WorldWideScience

Sample records for 53r gene highly

  1. Clofarabine Has Apoptotic Effect on T47D Breast Cancer Cell Line via P53R2 Gene Expression

    OpenAIRE

    Mohammad Rahmati-Yamchi; Nosratollah Zarghami; Hojjatollah Nozad Charoudeh; Yasin Ahmadi; Behzad Baradaran; Mohammad Khalaj-Kondori; Morteza Milani; Abolfazl Akbarzadeh; Maghsud Shaker; Mohammad Pourhassan-Moghaddam

    2015-01-01

    Purpose: Clofarabine, a purine nucleoside analogue and inhibitor of Ribonucleotide Reductase (RR), is used for treatment of leukemia. Clofarabine-induced defect in DNA replication, induces p53 and subsequently P53R2 genes as subunit of RR. clofarabine deregulated P53R2 gene expression leading to the elevated levels of P53R2 which impose resistance to DNA damaging drugs. In this study the apoptotic and cytotoxic effects of clofarabine has been investigated on breast cancer ce...

  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

    Directory of Open Access Journals (Sweden)

    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. Synergism between clofarabine and decitabine through p53R2: A pharmacodynamic drug-drug interaction modeling

    OpenAIRE

    Thudium, Karen E.; Ghoshal, Sampa; Fetterly, Gerald J.; Den Haese, Jason P.; Karpf, Adam R.; Wetzler, Meir

    2012-01-01

    Clofarabine (CLO), a purine nucleoside analog with promising efficacy in acute myeloid leukemia (AML), inhibits the ribonucleotide reductase, p53R2. We have shown that p53R2 mRNA is up-regulated by decitabine (DEC), another drug with promising activity in AML. We developed a pharmacodynamic model to characterize the interaction between CLO and DEC on an AML cell line and down-regulated p53R2 protein to understand its role. These results confirm a role for p53R2 in both CLO and DEC mechanism o...

  5. MEK2 regulates ribonucleotide reductase activity through functional interaction with ribonucleotide reductase small subunit p53R2.

    Science.gov (United States)

    Piao, Chunmei; Youn, Cha-Kyung; Jin, Min; Yoon, Sang Pil; Chang, In-Youb; Lee, Jung Hee; You, Ho Jin

    2012-09-01

    The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRNA, dramatically decreased the serum-stimulated RNR activity. Moreover, p53R2 siRNA, but not R2 siRNA, significantly inhibits serum-stimulated RNR activity, indicating that p53R2 is specifically regulated by a MEK2-dependent pathway. Co-immunoprecipitation analyses revealed that the MEK2 segment comprising amino acids 65-171 is critical for p53R2-MEK2 interaction, and the binding domain of MEK2 is required for MEK2-mediated increased RNR activity. Phosphorylation of MEK1/2 was greatly augmented by ionizing radiation, and RNR activity was concurrently increased. Ionizing radiation-induced RNR activity was markedly attenuated by transfection of MEK2 or p53R2 siRNA, but not R2 siRNA. These data show that MEK2 is an endogenous regulator of p53R2 and suggest that MEK2 may associate with p53R2 and upregulate its activity. PMID:22895183

  6. MEK2 regulates ribonucleotide reductase activity through functional interaction with ribonucleotide reductase small subunit p53R2

    OpenAIRE

    Piao, Chunmei; Youn, Cha-Kyung; Jin, Min; Yoon, Sang Pil; Chang, In-Youb; Lee, Jung Hee; You, Ho Jin

    2012-01-01

    The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRN...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

  9. 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.

  10. 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.

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    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/.

  13. Mitochondrial tRNA gene translocations in highly eusocial bees

    Directory of Open Access Journals (Sweden)

    Daniela Silvestre

    2006-01-01

    Full Text Available Mitochondrial gene rearrangement events, especially involving tRNA genes, have been described more frequently as more complete mitochondrial genome sequences are becoming available. In the present work, we analyzed mitochondrial tRNA gene rearrangements between two bee species belonging to the tribes Apini and Meliponini within the "corbiculate Apidae". Eleven tRNA genes are in different genome positions or strands. The molecular events responsible for each translocation are explained. Considering the high number of rearrangements observed, the data presented here contradict the general rule of high gene order conservation among closely related organisms, and also represent a powerful molecular tool to help solve questions about phylogeny and evolution in bees.

  14. Genes related to high temperature tolerance during maize seed germination.

    Science.gov (United States)

    Dutra, S M F; Von Pinho, E V R; Santos, H O; Lima, A C; Von Pinho, R G; Carvalho, M L M

    2015-01-01

    The identification of genes related to heat tolerance is fundamental for the development of high-quality seeds that are tolerant to heat stress condition. The objective of this study was to evaluate maize lineages and the gene expression involved in high temperature tolerance during germination using physiological tests, proteomics, and transcriptome analysis. Seeds from six maize lineages (30, 44, 54, 63, 64, and 91) with different levels of tolerance to high temperatures were used. Lineages 54 and 91 were observed to be more tolerant to high temperature conditions. The highest expression of α-amylase was observed in maize seeds from lineages 30 and 91 that were subjected to controlled deterioration. The highest expression of α-amylase was observed in maize seeds from lineages 30 and 91 that were subjected to controlled deterioration; with the controlled deterioration, the highest level of gene expression did not occur in the most tolerant materials; the association of lower expression of genes involved in heat-resistant protein systems was observed in seeds from lineage 44, which were more susceptible to high temperatures, and the highest gene expression of LEA D-34, ZmAN13, and AOX-1 was observed in seeds from lineage 64 when submitted to controlled deterioration. PMID:26782452

