Microarray analysis presented the general profile of differential expressed genes at the tomato developmental stage of 7DPB (days post breaker). One hundred and forty three differential expression genes were identified according to the following criterion: the average changes were no less than 1.5 folds with q-value (similar to FDR) less than 0.05 or changes were no less than 1.5 folds in all three biological replications. Most of the differential expressed genes were mainly involved in metabolism, response to stimulus, biosynthesis, development and regulation. Particularly, we discussed the genes involved in protein metabolism, response to unfolded protein, carotenoid biosynthesis and photosynthesis that might be related to the fruit development and the accumulation of lycopene. What's more, we conducted QRT-PCR validation of five key genes (Fps, CrtL-b, CrtR-b, Zep and Nxs) in the lycopene biosynthesis pathway through time courses and that provided the direct molecular evidence for the hl phenotype. Our results demonstrate that long-term space flight, as a rarely used tool, can positively cause some beneficial mutations in the seeds and thus to help to generate a high quality variety, combined with ground selections.

  16. Coordinated gene expression in adipose tissue and liver differs between cows with high or low NEFA concentrations in early lactation.

    Science.gov (United States)

    van Dorland, H A; Sadri, H; Morel, I; Bruckmaier, R M

    2012-02-01

    Dairy cows with high and low plasma non-esterified fatty acid (NEFA) concentrations in early lactation were compared for plasma parameters and mRNA expression of genes in liver and subcutaneous adipose tissue. The study involved 16 multiparous dairy cows with a plasma NEFA concentration of >500 μmol/l [n = 8, high NEFA (HNEFA)] and Subcutaneous adipose and liver tissues were analysed for mRNA abundance by real-time qRT-PCR encoding parameters related to lipid metabolism. Results showed that mean daily milk yield and milk fat quantity were higher in HNEFA than in LNEFA cows (p carnitine palmitoyltransferase 2 and very long chain acyl-coenzyme A dehydrogenase) and ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2) were lower in HNEFA than in LNEFA cows. No differences between the two groups were observed for mRNA expression of genes in adipose tissue. The number of calculated significant correlation coefficients (moderately strong) between parameters in the liver and in adipose tissue was nearly similar on +1d, and higher for HNEFA compared with LNEFA cows in +3wk. In conclusion, dairy cows with high compared with low plasma NEFA concentrations in early lactation show differentially synchronized mRNA expression of genes in adipose tissue and liver in +3wk that suggests a different orchestrated homeorhetic regulation of lipid metabolism.

  17. Whole genome and global gene expression analyses of the model mushroom Flammulina velutipes reveal a high capacity for lignocellulose degradation.

    Directory of Open Access Journals (Sweden)

    Young-Jin Park

    Full Text Available Flammulina velutipes is a fungus with health and medicinal benefits that has been used for consumption and cultivation in East Asia. F. velutipes is also known to degrade lignocellulose and produce ethanol. The overlapping interests of mushroom production and wood bioconversion make F. velutipes an attractive new model for fungal wood related studies. Here, we present the complete sequence of the F. velutipes genome. This is the first sequenced genome for a commercially produced edible mushroom that also degrades wood. The 35.6-Mb genome contained 12,218 predicted protein-encoding genes and 287 tRNA genes assembled into 11 scaffolds corresponding with the 11 chromosomes of strain KACC42780. The 88.4-kb mitochondrial genome contained 35 genes. Well-developed wood degrading machinery with strong potential for lignin degradation (69 auxiliary activities, formerly FOLymes and carbohydrate degradation (392 CAZymes, along with 58 alcohol dehydrogenase genes were highly expressed in the mycelium, demonstrating the potential application of this organism to bioethanol production. Thus, the newly uncovered wood degrading capacity and sequential nature of this process in F. velutipes, offer interesting possibilities for more detailed studies on either lignin or (hemi- cellulose degradation in complex wood substrates. The mutual interest in wood degradation by the mushroom industry and (ligno-cellulose biomass related industries further increase the significance of F. velutipes as a new model.

  18. Whole genome and global gene expression analyses of the model mushroom Flammulina velutipes reveal a high capacity for lignocellulose degradation.

    Science.gov (United States)

    Park, Young-Jin; Baek, Jeong Hun; Lee, Seonwook; Kim, Changhoon; Rhee, Hwanseok; Kim, Hyungtae; Seo, Jeong-Sun; Park, Hae-Ran; Yoon, Dae-Eun; Nam, Jae-Young; Kim, Hong-Il; Kim, Jong-Guk; Yoon, Hyeokjun; Kang, Hee-Wan; Cho, Jae-Yong; Song, Eun-Sung; Sung, Gi-Ho; Yoo, Young-Bok; Lee, Chang-Soo; Lee, Byoung-Moo; Kong, Won-Sik

    2014-01-01

    Flammulina velutipes is a fungus with health and medicinal benefits that has been used for consumption and cultivation in East Asia. F. velutipes is also known to degrade lignocellulose and produce ethanol. The overlapping interests of mushroom production and wood bioconversion make F. velutipes an attractive new model for fungal wood related studies. Here, we present the complete sequence of the F. velutipes genome. This is the first sequenced genome for a commercially produced edible mushroom that also degrades wood. The 35.6-Mb genome contained 12,218 predicted protein-encoding genes and 287 tRNA genes assembled into 11 scaffolds corresponding with the 11 chromosomes of strain KACC42780. The 88.4-kb mitochondrial genome contained 35 genes. Well-developed wood degrading machinery with strong potential for lignin degradation (69 auxiliary activities, formerly FOLymes) and carbohydrate degradation (392 CAZymes), along with 58 alcohol dehydrogenase genes were highly expressed in the mycelium, demonstrating the potential application of this organism to bioethanol production. Thus, the newly uncovered wood degrading capacity and sequential nature of this process in F. velutipes, offer interesting possibilities for more detailed studies on either lignin or (hemi-) cellulose degradation in complex wood substrates. The mutual interest in wood degradation by the mushroom industry and (ligno-)cellulose biomass related industries further increase the significance of F. velutipes as a new model.

  19. Molecular cloning of C4-specific Ppc gene of sorghum and its high level expression in transgenic rice

    Institute of Scientific and Technical Information of China (English)

    ZHANG Fang; CHI Wei; WANG Qiang; ZHANG Qide; WU Naihu

    2003-01-01

    In order to improve the carbon-assimilation ability of C3 plants, we isolated a C4-specific photosynthetic enzyme gene, Ppc (encode phosphoenolpyruvate carboxylase, PEPCase) from the genome of the C4 plant, sorghum, and transformed rice with it. As shown by sequence analysis, the gene is composed of 10 exons and 9 introns, and the full-length transcript is 5989 bp long. A recombinant expression vector, p1301PEPC, was constructed by inserting the gene into a plasmid vector, pCAMBIA1301, which was then transformed into two japonica rice varieties, Nongken 58 and Zhonghua 10, using an Agrobacterium-mediated transformation system. PCR analysis, activity measurement of PEPCase, and protein-, RNA- and DNA-based hybridization all confirmed the successful integration of the C4-specific Ppc gene into the nuclear genome of rice and its high level expression. Physiological studies revealed the photosynthetic features characterizing C4 plants such as marked lowering of CO2 compensation point and photorespiration rate, and improved carboxylation efficiency. This study provides useful experimental materialsand opens up new avenues for further studies on improving photosynthetic efficiency of elite varieties of rice.

  20. A high-throughput fluorescence-based assay system for appetite-regulating gene and drug screening.

    Directory of Open Access Journals (Sweden)

    Yasuhito Shimada

    Full Text Available The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing countries. However, there are few agents currently approved for the treatment of obesity. Those that are available are mainly appetite suppressants and gastrointestinal fat blockers. We have developed a simple and rapid method for the measurement of the feeding volume of Danio rerio (zebrafish. This assay can be used to screen appetite suppressants and enhancers. In this study, zebrafish were fed viable paramecia that were fluorescently-labeled, and feeding volume was measured using a 96-well microplate reader. Gene expression analysis of brain-derived neurotrophic factor (bdnf, knockdown of appetite-regulating genes (neuropeptide Y, preproinsulin, melanocortin 4 receptor, agouti related protein, and cannabinoid receptor 1, and the administration of clinical appetite suppressants (fluoxetine, sibutramine, mazindol, phentermine, and rimonabant revealed the similarity among mechanisms regulating appetite in zebrafish and mammals. In combination with behavioral analysis, we were able to evaluate adverse effects on locomotor activities from gene knockdown and chemical treatments. In conclusion, we have developed an assay that uses zebrafish, which can be applied to high-throughput screening and target gene discovery for appetite suppressants and enhancers.

  1. Spectrum of mutations in sarcoglycan genes in the Mumbai region of western India: High prevalence of 525del T

    Directory of Open Access Journals (Sweden)

    Khadilkar Satish

    2009-01-01

    Full Text Available Background : While the clinical and immunocytochemical features of sarcoglycanopathies have been reported from India, genetic aspects have not been studied. There is large variation in the sarcoglycan mutations among the studied populations. Aim : To study the spectrum of mutations in sarcoglycan genes (SG. Materials and Methods : Patients fulfilling Bushby′s criteria for limb girdle muscular dystrophy were prospectively analyzed. Patients gave their medical history and underwent a clinical examination, serum creatine kinase estimation, electrophysiology, muscle biopsy with immunostaining for alpha, beta, gamma, and delta subunits and mutational analysis using denaturing high pressure liquid chromatography and direct sequencing. Results : Mutations in SG accounted for 26.4% of the cohort of limb girdle muscular dystrophy. The mean age of these 18 patients was 22.5 years. Generally, proximal weakness affected the flexor and adductor compartments of the lower and upper limbs. The clinical profile of various mutations was indistinguishable from each other. Gamma SG mutations were most common, seen in 8 patients, followed by delta SG mutation in 5 patients and alpha mutation in 4 patients, while only 1 patient had mutation in the beta sarcoglycan gene. The most prevalent mutation in the gamma SG gene was 525del T. This is of interest as the mutation has been known to exist only in specific populations. Conclusion : This study, the first mutational analysis of Indian patients with sarcoglycanopathies suggests gamma SG mutations were the most common and the most prevalent mutation in the gamma SG gene was 525del T.

  2. The Mx1 Gene Protects Mice against the Pandemic 1918 and Highly Lethal Human H5N1 Influenza Viruses▿

    OpenAIRE

    2007-01-01

    Mice carrying a wild-type Mx1 gene (Mx1+/+) differ from standard laboratory mice (Mx1−/−) in being highly resistant to infection with common laboratory strains of influenza A virus. We report that Mx1 also protects mice against the pandemic human 1918 influenza virus and a highly lethal human H5N1 strain from Vietnam. Resistance to H5N1 of Mx1+/+ but not Mx1−/− mice was enhanced if the animals were treated with a single dose of exogenous alpha interferon before infection. Thus, the interferon...

  3. A two-sample test for high-dimensional data with applications to gene-set testing

    CERN Document Server

    Chen, Song Xi; 10.1214/09-AOS716

    2010-01-01

    We propose a two-sample test for the means of high-dimensional data when the data dimension is much larger than the sample size. Hotelling's classical $T^2$ test does not work for this "large $p$, small $n$" situation. The proposed test does not require explicit conditions in the relationship between the data dimension and sample size. This offers much flexibility in analyzing high-dimensional data. An application of the proposed test is in testing significance for sets of genes which we demonstrate in an empirical study on a leukemia data set.

  4. High dietary zinc supplementation increases the occurrence of tetracycline and sulfonamide resistance genes in the intestine of weaned pigs

    OpenAIRE

    2015-01-01

    Background Dietary zinc oxide is used in pig nutrition to combat post weaning diarrhoea. Recent data suggests that high doses (2.5 g/kg feed) increase the bacterial antibiotic resistance development in weaned pigs. Therefore, the aim of this study was to investigate the development of enterobacterial antibiotic resistance genes in the intestinal tract of weaned pigs. Findings Weaned pigs were fed diets for 4 weeks containing 57 (low), 164 (intermediate) or 2425 (high) mg kg−1 analytical grade...

  5. Antibiotic resistant bacteria/genes dissemination in lacustrine sediments highly increased following cultural eutrophication of Lake Geneva (Switzerland).

    Science.gov (United States)

    Thevenon, Florian; Adatte, Thierry; Wildi, Walter; Poté, John

    2012-02-01

    This study investigates faecal indicator bacteria (FIB), multiple antibiotic resistant (MAR), and antibiotic resistance genes (ARGs), of sediment profiles from different parts of Lake Geneva (Switzerland) over the last decades. MARs consist to expose culturable Escherichia coli (EC) and Enterococcus (ENT) to mixed five antibiotics including Ampicillin, Tetracycline, Amoxicillin, Chloramphenicol and Erythromycin. Culture-independent is performed to assess the distribution of ARGs responsible for, β-lactams (blaTEM; Amoxicillin/Ampicillin), Streptomycin/Spectinomycin (aadA), Tetracycline (tet) Chloramphenicol (cmlA) and Vancomycin (van). Bacterial cultures reveal that in the sediments deposited following eutrophication of Lake Geneva in the 1970s, the percentage of MARs to five antibiotics varied from 0.12% to 4.6% and 0.016% to 11.6% of total culturable EC and ENT, respectively. In these organic-rich bacteria-contaminated sediments, the blaTEM resistant of FIB varied from 22% to 48% and 16% to 37% for EC and ENT respectively, whereas the positive PCR assays responsible for tested ARGs were observed for EC, ENT, and total DNA from all samples. The aadA resistance gene was amplified for all the sediment samples, including those not influenced by WWTP effluent water. Our results demonstrate that bacteria MARs and ARGs highly increased in the sediments contaminated with WWTP effluent following the cultural eutrophication of Lake Geneva. Hence, the human-induced changing limnological conditions highly enhanced the sediment microbial activity, and therein the spreading of antibiotic resistant bacteria and genes in this aquatic environment used to supply drinking water in a highly populated area. Furthermore, the presence of the antibiotic resistance gene aadA in all the studied samples points out a regional dissemination of this emerging contaminant in freshwater sediments since at least the late nineteenth century.

  6. Genomic signatures of local directional selection in a high gene flow marine organism; the Atlantic cod (Gadus morhua

    Directory of Open Access Journals (Sweden)

    Mittelholzer Christian

    2009-12-01

    Full Text Available Abstract Background Marine fishes have been shown to display low levels of genetic structuring and associated high levels of gene flow, suggesting shallow evolutionary trajectories and, possibly, limited or lacking adaptive divergence among local populations. We investigated variation in 98 gene-associated single nucleotide polymorphisms (SNPs for evidence of selection in local populations of Atlantic cod (Gadus morhua L. across the species distribution. Results Our global genome scan analysis identified eight outlier gene loci with very high statistical support, likely to be subject to directional selection in local demes, or closely linked to loci under selection. Likewise, on a regional south/north transect of central and eastern Atlantic populations, seven loci displayed strongly elevated levels of genetic differentiation. Selection patterns among populations appeared to be relatively widespread and complex, i.e. outlier loci were generally not only associated with one of a few divergent local populations. Even on a limited geographical scale between the proximate North Sea and Baltic Sea populations four loci displayed evidence of adaptive evolution. Temporal genome scan analysis applied to DNA from archived otoliths from a Faeroese population demonstrated stability of the intra-population variation over 24 years. An exploratory landscape genetic analysis was used to elucidate potential effects of the most likely environmental factors responsible for the signatures of local adaptation. We found that genetic variation at several of the outlier loci was better correlated with temperature and/or salinity conditions at spawning grounds at spawning time than with geographic distance per se. Conclusion These findings illustrate that adaptive population divergence may indeed be prevalent despite seemingly high levels of gene flow, as found in most marine fishes. Thus, results have important implications for our understanding of the interplay of

  7. Collinearity Analysis and High-Density Genetic Mapping of the Wheat Powdery Mildew Resistance Gene Pm40 in PI 672538

    Science.gov (United States)

    Fatima, Syeda Akash; Yang, Jiezhi; Chen, Wanquan; Liu, Taiguo; Hu, Yuting; Li, Qing; Guo, Jingwei; Zhang, Min; Lei, Li; Li, Xin; Tang, Shengwen; Luo, Peigao

    2016-01-01

    The wheat powdery mildew resistance gene Pm40, which is located on chromosomal arm 7BS, is effective against nearly all prevalent races of Blumeria graminis f. sp tritici (Bgt) in China and is carried by the common wheat germplasm PI 672538. A set of the F1, F2 and F2:3 populations from the cross of the resistant PI 672538 with the susceptible line L1034 were used to conduct genetic analysis of powdery mildew resistance and construct a high-density linkage map of the Pm40 gene. We constructed a high-density linkage genetic map with a total length of 6.18 cM and average spacing between markers of 0.48 cM.Pm40 is flanked by Xwmc335 and BF291338 at genetic distances of 0.58 cM and 0.26 cM, respectively, in deletion bin C-7BS-1-0.27. Comparative genomic analysis based on EST-STS markers established a high level of collinearity of the Pm40 genomic region with a 1.09-Mbp genomic region on Brachypodium chromosome 3, a 1.16-Mbp genomic region on rice chromosome 8, and a 1.62-Mbp genomic region on sorghum chromosome 7. We further anchored the Pm40 target intervals to the wheat genome sequence. A putative linear index of 85 wheat contigs containing 97 genes on 7BS was constructed. In total, 9 genes could be considered as candidates for the resistances to powdery mildew in the target genomic regions, which encoded proteins that were involved in the plant defense and response to pathogen attack. These results will facilitate the development of new markers for map-based cloning and marker-assisted selection of Pm40 in wheat breeding programs. PMID:27755575

  8. Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

    Science.gov (United States)

    Bilbao, R; Reay, D P; Hughes, T; Biermann, V; Volpers, C; Goldberg, L; Bergelson, J; Kochanek, S; Clemens, P R

    2003-10-01

    High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

  9. Collinearity Analysis and High-Density Genetic Mapping of the Wheat Powdery Mildew Resistance Gene Pm40 in PI 672538.

    Science.gov (United States)

    Zhong, Shengfu; Ma, Lixia; Fatima, Syeda Akash; Yang, Jiezhi; Chen, Wanquan; Liu, Taiguo; Hu, Yuting; Li, Qing; Guo, Jingwei; Zhang, Min; Lei, Li; Li, Xin; Tang, Shengwen; Luo, Peigao

    2016-01-01

    The wheat powdery mildew resistance gene Pm40, which is located on chromosomal arm 7BS, is effective against nearly all prevalent races of Blumeria graminis f. sp tritici (Bgt) in China and is carried by the common wheat germplasm PI 672538. A set of the F1, F2 and F2:3 populations from the cross of the resistant PI 672538 with the susceptible line L1034 were used to conduct genetic analysis of powdery mildew resistance and construct a high-density linkage map of the Pm40 gene. We constructed a high-density linkage genetic map with a total length of 6.18 cM and average spacing between markers of 0.48 cM.Pm40 is flanked by Xwmc335 and BF291338 at genetic distances of 0.58 cM and 0.26 cM, respectively, in deletion bin C-7BS-1-0.27. Comparative genomic analysis based on EST-STS markers established a high level of collinearity of the Pm40 genomic region with a 1.09-Mbp genomic region on Brachypodium chromosome 3, a 1.16-Mbp genomic region on rice chromosome 8, and a 1.62-Mbp genomic region on sorghum chromosome 7. We further anchored the Pm40 target intervals to the wheat genome sequence. A putative linear index of 85 wheat contigs containing 97 genes on 7BS was constructed. In total, 9 genes could be considered as candidates for the resistances to powdery mildew in the target genomic regions, which encoded proteins that were involved in the plant defense and response to pathogen attack. These results will facilitate the development of new markers for map-based cloning and marker-assisted selection of Pm40 in wheat breeding programs.

  10. High-efficiency gene transfer into skeletal muscle mediated by electric pulses

    DEFF Research Database (Denmark)

    Mir, L M; Bureau, M F; Gehl, J;

    1999-01-01

    Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. However, present DNA delivery technologies have to be improved with regard to both the level of expression and interindividual variability. We...... report very efficient plasmid DNA transfer in muscle fibers by using square-wave electric pulses of low field strength (less than 300 V/cm) and of long duration (more than 1 ms). Contrary to the electropermeabilization-induced uptake of small molecules into muscle fibers, plasmid DNA has to be present...... in the tissue during the electric pulses, suggesting a direct effect of the electric field on DNA during electrotransfer. This i.m. electrotransfer method increases reporter and therapeutic gene expression by several orders of magnitude in various muscles in mouse, rat, rabbit, and monkey. Moreover, i...

  11. A High Throughput Soybean Gene Identification System Developed using Soybean Yellow Common Mosaic Virus (SYCMV)

    OpenAIRE

    Seo, Eun–Young; Cho, Seunghee; Moon, Jae Sun; Gotoh, Takafumi; Goto, Takafumi; Kim, Hong Gi; Domier, Leslie L; Lim, Seungmo; Kim, Kil Hyun; Moon, Jung–Kyung; Hammond, John; Lim, Hyoun–Sub; Song, Ki Hak

    2015-01-01

    Soybean yellow common mosaic virus (SYCMV) was recently reported from Korea, and a subsequent survey of soybean fields found that SYCMV, Soybean yellow mottle mosaic virus (SYMMV), and Soybean mosaic virus (SMV) infections were widespread. SYCMV has recently been developed into a Virus Inducing Gene Silencing (VIGS) vector for use as a reverse genetics tool for soybean, and here we report a modified SYCMV VIGS vector containing a new restriction enzyme site in the 3’ non–coding region into wh...