  15. The human VH3b gene subfamily is highly polymorphic

    Energy Technology Data Exchange (ETDEWEB)

    Adderson, E.E.; Carroll, W.L. (Univ. of Utah, Salt Lake City, UT (United States)); Azmi, F.H.; Wilson, P.M.; Shackelford, P.G. (Washington Univ., St. Louis, MO (United States))

    1993-07-15

    The authors have previously shown that human antibody (Ab) directed against the capsular polysaccharide of the important bacterial pathogen, Haemophilus influenzae type b (Hib) is encoded by a small group of VH3 gene family members. The majority of anti-Hib PS Ab use members of the smaller VH3b subfamily. To examine directly the available human VH3 repertoire, they have used PCR to amplify and clone candidate germ-line VH3b H chain V region genes from two unrelated subjects from whom anti-Hib polysaccharide mAb had been previously obtained. A single functional VH3b germ-line gene was obtained from one subject. This gene is identical throughout the coding region to the previously identified gene 9.1. Twelve distinct VH3b germ-line sequences, 87.6-99.8% homologous to one another, were obtained from the second subject. One of these genes, LSG1.1, is also identical to the 9.1 germ-line gene, and a second, LSG6.1 is identical to a previously reported cDNA, M85. These germ-line VH3b genes are 82.7-94.1% homologous to rearranged anti-Hib PS VH3b segments obtained from these subjects. These findings further demonstrate that considerable polymorphism of VH segments exists in the human population. Despite the presence of very highly homologous VH elements in the germ line, particular genes are highly conserved within the outbred human population. 52 refs., 4 figs.

  16. Gene expression profiling in peanut using high density oligonucleotide microarrays

    Directory of Open Access Journals (Sweden)

    Burow Mark

    2009-06-01

    Full Text Available Abstract Background Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology. Results We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B, oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes. Conclusion The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues.

  17. Detecting key structural features within highly recombined genes.

    Directory of Open Access Journals (Sweden)

    John E Wertz

    2007-01-01

    Full Text Available 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

  18. 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.

  19. 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

  20. A novel method to identify high order gene-gene interactions in genome-wide association studies: Gene-based MDR

    OpenAIRE

    Oh Sohee; Lee Jaehoon; Kwon Min-Seok; Weir Bruce; Ha Kyooseob; Park Taesung

    2012-01-01

    Abstract Background Because common complex diseases are affected by multiple genes and environmental factors, it is essential to investigate gene-gene and/or gene-environment interactions to understand genetic architecture of complex diseases. After the great success of large scale genome-wide association (GWA) studies using the high density single nucleotide polymorphism (SNP) chips, the study of gene-gene interaction becomes a next challenge. Multifactor dimensionality reduction (MDR) analy...

  1. A mammalianized synthetic nitroreductase gene for high-level expression

    International Nuclear Information System (INIS)

    The nitroreductase/5-(azaridin-1-yl)-2,4-dinitrobenzamide (NTR/CB1954) enzyme/prodrug system is considered as a promising candidate for anti-cancer strategies by gene-directed enzyme prodrug therapy (GDEPT) and has recently entered clinical trials. It requires the genetic modification of tumor cells to express the E. coli enzyme nitroreductase that bioactivates the prodrug CB1954 to a powerful cytotoxin. This metabolite causes apoptotic cell death by DNA interstrand crosslinking. Enhancing the enzymatic NTR activity for CB1954 should improve the therapeutical potential of this enzyme-prodrug combination in cancer gene therapy. We performed de novo synthesis of the bacterial nitroreductase gene adapting codon usage to mammalian preferences. The synthetic gene was investigated for its expression efficacy and ability to sensitize mammalian cells to CB1954 using western blotting analysis and cytotoxicity assays. In our study, we detected cytoplasmic protein aggregates by expressing GFP-tagged NTR in COS-7 cells, suggesting an impaired translation by divergent codon usage between prokaryotes and eukaryotes. Therefore, we generated a synthetic variant of the nitroreductase gene, called ntro, adapted for high-level expression in mammalian cells. A total of 144 silent base substitutions were made within the bacterial ntr gene to change its codon usage to mammalian preferences. The codon-optimized ntro either tagged to gfp or c-myc showed higher expression levels in mammalian cell lines. Furthermore, the ntro rendered several cell lines ten times more sensitive to the prodrug CB1954 and also resulted in an improved bystander effect. Our results show that codon optimization overcomes expression limitations of the bacterial ntr gene in mammalian cells, thereby improving the NTR/CB1954 system at translational level for cancer gene therapy in humans

  2. High pressure-sensitive gene expression in Lactobacillus sanfranciscensis

    Directory of Open Access Journals (Sweden)

    Vogel R.F.

    2005-01-01

    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.

  3. 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.

  4. Conditional Gene Targeting in Mouse High Endothelial Venules

    OpenAIRE

    Kawashima, Hiroto; Hirakawa, Jotaro; Tobisawa, Yuki; Fukuda, Minoru; Saga, Yumiko

    2009-01-01

    High endothelial venules (HEVs) are specialized blood vessels of secondary lymphoid organs composed of endothelial cells with a characteristic cuboidal morphology. Lymphocytes selectively adhere to and migrate across HEVs to initiate immune responses. In this study, we established a novel transgenic mouse line expressing Cre recombinase under the transcriptional control of the gene encoding HEV-expressed sulfotransferase, N-acetylglucosamine-6-O-sulfotransferase 2 (GlcNAc6ST-2), using bacteri...

  5. 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.

  6. 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.

  7. α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (KrasG12D, and KrasG12D/tp53R270H) mice

    Science.gov (United States)

    Verma, Raj Kumar; Yu, Wei; Shrivastava, Anju; Shankar, Sharmila; Srivastava, Rakesh K.

    2016-01-01

    Activation of sonic hedgehog (Shh) in cancer stem cell (CSC) has been demonstrated with aggressiveness of pancreatic cancer. In order to enhance the biological activity of α-mangostin, we formulated mangostin-encapsulated PLGA nanoparticles (Mang-NPs) and examined the molecular mechanisms by which they inhibit human and KC mice (PdxCre;LSL-KrasG12D) pancreatic CSC characteristics in vitro, and pancreatic carcinogenesis in KPC (PdxCre;LSLKrasG12D;LSL-Trp53R172H) mice. Mang-NPs inhibited human and KrasG12D mice pancreatic CSC characteristics in vitro. Mang-NPs also inhibited EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Slug, and pluripotency maintaining factors Nanog, c-Myc, and Oct4. Furthermore, Mang-NPs inhibited the components of Shh pathway and Gli targets. In vivo, Mang-NPs inhibited the progression of pancreatic intraneoplasia to pancreatic ductal adenocarcinoma and liver metastasis in KPC mice. The inhibitory effects of Mang-NPs on carcinogenesis in KPC mice were associated with downregulation of pluripotency maintaining factors (c-Myc, Nanog and Oct4), stem cell markers (CD24 and CD133), components of Shh pathway (Gli1, Gli2, Patched1/2, and Smoothened), Gli targets (Bcl-2, XIAP and Cyclin D1), and EMT markers and transcription factors (N-cadherin, Slug, Snail and Zeb1), and upregulation of E-cadherin. Overall, our data suggest that Mang-NPs can inhibit pancreatic cancer growth, development and metastasis by targeting Shh pathway. PMID:27624879

  8. High-throughput approaches to understanding gene function and mapping network architecture in bacteria.

    Science.gov (United States)

    Brochado, Ana Rita; Typas, Athanasios

    2013-04-01

    Advances in sequencing technology have provided an unprecedented view of bacterial diversity, along with a daunting number of novel genes. Within this new reality lies the challenge of developing large-scale approaches to assign function to the new genes and place them in pathways. Here, we highlight recent advances on this front, focusing on how high-throughput gene-gene, gene-drug and drug-drug interactions can yield functional and mechanistic inferences in bacteria. PMID:23403119

  9. 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.

  10. High Gene Family Turnover Rates and Gene Space Adaptation in the Compact Genome of the Carnivorous Plant Utricularia gibba.

    Science.gov (United States)

    Carretero-Paulet, Lorenzo; Librado, Pablo; Chang, Tien-Hao; Ibarra-Laclette, Enrique; Herrera-Estrella, Luis; Rozas, Julio; Albert, Victor A

    2015-05-01

    Utricularia gibba is an aquatic carnivorous plant with highly specialized morphology, featuring fibrous floating networks of branches and leaf-like organs, no recognizable roots, and bladder traps that capture and digest prey. We recently described the compressed genome of U. gibba as sufficient to control the development and reproduction of a complex organism. We hypothesized intense deletion pressure as a mechanism whereby most noncoding DNA was deleted, despite evidence for three independent whole-genome duplications (WGDs). Here, we explore the impact of intense genome fractionation in the evolutionary dynamics of U. gibba's functional gene space. We analyze U. gibba gene family turnover by modeling gene gain/death rates under a maximum-likelihood statistical framework. In accord with our deletion pressure hypothesis, we show that the U. gibba gene death rate is significantly higher than those of four other eudicot species. Interestingly, the gene gain rate is also significantly higher, likely reflecting the occurrence of multiple WGDs and possibly also small-scale genome duplications. Gene ontology enrichment analyses of U. gibba-specific two-gene orthogroups, multigene orthogroups, and singletons highlight functions that may represent adaptations in an aquatic carnivorous plant. We further discuss two homeodomain transcription factor gene families (WOX and HDG/HDZIP-IV) showing conspicuous differential expansions and contractions in U. gibba. Our results 1) reconcile the compactness of the U. gibba genome with its accommodation of a typical number of genes for a plant genome, and 2) highlight the role of high gene family turnover in the evolutionary diversification of U. gibba's functional gene space and adaptations to its unique lifestyle and highly specialized body plan. PMID:25637935

  11. 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...