  12. 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...

  13. Natural Variation Identifies ICARUS1, a Universal Gene Required for Cell Proliferation and Growth at High Temperatures in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Wangsheng Zhu

    2015-05-01

    Full Text Available Plants are highly sensitive to environmental changes and even small variations in ambient temperature have severe consequences on their growth and development. Temperature affects multiple aspects of plant development, but the processes and mechanisms underlying thermo-sensitive growth responses are mostly unknown. Here we exploit natural variation in Arabidopsis thaliana to identify and characterize novel components and processes mediating thermo-sensitive growth responses in plants. Phenotypic screening of wild accessions identified several strains displaying pleiotropic growth defects, at cellular and organism levels, specifically at high ambient temperatures. Positional cloning and characterization of the underlying gene revealed that ICARUS1 (ICA1, which encodes a protein of the tRNAHis guanylyl transferase (Thg1 superfamily, is required for plant growth at high temperatures. Transcriptome and gene marker analyses together with DNA content measurements show that ICA1 loss-of-function results in down regulation of cell cycle associated genes at high temperatures, which is linked with a block in G2/M transition and endoreduplication. In addition, plants with mutations in ICA1 show enhanced sensitivity to DNA damage. Characterization of additional strains that carry lesions in ICA1, but display normal growth, shows that alternative splicing is likely to alleviate the deleterious effects of some natural mutations. Furthermore, analyses of worldwide and regional collections of natural accessions indicate that ICA1 loss-of-function has arisen several times independently, and that these occur at high frequency in some local populations. Overall our results suggest that ICA1-mediated-modulation of fundamental processes such as tRNAHis maturation, modify plant growth responses to temperature changes in a quantitative and reversible manner, in natural populations.

  14. Hypothalamic Leptin Gene Therapy Reduces Bone Marrow Adiposity in ob/ob Mice Fed Regular and High-Fat Diets

    Science.gov (United States)

    Lindenmaier, Laurence B.; Philbrick, Kenneth A.; Branscum, Adam J.; Kalra, Satya P.; Turner, Russell T.; Iwaniec, Urszula T.

    2016-01-01

    Low bone mass is often associated with elevated bone marrow adiposity. Since osteoblasts and adipocytes are derived from the same mesenchymal stem cell (MSC) progenitor, adipocyte formation may increase at the expense of osteoblast formation. Leptin is an adipocyte-derived hormone known to regulate energy and bone metabolism. Leptin deficiency and high-fat diet-induced obesity are associated with increased marrow adipose tissue (MAT) and reduced bone formation. Short-duration studies suggest that leptin treatment reduces MAT and increases bone formation in leptin-deficient ob/ob mice fed a regular diet. Here, we determined the long-duration impact of increased hypothalamic leptin on marrow adipocytes and osteoblasts in ob/ob mice following recombinant adeno-associated virus (rAAV) gene therapy. Eight- to 10-week-old male ob/ob mice were randomized into four groups: (1) untreated, (2) rAAV-Lep, (3) rAAV-green fluorescent protein (rAAV-GFP), or (4) pair-fed to rAAV-Lep. For vector administration, mice were injected intracerebroventricularly with either rAAV-leptin gene therapy (rAAV-Lep) or rAAV-GFP (9 × 107 particles) and maintained for 30 weeks. In a second study, the impact of increased hypothalamic leptin levels on MAT was determined in mice fed high-fat diets; ob/ob mice were randomized into two groups and treated with either rAAV-Lep or rAAV-GFP. At 7 weeks post-vector administration, half the mice in each group were switched to a high-fat diet for 8 weeks. Wild-type (WT) controls included age-matched mice fed regular or high-fat diet. High-fat diet resulted in a threefold increase in MAT in WT mice, whereas MAT was increased by leptin deficiency up to 50-fold. Hypothalamic leptin gene therapy increased osteoblast perimeter and osteoclast perimeter with minor change in cancellous bone architecture. The gene therapy decreased MAT levels in ob/ob mice fed regular or high-fat diet to values similar to WT mice fed regular diet. These findings suggest

  15. Bacterial niche-specific genome expansion is coupled with highly frequent gene disruptions in deep-sea sediments

    KAUST Repository

    Wang, Yong

    2011-12-21

    The complexity and dynamics of microbial metagenomes may be evaluated by genome size, gene duplication and the disruption rate between lineages. In this study, we pyrosequenced the metagenomes of microbes obtained from the brine and sediment of a deep-sea brine pool in the Red Sea to explore the possible genomic adaptations of the microbes in response to environmental changes. The microbes from the brine and sediments (both surface and deep layers) of the Atlantis II Deep brine pool had similar communities whereas the effective genome size varied from 7.4 Mb in the brine to more than 9 Mb in the sediment. This genome expansion in the sediment samples was due to gene duplication as evidenced by enrichment of the homologs. The duplicated genes were highly disrupted, on average by 47.6% and 70% for the surface and deep layers of the Atlantis II Deep sediment samples, respectively. The disruptive effects appeared to be mainly due to point mutations and frameshifts. In contrast, the homologs from the Atlantis II Deep brine sample were highly conserved and they maintained relatively small copy numbers. Likely, the adaptation of the microbes in the sediments was coupled with pseudogenizations and possibly functional diversifications of the paralogs in the expanded genomes. The maintenance of the pseudogenes in the large genomes is discussed. © 2011 Wang et al.

  16. Plasmodium falciparum antigenic variation. Mapping mosaic var gene sequences onto a network of shared, highly polymorphic sequence blocks.

    Science.gov (United States)

    Bull, Peter C; Buckee, Caroline O; Kyes, Sue; Kortok, Moses M; Thathy, Vandana; Guyah, Bernard; Stoute, José A; Newbold, Chris I; Marsh, Kevin

    2008-06-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, encoded by an extremely diverse gene family called var. Understanding of the genetic organization of var genes is hampered by sequence mosaicism that results from a long history of non-homologous recombination. Here we have used software designed to analyse social networks to visualize the relationships between large collections of short var sequences tags sampled from clinical parasite isolates. In this approach, two sequences are connected if they share one or more highly polymorphic sequence blocks. The results show that the majority of analysed sequences including several var-like sequences from the chimpanzee parasite Plasmodium reichenowi can be either directly or indirectly linked together in a single unbroken network. However, the network is highly structured and contains putative subgroups of recombining sequences. The major subgroup contains the previously described group A var genes, previously proposed to be genetically distinct. Another subgroup contains sequences found to be associated with rosetting, a parasite virulence phenotype. The mosaic structure of the sequences and their division into subgroups may reflect the conflicting problems of maximizing antigenic diversity and minimizing epitope sharing between variants while maintaining their host cell binding functions.

  17. Recycling Gene Carrier with High Efficiency and Low Toxicity Mediated by L-Cystine-Bridged Bis(β-cyclodextrin)s

    Science.gov (United States)

    Zhang, Yu-Hui; Chen, Yong; Zhang, Ying-Ming; Yang, Yang; Chen, Jia-Tong; Liu, Yu

    2014-12-01

    Constructing safe and effective gene delivery carriers is becoming highly desirable for gene therapy. Herein, a series of supramolecular crosslinking system were prepared through host-guest binding of adamantyl-modified low molecular weight of polyethyleneimine with L-cystine-bridged bis(β-cyclodextrin)s and characterized by 1H NMR titration, electron microscopy, zeta potential, dynamic light-scattering, gel electrophoresis, flow cytometry and confocal fluorescence microscopy. The results showed that these nanometersized supramolecular crosslinking systems exhibited higher DNA transfection efficiencies and lower cytotoxicity than the commercial DNA carrier gold standard (25 kDa bPEI) for both normal cells and cancer cells, giving a very high DNA transfection efficiency up to 54% for 293T cells. Significantly, this type of supramolecular crosslinking system possesses a number of enzyme-responsive disulfide bonds, which can be cleaved by reductive enzyme to promote the DNA release but recovered by oxidative enzyme to make the carrier renewable. These results demonstrate that these supramolecular crosslinking systems can be used as promising gene carriers.

  18. The HU regulon is composed of genes responding to anaerobiosis, acid stress, high osmolarity and SOS induction.

    Directory of Open Access Journals (Sweden)

    Jacques Oberto

    Full Text Available BACKGROUND: The Escherichia coli heterodimeric HU protein is a small DNA-bending protein associated with the bacterial nucleoid. It can introduce negative supercoils into closed circular DNA in the presence of topoisomerase I. Cells lacking HU grow very poorly and display many phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the transcription profile of every Escherichia coli gene in the absence of one or both HU subunits. This genome-wide in silico transcriptomic approach, performed in parallel with in vivo genetic experimentation, defined the HU regulon. This large regulon, which comprises 8% of the genome, is composed of four biologically relevant gene classes whose regulation responds to anaerobiosis, acid stress, high osmolarity, and SOS induction. CONCLUSIONS/SIGNIFICANCE: The regulation a large number of genes encoding enzymes involved in energy metabolism and catabolism pathways by HU explains the highly pleiotropic phenotype of HU-deficient cells. The uniform chromosomal distribution of the many operons regulated by HU strongly suggests that the transcriptional and nucleoid architectural functions of HU constitute two aspects of a unique protein-DNA interaction mechanism.

  19. Bacterial niche-specific genome expansion is coupled with highly frequent gene disruptions in deep-sea sediments.

    Directory of Open Access Journals (Sweden)

    Yong Wang

    Full Text Available The complexity and dynamics of microbial metagenomes may be evaluated by genome size, gene duplication and the disruption rate between lineages. In this study, we pyrosequenced the metagenomes of microbes obtained from the brine and sediment of a deep-sea brine pool in the Red Sea to explore the possible genomic adaptations of the microbes in response to environmental changes. The microbes from the brine and sediments (both surface and deep layers of the Atlantis II Deep brine pool had similar communities whereas the effective genome size varied from 7.4 Mb in the brine to more than 9 Mb in the sediment. This genome expansion in the sediment samples was due to gene duplication as evidenced by enrichment of the homologs. The duplicated genes were highly disrupted, on average by 47.6% and 70% for the surface and deep layers of the Atlantis II Deep sediment samples, respectively. The disruptive effects appeared to be mainly due to point mutations and frameshifts. In contrast, the homologs from the Atlantis II Deep brine sample were highly conserved and they maintained relatively small copy numbers. Likely, the adaptation of the microbes in the sediments was coupled with pseudogenizations and possibly functional diversifications of the paralogs in the expanded genomes. The maintenance of the pseudogenes in the large genomes is discussed.

  20. Limited contemporary gene flow and high self-replenishment drives peripheral isolation in an endemic coral reef fish.

    Science.gov (United States)

    van der Meer, Martin H; Horne, John B; Gardner, Michael G; Hobbs, Jean-Paul A; Pratchett, Morgan; van Herwerden, Lynne

    2013-06-01

    Extensive ongoing degradation of coral reef habitats worldwide has lead to declines in abundance of coral reef fishes and local extinction of some species. Those most vulnerable are ecological specialists and endemic species. Determining connectivity between locations is vital to understanding recovery and long-term persistence of these species following local extinction. This study explored population connectivity in the ecologically-specialized endemic three-striped butterflyfish (Chaetodon tricinctus) using mt and msatDNA (nuclear microsatellites) to distinguish evolutionary versus contemporary gene flow, estimate self-replenishment and measure genetic diversity among locations at the remote Australian offshore coral reefs of Middleton Reef (MR), Elizabeth Reef (ER), Lord Howe Island (LHI), and Norfolk Island (NI). Mt and msatDNA suggested genetic differentiation of the most peripheral location (NI) from the remaining three locations (MR, ER, LHI). Despite high levels of mtDNA gene flow, there is limited msatDNA gene flow with evidence of high levels of self-replenishment (≥76%) at all four locations. Taken together, this suggests prolonged population recovery times following population declines. The peripheral population (NI) is most vulnerable to local extinction due to its relative isolation, extreme levels of self-replenishment (95%), and low contemporary abundance.

  1. Gene Flow Results in High Genetic Similarity Between Sibiraea (Rosaceae species in the Qinghai-Tibetan Plateau

    Directory of Open Access Journals (Sweden)

    Peng-Cheng Fu

    2016-10-01

    Full Text Available Studying closely related species and divergent populations provides insight into the process of speciation. Previous studies showed that the Sibiraea complex's evolutionary history on the Qinghai-Tibetan Plateau (QTP was confusing and could not be distinguishable on the molecular level. In this study, the genetic structure and gene flow of S. laevigata and S. angustata on the QTP was examined across 45 populations using 8 microsatellite loci. Microsatellites revealed high genetic diversity in Sibiraea populations. Most of the variance was detected within populations (87.45% rather than between species (4.39%. We found no significant correlations between genetic and geographical distances among populations. Bayesian cluster analysis grouped all individuals in the sympatric area of Sibiraea into one cluster and other individuals of S. angustata into another. Divergence history analysis based on the approximate Bayesian computation method indicated that the populations of S. angustata at the sympatric area derived from the admixture of 2 species. The assignment test assigned all individuals to populations of their own species rather than its congeneric species. Consistently, intraspecies were detected rather than interspecies first-generation migrants. The bidirectional gene flow in long-term patterns between the 2 species was asymmetric, with more from S. angustata to S. laevigata. In conclusion, the Sibiraea complex was distinguishable on the molecular level using microsatellite loci. We found that the high genetic similarity of these 2 species resulted from huge bidirectional gene flow, especially on the sympatric area where population admixtures between the species occurred.

  2. Identification of rat mammary tumor-1 gene (RMT-1), which is highly expressed in rat mammary tumors.

    Science.gov (United States)

    Chiou, S; Yoo, J; Loh, K C; Guzman, R C; Gopinath, G R; Rajkumar, L; Chou, Y C; Yang, J; Popescu, N C; Nandi, S

    2001-12-10

    Full-term pregnancy early in life results in a permanent reduction in lifetime breast cancer risk in women. Parous rats and mice are also refractory to chemical carcinogenesis. Therefore, investigation of the differences between mammary glands from virgin and parous rats would provide valuable information regarding the protective effects of early full-term pregnancy. In this report, we examined the gene expression patterns in mammary glands from virgin and parous Lewis rats. Using differential display technology, a novel 4.2 kb cDNA, designated rat mammary tumor-1 (RMT-1) was isolated. Northern blot analysis of RMT-1 showed that RMT-1 expression was higher in the pre-pubertal and pubertal stages during rat mammary gland development while it was down-regulated in mammary glands from mature virgin and parous rats. RMT-1 expression was highest in rat mammary cancers compared with either the mammary glands of virgin or parous rats. At the Northern blot sensitivity level, RMT-1 expression was found only in the mammary gland. Northern blot analysis also showed that the expression of this gene was found in 74% of N-methyl-nitrosourea (MNU)-induced mammary cancers while it was not found in MNU-induced cancers from other organs. The examination of the RMT-1 gene structure revealed that it consists of five exons spanning 5.9 kb. Using fluorescence in situ hybridization, the gene was localized on rat chromosome 1 band q 43-51. The present data show that there is a correlation between high RMT-1 expression and rat mammary carcinogenesis or decreased RMT-1 expression and parity associated refractoriness to chemically induced mammary carcinogenesis. However, whether or not RMT-1 gene has a functional role in these processes remains to be investigated.

  3. Characterization of polymorphisms and isoforms of the Clostridium perfringens phospholipase C gene (plc) reveals high genetic diversity.

    Science.gov (United States)

    Siqueira, Flávia F; Almeida, Marcelle O; Barroca, Tatiana M; Horta, Carolina C R; Carmo, Anderson O; Silva, Rodrigo O S; Pires, Prhiscylla S; Lobato, Francisco C F; Kalapothakis, Evanguedes

    2012-10-12

    Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is encoded by the plc gene and has been implicated in several diseases; however, only a few studies have described polymorphisms in this gene. The aim of this study was to analyze polymorphisms in the Cp-PLC nucleotide and amino acid sequences obtained from isolates from different regions and to compare them to Clostridium phospholipase C sequences deposited in the NCBI database. Environmental samples (sediment, poultry feed, sawdust) and stool samples (from poultry, bovine, swine, horse, caprine, bird, dog, rabbit, toucan) were collected from healthy and sick animals. A total of 73 isolates were analyzed with the majority of samples belonging to the toxin type A subtype and possessing the gene encoding for the beta-2 toxin. Comparison of plc gene sequences from respective isolates revealed a high genetic diversity in the nucleotide sequences of mature Cp-PLC. Sequence comparisons identified 30 amino acid substitutions and 34 isoforms including some isoforms with substitutions in amino acids critical to toxin function. Comparison of sequences obtained in this study to Cp-PLC sequences obtained from the NCBI database resulted in the identification of 11 common haplotypes and 22 new isoforms. Phylogenetic analysis of phospholipase C sequences obtained from other Clostridium species identified relationships previously described. This report describes a broad characterization of the genetic diversity in the C. perfringens plc gene resulting in the identification of various isoforms. A better understanding of sequences encoding phospholipase C isoforms may reveal changes associated with protein function and C. perfringens virulence.

  4. Responses in colonic microbial community and gene expression of pigs to a long-term high resistant starch diet

    Directory of Open Access Journals (Sweden)

    Yue eSun

    2015-08-01

    Full Text Available Intake of raw potato starch (RPS has been associated with various intestinal health benefits, but knowledge of its mechanism in a long-term is limited. The aim of this study was to investigate the effects of long-term intake of RPS on microbial composition, genes expression profiles in the colon of pigs. Thirty-six Duroc × Landrace × Large White growing barrows were randomly allocated to corn starch (CS and RPS groups with a randomized block design. Each group consisted of six replicates (pens, with three pigs per pen. Pigs in the CS group were offered a corn/soybean-based diet, while pigs in the RPS group were put on a diet in which 230 g/kg (growing period or 280 g/kg (finishing period purified corn starch was replaced with purified RPS during a 100-day trial. Real-time PCR assay showed that RPS significantly decreased the number of total bacteria in the colonic digesta. MiSeq sequencing of the V3-V4 region of the 16S rRNA genes showed that RPS significantly decreased the relative abundance of Clostridium, Treponema, Oscillospira, Phascolarctobacterium, RC9 gut group, and S24-7-related operational taxonomic units (OTUs, and increased the relative abundance of Turicibacter, Blautia, Ruminococcus, Coprococcus, Marvinbryantia, and Ruminococcus bromii-related OTUs in colonic digesta and mucosa. Analysis of the colonic transcriptome profiles revealed that the RPS diet changed the colonic expression profile of the host genes mainly involved in immune response pathways. RPS significantly increased proinflammartory cytokine IL-1β gene expression and suppressed genes involved in lysosome. Our findings suggest that long-term intake of high resistant starch (RS diet may result in both positive and negative roles in gut health.

  5. Changes of microbial community structures and functional genes during biodegradation of phenolic compounds under high salt condition

    Institute of Scientific and Technical Information of China (English)

    WANG Ping; QU Yuanyuan; ZHOU Jiti

    2009-01-01

    The changes of microbial community structures and functional genes during the biodegradation of single phenol and phenol plus p-cresol under high salt condition were explored.It was found that the phenol-fed system (PFS) exhibited stronger degrading abilities and more stable biomass than that of the phenol plus p-cresol-fed system (PCFS).The microbial community structures were revealed by a modern DNA fingerprint technique,ribosomal intergenic spacer analysis (RISA).The results indicated that the microbial community of PFS changed obviously when gradually increased phenol concentration,while PCFS showed a little change.16S rRNA sequence analysis of the major bands showed that Alcanivorax sp.genus was predominant species during phenolic compounds degradation.Furthermore,amplified functional DNA restriction analysis (AFDRA) on phenol hydroxylase genes showed that the fingerprints were substantially different in the two systems,and the fingerprints were not the same during the different operational periods.

  6. Genomic signatures of local directional selection in a high gene flow marine organism, the Atlantic cod (Gadus morhua)

    DEFF Research Database (Denmark)

    Eg Nielsen, Einar; Hansen, Jakob Hemmer; Poulsen, Nina Aagaard;

    2009-01-01

    Background: Marine fishes have been shown to display low levels of genetic structuring and associated high levels of gene flow, suggesting shallow evolutionary trajectories and, possibly, limited or lacking adaptive divergence among local populations. We investigated variation in 98 gene...... selection in local demes, or closely linked to loci under selection. Likewise, on a regional south/north transect of central and eastern Atlantic populations, seven loci displayed strongly elevated levels of genetic differentiation. Selection patterns among populations appeared to be relatively widespread...... archived otoliths from a Faeroese population demonstrated stability of the intra-population variation over 24 years. An exploratory landscape genetic analysis was used to elucidate potential effects of the most likely environmental factors responsible for the signatures of local adaptation. We found...

  7. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    Science.gov (United States)

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

  8. High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia

    DEFF Research Database (Denmark)

    Kristensen, Louise; Kielsgaard Kristensen, Thomas; Abildgaard, Niels;

    2015-01-01

    Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world. Autophagy is a highly conserved process in eukaryotic cells. In CLL autophagy is involved in mediating the effect of chemotherapy but the role of autophagy in CLL pathogenesis remains unknown....... In the present study, we used real-time RT-PCR to analyze expression of the PIK3C3, PIK3R4, and BECN1 genes. These genes encode the components of the PI3K core complex, which is central to initiation of autophagy. A consecutive series of 149 well-characterized CLL cases from Region of Southern Denmark were...... on the role of autophagy in CLL, and they may further represent targets of treatment....