  12. Consciousness of evolution and human gene in German high school students

    OpenAIRE

    Kamizono, Kohtaro

    2004-01-01

    An association map of evolution (Vererbung) indicates two meanings, 'money' and 'gene', among high school students in the Rhein district in Germany. What the students know about evolution includes biological words, social development and alteration of planets in transferred meaning. High school students in Germany know more about human gene than Japanese students at Nagasaki University. What high school students know about human gene seems to depend on the media. Over 20% among the testees in...

  13. 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. PMID:26747053

  14. 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.

  15. Gene transcriptional profiles in human lymphoblastoid cells with low and high doses of irradiation

    International Nuclear Information System (INIS)

    Objective: To compare the gene expression difference between 0.1 and 5 Gy X-ray irradiated cells,and to explore its possible mechanism. Methods: A cDNA microarray corresponding to 45033 human genes was used to analyze the transcriptional profiles of normal human lymphoblastoid AHH-1 cells at 4 h after 0.1 or 5 Gy irradiation. The genes with a fold change ≥ 2.0 were identified as the differentially expressed genes. real-lime PCR and Western blot were used to confirm the expression of PERP. Results: The microarray assay showed that there were 760 up-regulated genes and 1222 down-regulated genes in the cells at 0.1 Gy, while there were 744 genes down-regulated and 457 genes up-regulated in the cells at 5 Gy. In addition, 55 genes were commonly up-regulated and 339 genes commonly down-regulated at 0.1 and 5 Gy. The predominant biological processes of the differential genes responding to low-dose radiation include cell-cell signaling transduction and DNA damage response, and the altered genes after 5 Gy irradiation were related to cell proliferation, differentiation, and apoptosis. Moreover, the expression of PERP gene was down regulated, which was consistent with the data of microarray assay. Conclusions: The quantitative and qualitative differences in the gene expressions may contribute to the diverse biological effects induced by low or high doses of ionizing radiation. (authors)

  16. High Order Gene-Gene Interactions in Eight Single Nucleotide Polymorphisms of Renin-Angiotensin System Genes for Hypertension Association Study

    Directory of Open Access Journals (Sweden)

    Cheng-Hong Yang

    2015-01-01

    Full Text Available Several single nucleotide polymorphisms (SNPs of renin-angiotensin system (RAS genes are associated with hypertension (HT but most of them are focusing on single locus effects. Here, we introduce an unbalanced function based on multifactor dimensionality reduction (MDR for multiloci genotypes to detect high order gene-gene (SNP-SNP interaction in unbalanced cases and controls of HT data. Eight SNPs of three RAS genes (angiotensinogen, AGT; angiotensin-converting enzyme, ACE; angiotensin II type 1 receptor, AT1R in HT and non-HT subjects were included that showed no significant genotype differences. In 2- to 6-locus models of the SNP-SNP interaction, the SNPs of AGT and ACE genes were associated with hypertension (bootstrapping odds ratio [Boot-OR] = 1.972~3.785; 95%, confidence interval (CI 1.26~6.21; P<0.005. In 7- and 8-locus model, SNP A1166C of AT1R gene is joined to improve the maximum Boot-OR values of 4.050 to 4.483; CI = 2.49 to 7.29; P<1.63E−08. In conclusion, the epistasis networks are identified by eight SNP-SNP interaction models. AGT, ACE, and AT1R genes have overall effects with susceptibility to hypertension, where the SNPs of ACE have a mainly hypertension-associated effect and show an interacting effect to SNPs of AGT and AT1R genes.

  17. Structural relationships between highly conserved elements and genes in vertebrate genomes.

    Directory of Open Access Journals (Sweden)

    Hong Sun

    Full Text Available Large numbers of sequence elements have been identified to be highly conserved among vertebrate genomes. These highly conserved elements (HCEs are often located in or around genes that are involved in transcription regulation and early development. They have been shown to be involved in cis-regulatory activities through both in vivo and additional computational studies. We have investigated the structural relationships between such elements and genes in six vertebrate genomes human, mouse, rat, chicken, zebrafish and tetraodon and detected several thousand cases of conserved HCE-gene associations, and also cases of HCEs with no common target genes. A few examples underscore the potential significance of our findings about several individual genes. We found that the conserved association between HCE/HCEs and gene/genes are not restricted to elements by their absolute distance on the genome. Notably, long-range associations were identified and the molecular functions of the associated genes do not show any particular overrepresentation of the functional categories previously reported. HCEs in close proximity are found to be linked with different set of gene/genes. The results reflect the highly complex correlation between HCEs and their putative target genes.

  18. Evaluation of high-throughput functional categorization of human disease genes

    Directory of Open Access Journals (Sweden)