  9. High DNA-Binding Affinity and Gene-Transfection Efficacy of Bioreducible Cationic Nanomicelles with a Fluorinated Core.

    Science.gov (United States)

    Wang, Long-Hai; Wu, De-Cheng; Xu, Hang-Xun; You, Ye-Zi

    2016-01-11

    During the last two decades, cationic polymers have become one of the most promising synthetic vectors for gene transfection. However, the weak interactions formed between DNA and cationic polymers result in low transfection efficacy. Furthermore, the polyplexes formed between cationic polymers and DNA generally exhibit poor stability and toxicity because of the large excess of cationic polymer typically required for complete DNA condensation. Herein, we report the preparation of a novel class of bioreducible cationic nanomicelles by the use of disulfide bonds to connect the cationic shell to the fluorocarbon core. These bioreducible nanomicelles form strong interactions with DNA and completely condense DNA at an N/P ratio of 1. The resulting nanomicelle/DNA polyplexes exhibited high biocompatibility and performed very effectively as a gene-delivery system.

  10. High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris using codon optimization.

    Science.gov (United States)

    Gao, Zhaowei; Li, Zhuofu; Zhang, Yuhong; Huang, Huoqing; Li, Mu; Zhou, Liwei; Tang, Yunming; Yao, Bin; Zhang, Wei

    2012-03-01

    The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35-40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml(-1) (2.5 g protein l(-1)) in a 3 l fermentor--410% higher than GOD-w (148 U ml(-1)), and thus is a low-cost alternative for the bread baking industry.

  11. High-sensitivity Mass Spectrometry for Probing Gene Translation in Single Embryonic Cells in the Early Frog (Xenopus Embryo

    Directory of Open Access Journals (Sweden)

    Camille Lombard-Banek

    2016-10-01

    Full Text Available Direct measurement of protein expression with single-cell resolution promises to deepen the understanding of basic molecular processes during normal and impaired development. High-resolution mass spectrometry provides detailed coverage of the proteomic composition of large numbers of cells. Here we discuss recent mass spectrometry developments based on single-cell capillary electrophoresis that extend discovery proteomics to sufficient sensitivity to enable the measurement of proteins in single cells. The single-cell mass spectrometry system is used to detect a large number of proteins in single embryonic cells in blastomeres in the 16-cell embryo of the South African clawed frog (Xenopus laevis that give rise to distinct tissue types. Single-cell measurements of protein expression provide complementary information on gene transcription during early development of the vertebrate embryo, raising a potential to understand how differential gene expression coordinates normal cell heterogeneity during development.

  12. Progress Towards Genetics and Breeding for Minor Genes Based Resistance to Ug99 and Other Rusts in CIMMYT High-Yielding Spring Wheat

    Institute of Scientific and Technical Information of China (English)

    Ravi Prakash Singh; Sybil Herrera-Foessel; Julio Huerta-Espino; Sukhwinder Singh; Sridhar Bhavani; Caixia Lan; and Bhoja Raj Basnet

    2014-01-01

    Wheat rusts continue to cause signiifcant losses worldwide despite major efforts given to their genetic control. This is due to frequent evolution and selection of virulence in pathogen overcoming the deployed race-speciifc resistance genes. Although the life of effective race-speciifc resistance genes can be prolonged by using gene combinations, an alternative approach being implemented at CIMMYT is to deploy varieties that posses adult plant resistance (APR) based on combinations of minor, slow rusting genes. When present alone, the APR genes do not confer adequate resistance especially under high disease pressure; however, combinations of 4 or 5 minor genes usually result in “near-immunity” or a high level of resistance. Although only a few APR genes are catalogued, various APR QTLs are now known and could lead to further characterization of additional genes. Four characterized genes have pleiotropic effects in conferring partial APR to all 3 rusts and powdery mildew, thus simplifying the task of breeding wheat varieties that are resistant to multiple diseases. Signiifcant progress was made recently in developing high-yielding wheat germplasm that possesses high levels of APR to all three rusts by implementing a Mexico-Kenya shuttle breeding scheme. Parents with APR to Ug99 were hybridized with high-yielding parents that had adequate to high levels of APR to leaf rust and yellow rust. Segregating populations and advanced lines from these crosses were selected under high rust pressures in Mexico (leaf rust and yellow rust) and Kenya (Ug99 stem rust and yellow rust) to identify high-yielding progenies that possess high to adequate APR to all three rusts. International distribution of these high-yielding wheats is underway through CIMMYT international yield trials and screening nurseries. It is expected that several wheat varieties with APR to three rusts will be released and grown in various countries in the near-future that will allow determining the

  13. Molecular gate keepers succumb to gene aberrations in colorectal cancer in Kashmiri population, revealing a high incidence area

    Directory of Open Access Journals (Sweden)

    Sameer A

    2009-01-01

    Full Text Available Background/Aim: Colorectal cancer (CRC is one of the leading malignancies worldwide and has been reported to show geographical variation in its incidence, even within areas of ethnic homogeneity. The aim of this study was to identify p53 and K-ras gene mutations in CRC patients in a Kashmiri population, and to assess whether these mutations are linked with clinicopathological parameters. Materials and Methods: Paired tumor and normal tissue samples from a consecutive series of 53 patients undergoing resective surgery for CRC were prospectively studied for p53 and K-ras gene mutations by PCR/single strand conformation polymorphism (SSCP. Results: Less than half (45%, 19/42 of the patients presented mutations in the p53 gene. Twenty eight mutations were found in the p53 gene, which comprised of 23 substitutions (17 transitions + 6 transversions, and five insertions. The 23 substitutions constituted 18 missense mutations, two nonsense mutations, and three silent mutations. Of the 28 mutations (7.14% observed in this study, 2 were not previously reported for CRC samples and were identified as novel p53 mutations. A few patients (22.64%, 12/53 presented with mutations in K-ras, constituting 13 missense mutations, out of which 11 were G→A transitions, one was a G→C transversion, and one a G→T transversion. More than half (61.5% of the mutations occurred in codon 12 whereas a few (38.5% occurred in codon 13. One tumor contained missense mutations in both codons. Comparison of the mutation profiles of our patients with those of other ethnic populations and regions reflected both differences and similarities, indicating co-exposure to a unique set of risk factors. Conclusion: Mutations of the p53 and K-ras genes are some of the most common genetic changes in the development of human CRC. The high frequency of p53 gene mutations implicates p53 as a predominant factor for CRC in the high-risk ethnic Kashmiri population.

  14. Gene expression in the scleractinian Acropora microphthalma exposed to high solar irradiance reveals elements of photoprotection and coral bleaching.

    Directory of Open Access Journals (Sweden)

    Antonio Starcevic

    Full Text Available BACKGROUND: The success of tropical reef-building corals depends on the metabolic co-operation between the animal host and the photosynthetic performance of endosymbiotic algae residing within its cells. To examine the molecular response of the coral Acropora microphthalma to high levels of solar irradiance, a cDNA library was constructed by PCR-based suppression subtractive hybridisation (PCR-SSH from mRNA obtained by transplantation of a colony from a depth of 12.7 m to near-surface solar irradiance, during which the coral became noticeably paler from loss of endosymbionts in sun-exposed tissues. METHODOLOGY/PRINCIPAL FINDINGS: A novel approach to sequence annotation of the cDNA library gave genetic evidence for a hypothetical biosynthetic pathway branching from the shikimic acid pathway that leads to the formation of 4-deoxygadusol. This metabolite is a potent antioxidant and expected precursor of the UV-protective mycosporine-like amino acids (MAAs, which serve as sunscreens in coral phototrophic symbiosis. Empirical PCR based evidence further upholds the contention that the biosynthesis of these MAA sunscreens is a 'shared metabolic adaptation' between the symbiotic partners. Additionally, gene expression induced by enhanced solar irradiance reveals a cellular mechanism of light-induced coral bleaching that invokes a Ca(2+-binding synaptotagmin-like regulator of SNARE protein assembly of phagosomal exocytosis, whereby algal partners are lost from the symbiosis. CONCLUSIONS/SIGNIFICANCE: Bioinformatics analyses of DNA sequences obtained by differential gene expression of a coral exposed to high solar irradiance has revealed the identification of putative genes encoding key steps of the MAA biosynthetic pathway. Revealed also by this treatment are genes that implicate exocytosis as a cellular process contributing to a breakdown in the metabolically essential partnership between the coral host and endosymbiotic algae, which manifests as coral

  15. Analysis of hepatic gene expression profile in a spontaneous mouse model of type 2 diabetes under a high sucrose diet.

    Science.gov (United States)

    Nojima, Koji; Sugimoto, Ken; Ueda, Hironori; Babaya, Naru; Ikegami, Hiroshi; Rakugi, Hiromi

    2013-01-01

    Both genetic factors and diabetogenic environmental factors, such as a high-sucrose diet (HSD), are involved in the development of type 2 diabetes. In this study, the Nagoya-Shibata-Yasuda (NSY) mouse, an animal model of type 2 diabetes and C3H mice used as controls, were fed a HSD, a high-fat diet (HFD) or a regular diet (RD) from weaning. In C3H mice, HFD significantly increased body weight gain, but maintained glucose tolerance. In contrast, in NSY mice, HSD resulted in increased body weight gain and liver steatosis and increased glucose intolerance to a greater extent than HFD. Furthermore, we performed DNA microarray analysis to detect differences in hepatic gene expression levels in both strains under HSD. We then performed RT-PCR analysis on selected genes to evaluate basal expression level under RD and changes under HSD conditions. HSD-fed NSY, but not C3H mice, exhibited increased hepatic expression levels of Pparg2, an isoform of Pparg as well as G0s2, a target of Pparg, which are known to be adipocyte-specific genes. Compared to RD-fed C3H mice, hepatic expression levels of Kat2b (transcriptional regulation), Hsd3b5 (steroid hormone metabolism) and Cyp7b1 (bile acid metabolism) were initially lower in RD-fed NSY mice, and were further decreased in HSD-fed NSY mice. Expression of Metallothionein (Mt1) and Metallothionein 2 (Mt2) was significantly lower in NSY mice compared to C3H mice, irrespective of dietary condition. These data suggest that elucidation of this heterogeneity in response to HSD might contribute to further understanding of the gene-environment interactions leading to diabetes in humans.

  16. Bulk segregant analysis by high-throughput sequencing reveals a novel xylose utilization gene from Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Jared W Wenger

    2010-05-01

    Full Text Available Fermentation of xylose is a fundamental requirement for the efficient production of ethanol from lignocellulosic biomass sources. Although they aggressively ferment hexoses, it has long been thought that native Saccharomyces cerevisiae strains cannot grow fermentatively or non-fermentatively on xylose. Population surveys have uncovered a few naturally occurring strains that are weakly xylose-positive, and some S. cerevisiae have been genetically engineered to ferment xylose, but no strain, either natural or engineered, has yet been reported to ferment xylose as efficiently as glucose. Here, we used a medium-throughput screen to identify Saccharomyces strains that can increase in optical density when xylose is presented as the sole carbon source. We identified 38 strains that have this xylose utilization phenotype, including strains of S. cerevisiae, other sensu stricto members, and hybrids between them. All the S. cerevisiae xylose-utilizing strains we identified are wine yeasts, and for those that could produce meiotic progeny, the xylose phenotype segregates as a single gene trait. We mapped this gene by Bulk Segregant Analysis (BSA using tiling microarrays and high-throughput sequencing. The gene is a putative xylitol dehydrogenase, which we name XDH1, and is located in the subtelomeric region of the right end of chromosome XV in a region not present in the S288c reference genome. We further characterized the xylose phenotype by performing gene expression microarrays and by genetically dissecting the endogenous Saccharomyces xylose pathway. We have demonstrated that natural S. cerevisiae yeasts are capable of utilizing xylose as the sole carbon source, characterized the genetic basis for this trait as well as the endogenous xylose utilization pathway, and demonstrated the feasibility of BSA using high-throughput sequencing.

  17. Overexpression of a modiifed AM79 aroA gene in transgenic maize confers high tolerance to glyphosate

    Institute of Scientific and Technical Information of China (English)

    REN Zhen-jing; CAO Gao-yi; ZHANG Yu-wen; LIU Yan; LIU Yun-jun

    2015-01-01

    It has previously been shown that a bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene AM79 aroA can be a candidate gene to develop glyphosate-tolerant transgenic crops (Cao et al. 2012). In this study, AM79 aroA was redesigned using the plant biased codons and eliminating the motifs which would lead to the instability of mRNA, to create a synthetic gene that would be expressed highly in plant cel s. The redesigned and artiifcial y synthesized gene, named as mAM79, was cloned into plant expression vector pM3301UbiSpAM79, where mAM79 is fused with signal peptide sequence of pea rib-1,5-bisphospate carboxylase (rbcS) smal subunit and control ed by ubiquitin promoter. The plasmid was transformed into maize (Zea mays) immature embryos using Agrobacterium-mediated transformation method. Total 74 regenerated plants were obtained and PCR analysis showed that these transgenic plants had the integration of mAM79. Southern blot analysis was performed on the genomic DNA from four transgenic lines, and the result showed that one or two copies of mAM79 were integrated into maize genome. RT-PCR analysis result indicated that mAM79 was highly transcribed in transgenic maize plants. When sprayed with glyphosate, transgenic maize line AM85 and AM72 could tolerate 4-fold of commercial usage of glyphosate;however, al the non-transgenic maize plants were kil ed by glyphosate. The results in this study conifrmed that mAM79 could be used to develop glyphosate-tolerant maize, and the obtained transgenic maize lines could be used for the breeding of glyphosate-tolerant maize.

  18. Intergenic DNA sequences from the human X chromosome reveal high rates of global gene flow

    Directory of Open Access Journals (Sweden)

    Wall Jeffrey D

    2008-11-01

    Full Text Available Abstract Background Despite intensive efforts devoted to collecting human polymorphism data, little is known about the role of gene flow in the ancestry of human populations. This is partly because most analyses have applied one of two simple models of population structure, the island model or the splitting model, which make unrealistic biological assumptions. Results Here, we analyze 98-kb of DNA sequence from 20 independently evolving intergenic regions on the X chromosome in a sample of 90 humans from six globally diverse populations. We employ an isolation-with-migration (IM model, which assumes that populations split and subsequently exchange migrants, to independently estimate effective population sizes and migration rates. While the maximum effective size of modern humans is estimated at ~10,000, individual populations vary substantially in size, with African populations tending to be larger (2,300–9,000 than non-African populations (300–3,300. We estimate mean rates of bidirectional gene flow at 4.8 × 10-4/generation. Bidirectional migration rates are ~5-fold higher among non-African populations (1.5 × 10-3 than among African populations (2.7 × 10-4. Interestingly, because effective sizes and migration rates are inversely related in African and non-African populations, population migration rates are similar within Africa and Eurasia (e.g., global mean Nm = 2.4. Conclusion We conclude that gene flow has played an important role in structuring global human populations and that migration rates should be incorporated as critical parameters in models of human demography.

  19. Nicotianamine synthase overexpression positively modulates iron homeostasis-related genes in high iron rice.

    Science.gov (United States)

    Wang, Meng; Gruissem, Wilhelm; Bhullar, Navreet K

    2013-01-01

    Nearly one-third of the world population, mostly women and children, suffer from iron malnutrition and its consequences, such as anemia or impaired mental development. Biofortification of rice, which is a staple crop for nearly half of the world's population, can significantly contribute in alleviating iron deficiency. NFP rice (transgenic rice expressing nicotianamine synthase, ferritin and phytase genes) has a more than six-fold increase in iron content in polished rice grains, resulting from the synergistic action of nicotianamine synthase (NAS) and ferritin transgenes. We investigated iron homeostasis in NFP plants by analyzing the expression of 28 endogenous rice genes known to be involved in the homeostasis of iron and other metals, in iron-deficient and iron-sufficient conditions. RNA was collected from different tissues (roots, flag leaves, grains) and at three developmental stages during grain filling. NFP plants showed increased sensitivity to iron-deficiency conditions and changes in the expression of endogenous genes involved in nicotianamine (NA) metabolism, in comparison to their non-transgenic siblings (NTS). Elevated transcript levels were detected in NFP plants for several iron transporters. In contrast, expression of OsYSL2, which encodes a member of yellow stripe like protein family, and a transporter of the NA-Fe(II) complex was reduced in NFP plants under low iron conditions, indicating that expression of OsYSL2 is regulated by the endogenous iron status. Expression of the transgenes did not significantly affect overall iron homeostasis in NFP plants, which establishes the engineered push-pull mechanism as a suitable strategy to increase rice endosperm iron content.

  20. Nicotianamine synthase overexpression positively modulates iron homeostasis-related genes in high iron rice

    Directory of Open Access Journals (Sweden)

    Meng eWang

    2013-05-01

    Full Text Available Nearly one-third of the world population, mostly women and children, suffer from iron malnutrition and its consequences, such as anemia or impaired mental development. Biofortification of rice, which is a staple crop for nearly half of the world’s population, can significantly contribute in alleviating iron deficiency. NFP rice (transgenic rice expressing nicotianamine synthase, ferritin and phytase genes has a more than six-fold increase in iron content in polished rice grains, resulting from the synergistic action of nicotianamine synthase (NAS and ferritin transgenes. We investigated iron homeostasis in NFP plants by analyzing the expression of 28 endogenous rice genes known to be involved in the homeostasis of iron and other metals, in iron-deficient and iron-sufficient conditions. RNA was collected from different tissues (roots, flag leaves, grains and at three developmental stages during grain filling. NFP plants showed increased sensitivity to iron-deficiency conditions and changes in the expression of endogenous genes involved in nicotianamine (NA metabolism, in comparison to their non-transgenic siblings. Elevated transcript levels were detected in NFP plants for several iron transporters. In contrast, expression of OsYSL2, which encodes a member of Yellow Stripe-like protein family, and a transporter of the NA-Fe(II complex was reduced in NFP plants under low iron conditions, indicating that expression of OsYSL2 is regulated by the endogenous iron status. Expression of the transgenes did not significantly affect overall iron homeostasis in NFP plants, which establishes the engineered push-pull mechanism as a suitable strategy to increase rice endosperm iron content.

  1. Distribution of apolipoprotein E gene polymorphism in students and in high-educated elderly from Serbia

    Directory of Open Access Journals (Sweden)

    Maksimović Nela

    2013-01-01

    Full Text Available Apolipoprotein E (ApoE play important role in lipid metabolism and in processes of remodeling and reparation in central nervous system. Three common ApoE isoforms, ApoE2, ApoE3 and ApoE4, show strong genetic determination by ε2, ε3, and ε4 allele. In human genome gene encoding Apolipoprotein E (APOE is located on cromosome 19, and ε2/ε3/ε4 haplotype system is defined by 2 non-synonymous single nucleotide polymorphisms (SNPs in the APOE exon 4. The frequency of the three APOE alleles and corresponding genotypes varies across human populations, with possible clinical implications. At least, variable distribution of ε4 allele may contribute to the regional risk of cardiovascular and Alzheimer’s diseases. Allele-frequency comparisons between younger and older populations suggest an effect of APOE on mortality, but these data are not consistently confirmed. In the present study we have analyzed the distribution of APOE gene polymorphism in a group of University students and retained University professors living in Serbia. After DNA extraction from peripheral blood samples, the APOE genotype was determined by polymerase chain reaction (PCR followed with HhaI restriction digestion. We found no statistically significant difference in alleles and genotypes distribution between younger and elder group of participants. Also, there was no significant difference compared to APOE data previously obtained in YUSAD cohort of healthy school children (15 y of age from different regions of Serbia. In both of our groups, as well as in YUSAD cohort, frequency of APOE ε4 allele was <10%. The observed frequencies are lower than in neighboring countries, but similar with Spanish data and some Asian populations. Our results do not support important role of APOE ε4 in the morbidity and mortality in Serbian population, but gene-environmental-social interactions should be considered. [Projekat Ministarstva nauke Republike Srbije, br. ON175091

  2. Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3 in Thai malignant lymphoma by High Resolution Melting curve analysis

    Directory of Open Access Journals (Sweden)

    Pongpruttipan Tawatchai

    2010-05-01

    Full Text Available Abstract Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Detection of antigen receptor gene rearrangement including T cell receptor (TCR and immunoglobulin heavy chain (IgH by polymerase chain reaction followed by heteroduplex has currently become standard whereas fluorescent fragment analysis (GeneScan has been used for confirmation test. In this study, three techniques had been compared: thermocycler polymerase chain reaction (PCR followed by heteroduplex and polyacrylamide gel electrophoresis, GeneScan analysis, and real time PCR with High Resolution Melting curve analysis (HRM. The comparison was carried out with DNA extracted from paraffin embedded tissues diagnosed as B- cell non-Hodgkin lymphoma. Specific PCR primers sequences for IgH gene variable region 3, including fluorescence labeled IgH primers were used and results were compared with HRM. In conclusion, the detection IgH gene rearrangement by HRM in the LightCycler System showed potential for distinguishing monoclonality from polyclonality in B-cell non-Hodgkin lymphoma. Introduction Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The incidence rate as reported by Ministry of Public Health is 3.1 per 100,000 population in female whereas the rate in male is 4.5 per 100,000 population 1. At Siriraj Hospital, the new cases diagnosed as malignant lymphoma were 214.6 cases/year 2. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Therefore, detection of antigen receptor gene rearrangement including T cell receptor (TCR and immunoglobulin heavy chain (IgH by polymerase chain reaction (PCR assay has recently become a standard laboratory test for discrimination of reactive from malignant clonal

  3. High Throughput Synthesis and Screening for Agents Inhibiting Androgen Receptor Mediated Gene Transcription

    Science.gov (United States)

    2005-02-01

    molecule Soc. 2002, 124, 544. agonists. Bioorg. Med. Chem. 2001, 9, 557. 33. Berg , T.; Cohen, S. B.; Desharnais, J.; Sonderegger, C.; 18. Chen, Y.; Bilban...Kaldor, M. G. Siegel, Cure. Opin. C/tern. Biol. 1997, 1, [80] T. Berg , S. B. Cohen, J. Desharnais, C. Sonderegger, D. J. 101-106. Maslyar, J. Goldberg...251, 1211 [322] l. A. Wilson, L K. Joliffe, Curr. Opin. Struct. Biol. 1999,9,696- 1217. 702 [289] E. M. Blackwood, B. Luscher , R. N. Eisenman, Genes

  4. GeoChip-based insights into the microbial functional gene repertoire of marine sponges (high microbial abundance, low microbial abundance) and seawater

    KAUST Repository

    Bayer, Kristina

    2015-01-08

    The GeoChip 4.2 gene array was employed to interrogate the microbial functional gene repertoire of sponges and seawater collected from the Red Sea and the Mediterranean. Complementary amplicon sequencing confirmed the microbial community composition characteristic of high microbial abundance (HMA) and low microbial abundance (LMA) sponges. By use of GeoChip, altogether 20 273 probes encoding for 627 functional genes and representing 16 gene categories were identified. Minimum curvilinear embedding analyses revealed a clear separation between the samples. The HMA/LMA dichotomy was stronger than any possible geographic pattern, which is shown here for the first time on the level of functional genes. However, upon inspection of individual genes, very few specific differences were discernible. Differences were related to microbial ammonia oxidation, ammonification, and archaeal autotrophic carbon fixation (higher gene abundance in sponges over seawater) as well as denitrification and radiation-stress-related genes (lower gene abundance in sponges over seawater). Except for few documented specific differences the functional gene repertoire between the different sources appeared largely similar. This study expands previous reports in that functional gene convergence is not only reported between HMA and LMA sponges but also between sponges and seawater.

  5. A 454 multiplex sequencing method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies

    Directory of Open Access Journals (Sweden)

    Charbonnel Nathalie

    2010-05-01

    Full Text Available Abstract Background High-throughput sequencing technologies offer new perspectives for biomedical, agronomical and evolutionary research. Promising progresses now concern the application of these technologies to large-scale studies of genetic variation. Such studies require the genotyping of high numbers of samples. This is theoretically possible using 454 pyrosequencing, which generates billions of base pairs of sequence data. However several challenges arise: first in the attribution of each read produced to its original sample, and second, in bioinformatic analyses to distinguish true from artifactual sequence variation. This pilot study proposes a new application for the 454 GS FLX platform, allowing the individual genotyping of thousands of samples in one run. A probabilistic model has been developed to demonstrate the reliability of this method. Results DNA amplicons from 1,710 rodent samples were individually barcoded using a combination of tags located in forward and reverse primers. Amplicons consisted in 222 bp fragments corresponding to DRB exon 2, a highly polymorphic gene in mammals. A total of 221,789 reads were obtained, of which 153,349 were finally assigned to original samples. Rules based on a probabilistic model and a four-step procedure, were developed to validate sequences and provide a confidence level for each genotype. The method gave promising results, with the genotyping of DRB exon 2 sequences for 1,407 samples from 24 different rodent species and the sequencing of 392 variants in one half of a 454 run. Using replicates, we estimated that the reproducibility of genotyping reached 95%. Conclusions This new approach is a promising alternative to classical methods involving electrophoresis-based techniques for variant separation and cloning-sequencing for sequence determination. The 454 system is less costly and time consuming and may enhance the reliability of genotypes obtained when high numbers of samples are studied

  6. Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages

    KAUST Repository

    Lescot, Magali

    2015-11-27

    Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage.

  7. Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages.

    Science.gov (United States)

    Lescot, Magali; Hingamp, Pascal; Kojima, Kenji K; Villar, Emilie; Romac, Sarah; Veluchamy, Alaguraj; Boccara, Martine; Jaillon, Olivier; Iudicone, Daniele; Bowler, Chris; Wincker, Patrick; Claverie, Jean-Michel; Ogata, Hiroyuki

    2016-05-01

    Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage.

  8. High resolution genetic map of the adenomatous polyposis coli gene (APC) region

    Energy Technology Data Exchange (ETDEWEB)

    Olschwang, S.; Laurent-Puig, P.; Melot, T. [Institut Curie, Paris (France)

    1995-05-08

    Familial adenomatous polyposis coli (APC) is a dominantly inherited colorectal cancer susceptibility disease caused by mutation in a gene called APC located on chromosome 5q21. Presymptomatic diagnosis of this condition is recommended because it enables restriction of the efficient but demanding prevention program to those relatives that are genetically affected. The large size of the APC gene makes the direct search for the casual alteration difficult to implement in routine diagnostic laboratories. Because APC appears to be genetically homogeneous with alteration in a single locus causing the disease, cosegregation analysis may represent an alternative efficient method for presymptomatic diagnosis. However, the reliability of the risk estimation by linkage analysis in APC families is hampered by the lack of a short range genetic map of the APC locus. A combined approach including genotyping of 65 APC families, analysis of the CEPH database, and complementary typing of both APC and CEPH families has made it possible to derive the following genetic map: Centromere-[D5S82-D5S49]-0.02-D5S122-0.01-D5S136-0.01-D5S135-0.02-[APC-D5S346-MCC]-0.04-[D5S81-D5S64]-Telomere. This order, which differs from previously proposed genetic maps, is fully compatible with recent physical mapping data. These data should contribute to increase the reliability of the presymptomatic test for APC. 42 refs., 1 fig., 3 tabs.

  9. Hypermethylation Status of E-Cadherin Gene in Gastric Cancer Patients in a High Incidence Area.

    Science.gov (United States)

    Rashid, Haroon; Alam, Khursheed; Afroze, Dil; Yousuf, Adfar; Banday, Manzoor; Kawoosa, Fizalah

    2016-01-01

    Gastric cancer (GC) is the fourth most prevalant cancer and the second leading cause of cancer-related mortality worldwide. As in other cancers gastric carcinogenesis is multifactorial involving environmental, genetic and epigenetic components. Epigenetic silencing due to hypermethylation of tumour suppressor genes is one of the key events in gastric carcinogenesis. This study was aimed to analyse the hypermethylation status of the E-Cadherin (CDH1) gene promoter in GCs in the ethnic Kashmiri population. In this study a total of 80 GC patients were recruited. Hypermethylation in tumour tissue was detected by methylation specific PCR (MS-PCR). Hypermethylation of CDH1 promoter was observed in 52 (65%) of gastric carcinoma cases which was significantly much higher than adjacent normal tissue [p≤0.0001]. Further the frequency of CDH1 promoter methylation was significantly different with intestinal and diffuse types of gastric cancer [55.7% vs 82.1%; CDH1 hypermethylation [P≤0.05]. Thus the current data indicate a vital role of epigenetic alteration of CDH1 in the causation and development of gastric cancer, particularly of diffuse type, in our population.

  10. Altruism can proliferate through population viscosity despite high random gene flow.

    Directory of Open Access Journals (Sweden)

    Roberto H Schonmann

    Full Text Available The ways in which natural selection can allow the proliferation of cooperative behavior have long been seen as a central problem in evolutionary biology. Most of the literature has focused on interactions between pairs of individuals and on linear public goods games. This emphasis has led to the conclusion that even modest levels of migration would pose a serious problem to the spread of altruism through population viscosity in group structured populations. Here we challenge this conclusion, by analyzing evolution in a framework which allows for complex group interactions and random migration among groups. We conclude that contingent forms of strong altruism that benefits equally all group members, regardless of kinship and without greenbeard effects, can spread when rare under realistic group sizes and levels of migration, due to the assortment of genes resulting only from population viscosity. Our analysis combines group-centric and gene-centric perspectives, allows for arbitrary strength of selection, and leads to extensions of Hamilton's rule for the spread of altruistic alleles, applicable under broad conditions.

  11. Genetic Analysis and Preliminary Mapping of a Highly Male-Sterile Gene in Foxtail Millet (Setaria italica L. Beauv.) Using SSR Markers

    Institute of Scientific and Technical Information of China (English)

    WANG Jun; DIAO Xian-min; GUO Ping-yi; WANG Zhi-lan; YANG Hui-qing; YUAN Feng; GUO Er-hu; TIAN Gang; AN Yuan-huai; LI Hui-xia; WANG Yu-wen

    2013-01-01

    Breeding of male-sterile lines has become the mainstream for the heterosis utilization in foxtail millet, but the genetic basis of most male-sterile lines used for the hybrid is still an area to be elucidated. In this study, a highly male-sterile line Gao146A was investigated. Genetic analysis indicated that the highly male-sterile phenotype was controlled by a single recessive gene a single recessive gene. Using F2 population derived from cross Gao146A/K103, one gene controlling the highly male-sterility, tentatively named asms1, which linked to SSR marker b234 with genetic distance of 16.7 cM, was mapped on the chromosome VI. These results not only laid the foundation for ifne mapping of this highly male-sterile gene, but also helped to accelerate the improvement of highly male-sterile lines by using molecular marker assisted breeding method.

  12. Bacterial community structure in High-Arctic snow and freshwater as revealed by pyrosequencing of 16S rRNA genes and cultivation

    DEFF Research Database (Denmark)

    Møller, Annette K.; Søborg, Ditte A.; Abu Al-Soud, Waleed;

    2013-01-01

    The bacterial community structures in High-Arctic snow over sea ice and an ice-covered freshwater lake were examined by pyrosequencing of 16S rRNA genes and 16S rRNA gene sequencing of cultivated isolates. Both the pyrosequence and cultivation data indicated that the phylogenetic composition...

  13. High-level, erythroid specific, expression of the human α-globin gene in transgenic mice and the production of human haemoglobin in murine erythrocytes.

    NARCIS (Netherlands)

    O. Hanscombe (Olivia); M. Vidal; J. Kaeda; L. Luzzatto; D.R. Greaves (David); F.G. Grosveld (Frank)

    1989-01-01

    textabstractUsing the dominant control region (DCR) sequences that flank the beta-globin gene locus, we have been able to achieve high-level expression of the human alpha-globin gene in transgenic mice. Expression in fetal liver and blood is copy number dependent and at levels comparable to that of

  14. D-Xylose suppresses adipogenesis and regulates lipid metabolism genes in high-fat diet-induced obese mice.

    Science.gov (United States)

    Lim, Eunjin; Lim, Ji Ye; Shin, Jae-Ho; Seok, Pu Reum; Jung, Sangwon; Yoo, Sang-Ho; Kim, Yuri

    2015-07-01

    D-Xylose, a natural pentose, has been reported to reduce postprandial glucose levels, although its effect on lipid metabolism has not been investigated. Therefore, this study hypothesized that d-xylose, as an alternative sweetener, suppresses adipogenesis and lipid metabolism by regulating blood lipid profiles, blood glucose levels, and related gene expression in high-fat diet (HFD)-induced obese mice. Mice were fed a normal diet, a 60% HFD diet, or an HFD with 5% or 10% of the total sucrose content supplemented with d-xylose (Xylo 5 and Xylo 10 diets, respectively). Weight gain, food intake, and serum lipid levels for each group were measured. After 12 weeks, histopathology of liver sections and assays of gene expression related to adipogenesis and lipid metabolism in visceral fat and liver tissues were analyzed. Body weight gain; fasting blood glucose levels; weights of subcutaneous and visceral adipose tissues; and serum biochemical markers, including total cholesterol and low-density lipoprotein cholesterol, low-/high-density lipoprotein, and total cholesterol/high-density lipoprotein, were significantly lowered in the Xylo 5 and Xylo 10 groups. In addition, d-xylose supplementation resulted in the down-regulation of adipogenesis-related genes, including sterol regulatory element-binding protein 1C, fatty acid synthase, adipocyte protein 2, and CCAAT/enhancer-binding protein α in visceral adipose tissues. Histopathologically, Xylo 5 and Xylo 10 supplementation reduced HFD-induced fat accumulation in the liver and decreased expressions of fatty acid synthase and peroxisome proliferator-activated receptor γ. D-Xylose supplementation also enhanced lipid oxidation by increasing expressions of carnitine palmitoyltransferase 1A; cytochrome P450, family 4, subfamily a, polypeptide 10; and acyl-CoA oxidase. In conclusion, our finding suggests that d-xylose may help prevent or attenuate the progression of obesity-related metabolic disorders by alleviating adipogenesis

  15. RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond

    Science.gov (United States)

    Gama-Castro, Socorro; Salgado, Heladia; Santos-Zavaleta, Alberto; Ledezma-Tejeida, Daniela; Muñiz-Rascado, Luis; García-Sotelo, Jair Santiago; Alquicira-Hernández, Kevin; Martínez-Flores, Irma; Pannier, Lucia; Castro-Mondragón, Jaime Abraham; Medina-Rivera, Alejandra; Solano-Lira, Hilda; Bonavides-Martínez, César; Pérez-Rueda, Ernesto; Alquicira-Hernández, Shirley; Porrón-Sotelo, Liliana; López-Fuentes, Alejandra; Hernández-Koutoucheva, Anastasia; Moral-Chávez, Víctor Del; Rinaldi, Fabio; Collado-Vides, Julio

    2016-01-01

    RegulonDB (http://regulondb.ccg.unam.mx) is one of the most useful and important resources on bacterial gene regulation,as it integrates the scattered scientific knowledge of the best-characterized organism, Escherichia coli K-12, in a database that organizes large amounts of data. Its electronic format enables researchers to compare their results with the legacy of previous knowledge and supports bioinformatics tools and model building. Here, we summarize our progress with RegulonDB since our last Nucleic Acids Research publication describing RegulonDB, in 2013. In addition to maintaining curation up-to-date, we report a collection of 232 interactions with small RNAs affecting 192 genes, and the complete repertoire of 189 Elementary Genetic Sensory-Response units (GENSOR units), integrating the signal, regulatory interactions, and metabolic pathways they govern. These additions represent major progress to a higher level of understanding of regulated processes. We have updated the computationally predicted transcription factors, which total 304 (184 with experimental evidence and 120 from computational predictions); we updated our position-weight matrices and have included tools for clustering them in evolutionary families. We describe our semiautomatic strategy to accelerate curation, including datasets from high-throughput experiments, a novel coexpression distance to search for ‘neighborhood’ genes to known operons and regulons, and computational developments. PMID:26527724

  16. High-throughput sequencing identification of genes involved with Varroa destructor resistance in the eastern honeybee, Apis cerana.

    Science.gov (United States)

    Ji, T; Yin, L; Liu, Z; Shen, F; Shen, J

    2014-10-31

    Varroa destructor is the greatest threat to the honeybee Apis mellifera worldwide, while it rarely causes serious harm to its native host, the Eastern honeybee Apis cerana. The genetic mechanisms underlying the resistance of A. cerana to Varroa remain unclear. Thus, understanding the molecular mechanism of resistance to Varroa may provide useful insights for reducing this disease in other organisms. In this study, the transcriptomes of two A. cerana colonies were sequenced using the Illumina Solexa sequencing method. One colony was highly affected by mites, whereas the other colony displayed strong resistance to V. destructor. We determined differences in gene expression in the two colonies after challenging the colonies with V. destructor. After de novo transcriptome assembly, we obtained 91,172 unigenes for A. cerana and found that 288 differentially expressed genes varied by more than 15-fold. A total of 277 unigenes were present at higher levels in the non-affected colony. Genes involved in resistance to Varroa included unigenes related to skeletal muscle movement, olfactory sensitivity, and transcription factors. This suggests that hygienic behavior and grooming behavior may play important roles in the resistance to Varroa.

  17. A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes.

    Science.gov (United States)

    Schmid-Burgk, Jonathan L; Schmidt, Tobias; Kaiser, Vera; Höning, Klara; Hornung, Veit

    2013-01-01

    Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DNA overhangs suitable for LIC. Assembly of TAL effector arrays requires only the combinatorial mixing of fluids and has exceptional fidelity. TAL effector nucleases (TALENs) produced by this method had high genome-editing activity at endogenous loci in HEK 293T cells (64% were active). To maximize throughput, we generated a comprehensive 5-mer TAL effector repeat unit fragment library that allows automated assembly of >600 TALEN genes in a single day. Given its simplicity, throughput and fidelity, LIC assembly will permit the generation of TAL effector gene libraries for large-scale functional genomics studies.

  18. Directed engineering of a high-expression chimeric transgene as a strategy for gene therapy of hemophilia A.

    Science.gov (United States)

    Doering, Christopher B; Denning, Gabriela; Dooriss, Kerry; Gangadharan, Bagirath; Johnston, Jennifer M; Kerstann, Keith W; McCarty, David A; Spencer, H Trent

    2009-07-01

    Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

  19. Population genetic studies revealed local adaptation in a high gene-flow marine fish, the small yellow croaker (Larimichthys polyactis.

    Directory of Open Access Journals (Sweden)

    Le Wang

    Full Text Available The genetic differentiation of many marine fish species is low. Yet local adaptation may be common in marine fish species as the vast and changing marine environment provides more chances for natural selection. Here, we used anonymous as well as known protein gene linked microsatellites and mitochondrial DNA to detect the population structure of the small yellow croaker (Larimichthys polyactis in the Northwest Pacific marginal seas. Among these loci, we detected at least two microsatellites, anonymous H16 and HSP27 to be clearly under diversifying selection in outlier tests. Sequence cloning and analysis revealed that H16 was located in the intron of BAHCC1 gene. Landscape genetic analysis showed that H16 mutations were significantly associated with temperature, which further supported the diversifying selection at this locus. These marker types presented different patterns of population structure: (i mitochondrial DNA phylogeny showed no evidence of genetic divergence and demonstrated only one glacial linage; (ii population differentiation using putatively neutral microsatellites presented a pattern of high gene flow in the L. polyactis. In addition, several genetic barriers were identified; (iii the population differentiation pattern revealed by loci under diversifying selection was rather different from that revealed by putatively neutral loci. The results above suggest local adaptation in the small yellow croaker. In summary, population genetic studies based on different marker types disentangle the effects of demographic history, migration, genetic drift and local adaptation on population structure and also provide valuable new insights for the design of management strategies in L. polyactis.

  20. A high-resolution linkage map of the citrus tristeza virus resistance gene region in Poncirus trifoliata (L.) Raf.

    Science.gov (United States)

    Fang, D Q; Federici, C T; Roose, M L

    1998-10-01

    Resistance to citrus tristeza virus (CTV) was evaluated in 554 progeny of 10 populations derived from Poncirus trifoliata. A dominant gene (Ctv) controlled CTV resistance in P. trifoliata. Twenty-one dominant PCR-based DNA markers were identified as linked to Ctv by bulked segregant analysis. Of the 11 closest markers to Ctv, only 2 segregated in all populations. Ten of these markers were cloned and sequenced, and codominant RFLP markers were developed. Seven RFLP markers were then evaluated in 10 populations. Marker orders were consistent in all linkage maps based on data of single populations or on combined data of populations with similar segregation patterns. In a consensus map, the six closest marker loci spanned 5.3 cM of the Ctv region. Z16 cosegregated with Ctv. C19 and AD08 flanked Ctv at distances of 0.5 and 0.8 cM, respectively. These 3 markers were present as single copies in the Poncirus genome, and could be used directly for bacterial artificial chromosome library screening to initiate a walk toward Ctv. BLAST searches of the GenBank database revealed high sequence similarities between 2 markers and known plant disease resistance genes, indicating that a resistance gene cluster exists in the Ctv region in P. trifoliata.

  1. Isoflavone Regulates Lipid Metabolism via Expression of Related Genes in OVX Rats Fed on a High-fat Diet

    Institute of Scientific and Technical Information of China (English)

    XIAO-LIN NA; JUNKO EZAKI; FUMIE SUGIYAMA; HONG-BIN CUI; YOSHIKO ISHIMI

    2008-01-01

    Objective To investigate the effects of isoflavone on body weight, fat mass, and gene expression in relation to lipid metabolism. Methods Thirty-six female SD rats were variectomized or sham-operated and fed on a high-fat diet. Two months later, abdominal incision was made, blood was collected to separate serum, and the liver and adipose tissue were immediately collected and weighed. Some portions of these tissues were frozen in liquid nitrogen and stored at -80℃. Results Ovariectomy (OVX) with a high-fat diet could induce obesity in rats, while treatment with isoflavone significantly inhibited the increase in body weight and fat mass in abdomen. Serum total cholesterol and leptin were significantly decreased in isoflavone group, compared with the OVX group. The mRNA expression of liver fatty acid synthase (FAS) in the OVX group was significantly higher than that in sham-operated group, while this difference was not observed in the isoflavone group. The mRNA expression of liver hormone-sensitive lipase (HSL) in the OVX rats tended to be lower than that in the sham-operated rats. Furthermore, a large amount of isoflavone maintained the mRNA expression at a sham level. Conclusion lsoflavone may prevent obesity induced by ovariectomy with a high-fat diet, in part by modulating gene expression related to lipid metabolism.