    Li Jianrong

    2007-05-01

    Full Text Available Abstract Background Biological data that are well-organized by an ontology, such as Gene Ontology, enables high-throughput availability of the semantic web. It can also be used to facilitate high throughput classification of biomedical information. However, to our knowledge, no evaluation has been published on automating classifications of human diseases genes using Gene Ontology. In this study, we evaluate automated classifications of well-defined human disease genes using their Gene Ontology annotations and compared them to a gold standard. This gold standard was independently conceived by Valle's research group, and contains 923 human disease genes organized in 14 categories of protein function. Results Two automated methods were applied to investigate the classification of human disease genes into independently pre-defined categories of protein function. One method used the structure of Gene Ontology by pre-selecting 74 Gene Ontology terms assigned to 11 protein function categories. The second method was based on the similarity of human disease genes clustered according to the information-theoretic distance of their Gene Ontology annotations. Compared to the categorization of human disease genes found in the gold standard, our automated methods can achieve an overall 56% and 47% precision with 62% and 71% recall respectively. However, approximately 15% of the studied human disease genes remain without GO annotations. Conclusion Automated methods can recapitulate a significant portion of classification of the human disease genes. The method using information-theoretic distance performs slightly better on the precision with some loss in recall. For some protein function categories, such as 'hormone' and 'transcription factor', the automated methods perform particularly well, achieving precision and recall levels above 75%. In summary, this study demonstrates that for semantic webs, methods to automatically classify or analyze a majority of

  19. 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.

  20. A general strategy to achieve ultra-high gene transfection efficiency using lipid-nanoparticle composites.

    Science.gov (United States)

    Vankayala, Raviraj; Chiang, Chi-Shiun; Chao, Jui-I; Yuan, Chiun-Jye; Lin, Shyr-Yeu; Hwang, Kuo Chu

    2014-09-01

    Gene therapy provides a new hope for previously "incurable" diseases. Low gene transfection efficiency, however, is the bottle-neck to the success of gene therapy. It is very challenging to develop non-viral nanocarriers to achieve ultra-high gene transfection efficiencies. Herein, we report a novel design of "tight binding-but-detachable" lipid-nanoparticle composite to achieve ultrahigh gene transfection efficiencies of 60∼82%, approaching the best value (∼90%) obtained using viral vectors. We show that Fe@CNPs nanoparticles coated with LP-2000 lipid molecules can be used as gene carriers to achieve ultra-high (60-80%) gene transfection efficiencies in HeLa, U-87MG, and TRAMP-C1 cells. In contrast, Fe@CNPs having surface-covalently bound N,N,N-trimethyl-N-2-methacryloxyethyl ammonium chloride (TMAEA) oligomers can only achieve low (23-28%) gene transfection efficiencies. Similarly ultrahigh gene transfection/expression was also observed in zebrafish model using lipid-coated Fe@CNPs as gene carriers. Evidences for tight binding and detachability of DNA from lipid-nanoparticle nanocarriers will be presented. PMID:24973297

  1. 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

    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 17β-estradiol, equol, Tween 80, and milk high and low in isoflavone content for a week. Gene expression was analyzed using an array q......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...

  2. 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.

  3. 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...

  4. 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 provides...

  5. HIPMap: A High-Throughput Imaging Method for Mapping Spatial Gene Positions.

    Science.gov (United States)

    Shachar, Sigal; Pegoraro, Gianluca; Misteli, Tom

    2015-01-01

    The three-dimensional organization of genes inside the cell nucleus affects their functions including DNA transcription, replication, and repair. A major goal in the field of nuclear architecture is to determine what cellular factors establish and maintain the position of individual genes. Here, we describe HIPMap, a high-throughput imaging and analysis pipeline for the mapping of endogenous gene loci within the 3D space of the nucleus. HIPMap can be used for a variety of applications including screening, mapping translocations, validating chromosome conformation capture data, probing DNA-protein interactions, and interrogation of the relationship of gene expression with localization. PMID:26472748

  6. Highly Expressed Genes within Hippocampal Sector CA1: Implications for the Physiology of Memory

    OpenAIRE

    Michael A. Meyer

    2014-01-01

    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 id...

  7. 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.

  8. Gene expression in mammalian cells after exposure to 95 MeV/amu argon ions

    Science.gov (United States)

    Arenz, Andrea; Hellweg, Christine E.; Meier, Matthias M.; Baumstark-Khan, Christa

    High LET radiations, such as heavy ions or neutrons, have an increased biological effectiveness compared to X-rays for gene mutation, genomic instability and carcinogenesis. Estimating the biological risks from space radiation encountered by cosmonauts will continue to influence long term duration in space, such as the planned mission to Mars. The human radiation responsive genes CDKN1A (p21/WAF), GADD45α (GADD45), GADD45β (MyD118), RRM2b (p53R2) and BRCA2 (FancD1), involved in cell cycle control or damage repair, were screened for gene expression changes in MCF-7 cells by quantitative real-time reverse transcription PCR (qRT-PCR) assay, using cDNA obtained from total RNA isolated at various time points after irradiation with accelerated doses of 36-argon ions and X-rays. Examination of the expression profiles 2 and 12 h after exposure reveals a pattern consistent with a population of cells in the early response to DNA damage and invoking cell stress responses. Interesting new data showing different expression patterns according to the gene and the type of ionizing radiation used could be obtained. Results show, that the signaling and repair activities induced after heavy ion or X-ray exposure are not the same and gene expression patterns may become useful indicators for distinguishing different types of radiation in relation to their biological effects.