  2. Gene expression profiling by high throughput sequencing to determine signatures for the bovine receptive uterus at early gestation

    Directory of Open Access Journals (Sweden)

    Veerle Van Hoeck

    2015-09-01

    Full Text Available The uterus plays a central role among the reproductive tissues in the context of early embryo-maternal communication and a successful pregnancy depends on a complex series of endometrial molecular and cellular events. The factors responsible for the initial interaction between maternal and embryonic tissues, leading to the establishment of pregnancy, remain poorly understood. In this context, Illumina's next-generation sequencing technology has been used to discover the uterine transcriptome signature that is favourable for ongoing pregnancy. More specifically, the present report documents on a retrospective in vivo study in which data on pregnancy outcome were linked to uterine gene expression signatures on day 6 (bovine model. Using the RNA-Seq method, 14.654 reference genes were effectively analysed for differential expression between pregnant and non-pregnant uterine tissue. Transcriptome data revealed that 216 genes were differently expressed when comparing uterine tissue from pregnant and non-pregnant cows. All read sequences were deposited in the Sequence Read Archive (SRA of the NCBI (http://www.ncbi.nlm.nih.gov/sra. An overview of the gene expression data has been deposited in NCBI's Gene Expression Omnibus (GEO and is accessible through GEO Series accession number GSE65117. This allows the research community to enhance reproducibility and allows for new discoveries by comparing datasets of signatures linked to receptivity and/or pregnancy success. The resulting information can serve as tool to identify valuable and urgently needed biomarkers for scoring maternal receptivity and even for accurate detection of early pregnancy, which is a matter of cross-species interest. Beyond gene expression analysis as a marker tool, the RNA-Seq information on pregnant uterine tissue can be used to gain novel mechanistic insights, such as by identifying alternative splicing events, allele-specific expression, and rare and novel transcripts that might

  3. Transfer of Bt-toxin protein gene into maize by high-velocity microprojectile bombardments and regeneration of transgenic plants

    Institute of Scientific and Technical Information of China (English)

    王国英; 杜天兵; 张宏; 谢友菊; 戴景瑞; 米景九; 李太源; 田颖川; 乔利亚; 莽克强

    1995-01-01

    Bt-toxin protein gene was successfully transferred into maize by the microprojectile bombard-ments of cell suspension,embryogenic calli and immature embryos with a Chinese-made particle gun(JQ-700).Although the bombarded embryogenic calli and immature embryos produced less mean transformants per dishthan the cell suspensions,they were the suitable materials for maize transformation because their culture andregeneration have been achieved in most maize cultivars.The evaluation on the resistance of transgenic plantsto corn borer shows the significant difference between them,from highly resistant to susceptible.

  4. O 6 -methylguanine DNA methyltransferase gene promoter methylation in high-grade gliomas: A review of current status

    Directory of Open Access Journals (Sweden)

    Vaishali Suri

    2011-01-01

    Full Text Available Assessment of promoter methylation of the O 6 -methylguanine DNA methyltransferase (MGMT gene has recently gained importance in molecular profiling of high-grade gliomas. It has emerged not only as an important prognostic marker but also as a predictive marker for response to temozolomide in patients with newly diagnosed glioblastoma. Further, recent studies indicate that MGMT promoter methylation has strong prognostic relevance even in anaplastic (grade III gliomas, irrespective of therapy (chemotherapy or radiotherapy. This article provides an overview of its use as a predictive and prognostic biomarker, as well as the methods employed for its assessment and use in therapeutic decision making.

  5. Highly luminescent and cytocompatible cationic Ag2S NIR-emitting quantum dots for optical imaging and gene transfection

    OpenAIRE

    Duman, Fatma Demir; Hocaoğlu, İbrahim; Hocaoglu, Ibrahim; Öztürk, Deniz Gülfem; Ozturk, Deniz Gulfem; Gözüaçık, Devrim; Gozuacik, Devrim; Kiraz, Alper; Yağcı Acar, Havva; Yagci Acar, Havva

    2015-01-01

    Nanoscale PAPER Cite this: Nanoscale, 2015, 7, 11352 Received 12th January 2015, Accepted 23rd May 2015 DOI: 10.1039/c5nr00189g www.rsc.org/nanoscale Highly luminescent and cytocompatible cationic Ag2S NIR-emitting quantum dots for optical imaging and gene transfection† Fatma Demir Duman,a Ibrahim Hocaoglu,a Deniz Gulfem Ozturk,b Devrim Gozuacik,b Alper Kiraza,c and Havva Yagci Acar*a,d,e The development of non-toxic theranostic nanoparticles capable of del...

  6. Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

    Science.gov (United States)

    Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

    2015-02-01

    Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line.

  7. piggyBac as a high-capacity transgenesis and gene-therapy vector in human cells and mice

    OpenAIRE

    Rongbo Li; Yuan Zhuang; Min Han; Tian Xu; Xiaohui Wu

    2013-01-01

    SUMMARY The stable genomic integration and expression of a large transgene is a major hurdle in gene therapy. We show that the modified piggyBac (PB) transposon system can be used to introduce a 207 kb genomic DNA fragment containing the RORγ/γt locus into human cells and mice. PB-mediated transgenesis results in a single copy of a stably inherited and expressed transgene. These results indicate that PB could serve as an effective high-capacity vector for functional analysis of the mammalian ...

  8. High Genetic Differentiation among European White Oak Species (Quercus spp. at a Dehydrin Gene

    Directory of Open Access Journals (Sweden)

    Iacob CRĂCIUNESC

    2015-12-01

    Full Text Available Dehydryn genes are involved in plant response to environmental stress and may be useful to examine functional diversity in relation to adaptive variation. Recently, a dehydrin gene (DHN3 was isolated in Quercus petraea and showed little differentiation between populations of the same species in an altitudinal transect. In the present study, inter- and intraspecific differentiation patterns in closely related and interfertile oaks were investigated for the first time at the DHN3 locus. A four-oak-species stand (Quercus frainetto Ten., Q. petraea (Matt. Liebl., Q. pubescens Willd., Q. robur L. and two populations for each of five white oak species (Q. frainetto Ten., Q. petraea (Matt. Liebl., Q. pubescens Willd., Q. robur L. and Q. pedunculiflora K. Koch were analyzed. Three alleles shared by all five oak species were observed. However, only two alleles were present in each population, but with different frequencies according to the species. At population level, all interspecific pairs of populations showed significant differentiation, except for pure Q. robur and Q. pedunculiflora populations. In contrast, no significant differentiation (p > 0.05 was found among conspecific populations. The DHN3 locus proved to be very useful to differentiate Q. frainetto and Q. pubescens from Q. pedunculiflora (FST = 0.914 and 0.660, respectively and Q. robur (FST = 0.858 and 0.633, respectively. As expected, the lowest level of differentiation was detected between the most closely related species, Q. robur and Q. pedunculiflora (FST = 0.020. Our results suggest that DHN3 can be an important genetic marker for differentiating among European white oak species.

  9. Differential epigenetic regulation of TOX subfamily high mobility group box genes in lung and breast cancers.

    Directory of Open Access Journals (Sweden)

    Mathewos Tessema

    Full Text Available Aberrant cytosine methylation affects regulation of hundreds of genes during cancer development. In this study, a novel aberrantly hypermethylated CpG island in cancer was discovered within the TOX2 promoter. TOX2 was unmethylated in normal cells but 28% lung (n = 190 and 23% breast (n = 80 tumors were methylated. Expression of two novel TOX2 transcripts identified was significantly reduced in primary lung tumors than distant normal lung (p<0.05. These transcripts were silenced in methylated lung and breast cancer cells and 5-Aza-2-deoxycytidine treatment re-expressed both. Extension of these assays to TOX, TOX3, and TOX4 genes that share similar genomic structure and protein homology with TOX2 revealed distinct methylation profiles by smoking status, histology, and cancer type. TOX was almost exclusively methylated in breast (43% than lung (5% cancer, whereas TOX3 was frequently methylated in lung (58% than breast (30% tumors. TOX4 was unmethylated in all samples and showed the highest expression in normal lung. Compared to TOX4, expression of TOX, TOX2 and TOX3 in normal lung was 25, 44, and 88% lower, respectively, supporting the premise that reduced promoter activity confers increased susceptibility to methylation during lung carcinogenesis. Genome-wide assays revealed that siRNA-mediated TOX2 knockdown modulated multiple pathways while TOX3 inactivation targeted neuronal development and function. Although these knockdowns did not result in further phenotypic changes of lung cancer cells in vitro, the impact on tissue remodeling, inflammatory response, and cell differentiation pathways suggest a potential role for TOX2 in modulating tumor microenvironment.

  10. High prevalence of BRAF gene mutation in papillary thyroid carcinomas and thyroid tumor cell lines.

    Science.gov (United States)

    Xu, Xiulong; Quiros, Roderick M; Gattuso, Paolo; Ain, Kenneth B; Prinz, Richard A

    2003-08-01

    The RAS-RAF-MEK-ERK-MAP kinase pathway mediates the cellular response to extracellular signals that regulate cell proliferation, differentiation, and apoptosis. Mutation of the RAS proto-oncogene occurs in various thyroid neoplasms such as papillary thyroid carcinomas (PTCs), follicular thyroid adenomas and carcinomas. A second genetic alteration frequently involved in PTC is RET/PTC rearrangements. Recent studies have shown that BRAF, which is a downstream signaling molecule of RET and RAS, is frequently mutated in melanomas. This study tests whether BRAF is also mutated in thyroid tumors and cell lines. We analyzed BRAF gene mutation at codon 599 in thyroid tumors using mutant-allele-specific PCR and in 10 thyroid tumor cell lines by DNA sequencing of the PCR-amplified exon 15. We found that BRAF was mutated in 8 of 10 thyroid tumor cell lines, including 2 of 2 papillary carcinoma cell lines, 4 of 5 anaplastic carcinoma cell lines, 1 of 2 follicular carcinoma cell lines, and 1 follicular adenoma cell line. BRAF mutation at codon 599 was detected in 21 of 56 PTC (38%) but not in 18 follicular adenomas and 6 goiters. BRAF mutation occurred in PTC at a significantly higher frequency in male patients than in female patients. To test whether BRAF mutation may cooperate with RET/PTC rearrangements in the oncogenesis of PTC, we tested whether BRAF-mutated PTCs were also positive for RET/PTC rearrangements. Immunohistochemical staining was conducted to evaluate RET/PTC rearrangements by using two different anti-RET antibodies. Surprisingly, we found that a large number of BRAF-mutated PTCs (8 of 21) also expressed RET, indicating that the RET proto-oncogene is rearranged in these BRAF-mutated PTCs. These observations suggest that mutated BRAF gene may cooperate with RET/PTC to induce the oncogenesis of PTC.

  11. High-throughput sequencing of mGluR signaling pathway genes reveals enrichment of rare variants in autism.

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    Raymond J Kelleher

    Full Text Available Identification of common molecular pathways affected by genetic variation in autism is important for understanding disease pathogenesis and devising effective therapies. Here, we test the hypothesis that rare genetic variation in the metabotropic glutamate-receptor (mGluR signaling pathway contributes to autism susceptibility. Single-nucleotide variants in genes encoding components of the mGluR signaling pathway were identified by high-throughput multiplex sequencing of pooled samples from 290 non-syndromic autism cases and 300 ethnically matched controls on two independent next-generation platforms. This analysis revealed significant enrichment of rare functional variants in the mGluR pathway in autism cases. Higher burdens of rare, potentially deleterious variants were identified in autism cases for three pathway genes previously implicated in syndromic autism spectrum disorder, TSC1, TSC2, and SHANK3, suggesting that genetic variation in these genes also contributes to risk for non-syndromic autism. In addition, our analysis identified HOMER1, which encodes a postsynaptic density-localized scaffolding protein that interacts with Shank3 to regulate mGluR activity, as a novel autism-risk gene. Rare, potentially deleterious HOMER1 variants identified uniquely in the autism population affected functionally important protein regions or regulatory sequences and co-segregated closely with autism among children of affected families. We also identified rare ASD-associated coding variants predicted to have damaging effects on components of the Ras/MAPK cascade. Collectively, these findings suggest that altered signaling downstream of mGluRs contributes to the pathogenesis of non-syndromic autism.

  12. High temperature inhibits ascorbate recycling and light stimulation of the ascorbate pool in tomato despite increased expression of biosynthesis genes.

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    Capucine Massot

    Full Text Available Understanding how the fruit microclimate affects ascorbate (AsA biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31 °C and irradiance regimes (darkness or 150 µmol m(-2 s(-1. Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH. The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27 °C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12 °C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX. In contrast, at 31 °C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12 °C, light total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling.

  13. Characterization of the highly variable immune response gene family, He185/333, in the sea urchin, Heliocidaris erythrogramma.

    Science.gov (United States)

    Roth, Mattias O; Wilkins, Adam G; Cooke, Georgina M; Raftos, David A; Nair, Sham V

    2014-01-01

    This study characterizes the highly variable He185/333 genes, transcripts and proteins in coelomocytes of the sea urchin, Heliocidaris erythrogramma. Originally discovered in the purple sea urchin, Strongylocentrotus purpuratus, the products of this gene family participate in the anti-pathogen defenses of the host animals. Full-length He185/333 genes and transcripts are identified. Complete open reading frames of He185/333 homologues are analyzed as to their element structure, single nucleotide polymorphisms, indels and sequence repeats and are subjected to diversification analyses. The sequence elements that compose He185/333 are different to those identified for Sp185/333. Differences between Sp185/333 and He185/333 genes are also evident in the complexity of the sequences of the introns. He185/333 proteins show a diverse range of molecular weights on Western blots. The observed sizes and pIs of the proteins differ from predicted values, suggesting post-translational modifications and oligomerization. Immunofluorescence microscopy shows that He185/333 proteins are mainly located on the surface of coelomocyte subpopulations. Our data demonstrate that He185/333 bears the same substantial characteristics as their S. purpuratus homologues. However, we also identify several unique characteristics of He185/333 (such as novel element patterns, sequence repeats, distribution of positively-selected codons and introns), suggesting species-specific adaptations. All sequences in this publication have been submitted to Genbank (accession numbers JQ780171-JQ780321) and are listed in table S1.

  14. Characterization of the highly variable immune response gene family, He185/333, in the sea urchin, Heliocidaris erythrogramma.

    Directory of Open Access Journals (Sweden)

    Mattias O Roth

    Full Text Available This study characterizes the highly variable He185/333 genes, transcripts and proteins in coelomocytes of the sea urchin, Heliocidaris erythrogramma. Originally discovered in the purple sea urchin, Strongylocentrotus purpuratus, the products of this gene family participate in the anti-pathogen defenses of the host animals. Full-length He185/333 genes and transcripts are identified. Complete open reading frames of He185/333 homologues are analyzed as to their element structure, single nucleotide polymorphisms, indels and sequence repeats and are subjected to diversification analyses. The sequence elements that compose He185/333 are different to those identified for Sp185/333. Differences between Sp185/333 and He185/333 genes are also evident in the complexity of the sequences of the introns. He185/333 proteins show a diverse range of molecular weights on Western blots. The observed sizes and pIs of the proteins differ from predicted values, suggesting post-translational modifications and oligomerization. Immunofluorescence microscopy shows that He185/333 proteins are mainly located on the surface of coelomocyte subpopulations. Our data demonstrate that He185/333 bears the same substantial characteristics as their S. purpuratus homologues. However, we also identify several unique characteristics of He185/333 (such as novel element patterns, sequence repeats, distribution of positively-selected codons and introns, suggesting species-specific adaptations. All sequences in this publication have been submitted to Genbank (accession numbers JQ780171-JQ780321 and are listed in table S1.

  15. Full genotyping of a highly polymorphic human gene trait by time-resolved fluorescence resonance energy transfer.

    Directory of Open Access Journals (Sweden)

    Edoardo Totè

    Full Text Available The ability of detecting the subtle variations occurring, among different individuals, within specific DNA sequences encompassed in highly polymorphic genes discloses new applications in genomics and diagnostics. DQB1 is a gene of the HLA-II DQ locus of the Human Leukocyte Antigens (HLA system. The polymorphisms of the trait of the DQB1 gene including codons 52-57 modulate the susceptibility to a number of severe pathologies. Moreover, the donor-receiver tissue compatibility in bone marrow transplantations is routinely assessed through crossed genotyping of DQB and DQA. For the above reasons, the development of rapid, reliable and cost-effective typing technologies of DQB1 in general, and more specifically of the codons 52-57, is a relevant although challenging task. Quantitative assessment of the fluorescence resonance energy transfer (FRET efficiency between chromophores labelling the opposite ends of gene-specific oligonucleotide probes has proven to be a powerful tool to type DNA polymorphisms with single-nucleotide resolution. The FRET efficiency can be most conveniently quantified by applying a time-resolved fluorescence analysis methodology, i.e. time-correlated single-photon counting, which allows working on very diluted template specimens and in the presence of fluorescent contaminants. Here we present a full in-vitro characterization of the fluorescence responses of two probes when hybridized to oligonucleotide mixtures mimicking all the possible genotypes of the codons 52-57 trait of DQB1 (8 homozygous and 28 heterozygous. We show that each genotype can be effectively tagged by the combination of the fluorescence decay constants extrapolated from the data obtained with such probes.

  16. Genes associated to lactose metabolism illustrate the high diversity of Carnobacterium maltaromaticum

    DEFF Research Database (Denmark)

    Iskandar, Christelle F.; Cailliez-Grimal, Catherine; Rahman, Abdur

    2016-01-01

    The dairy population of Carnobacterium maltaromaticum is characterized by a high diversity suggesting a high diversity of the genetic traits linked to the dairy process. As lactose is the main carbon source in milk, the genetics of lactose metabolism was investigated in this LAB. Comparative...

  17. High-calorie diet exacerbates prostate neoplasia in mice with haploinsufficiency of Pten tumor suppressor gene

    Directory of Open Access Journals (Sweden)

    Jehnan Liu

    2015-03-01

    Conclusion: High-calorie diet promotes prostate cancer progression in the genetically susceptible Pten haploinsufficient mouse while preserving insulin sensitivity. This appears to be partly due to increased inflammatory response to high-caloric intake in addition to increased ability of insulin to promote lipogenesis.

  18. High-throughput identification of ionizing radiation-sensitive plant genes and development of radiation indicator plant and radiation sensing Genechip

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dong Sub; Kim, Jinbaek; Ha, Bokeun; Kim, Sang Hoon; Kim, Sunhee

    2013-05-15

    Physiological analysis of monocot model plant (rice) in response to ionizing radiation (cosmic-ray, gamma-ray, Ion beam). - Identification of antioxidant characters through cytochemical analysis. - Comparison of antioxidant activities in response to ionizing irradiation. - Evaluation of anthocyanin quantity in response to ionizing irradiation. Ionization energy response gene family analysis via bioinformatic validation. - Expression analysis of monocot and dicot gene families. - In silico and bioinformatic approach to elucidate gene function. Characterization and functional analysis of genes specifically expressed in response to ionizing irradiation (cosmic-ray, gamma-ray, Ion beam). - High throughput trancriptomic analysis of plants under ionizing radiation using microarray. - Promotor and cis-element analysis of genes specifically expressed in response to ionizing radiation. - Validation and function analysis of candidate genes. - Elucidation of plant mechanism of sensing and response to ionization energy. Development of bioindicator plants detecting ionization energy. - Cloning and identification of 'Radio marker genes (RMG)'. - Development of Over-expression (O/E) or Knock-out (K/O) plant using RMG. Development of Genechip as an ionization energy detector. - Expression profiling analysis of genes specifically expression in response to ionization energy. - Prepare high-conserved gene specific oligomer. - Development of ionization energy monitoring Genechip and application.

  19. Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction

    Science.gov (United States)

    Birla, Bhagyashree S.; Chou, Hui-Hsien

    2015-01-01

    Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly. PMID:26716828

  20. Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction.

    Directory of Open Access Journals (Sweden)

    Bhagyashree S Birla

    Full Text Available Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.

  1. Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction.

    Science.gov (United States)

    Birla, Bhagyashree S; Chou, Hui-Hsien

    2015-01-01

    Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.

  2. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth

    DEFF Research Database (Denmark)

    Jakočiūnė, Dzuiga; Herrero-Fresno, Ana; Jelsbak, Lotte;

    2016-01-01

    RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis...

  3. High-resolution linkage analyses to identify genes that influence Varroa sensitive hygiene behavior in honey bees.

    Science.gov (United States)

    Tsuruda, Jennifer M; Harris, Jeffrey W; Bourgeois, Lanie; Danka, Robert G; Hunt, Greg J

    2012-01-01

    Varroa mites (V. destructor) are a major threat to honey bees (Apis melilfera) and beekeeping worldwide and likely lead to colony decline if colonies are not treated. Most treatments involve chemical control of the mites; however, Varroa has evolved resistance to many of these miticides, leaving beekeepers with a limited number of alternatives. A non-chemical control method is highly desirable for numerous reasons including lack of chemical residues and decreased likelihood of resistance. Varroa sensitive hygiene behavior is one of two behaviors identified that are most important for controlling the growth of Varroa populations in bee hives. To identify genes influencing this trait, a study was conducted to map quantitative trait loci (QTL). Individual workers of a backcross family were observed and evaluated for their VSH behavior in a mite-infested observation hive. Bees that uncapped or removed pupae were identified. The genotypes for 1,340 informative single nucleotide polymorphisms were used to construct a high-resolution genetic map and interval mapping was used to analyze the association of the genotypes with the performance of Varroa sensitive hygiene. We identified one major QTL on chromosome 9 (LOD score = 3.21) and a suggestive QTL on chromosome 1 (LOD = 1.95). The QTL confidence interval on chromosome 9 contains the gene 'no receptor potential A' and a dopamine receptor. 'No receptor potential A' is involved in vision and olfaction in Drosophila, and dopamine signaling has been previously shown to be required for aversive olfactory learning in honey bees, which is probably necessary for identifying mites within brood cells. Further studies on these candidate genes may allow for breeding bees with this trait using marker-assisted selection.

  4. Mutations to Less-Preferred Synonymous Codons in a Highly Expressed Gene of Escherichia coli: Fitness and Epistatic Interactions.

    Directory of Open Access Journals (Sweden)

    David J Hauber

    Full Text Available Codon-tRNA coevolution to maximize protein production has been, until recently, the dominant hypothesis to explain codon-usage bias in highly expressed bacterial genes. Two predictions of this hypothesis are 1 selection is weak; and 2 similar silent replacements at different codons should have similar fitness consequence. We used an allele-replacement strategy to change five specific 3rd-codon-position (silent sites in the highly expressed Escherichia coli ribosomal protein gene rplQ from the wild type to a less-preferred alternative. We introduced the five mutations within a 10-codon region. Four of the silent sites were chosen to test the second prediction, with a CTG to CTA mutation being introduced at two closely linked leucine codons and an AAA to AAG mutation being introduced at two closely linked lysine codons. We also introduced a fifth silent mutation, a GTG to GTA mutation at a valine codon in the same genic region. We measured the fitness effect of the individual mutations by competing each single-mutant strain against the parental wild-type strain, using a disrupted form of the araA gene as a selectively neutral phenotypic marker to distinguish between strains in direct competition experiments. Three of the silent mutations had a fitness effect of |s| > 0.02, which is contradictory to the prediction that selection will be weak. The two leucine mutations had significantly different fitness effects, as did the two lysine mutations, contradictory to the prediction that similar mutations at different codons should have similar fitness effects. We also constructed a strain carrying all five silent mutations in combination. Its fitness effect was greater than that predicted from the individual fitness values, suggesting that negative synergistic epistasis acts on the combination allele.

  5. High Silicon Accumulation in the Shoot is Required for Down-Regulating the Expression of Si Transporter Genes in Rice.

    Science.gov (United States)

    Mitani-Ueno, Namiki; Yamaji, Naoki; Ma, Jian Feng

    2016-12-01

    Rice requires high silicon (Si) for its high and sustainable yield. The efficient uptake of Si in rice is mediated by two transporters OsLsi1 and OsLsi2, which function as influx and efflux transporters, respectively. Our previous studies showed that the mRNA expression levels of these transporter genes were down-regulated by Si. Herein we investigated the mechanism underlying regulation of OsLsi1 and OsLsi2 expression. There was a negative correlation between the expression level of OsLsi1 and OsLsi2 and shoot Si accumulation when the rice seedlings were exposed to different Si supply conditions. A split root experiment showed that the expression of both OsLsi1 and OsLsi2 was also down-regulated in half the roots without direct Si exposure when the other half of the roots were exposed to Si. Analysis with transgenic rice carrying different lengths of OsLsi1 promoter regions fused with green fluorescent protein (GFP) as a reporter gene revealed that the region responsible for the Si response of OsLsi1 expression is present between -327 to -292 in the promoter. However, this region was not associated with the tissue and cellular localization of OsLsi1. In conclusion, the Si-induced down-regulation of Si transporter genes is controlled by shoot Si, not root Si, and the region between -327 and -292 in the OsLsi1 promoter is involved in this regulation of OsLsi1 expression in rice.

  6. CluGene: A Bioinformatics Framework for the Identification of Co-Localized, Co-Expressed and Co-Regulated Genes Aimed at the Investigation of Transcriptional Regulatory Networks from High-Throughput Expression Data.

    Directory of Open Access Journals (Sweden)

    Tania Dottorini

    Full Text Available The full understanding of the mechanisms underlying transcriptional regulatory networks requires unravelling of complex causal relationships. Genome high-throughput technologies produce a huge amount of information pertaining gene expression and regulation; however, the complexity of the available data is often overwhelming and tools are needed to extract and organize the relevant information. This work starts from the assumption that the observation of co-occurrent events (in particular co-localization, co-expression and co-regulation may provide a powerful starting point to begin unravelling transcriptional regulatory networks. Co-expressed genes often imply shared functional pathways; co-expressed and functionally related genes are often co-localized, too; moreover, co-expressed and co-localized genes are also potential targets for co-regulation; finally, co-regulation seems more frequent for genes mapped to proximal chromosome regions. Despite the recognized importance of analysing co-occurrent events, no bioinformatics solution allowing the simultaneous analysis of co-expression, co-localization and co-regulation is currently available. Our work resulted in developing and valuating CluGene, a software providing tools to analyze multiple types of co-occurrences within a single interactive environment allowing the interactive investigation of combined co-expression, co-localization and co-regulation of genes. The use of CluGene will enhance the power of testing hypothesis and experimental approaches aimed at unravelling transcriptional regulatory networks. The software is freely available at http://bioinfolab.unipg.it/.

  7. High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis.

    Science.gov (United States)

    Sridharan, Vinod; Heimiller, Joseph; Robida, Mark D; Singh, Ravinder

    2016-01-01

    The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5' ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis.

  8. Sequence modification of merB gene and high organo-mercurial resistance of transgenic tobacco plants

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Mercury pollution has caused severe damage to environment and great attention has been paid to its control. Phytoremediation may become one of the most efficient measures to recover the polluted soil since it is economical, highly efficient and friendly to environment. In this report, plant genetic engineering methods were employed to modify the DNA sequence of merB genes that catalyze the conversion of organomercurals into ionic mercury. The modified merBhe genes were introduced into tobacco by Agrobacterium, and the resultant transgenic plants were verified by Southern and Northern hybridization. High level of organomercurial resistance was detected on progenies of transgenic plants, some of which were resistant to PMA (phenyl mercury acetate) of 2.5 ?mol/L whereas 0.1 ?mol/L PMA killed the seedlings of wild-type tobacco in soiless culrure. With the increase of PMA concentration, the inhibition of the seedling growth became apparent. This result makes it possible to breed mercury-resistant tobacco for phytoremediation of mercury-polluted soil.

  9. Large plasmids of Escherichia coli and Salmonella encode highly diverse arrays of accessory genes on common replicon families.

    Science.gov (United States)

    Williams, Laura E; Wireman, Joy; Hilliard, Valda C; Summers, Anne O

    2013-01-01

    Plasmids are important in evolution and adaptation of host bacteria, yet we lack a comprehensive picture of their own natural variation. We used replicon typing and RFLP analysis to assess diversity and distribution of plasmids in the ECOR, SARA, SARB and SARC reference collections of Escherichia coli and Salmonella. Plasmids, especially large (≥30 kb) plasmids, are abundant in these collections. Host species and genotype clearly impact plasmid prevalence; plasmids are more abundant in ECOR than SAR, but, within ECOR, subgroup B2 strains have the fewest large plasmids. The majority of large plasmids have unique RFLP patterns, suggesting high variation, even within dominant replicon families IncF and IncI1. We found only four conserved plasmid types within ECOR, none of which are widely distributed. Within SAR, conserved plasmid types are primarily serovar-specific, including a pSLT-like plasmid in 13 Typhimurium strains. Conservation of pSLT contrasts with variability of other plasmids, suggesting evolution of serovar-specific virulence plasmids is distinct from that of most enterobacterial plasmids. We sequenced a conserved serovar Heidelberg plasmid but did not detect virulence or antibiotic resistance genes. Our data illustrate the high degree of natural variation in large plasmids of E. coli and Salmonella, even among plasmids sharing backbone genes.

  10. Sports genetics: the PPARA gene and athletes’ high ability in endurance sports. A systematic review and meta-analysis

    Science.gov (United States)

    Tuvblad, C; Forero, DA

    2015-01-01

    A meta-analysis was performed with the aim of re-evaluating the role of the peroxisome proliferator activated receptor alpha (PPARA) gene intron 7 G/C polymorphism (rs4253778) in athletes’ high ability in endurance sports. Design: A meta-analysis of case control studies assessing the association between the G/C polymorphisms of the PPARA gene and endurance sports was conducted. The Cochrane Review Manager software was used to compare the genotype and allele frequencies between endurance athletes and controls to determine whether a genetic variant is more common in athletes than in the general population. Five studies, encompassing 760 endurance athletes and 1792 controls, fulfilled our inclusion criteria. The pooled odds ratio (and confidence intervals, CIs) for the G allele compared to the C allele was 1.65 (95% CI 1.39-1.96). The pooled OR for the GG genotype compared to the GC genotype was 1.79 (95% CI 1.44-2.22), and for the GG genotype compared to the CC genotype 2.37 (95% CI 1.40-3.99). There was no evidence of heterogeneity (I2 =0%) or of publication bias. Athletes with high ability in endurance sports had a higher frequency of the GG genotype and G allele. PMID:26985127

  11. High-resolution time series of Pseudomonas aeruginosa gene expression and rhamnolipid secretion through growth curve synchronization

    Directory of Open Access Journals (Sweden)

    Xavier João B

    2011-06-01

    Full Text Available Abstract Background Online spectrophotometric measurements allow monitoring dynamic biological processes with high-time resolution. Contrastingly, numerous other methods require laborious treatment of samples and can only be carried out offline. Integrating both types of measurement would allow analyzing biological processes more comprehensively. A typical example of this problem is acquiring quantitative data on rhamnolipid secretion by the opportunistic pathogen Pseudomonas aeruginosa. P. aeruginosa cell growth can be measured by optical density (OD600 and gene expression can be measured using reporter fusions with a fluorescent protein, allowing high time resolution monitoring. However, measuring the secreted rhamnolipid biosurfactants requires laborious sample processing, which makes this an offline measurement. Results Here, we propose a method to integrate growth curve data with endpoint measurements of secreted metabolites that is inspired by a model of exponential cell growth. If serial diluting an inoculum gives reproducible time series shifted in time, then time series of endpoint measurements can be reconstructed using calculated time shifts between dilutions. We illustrate the method using measured rhamnolipid secretion by P. aeruginosa as endpoint measurements and we integrate these measurements with high-resolution growth curves measured by OD600 and expression of rhamnolipid synthesis genes monitored using a reporter fusion. Two-fold serial dilution allowed integrating rhamnolipid measurements at a ~0.4 h-1 frequency with high-time resolved data measured at a 6 h-1 frequency. We show how this simple method can be used in combination with mutants lacking specific genes in the rhamnolipid synthesis or quorum sensing regulation to acquire rich dynamic data on P. aeruginosa virulence regulation. Additionally, the linear relation between the ratio of inocula and the time-shift between curves produces high-precision measurements of

  12. A New Advanced Backcross Tomato Population Enables High Resolution Leaf QTL Mapping and Gene Identification

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    Daniel Fulop

    2016-10-01

    Full Text Available Quantitative Trait Loci (QTL mapping is a powerful technique for dissecting the genetic basis of traits and species differences. Established tomato mapping populations between domesticated tomato (Solanum lycopersicum and its more distant interfertile relatives typically follow a near isogenic line (NIL design, such as the S. pennellii Introgression Line (IL population, with a single wild introgression per line in an otherwise domesticated genetic background. Here, we report on a new advanced backcross QTL mapping resource for tomato, derived from a cross between the M82 tomato cultivar and S. pennellii. This so-called Backcrossed Inbred Line (BIL population is comprised of a mix of BC2 and BC3 lines, with domesticated tomato as the recurrent parent. The BIL population is complementary to the existing S. pennellii IL population, with which it shares parents. Using the BILs, we mapped traits for leaf complexity, leaflet shape, and flowering time. We demonstrate the utility of the BILs for fine-mapping QTL, particularly QTL initially mapped in the ILs, by fine-mapping several QTL to single or few candidate genes. Moreover, we confirm the value of a backcrossed population with multiple introgressions per line, such as the BILs, for epistatic QTL mapping. Our work was further enabled by the development of our own statistical inference and visualization tools, namely a heterogeneous hidden Markov model for genotyping the lines, and by using state-of-the-art sparse regression techniques for QTL mapping.

  13. The Mx1 gene protects mice against the pandemic 1918 and highly lethal human H5N1 influenza viruses.

    Science.gov (United States)

    Tumpey, Terrence M; Szretter, Kristy J; Van Hoeven, Neal; Katz, Jacqueline M; Kochs, Georg; Haller, Otto; García-Sastre, Adolfo; Staeheli, Peter

    2007-10-01

    Mice carrying a wild-type Mx1 gene (Mx1+/+) differ from standard laboratory mice (Mx1-/-) in being highly resistant to infection with common laboratory strains of influenza A virus. We report that Mx1 also protects mice against the pandemic human 1918 influenza virus and a highly lethal human H5N1 strain from Vietnam. Resistance to H5N1 of Mx1+/+ but not Mx1-/- mice was enhanced if the animals were treated with a single dose of exogenous alpha interferon before infection. Thus, the interferon-induced resistance factor Mx1 represents a key component of the murine innate immune system that mediates protection against epidemic and pandemic influenza viruses.

  14. Investigating the relationship between UMODL1 gene polymorphisms and high myopia: a case–control study in Chinese

    Directory of Open Access Journals (Sweden)

    Zhu Miao-miao

    2012-08-01

    Full Text Available Abstract Background The UMODL1 gene was found to be associated with high myopia in Japanese. This study aimed to investigate this gene for association with high myopia in Chinese. Methods Two groups of unrelated Han Chinese from Hong Kong were recruited using the same criteria: Sample Set 1 comprising 356 controls (spherical equivalent, SE, within ±1 diopter or D and 356 cases (SE ≤ −8D, and Sample Set 2 comprising 394 controls and 526 cases. Fifty-nine tag single nucleotide polymorphisms (SNPs were selected and genotyped for Sample Set 1. Four SNPs were followed up with Sample Set 2. Both single-marker and haplotype analyses were performed with cases defined by different SE thresholds. Secondary phenotypes were also analyzed for association with genotypes. Results Data filtering left 57 SNPs for analysis. Single-marker analysis did not reveal any significant differences between cases and controls in the initial study. However, haplotype GCT for markers rs220168-rs220170-rs11911271 showed marginal significance (empirical P = 0.076; SE ≤ −12D for cases, but could not be replicated in the follow-up study. In contrast, non-synonymous SNP rs3819142 was associated with high myopia (SE ≤ −10D in the follow-up study, but could not be confirmed using Sample Set 1. The SNP rs2839471, positive in the original Japanese study, gave negative results in all our analyses. Exploratory analysis of secondary phenotypes indicated that allele C of rs220120 was associated with anterior chamber depth (adjusted P = 0.0460. Conclusions Common UMODL1 polymorphisms were unlikely to be important in the genetic susceptibility to high myopia in Han Chinese.

  15. High gene flow in reef fishes and its implications for ad-hoc no-take marine reserves.

    Science.gov (United States)

    Matias, Ambrocio Melvin A; Anticamara, Jonathan A; Quilang, Jonas P

    2013-10-01

    Integration of genetic connectivity information in effective marine reserve (MR) design is important in sustaining marine biodiversity. Here, genetic connectivity based on mitochondrial DNA (mtDNA) of three reef fish species, namely Epinephelus merra (n = 67; 32 from Bolinao, 14 from Alaminos, and 21 from Masinloc), Parupeneus multifasciatus (n = 23; 12 from Bolinao and 11 from Masinloc), and Odonus niger (n = 35; 21 from Mabini and 14 from Tingloy), sampled across western Luzon, Philippines, was inferred by assessing their genetic diversity, population genetic structure, and historical demography. The results show high haplotype and nucleotide diversity in the three species. Tests for population structure indicate high gene flow and no spatial genetic structuring for the three species. Mismatch analyses suggest unimodal distribution for E. merra and P. multifasciatus, but bimodal distribution for O. niger. Even with differences in mismatch distributions, all the three species exhibit low raggedness index indicating demographic population expansion. The bimodal distribution of O. niger could be attributed to the mixing of two isolated populations. High gene flow between sampling locations implies genetic exchanges and connectivity between many small MRs and fishing grounds in western Luzon, Philippines, at a scale similar to our study. This research is among the first few to elucidate the high genetic connectivity of reef fish communities across the Philippines (here western Luzon), but it also calls for more support (i.e. government and academia) for genetic research that aims to (1) understand the maintenance of megadiversity of the country and (2) search for effective biodiversity conservation options for the coral reefs.

  16. The mRNA cap-binding protein Cbc1 is required for high and timely expression of genes by promoting the accumulation of gene-specific activators at promoters.

    Science.gov (United States)

    Li, Tianlu; De Clercq, Nikki; Medina, Daniel A; Garre, Elena; Sunnerhagen, Per; Pérez-Ortín, José E; Alepuz, Paula

    2016-02-01

    The highly conserved Saccharomyces cerevisiae cap-binding protein Cbc1/Sto1 binds mRNA co-transcriptionally and acts as a key coordinator of mRNA fate. Recently, Cbc1 has also been implicated in transcription elongation and pre-initiation complex (PIC) formation. Previously, we described Cbc1 to be required for cell growth under osmotic stress and to mediate osmostress-induced translation reprogramming. Here, we observe delayed global transcription kinetics in cbc1Δ during osmotic stress that correlates with delayed recruitment of TBP and RNA polymerase II to osmo-induced promoters. Interestingly, we detect an interaction between Cbc1 and the MAPK Hog1, which controls most gene expression changes during osmostress, and observe that deletion of CBC1 delays the accumulation of the activator complex Hot1-Hog1 at osmostress promoters. Additionally, CBC1 deletion specifically reduces transcription rates of highly transcribed genes under non-stress conditions, such as ribosomal protein (RP) genes, while having low impact on transcription of weakly expressed genes. For RP genes, we show that recruitment of the specific activator Rap1, and subsequently TBP, to promoters is Cbc1-dependent. Altogether, our results indicate that binding of Cbc1 to the capped mRNAs is necessary for the accumulation of specific activators as well as PIC components at the promoters of genes whose expression requires high and rapid transcription.

  17. High expression of the circadian gene mPer2 diminishes the radiosensitivity of NIH 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chang, L.; Liu, Y.Y.; Zhu, B.; Li, Y.; Hua, H.; Wang, Y.H.; Zhang, J.; Jiang, Z.; Wang, Z.R. [Sichuan University, Chengdu (China). West China Medical Center. Health Ministry Key Lab. of Chronobiology], e-mail: wangzhengrong@126.com

    2009-10-15

    Period2 is a core circadian gene, which not only maintains the circadian rhythm of cells but also regulates some organic functions. We investigated the effects of mPeriod2 (mPer2) expression on radiosensitivity in normal mouse cells exposed to {sup 60}Co-{gamma}-rays. NIH 3T3 cells were treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to induce endogenous mPer2 expression or transfected with pcDNA3.1(+)-mPer2 and irradiated with {sup 6}0Co-{gamma}-rays, and then analyzed by several methods such as flow cytometry, colony formation assay, RT-PCR, and immunohistochemistry. Flow cytometry and colony formation assay revealed that irradiated NIH 3T3 cells expressing high levels of mPer2 showed a lower death rate (TPA: 24 h 4.3% vs 12 h 6.8% and control 9.4%; transfection: pcDNA3.1-mPer2 3.7% vs pcDNA3.1 11.3% and control 8.2%), more proliferation and clonogenic survival (TPA: 121.7 {+-} 6.51 vs 66.0 {+-} 3.51 and 67.7 {+-} 7.37; transfection: 121.7 {+-} 6.50 vs 65.3 {+-} 3.51 and 69.0 {+-} 4.58) both when treated with TPA and transfected with mPer2. RT-PCR analysis showed an increased expression of bax, bcl-2, p53, cmyc, mre11, and nbs1, and an increased proportionality of bcl-2/bax in the irradiated cells at peak mPer2 expression compared with cells at trough mPer2 expression and control cells. However, no significant difference in rad50 expression was observed among the three groups of cells. Immunohistochemistry also showed increased protein levels of P53, BAX and proliferating cell nuclear antigen in irradiated cells with peak mPer2 levels. Thus, high expression of the circadian gene mPer2 may reduce the radiosensitivity of NIH 3T3 cells. For this effect, mPer2 may directly or indirectly regulate the expressions of cell proliferation- and apoptosis-related genes and DNA repair-related genes. (author)

  18. Total alpha-globin gene cluster deletion has high frequency in Filipinos

    Energy Technology Data Exchange (ETDEWEB)

    Hunt, J.A.; Haruyama, A.Z.; Chu, B.M. [Kapiolani Medical Center, Honolulu, HI (United States)] [and others

    1994-09-01

    Most {alpha}-thalassemias [Thal] are due to large deletions. In Southeast Asians, the (--{sup SEA}) double {alpha}-globin gene deletion is common, 3 (--{sup Tot}) total {alpha}-globin cluster deletions are known: Filipino (--{sup Fil}), Thai (--{sup Thai}), and Chinese (--{sup Chin}). In a Hawaii Thal project, provisional diagnosis of {alpha}-Thal-1 heterozygotes was based on microcytosis, normal isoelectric focusing, and no iron deficiency. One in 10 unselected Filipinos was an {alpha}-Thal-1 heterozygote, 2/3 of these had a (--{sup Tot}) deletion: a {var_sigma}-cDNA probe consistently showed fainter intensity of the constant 5.5 kb {var_sigma}{sub 2} BamHI band, with no heterzygosity for {var_sigma}-globin region polymorphisms; {alpha}-cDNA or {var_sigma}-cDNA probes showed no BamHI or BglII bands diagnostic of the (--{sup SEA}) deletion; bands for the (-{alpha}) {alpha}-Thal-2 single {alpha}-globin deletions were only seen in Hb H cases. A reliable monoclonal anti-{var_sigma}-peptide antibody test for the (--{sup SEA}) deletion was always negative in (--{sup Tot}) samples. Southern digests with the Lo probe, a gift from D. Higgs of Oxford Univ., confirmed that 49 of 50 (--{sup Tot}) chromosomes in Filipinos were (--{sup Fil}). Of 20 {alpha}-Thal-1 hydrops born to Filipinos, 11 were (--{sup Fil}/--{sup SEA}) compound heterozygotes; 9 were (--{sup SEA}/--{sup SEA}) homozygotes, but none was a (--{sup Fil}/--{sup Fil}).