  9. Electronic Sorting of Immune Cell Subpopulations Based on Highly Plastic Genes.

    Science.gov (United States)

    Wang, Pingzhang; Han, Wenling; Ma, Dalong

    2016-07-15

    Immune cells are highly heterogeneous and plastic with regard to gene expression and cell phenotype. In this study, we categorized genes into those with low and high gene plasticity, and those categories revealed different functions and applications. We proposed that highly plastic genes could be suited for the labeling of immune cell subpopulations; thus, novel immune cell subpopulations could be identified by gene plasticity analysis. For this purpose, we systematically analyzed highly plastic genes in human and mouse immune cells. In total, 1,379 human and 883 mouse genes were identified as being extremely plastic. We also expanded our previous immunoinformatic method, electronic sorting, which surveys big data to perform virtual analysis. This approach used correlation analysis and took dosage changes into account, which allowed us to identify the differentially expressed genes. A test with human CD4(+) T cells supported the method's feasibility, effectiveness, and predictability. For example, with the use of human nonregulatory T cells, we found that FOXP3(hi)CD4(+) T cells were highly expressive of certain known molecules, such as CD25 and CTLA4, and that this process of investigation did not require isolating or inducing these immune cells in vitro. Therefore, the sorting process helped us to discover the potential signature genes or marker molecules and to conduct functional evaluations for immune cell subpopulations. Finally, in human CD4(+) T cells, 747 potential immune cell subpopulations and their candidate signature genes were identified, which provides a useful resource for big data-driven knowledge discoveries. PMID:27288532

  10. 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.

  11. 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

  12. High-throughput Microarray Detection of Vomeronasal Receptor Gene Expression in Rodents

    Directory of Open Access Journals (Sweden)

    Xiaohong Zhang

    2010-11-01

    Full Text Available We performed comprehensive data mining to explore the vomeronasal receptor (V1R & V2R repertoires in mouse and rat using the mm5 and rn3 genome, respectively. This bioinformatic analysis was followed by investigation of gene expression using a custom designed high-density oligonucleotide array containing all of these receptors and other selected genes of interest. This array enabled us to detect the specific expression of V1R and V2Rs which were previously identified solely based on computational prediction from gene sequence data, thereby establishing that these genes are indeed part of the vomeronasal system, especially the V2Rs. 168 V1Rs and 98 V2Rs were detected to be highly enriched in mouse vomeronasal organ (VNO, and 108 V1Rs and 87 V2Rs in rat VNO. We monitored the expression profile of mouse VR genes in other non-VNO tissues with the result that some VR genes were re-designated as VR-like genes based on their non-olfactory expression pattern. Temporal expression profiles for mouse VR genes were characterized and their patterns were classified, revealing the developmental dynamics of these so-called pheromone receptors. We found numerous patterns of temporal expression which indicate possible behavior-related functions. The uneven composition of VR genes in certain patterns suggests a functional differentiation between the two types of VR genes. We found the coherence between VR genes and transcription factors in terms of their temporal expression patterns. In situ hybridization experiments were performed to evaluate the cell number change over time for selected receptor genes.

  13. 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

    2016-01-01

    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. PMID:27154141

  14. 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....

  15. 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

  16. 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

  17. 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.

  18. Altered gene expression in highly purified enterocytes from patients with active coeliac disease

    Directory of Open Access Journals (Sweden)

    Jackson John

    2008-08-01

    Full Text Available Abstract Background Coeliac disease is a multifactorial inflammatory disorder of the intestine caused by ingestion of gluten in genetically susceptible individuals. Genes within the HLA-DQ locus are considered to contribute some 40% of the genetic influence on this disease. However, information on other disease causing genes is sparse. Since enterocytes are considered to play a central role in coeliac pathology, the aim of this study was to examine gene expression in a highly purified isolate of these cells taken from patients with active disease. Epithelial cells were isolated from duodenal biopsies taken from five coeliac patients with active disease and five non-coeliac control subjects. Contaminating T cells were removed by magnetic sorting. The gene expression profile of the cells was examined using microarray analysis. Validation of significantly altered genes was performed by real-time RT-PCR and immunohistochemistry. Results Enterocyte suspensions of high purity (98–99% were isolated from intestinal biopsies. Of the 3,800 genes investigated, 102 genes were found to have significantly altered expression between coeliac disease patients and controls (p Conclusion This study provides a profile of the molecular changes that occur in the intestinal epithelium of coeliac patients with active disease. Novel candidate genes were revealed which highlight the contribution of the epithelial cell to the pathogenesis of coeliac disease.

  19. 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.

  20. 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

  1. Lung gene therapy with highly compacted DNA nanoparticles that overcome the mucus barrier.

    Science.gov (United States)

    Suk, Jung Soo; Kim, Anthony J; Trehan, Kanika; Schneider, Craig S; Cebotaru, Liudmila; Woodward, Owen M; Boylan, Nicholas J; Boyle, Michael P; Lai, Samuel K; Guggino, William B; Hanes, Justin

    2014-03-28

    Inhaled gene carriers must penetrate the highly viscoelastic and adhesive mucus barrier in the airway in order to overcome rapid mucociliary clearance and reach the underlying epithelium; however, even the most widely used viral gene carriers are unable to efficiently do so. We developed two polymeric gene carriers that compact plasmid DNA into small and highly stable nanoparticles with dense polyethylene glycol (PEG) surface coatings. These highly compacted, densely PEG-coated DNA nanoparticles rapidly penetrate human cystic fibrosis (CF) mucus ex vivo and mouse airway mucus ex situ. Intranasal administration of the mucus penetrating DNA nanoparticles greatly enhanced particle distribution, retention and gene transfer in the mouse lung airways compared to conventional gene carriers. Successful delivery of a full-length plasmid encoding the cystic fibrosis transmembrane conductance regulator protein was achieved in the mouse lungs and airway cells, including a primary culture of mucus-covered human airway epithelium grown at air-liquid interface, without causing acute inflammation or toxicity. Highly compacted mucus penetrating DNA nanoparticles hold promise for lung gene therapy. PMID:24440664