  19. Comprehensive high-resolution analysis of the role of an Arabidopsis gene family in RNA editing.

    Directory of Open Access Journals (Sweden)

    Stéphane Bentolila

    2013-06-01

    Full Text Available In flowering plants, mitochondrial and chloroplast mRNAs are edited by C-to-U base modification. In plant organelles, RNA editing appears to be generally a correcting mechanism that restores the proper function of the encoded product. Members of the Arabidopsis RNA editing-Interacting Protein (RIP family have been recently shown to be essential components of the plant editing machinery. We report the use of a strand- and transcript-specific RNA-seq method (STS-PCRseq to explore the effect of mutation or silencing of every RIP gene on plant organelle editing. We confirm RIP1 to be a major editing factor that controls the editing extent of 75% of the mitochondrial sites and 20% of the plastid C targets of editing. The quantitative nature of RNA sequencing allows the precise determination of overlapping effects of RIP factors on RNA editing. Over 85% of the sites under the influence of RIP3 and RIP8, two moderately important mitochondrial factors, are also controlled by RIP1. Previously uncharacterized RIP family members were found to have only a slight effect on RNA editing. The preferential location of editing sites controlled by RIP7 on some transcripts suggests an RNA metabolism function for this factor other than editing. In addition to a complete characterization of the RIP factors for their effect on RNA editing, our study highlights the potential of RNA-seq for studying plant organelle editing. Unlike previous attempts to use RNA-seq to analyze RNA editing extent, our methodology focuses on sequencing of organelle cDNAs corresponding to known transcripts. As a result, the depth of coverage of each editing site reaches unprecedented values, assuring a reliable measurement of editing extent and the detection of numerous new sites. This strategy can be applied to the study of RNA editing in any organism.

  20. Comprehensive high-resolution analysis of the role of an Arabidopsis gene family in RNA editing.

    Science.gov (United States)

    Bentolila, Stéphane; Oh, Julyun; Hanson, Maureen R; Bukowski, Robert

    2013-06-01

    In flowering plants, mitochondrial and chloroplast mRNAs are edited by C-to-U base modification. In plant organelles, RNA editing appears to be generally a correcting mechanism that restores the proper function of the encoded product. Members of the Arabidopsis RNA editing-Interacting Protein (RIP) family have been recently shown to be essential components of the plant editing machinery. We report the use of a strand- and transcript-specific RNA-seq method (STS-PCRseq) to explore the effect of mutation or silencing of every RIP gene on plant organelle editing. We confirm RIP1 to be a major editing factor that controls the editing extent of 75% of the mitochondrial sites and 20% of the plastid C targets of editing. The quantitative nature of RNA sequencing allows the precise determination of overlapping effects of RIP factors on RNA editing. Over 85% of the sites under the influence of RIP3 and RIP8, two moderately important mitochondrial factors, are also controlled by RIP1. Previously uncharacterized RIP family members were found to have only a slight effect on RNA editing. The preferential location of editing sites controlled by RIP7 on some transcripts suggests an RNA metabolism function for this factor other than editing. In addition to a complete characterization of the RIP factors for their effect on RNA editing, our study highlights the potential of RNA-seq for studying plant organelle editing. Unlike previous attempts to use RNA-seq to analyze RNA editing extent, our methodology focuses on sequencing of organelle cDNAs corresponding to known transcripts. As a result, the depth of coverage of each editing site reaches unprecedented values, assuring a reliable measurement of editing extent and the detection of numerous new sites. This strategy can be applied to the study of RNA editing in any organism.

  1. Low carbohydrate/high-fat diet attenuates cardiac hypertrophy, remodeling, and altered gene expression in hypertension

    Science.gov (United States)

    The effects of dietary fat intake on the development of left ventricular hypertrophy and accompanying structural and molecular remodeling in response to hypertension are not understood. The present study compared the effects of a high-fat versus a low-fat diet on development of left ventricular hype...

  2. Thermal stress induces changes in gene expression and blood parameters in high and low feed efficiency meat quail.

    Science.gov (United States)

    Gasparino, Eliane; Voltolini, Débora Marques; Del Vesco, Ana Paula; Marcato, Simara Marcia; Zancanela, Vittor; de Oliveira Grieser, Daiane; de Souza Khatlab, Angélica; Guimarães, Simone Eliza Facioni; de Oliveira Neto, Adhemar Rodriges

    2015-05-01

    In this study, we analysed markers of stress, plasma creatinine and T3 content, and insulin-like growth factor I (IGF-I), growth hormone receptor (GHR), uncoupling protein (UCP), adenine nucleotide translocase (ANT) and cytochrome c oxidase subunit III (COX III) mRNA expression in the liver and muscle of high (0.22 g/g) and low (0.14 g/g) feed efficiency (FE) meat quail at three different air temperatures, comfortable, heat and cold stress, for 24 h. High FE quail presented higher plasma T3 and lower creatinine levels. IGF-I mRNA expression was higher in the livers of high FE quail than in the livers of low FE quail under both comfortable and cold stress conditions. In the muscle, regardless of the environment, high FE birds showed higher IGF-I mRNA expression. High FE birds also showed higher GHR mRNA expression under comfortable conditions. Regarding the environment, higher expression was observed in birds at comfortable conditions, and lower expression in birds under heat stress. UCP mRNA expression in the liver was lower in high FE birds and higher under heat stress compared with the other conditions. Low and high FE birds showed greater ANT mRNA expression in the muscle under cold stress. Greater mRNA COX III expressions were observed in the liver and muscle of quails under comfortable conditions. Our results suggest that temperature affects the expression of genes related to growth and mitochondrial energy production, and quail with different FEs respond differently to environmental stimuli. In comfortable conditions, high FE animals show higher IGF-I mRNA expression and plasma T3 and lower creatinine content.

  3. Analysis of High-altitude Syndrome and the Underlying Gene Polymorphisms Associated with Acute Mountain Sickness after a Rapid Ascent to High-altitude

    Science.gov (United States)

    Yu, Jie; Zeng, Ying; Chen, Guozhu; Bian, Shizhu; Qiu, Youzhu; Liu, Xi; Xu, Baida; Song, Pan; Zhang, Jihang; Qin, Jun; Huang, Lan

    2016-12-01

    To investigated the objective indicators and potential genotypes for acute mountain sickness (AMS). 176 male subjects were evaluated for symptoms scores and physiological parameters at 3700 m. EPAS1 gene polymorphisms were explored and verified effects of potential genotypes on pulmonary function by inhaled budesonide. The incidence of AMS was 53.98% (95/176). The individuals who suffered from headache with anxiety and greater changes in heart rate (HR), the forced vital capacity (FVC), and mean flow velocity of basilar artery (Vm-BA), all of which were likely to develop AMS. The rs4953348 polymorphism of EPAS1 gene had a significant correlation with the SaO2 level and AMS, and a significant difference in the AG and GG genotype distribution between the AMS and non-AMS groups. The spirometric parameters were significantly lower, but HR (P = 0.036) and Vm-BA (P = 0.042) significantly higher in the AMS subjects with the G allele than those with the A allele. In summary, changes in HR (≥82 beats/min), FVC (≤4.2 Lt) and Vm-BA (≥43 cm/s) levels may serve as predictors for diagnosing AMS accompanied by high-altitude syndrome. The A allele of rs4953348 is a protective factor for AMS through HR and Vm-BA compensation, while the G allele may contribute to hypoxic pulmonary hypertension in AMS.

  4. Molecular genetics of X chromosome-linked color vision among populations of African and Japanese ancestry: High frequency of a shortened red pigment gene among Afro-Americans

    Energy Technology Data Exchange (ETDEWEB)

    Joergensen, A.L.; Deeb, S.S.; Motulsky, A.G. (Univ. of Washington, Seattle (USA))

    1990-09-01

    Red-green color vision in humans is mediated by the X chromosome-linked highly homologous red and green pigment genes. Color vision defects are caused by deletions and fusions involving these genes. However, the authors found the frequency of molecular abnormalities among Caucasians to be twice as high as that of phenotypic color vision defects. Among Japanese the frequency of phenotypic and molecular color vision defects was similar. Among Afro-Americans, molecular defects were at least five times more frequent than phenotypic color vision defects. In addition, 35% of Afro-Americans, 2% of Japanese, and <1% of Caucasians had a shortened red pigment gene not associated with phenotpyic color vision defects. This gene lacked 1.9 kilobases in its first intron and had the identical size as the green pigment gene from which it presumably originated by gene conversion in an ancestral African population. This gene and the closely linked glucose-6-phosphate dehydrogenase A{sup +} variant were in linkage equilibrium. A model for the evolutionary origin of the color vision pigment genes in higher primates is portrayed.

  5. [Two-step synthesis of the full length Aspergillus niger lipase gene lipA leads to high-level expression in Pichia pastoris].

    Science.gov (United States)

    Yang, Jiangke; Yan, Xiangxiang; Zhang, Zhengping; Jiang, Xueqing; Yan, Yunjun

    2009-03-01

    Aspergillus niger lipases are important biocatalysis widely used in industries for food processing and pharmaceutical preparation. High-level expression recombinants can lead to cost effective lipase large scale production. Full length gene synthesis is an efficient measure to enhance the expression level of the gene. In order to reduce the non-specific binding between oligonucleotides and bases mutation caused by the complicate secondary structure of DNA and excessive PCR amplification, a frequently phenomenon in one-step gene synthesis, we used a two-step method including assembly PCR (A-PCR) and digestion-ligation step to synthesis Aspergillus niger lipase gene lipA. Assisted by DNA2.0 and Gene2Oliga software, we optimized the codon usage and secondary structure of RNA and induced enzyme sites Cla I (237 site) and Pst I (475 site) into the gene. In the first step, fragments F1 (237 bp), F2 (238 bp) and F3 (422 bp) were separately synthesized by assembly PCR. In the second step, fragments F1, F2 and F3 were separately digested by Cla I and Pst I, and then ligated into a full length lipA gene. Two-step method efficiently enhanced successful ratio for full-length gene synthesis and dispersed the risk for gene redesign. The synthesized gene was cloned into pPIC9K vector and transferred into Pichia pastoris. After methanol inducement, the expression level of the codon optimized lipA-syn gene reached 176.0 U/mL, 10.8-fold of the original lipA gene (16.3 U/mL) in Pichia pastoris GS1115. The recombinant offers the possibility for lipase large-scale production.

  6. A high protein diet during pregnancy affects hepatic gene expression of energy sensing pathways along ontogenesis in a porcine model.

    Directory of Open Access Journals (Sweden)

    Michael Oster

    Full Text Available In rodent models and in humans the impact of gestational diets on the offspring's phenotype was shown experimentally and epidemiologically. The underlying programming of fetal development was shown to be associated with an increased risk of degenerative diseases in adulthood, including the metabolic syndrome. There are clues that diet-dependent modifications of the metabolism during fetal life can persist until adulthood. This leads to the hypothesis that the offspring's transcriptomes show short-term and long-term changes depending on the maternal diet. To this end pregnant German landrace gilts were fed either a high protein diet (HP, 30% CP or an adequate protein diet (AP, 12% CP throughout pregnancy. Hepatic transcriptome profiles of the offspring were analyzed at prenatal (94 dpc and postnatal stages (1, 28, 188 dpn. Depending on the gestational dietary exposure, mRNA expression levels of genes related to energy metabolism, N-metabolism, growth factor signaling pathways, lipid metabolism, nucleic acid metabolism and stress/immune response were affected either in a short-term or in a long-term manner. Gene expression profiles at fetal stage 94 dpc were almost unchanged between the diets. The gestational HP diet affected the hepatic expression profiles at prenatal and postnatal stages. The effects encompassed a modulation of the genome in terms of an altered responsiveness of energy and nutrient sensing pathways. Differential expression of genes related to energy production and nutrient utilization contribute to the maintenance of development and growth performance within physiological norms, however the modulation of these pathways may be accompanied by a predisposition for metabolic disturbances up to adult stages.

  7. A novel frameshift mutation in BLM gene associated with high sister chromatid exchanges (SCE) in heterozygous family members.

    Science.gov (United States)

    Ben Salah, Ghada; Hadj Salem, Ikhlas; Masmoudi, Abderrahmen; Kallabi, Fakhri; Turki, Hamida; Fakhfakh, Faiza; Ayadi, Hamadi; Kamoun, Hassen

    2014-11-01

    The Bloom syndrome (BS) is an autosomic recessive disorder comprising a wide range of abnormalities, including stunted growth, immunodeficiency, sun sensitivity and increased frequency of various types of cancer. Bloom syndrome cells display a high level of genetic instability, including a 10-fold increase in the sister chromatid exchanges (SCE) level. Bloom syndrome arises through mutations in both alleles of the BLM gene, which was identified as a member of the RecQ helicase family. In this study, we screened a Tunisian family with three BS patients. Cytogenetic analysis showed several chromosomal aberrations, and an approximately 14-fold elevated SCE frequency in BS cells. A significant increase in SCE frequency was observed in some family members but not reaching the BS patients values, leading to suggest that this could be due to the heterozygous profile. Microsatellite genotyping using four fluorescent dye-labeled microsatellite markers revealed evidence of linkage to BLM locus and the healthy members, sharing higher SCE frequency, showed heterozygous haplotypes as expected. Additionally, the direct BLM gene sequencing identified a novel homozygous frameshift mutation c.3617-3619delAA (p.K1207fsX9) in BS patients and a heterozygous BLM mutation in the family members with higher SCE frequency. Our findings suggest that this latter mutation likely leads to a reduced BLM activity explaining the homologous recombination repair defect and, therefore, the increase in SCE. Based on the present data, the screening of this mutation could contribute to the rapid diagnosis of BS. The genetic confirmation of the mutation in BLM gene provides crucial information for genetic counseling and prenatal diagnosis.

  8. Gene discovery and molecular marker development, based on high-throughput transcript sequencing of Paspalum dilatatum Poir.

    Directory of Open Access Journals (Sweden)

    Andrea Giordano

    Full Text Available BACKGROUND: Paspalum dilatatum Poir. (common name dallisgrass is a native grass species of South America, with special relevance to dairy and red meat production. P. dilatatum exhibits higher forage quality than other C4 forage grasses and is tolerant to frost and water stress. This species is predominantly cultivated in an apomictic monoculture, with an inherent high risk that biotic and abiotic stresses could potentially devastate productivity. Therefore, advanced breeding strategies that characterise and use available genetic diversity, or assess germplasm collections effectively are required to deliver advanced cultivars for production systems. However, there are limited genomic resources available for this forage grass species. RESULTS: Transcriptome sequencing using second-generation sequencing platforms has been employed using pooled RNA from different tissues (stems, roots, leaves and inflorescences at the final reproductive stage of P. dilatatum cultivar Primo. A total of 324,695 sequence reads were obtained, corresponding to c. 102 Mbp. The sequences were assembled, generating 20,169 contigs of a combined length of 9,336,138 nucleotides. The contigs were BLAST analysed against the fully sequenced grass species of Oryza sativa subsp. japonica, Brachypodium distachyon, the closely related Sorghum bicolor and foxtail millet (Setaria italica genomes as well as against the UniRef 90 protein database allowing a comprehensive gene ontology analysis to be performed. The contigs generated from the transcript sequencing were also analysed for the presence of simple sequence repeats (SSRs. A total of 2,339 SSR motifs were identified within 1,989 contigs and corresponding primer pairs were designed. Empirical validation of a cohort of 96 SSRs was performed, with 34% being polymorphic between sexual and apomictic biotypes. CONCLUSIONS: The development of genetic and genomic resources for P. dilatatum will contribute to gene discovery and expression

  9. Ancient exaptation of a CORE-SINE retroposon into a highly conserved mammalian neuronal enhancer of the proopiomelanocortin gene.

    Directory of Open Access Journals (Sweden)

    Andrea M Santangelo

    2007-10-01

    Full Text Available The proopiomelanocortin gene (POMC is expressed in the pituitary gland and the ventral hypothalamus of all jawed vertebrates, producing several bioactive peptides that function as peripheral hormones or central neuropeptides, respectively. We have recently determined that mouse and human POMC expression in the hypothalamus is conferred by the action of two 5' distal and unrelated enhancers, nPE1 and nPE2. To investigate the evolutionary origin of the neuronal enhancer nPE2, we searched available vertebrate genome databases and determined that nPE2 is a highly conserved element in placentals, marsupials, and monotremes, whereas it is absent in nonmammalian vertebrates. Following an in silico paleogenomic strategy based on genome-wide searches for paralog sequences, we discovered that opossum and wallaby nPE2 sequences are highly similar to members of the superfamily of CORE-short interspersed nucleotide element (SINE retroposons, in particular to MAR1 retroposons that are widely present in marsupial genomes. Thus, the neuronal enhancer nPE2 originated from the exaptation of a CORE-SINE retroposon in the lineage leading to mammals and remained under purifying selection in all mammalian orders for the last 170 million years. Expression studies performed in transgenic mice showed that two nonadjacent nPE2 subregions are essential to drive reporter gene expression into POMC hypothalamic neurons, providing the first functional example of an exapted enhancer derived from an ancient CORE-SINE retroposon. In addition, we found that this CORE-SINE family of retroposons is likely to still be active in American and Australian marsupial genomes and that several highly conserved exonic, intronic and intergenic sequences in the human genome originated from the exaptation of CORE-SINE retroposons. Together, our results provide clear evidence of the functional novelties that transposed elements contributed to their host genomes throughout evolution.

  10. A novel bisulfite-microfluidic temperature gradient capillary electrophoresis platform for highly sensitive detection of gene promoter methylation.

    Science.gov (United States)

    Zhang, Huidan; Shan, Lianfeng; Wang, Xiaonan; Ma, Qian; Fang, Jin

    2013-04-15

    The hypermethylated tumor suppressor gene promoters are widely recognized as promising markers for cancer screening and ideal targets for cancer therapy, however, a major obstacle in their clinical study is highly sensitive screening. To address this limitation, we developed a novel bisulfite-microfluidic temperature gradient capillary electrophoresis (bisulfite-μTGCE) platform for gene methylation analysis by combining bisulfite treatment and slantwise radiative heating system-based μTGCE. Bisulfite-treated genomic DNA (gDNA) was amplified with universal primers for both methylated and unmethylated sequences, and introduced into glass microfluidic chip to perform electrophorectic separation under a continuous temperature gradient based on the formation of heteroduplexes. Eight CDKN2A promoter model fragments with different number and location of methylation sites were prepared and successfully analyzed according to their electrophoretic peak patterns, with high stability, picoliter-scale sample consumption, and significantly increased detection speed. The bisulfite-μTGCE could detect methylated gDNA with a detection limit of 7.5pg, and could distinguish as low as 0.1% methylation level in CDKN2A in an unmethylated background. Detection of seven colorectal cancer (CRC) cell lines with known and unknown methylation statuses of CDKN2A promoter and 20 tumor tissues derived from CRC patients demonstrated the capability of detecting hypermethylation in real-world samples. The wider adaptation of this platform was further supported by the detection of the CDKN2A and MLH1 promoters' methylation statuses in combination. This highly sensitive, fast, and low-consumption platform for methylation detection shows great potential for future clinical applications.

  11. Establishment of Stable High Expression Cell Line with Green Fluorescent Protein and Resistance Genes

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shengtao; LIU Wenli; HE Peigen; GONG Feili; YANG Dongliang

    2006-01-01

    In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

  12. Involvement of adenosine monophosphate-activated protein kinase in the influence of timed high-fat evening diet on the hepatic clock and lipogenic gene expression in mice.