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Cyclen-Based Cationic Lipids for Highly Efficient Gene Delivery towards Tumor Cells

    OpenAIRE

    Huang, Qing-Dong; Zhong, Guo-Xing; Zhang, Yang; Ren, Jiang; Fu, Yun; Zhang, Ji; ZHU, WEN; Yu, Xiao-Qi

    2011-01-01

    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 plas...

  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)

    Jakočiūnė, Dzuiga; 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. A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes

    Directory of Open Access Journals (Sweden)

    Craig W. Herbold

    2015-07-01

    Full Text Available High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse and high quality sets of amplicon sequence data for modern studies in microbial ecology.

  12. 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,...

  13. 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. PMID:26462458

  14. Identification of gene mutation in patients with osteogenesis imperfect using high resolution melting analysis.

    Science.gov (United States)

    Wang, Jianhai; Ren, Xiuzhi; Bai, Xue; Zhang, Tianke; Wang, Yi; Li, Keqiu; Li, Guang

    2015-01-01

    Osteogenesis imperfecta (OI), a congenital bone disorder, is caused by mutations in COL1A1 and COL1A2 genes, leading to deficiency of type I collagen. The high resolution melting (HRM) analysis has been used for detecting mutations, polymorphisms and epigenetic alteration in double-stranded DNAs. This study was to evaluate the potential application of HRM analysis for identifying gene mutations in patients with OI. This study included four children with OI and their parents and fifty normal people as controls. Blood samples were collected for HRM analysis of PCR-amplified exons and flanking DNA sequences of COL1A1 and COL1A2 genes. Direct gene sequencing was performed to validate HRM-identified gene mutations. As compared to controls, HRM analysis of samples form children with OI showed abnormal melting curves in exons 11 and 33-34 of the COL1A1 gene and exons 19 and 48 of the COL1A2 gene, which indicates the presence of heterozygous mutations in COL1A1 and COL1A2 genes. In addition to two known mutations in the COL1A2 gene, c.982G > A and c.3197G > T, sequencing analysis identified two novel mutations in the COL1A1 gene, c.2321delC and c.768dupC mutations, which function as premature stop codons. These results support future studies of applying HRM analysis as a diagnostic approach for OI. PMID:26307460

  15. Active maize genes are unmodified and flanked by diverse classes of modified, highly repetitive DNA.

    Science.gov (United States)

    Bennetzen, J L; Schrick, K; Springer, P S; Brown, W E; SanMiguel, P

    1994-08-01

    We have characterized the copy number, organization, and genomic modification of DNA sequences within and flanking several maize genes. We found that highly repetitive DNA sequences were tightly linked to most of these genes. The highly repetitive sequences were not found within the coding regions but could be found within 6 kb either 3' or 5' to the structural genes. These highly repetitive regions were each composed of unique combinations of different short repetitive sequences. Highly repetitive DNA blocks were not interrupted by any detected single copy DNA. The 13 classes of highly repetitive DNA identified were found to vary little between diverse Zea isolates. The level of DNA methylation in and near these genes was determined by scoring the digestibility of 63 recognition/cleavage sites with restriction enzymes that were sensitive to 5-methylation of cytosines in the sequences 5'-CG-3' and 5'-CNG-3'. All but four of these sites were digestible in chromosomal DNA. The four undigested sites were localized to extragenic DNA within or near highly repetitive DNA, while the other 59 sites were in low copy number DNAs. Pulsed field gel analysis indicated that the majority of cytosine modified tracts range from 20 to 200 kb in size. Single copy sequences hybridized to the unmodified domains, while highly repetitive sequences hybridized to the modified regions. Middle repetitive sequences were found in both domains. PMID:7958822

  16. 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.

  17. 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.

  18. 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.

  19. 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. PMID:27276034

  20. 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.

  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. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

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

    International Nuclear Information System (INIS)

    Isoflavones have generated much attention due to their potential positive effects in various diseases. Phyto estrogens especially equol can be found in bovine milk, as feed ration for dairy cows is comprised of plants containing phyto estrogens. 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 17βestradiol, 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 17β-estradiol up regulated 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.

  8. 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

  9. 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.

  10. 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.