    Science.gov (United States)

    Huang, Yan; Zhu, Zengyan; Xie, Meilin; Xue, Jie

    2015-09-01

    A high-fat diet may result in changes in hepatic clock gene expression, but potential mechanisms are not yet elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that is recognized as a key regulator of energy metabolism and certain clock genes. Therefore, we hypothesized that AMPK may be involved in the alteration of hepatic clock gene expression under a high-fat environment. This study aimed to examine the effects of timed high-fat evening diet on the activity of hepatic AMPK, clock genes, and lipogenic genes. Mice with hyperlipidemic fatty livers were induced by orally administering high-fat milk via gavage every evening (19:00-20:00) for 6 weeks. Results showed that timed high-fat diet in the evening not only decreased the hepatic AMPK protein expression and activity but also disturbed its circadian rhythm. Accordingly, the hepatic clock genes, including clock, brain-muscle-Arnt-like 1, cryptochrome 2, and period 2, exhibited prominent changes in their expression rhythms and/or amplitudes. The diurnal rhythms of the messenger RNA expression of peroxisome proliferator-activated receptorα, acetyl-CoA carboxylase 1α, and carnitine palmitoyltransferase 1 were also disrupted; the amplitude of peroxisome proliferator-activated receptorγcoactivator 1α was significantly decreased at 3 time points, and fatty liver was observed. These findings demonstrate that timed high-fat diet at night can change hepatic AMPK protein levels, activity, and circadian rhythm, which may subsequently alter the circadian expression of several hepatic clock genes and finally result in the disorder of hepatic lipogenic gene expression and the formation of fatty liver.

  13. RNAi validation of resistance genes and their interactions in the highly DDT-resistant 91-R strain of Drosophila melanogaster.

    Science.gov (United States)

    Gellatly, Kyle J; Yoon, Kyong Sup; Doherty, Jeffery J; Sun, Weilin; Pittendrigh, Barry R; Clark, J Marshall

    2015-06-01

    4,4'-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will increase in terms of level and extent. Drosophila melanogaster is a model dipteran that has many available genetic tools, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold), however, there is no mechanistic scheme that accounts for this level of resistance. Recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms in the 91-R strain. Their interactions, however, remain unclear. Use of UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has validated the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (Mdr50, Mdr65, and Mrp1) involved in DDT resistance. Further, increased sensitivity to DDT in the 91-R strain after intra-abdominal dsRNA injection for Mdr50, Mdr65, and Mrp1 was determined by a DDT contact bioassay, directly implicating these genes in DDT efflux and resistance.

  14. Ectomycorrhiza-mediated repression of the high-affinity ammonium importer gene AmAMT2 in Amanita muscaria.

    Science.gov (United States)

    Willmann, Anita; Weiss, Michael; Nehls, Uwe

    2007-02-01

    A main function of ectomycorrhizas, a symbiosis between certain soil fungi and fine roots of woody plants, is the exchange of plant-derived carbohydrates for fungus-derived nutrients. As it is required in large amounts, nitrogen is of special interest. A gene (AmAMT2) coding for a putative fungal ammonium importer was identified in an EST project of functional Amanita muscaria/poplar ectomycorrhizas. Heterologous expression of the entire AmAMT2 coding region in yeast revealed the corresponding protein to be a high-affinity ammonium importer. In axenically grown Amanita hyphae AmAMT2 expression was strongly repressed by nitrogen, independent of whether the offered nitrogen source was transported by AmAMT2 or not. In functional ectomycorrhizas the AmAMT2 transcript level was further decreased in both hyphal networks (sheath and Hartig net), while extraradical hyphae revealed strong gene expression. Together our data suggest that (1) AmAMT2 expression is regulated by the endogenous nitrogen content of hyphae and (2) fungal hyphae in ectomycorrhizas are well supported with nitrogen even when the extraradical mycelium is nitrogen limited. As a consequence of AmAMT2 repression in mycorrhizas, ammonium can be suggested as a potential nitrogen source delivered by fungal hyphae in symbiosis.

  15. High-frequency stimulation induces gradual immediate early gene expression in maturing adult-generated hippocampal granule cells.

    Science.gov (United States)

    Jungenitz, Tassilo; Radic, Tijana; Jedlicka, Peter; Schwarzacher, Stephan W

    2014-07-01

    Increasing evidence shows that adult neurogenesis of hippocampal granule cells is advantageous for learning and memory. We examined at which stage of structural maturation and age new granule cells can be activated by strong synaptic stimulation. High-frequency stimulation of the perforant pathway in urethane-anesthetized rats elicited expression of the immediate early genes c-fos, Arc, zif268 and pCREB133 in almost 100% of mature, calbindin-positive granule cells. In contrast, it failed to induce immediate early gene expression in immature doublecortin-positive granule cells. Furthermore, doublecortin-positive neurons did not react with c-fos or Arc expression to mild theta-burst stimulation or novel environment exposure. Endogenous expression of pCREB133 was increasingly present in young cells with more elaborated dendrites, revealing a close correlation to structural maturation. Labeling with bromodeoxyuridine revealed cell age dependence of stimulation-induced c-fos, Arc and zif268 expression, with only a few cells reacting at 21 days, but with up to 75% of cells activated at 35-77 days of cell age. Our results indicate an increasing synaptic integration of maturing granule cells, starting at 21 days of cell age, but suggest a lack of ability to respond to activation with synaptic potentiation on the transcriptional level as long as immature cells express doublecortin.

  16. Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus.

    Science.gov (United States)

    Arazoe, Takayuki; Miyoshi, Kennosuke; Yamato, Tohru; Ogawa, Tetsuo; Ohsato, Shuichi; Arie, Tsutomu; Kuwata, Shigeru

    2015-12-01

    CRISPR/Cas-derived RNA-guided nucleases (RGNs) that can generate DNA double-strand breaks (DSBs) at a specific sequence are widely used for targeted genome editing by induction of DSB repair in many organisms. The CRISPR/Cas system consists of two components: a single Cas9 nuclease and a single-guide RNA (sgRNA). Therefore, the system for constructing RGNs is simple and efficient, but the utilization of RGNs in filamentous fungi has not been validated. In this study, we established the CRISPR/Cas system in the model filamentous fungus, Pyricularia oryzae, using Cas9 that was codon-optimized for filamentous fungi, and the endogenous RNA polymerase (RNAP) III U6 promoter and a RNAP II fungal promoter for the expression of the sgRNA. We further demonstrated that RGNs could recognize the desired sequences and edit endogenous genes through homologous recombination-mediated targeted gene replacement with high efficiency. Our system will open the way for the development of various CRISPR/Cas-based applications in filamentous fungi.

  17. High frequency of mutations in codon 98 of the peripheral myelin protein Po gene in 20 French CMT1 patients

    Energy Technology Data Exchange (ETDEWEB)

    Rougher, H.; LeGuern, E. Gouider, R. [and others

    1996-03-01

    Charcot-Marie-Tooth disease, characterized by distal muscle weakness and amyotrophy, decreased or absent tendon reflexes, and high arched feet, is the most common inherited peripheral neuropathy, with a prevalence of 1 in 2,500. Two types of CMT have been distinguished on the basis of nerve conduction velocities. CMT type 1 is the most frequent, with markedly slowed velocities ({<=}40 m/s) associated with hypertrophic onion bulb changes on nerve biopsy. Autosomal dominant CMT1 is genetically heterogeneous: CMT1A is caused by a 1.5-Mb duplication in 17p11.2 and, more rarely, by a point mutation in tha PMP22 (peripheral myelin protein, 22 kD) gene located in the duplicated region; CMT1B results from mutations in the Po (peripheral myelin protein zero) gene in 1q22-23. Forty-five percent (7/16) of the published mutations associated with CMT1 occur in exon 3 of Po. In order to determine the cause of CMT1 in 20 unrelated patients without 17p11.2 duplications, mutations were sought in exon 3 of Po with three techniques: nonradioactive SSCP, automated sequencing, and PCR enzymatic restriction. 18 refs., 2 figs.

  18. Seizure-related 6,a brain-specific expression gene,is highly expressed in the human cerebellum

    Institute of Scientific and Technical Information of China (English)

    Jianming Jiang; Long Yu; Yangtai Guan; Zhiliang Yu; Xinghua Huang; Xiaosong Chen; Lisha Tang; Xianning Zhang

    2010-01-01

    Epilepsy is a complex,Mendelian disease,and most cases are sporadic.Genomic comparisons of tissue from identified monogenic epilepsies with multigenic and acquired syndromes could ultimately reveal crucial molecular neuropathology for an epileptic phenotype.In the present study,a novel gene,human seizure-related(hSEZ)-6,was isolated from a human brain cDNA library.hSEZ-6 comprises 17 exons and spans a region of at least 55.6 kb,which was localized to 17q12 by radiation hybridization,hSEZ-6 exhibits two isoform types,hSEZ-6A and hSEZ-6B,which encode996 and 995 amino acids,respectively.The two putative hSEZ-6 proteins contain similar motifs and share 82% and 84% identity with mouse SEZ-6A protein,whose expression level increased in mouse cerebral cortex-derived cells treated with a convulsant drug,pentylentetrazole.Northern blot analysis demonstrated that hSEZ-6 is expressed highly in the cerebellum and in nucleus of the extrapyramidal system,such as the caudate nucleus and putamen.Reverse transcription polymerase chain reaction revealed that hSEZ-6 is expressed in neurons rather than gliocytes,which suggests that hSEZ-6 is a seizure-related gene.

  19. High gene flow due to pelagic larval dispersal among South Pacific archipelagos in two amphidromous gastropods (Neritomorpha: Neritidae).

    Science.gov (United States)

    Crandall, E D; Taffel, J R; Barber, P H

    2010-06-01

    The freshwater stream fauna of tropical oceanic islands is dominated by amphidromous species, whose larvae are transported to the ocean and develop in the plankton before recruiting back to freshwater habitat as juveniles. Because stream habitat is relatively scarce and unstable on oceanic islands, this life history would seem to favor either the retention of larvae to their natal streams, or the ability to delay metamorphosis until new habitat is encountered. To distinguish between these hypotheses, we used population genetic methods to estimate larval dispersal among five South Pacific archipelagos in two amphidromous species of Neritid gastropod (Neritina canalis and Neripteron dilatatus). Sequence data from mitochondrial cytochrome oxidase I (COI) revealed that neither species is genetically structured throughout the Western Pacific, suggesting that their larvae have a pelagic larval duration (PLD) of at least 8 weeks, longer than many marine species. In addition, the two species have recently colonized isolated Central Pacific archipelagos in three independent events. Since colonization, there has been little or no gene flow between the Western and Central Pacific archipelagos in N. canalis, and high levels of gene flow across the same region in N. dilatatus. Both species show departures from neutrality and recent dates for colonization of the Central Pacific archipelagos, which is consistent with frequent extinction and recolonization of stream populations in this area. Similar results from other amphidromous species suggest that unstable freshwater habitats promote long-distance dispersal capabilities.

  20. Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene.

    Science.gov (United States)

    Costa, Jean-Marc; Cabaret, Odile; Moukoury, Sandrine; Bretagne, Stéphane

    2011-09-01

    Genetic studies of the protozoan parasite Toxoplasma gondii have identified three main distinct types according to virulence in some hosts. Several methods have been developed to differentiate genotypes currently dominated by microsatellite markers targeting single-copy loci. We analyzed the possibility of using the 35-fold repetitive B1 gene via high-resolution melting (HRM) curve analysis. Sequencing of the B1 gene of 14 reference strains (four Type I, six Type II, and four Type III strains) identified 18 single nucleotide polymorphisms (SNP). Primers were designed to amplify eight of them for HRM analysis and for relative quantification of each nucleotide variation using SNaPshot mini-sequencing. Genotyping with five microsatellite markers was performed for comparison. Two to four HRM profiles were obtained depending on the SNP tested. The differences observed relied on the different ratios of nucleotides at the SNP locus as evidenced via SNaPshot mini-sequencing. The three main lineages could be distinguished by using several HRM profiles. Some HRM profiles proved more informative than the analysis based on five microsatellite markers, showing additional differences in Type I and Type II strains. Using HRM analysis, we obtained at least an equally good discrimination of the main lineages than that based on five microsatellite markers.

  1. Cloning and Expression of Highly Pathogenic Avian Influenza Virus Full-Length Nonstructural Gene in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    M. B. Abubakar

    2011-01-01

    Full Text Available Avian influenza (AI is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1 was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1 avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1.

  2. Improvement of L-valine production at high temperature in Brevibacterium flavum by overexpressing ilvEBNrC genes.

    Science.gov (United States)

    Hou, Xiaohu; Ge, Xiangyang; Wu, Di; Qian, He; Zhang, Weiguo

    2012-01-01

    Brevibacterium flavum ATCC14067 was engineered for L: -valine production by overexpression of different ilv genes; the ilvEBN(r)C genes from B. flavum NV128 provided the best candidate for L: -valine production. In traditional fermentation, L: -valine production reached 30.08 ± 0.92 g/L at 31°C in 72 h with a low conversion efficiency of 0.129 g/g. To further improve the L: -valine production and conversion efficiency based on the optimum temperatures of L: -valine biosynthesis enzymes (above 35°C) and the thermotolerance of B. flavum, the fermentation temperature was increased to 34, 37, and 40°C. As a result, higher metabolic rate and L: -valine biosynthesis enzymes activity were obtained at high temperature, and the maximum L: -valine production, conversion efficiency, and specific L: -valine production rate reached 38.08 ± 1.32 g/L, 0.241 g/g, and 0.133 g g(-1) h(-1), respectively, at 37°C in 48 h fermentation. The strategy for enhancing L: -valine production by overexpression of key enzymes in thermotolerant strains may provide an alternative approach to enhance branched-chain amino acids production with other strains.

  3. High-level expression of whiG——A key gene for Streptomyces differentiation in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    谭华荣; 田宇清; 杨海花; 吴畏; 董可宁; K. F. Chater and M. J. Buttner

    1996-01-01

    Six nucleotides located in the region of translation start site of whiG were changed. whiG was amplified by PCR technique. Reformed sequences were determined. This gene was directly subcloned into expression vector pET11c containing strong T7 promoter, and the recombinant plasmid was introduced into E. coli BL21(DE3), which could be induced by IPTG to produce T7 RNA polymerase. The SDS-PAGE result showed that whiG highly expressed in E. coli BL21(DE3), and the yield of whiG product was about 20% of insoluble proteins in cell. whiG product (σwhiG) was further identified by Western blot hybridization after making its antibody. whiG gene was subcloned into Streptomyces plasmid pIJ6021, and then it was introduced into sporulation deficient mutant C71 from Streptomyces coelicolor. The result showed that C71 could restore sporulation and σwhiG has biological functions.

  4. High-Throughput DNA Array for SNP Detection of KRAS Gene Using a Centrifugal Microfluidic Device.

    Science.gov (United States)

    Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    Here, we describe detection of single nucleotide polymorphism (SNP) in genomic DNA samples using a NanoBioArray (NBA) chip. Fast DNA hybridization is achieved in the chip when target DNAs are introduced to the surface-arrayed probes using centrifugal force. Gold nanoparticles (AuNPs) are used to assist SNP detection at room temperature. The parallel setting of sample introduction in the spiral channels of the NBA chip enables multiple analyses on many samples, resulting in a technique appropriate for high-throughput SNP detection. The experimental procedure, including chip fabrication, probe array printing, DNA amplification, hybridization, signal detection, and data analysis, is described in detail.

  5. Expression of genes controlling fat deposition in two genetically diverse beef cattle breeds fed high or low silage diets

    Science.gov (United States)

    2013-01-01

    Background Both genetic background and finishing system can alter fat deposition, thus indicating their influence on adipogenic and lipogenic factors. However, the molecular mechanisms underlying fat deposition and fatty acid composition in beef cattle are not fully understood. This study aimed to assess the effect of breed and dietary silage level on the expression patterns of key genes controlling lipid metabolism in subcutaneous adipose tissue (SAT) and longissimus lumborum (LL) muscle of cattle. To that purpose, forty bulls from two genetically diverse Portuguese bovine breeds with distinct maturity rates, Alentejana and Barrosã, were selected and fed either low (30% maize silage/70% concentrate) or high silage (70% maize silage/30% concentrate) diets. Results The results suggested that enhanced deposition of fatty acids in the SAT from Barrosã bulls, when compared to Alentejana, could be due to higher expression levels of lipogenesis (SCD and LPL) and β-oxidation (CRAT) related genes. Our results also indicated that SREBF1 expression in the SAT is increased by feeding the low silage diet. Together, these results point out to a higher lipid turnover in the SAT of Barrosã bulls when compared to Alentejana. In turn, lipid deposition in the LL muscle is related to the expression of adipogenic (PPARG and FABP4) and lipogenic (ACACA and SCD) genes. The positive correlation between ACACA expression levels and total lipids, as well trans fatty acids, points to ACACA as a major player in intramuscular deposition in ruminants. Moreover, results reinforce the role of FABP4 in intramuscular fat development and the SAT as the major site for lipid metabolism in ruminants. Conclusions Overall, the results showed that SAT and LL muscle fatty acid composition are mostly dependent on the genetic background. In addition, dietary silage level impacted on muscle lipid metabolism to a greater extent than on that of SAT, as evaluated by gene expression levels of adipogenic and

  6. The Effect of a High-Protein Diet and Exercise on Cardiac AQP7 and GLUT4 Gene Expression.

    Science.gov (United States)

    Palabiyik, Orkide; Karaca, Aziz; Taştekin, Ebru; Yamasan, Bilge Eren; Tokuç, Burcu; Sipahi, Tammam; Vardar, Selma Arzu

    2016-10-01

    High-protein (HP) diets are commonly consumed by athletes despite their potential health hazard, which is postulated to enforce a negative effect on bone and renal health. However, its effects on heart have not been known yet. Aquaporin-7 (AQP7) is an aquaglyceroporin that facilitates glycerol and water transport. Glycerol is an important cardiac energy production substrate, especially during exercise, in conjunction with fatty acids and glucose. Glucose transporter 4 (GLUT4) is an insulin-sensitive glucose transporter in heart. We aimed to investigate the effect of HPD on AQP7 and GLUT4 levels in the rat heart subjected to exercise. Male Sprague-Dawley rats were divided into control (n = 12), exercise (E) training (n = 10), HPD (n = 12), and HPD-E training (n = 9) groups. The HPD groups were fed a 45 % protein-containing diet 5 weeks. The HPD-E and E groups were performed the treadmill exercise during the 5-week study period. Real-time polymerase chain reaction and immunohistochemistry techniques were used to determine the gene expression and localization of AQP7 and GLUT4 in heart tissue. Results of relative gene expression were calculated by the 'Pfaffl' mathematical method using the REST program. Differences in AQP7 and GLUT4 gene expression were expressed as fold change compared to the control group. Heart weight/tibia ratio and ventricular wall thickness were evaluated as markers of cardiac hypertrophy. Further, serum glucose, glycerol, and insulin levels were also measured. AQP7 gene expression was found to be increased in the E (3.47-fold, p GLUT4 showed a significant increase in the E (2.16-fold, p GLUT4 protein expression was significantly increased in the E, HPD, and HPD-E groups compared to the control group (p = 0.024, p GLUT4 expression in rat heart.

  7. High levan accumulation in transgenic tobacco plants expressing the Gluconacetobacter diazotrophicus levansucrase gene.

    Science.gov (United States)

    Banguela, Alexander; Arrieta, Juan G; Rodríguez, Raisa; Trujillo, Luis E; Menéndez, Carmen; Hernández, Lázaro

    2011-06-10

    Bacterial levansucrase (EC 2.4.1.10) converts sucrose into non-linear levan consisting of long β(2,6)-linked fructosyl chains with β(2,1) branches. Bacterial levan has wide food and non-food applications, but its production in industrial reactors is costly and low yielding. Here, we report the constitutive expression of Gluconacetobacter diazotrophicus levansucrase (LsdA) fused to the vacuolar targeting pre-pro-peptide of onion sucrose:sucrose 1-fructosyltransferase (1-SST) in tobacco, a crop that does not naturally produce fructans. In the transgenic plants, levan with degree of polymerization above 10(4) fructosyl units was detected in leaves, stem, root, and flowers, but not in seeds. High levan accumulation in leaves led to gradual phenotypic alterations that increased with plant age through the flowering stage. In the transgenic lines, the fructan content in mature leaves varied from 10 to 70% of total dry weight. No oligofructans were stored in the plant organs, although the in vitro reaction of transgenic LsdA with sucrose yielded β(2,1)-linked FOS and levan. Transgenic lines with levan representing up to 30mgg(-1) of fresh leaf weight produced viable seeds and the polymer accumulation remained stable in the tested T1 and T2 progenies. The lsdA-expressing tobacco represents an alternative source of highly polymerized levan.

  8. Identification of Differential Gene Expression Patterns after Acute Exposure to High and Low Doses of Low-LET Ionizing Radiation in a Reconstituted Human Skin Tissue

    Energy Technology Data Exchange (ETDEWEB)

    Tilton, Susan C.; Markillie, Lye Meng; Hays, Spencer; Taylor, Ronald C.; Stenoien, David L.

    2016-11-01

    Our goal here was to identify dose and temporal dependent radiation responses in a complex tissue, reconstituted human skin. Direct sequencing of RNA (RNA-seq) was used to quantify altered transcripts following exposure to 0.1, 2 and 10 Gy of ionizing radiation at 3 and 8 hours. These doses include a low dose in the range of some medical diagnostic procedures (0.1 Gy), a dose typically received during radiotherapy (2.0 Gy) and a lethal dose (10 Gy). These doses could be received after an intentional or accidental radiation exposure and biomarkers