  11. Preventing High Fat Diet-induced Obesity and Improving Insulin Sensitivity through Neuregulin 4 Gene Transfer.

    Science.gov (United States)

    Ma, Yongjie; Gao, Mingming; Liu, Dexi

    2016-01-01

    Neuregulin 4 (NRG4), an epidermal growth factor-like signaling molecule, plays an important role in cell-to-cell communication during tissue development. Its function to regulate energy metabolism has recently been reported. This current study was designed to assess the preventive and therapeutic effects of NRG4 overexpression on high fat diet (HFD)-induced obesity. Using the hydrodynamic gene transfer method, we demonstrate that Nrg4 gene transfer in mice suppressed the development of diet-induced obesity, but did not affect pre-existing adiposity and body weight in obese mice. Nrg4 gene transfer curbed HFD-induced hepatic steatosis by inhibiting lipogenesis and PPARγ-mediated lipid storage. Concurrently, overexpression of NRG4 reduced chronic inflammation in both preventive and treatment studies, evidenced by lower mRNA levels of macrophage marker genes including F4/80, Cd68, Cd11b, Cd11c, and macrophage chemokine Mcp1, resulting in improved insulin sensitivity. Collectively, these results demonstrate that overexpression of the Nrg4 gene by hydrodynamic gene delivery prevents HFD-induced weight gain and fatty liver, alleviates obesity-induced chronic inflammation and insulin resistance, and supports the health benefits of NRG4 in managing obesity and obesity-associated metabolic disorders. PMID:27184920

  12. Putative cis-regulatory elements in genes highly expressed in rice sperm cells

    Directory of Open Access Journals (Sweden)

    Singh Mohan B

    2011-09-01

    Full Text Available Abstract Background The male germ line in flowering plants is initiated within developing pollen grains via asymmetric division. The smaller cell then becomes totally encased within a much larger vegetative cell, forming a unique "cell within a cell structure". The generative cell subsequently divides to give rise to two non-motile diminutive sperm cells, which take part in double fertilization and lead to the seed set. Sperm cells are difficult to investigate because of their presence within the confines of the larger vegetative cell. However, recently developed techniques for the isolation of rice sperm cells and the fully annotated rice genome sequence have allowed for the characterization of the transcriptional repertoire of sperm cells. Microarray gene expression data has identified a subset of rice genes that show unique or highly preferential expression in sperm cells. This information has led to the identification of cis-regulatory elements (CREs, which are conserved in sperm-expressed genes and are putatively associated with the control of cell-specific expression. Findings We aimed to identify the CREs associated with rice sperm cell-specific gene expression data using in silico prediction tools. We analyzed 1-kb upstream regions of the top 40 sperm cell co-expressed genes for over-represented conserved and novel motifs. Analysis of upstream regions with the SIGNALSCAN program with the PLACE database, MEME and the Mclip tool helped to find combinatorial sets of known transcriptional factor-binding sites along with two novel motifs putatively associated with the co-expression of sperm cell-specific genes. Conclusions Our data shows the occurrence of novel motifs, which are putative CREs and are likely targets of transcriptional factors regulating sperm cell gene expression. These motifs can be used to design the experimental verification of regulatory elements and the identification of transcriptional factors that regulate sperm cell

  13. 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)

  14. 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

    2013-01-01

    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. PMID:23630316

  15. 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...... 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...

  16. Discovering biclusters in gene expression data based on high-dimensional linear geometries

    Directory of Open Access Journals (Sweden)

    Liew Alan

    2008-04-01

    Full Text Available Abstract Background In DNA microarray experiments, discovering groups of genes that share similar transcriptional characteristics is instrumental in functional annotation, tissue classification and motif identification. However, in many situations a subset of genes only exhibits consistent pattern over a subset of conditions. Conventional clustering algorithms that deal with the entire row or column in an expression matrix would therefore fail to detect these useful patterns in the data. Recently, biclustering has been proposed to detect a subset of genes exhibiting consistent pattern over a subset of conditions. However, most existing biclustering algorithms are based on searching for sub-matrices within a data matrix by optimizing certain heuristically defined merit functions. Moreover, most of these algorithms can only detect a restricted set of bicluster patterns. Results In this paper, we present a novel geometric perspective for the biclustering problem. The biclustering process is interpreted as the detection of linear geometries in a high dimensional data space. Such a new perspective views biclusters with different patterns as hyperplanes in a high dimensional space, and allows us to handle different types of linear patterns simultaneously by matching a specific set of linear geometries. This geometric viewpoint also inspires us to propose a generic bicluster pattern, i.e. the linear coherent model that unifies the seemingly incompatible additive and multiplicative bicluster models. As a particular realization of our framework, we have implemented a Hough transform-based hyperplane detection algorithm. The experimental results on human lymphoma gene expression dataset show that our algorithm can find biologically significant subsets of genes. Conclusion We have proposed a novel geometric interpretation of the biclustering problem. We have shown that many common types of bicluster are just different spatial arrangements of hyperplanes in

  17. SNP-based high density genetic map and mapping of btwd1 dwarfing gene in barley.

    Science.gov (United States)

    Ren, Xifeng; Wang, Jibin; Liu, Lipan; Sun, Genlou; Li, Chengdao; Luo, Hong; Sun, Dongfa

    2016-01-01

    A high-density linkage map is a valuable tool for functional genomics and breeding. A newly developed sequence-based marker technology, restriction site associated DNA (RAD) sequencing, has been proven to be powerful for the rapid discovery and genotyping of genome-wide single nucleotide polymorphism (SNP) markers and for the high-density genetic map construction. The objective of this research was to construct a high-density genetic map of barley using RAD sequencing. 1894 high-quality SNP markers were developed and mapped onto all seven chromosomes together with 68 SSR markers. These 1962 markers constituted a total genetic length of 1375.8 cM and an average of 0.7 cM between adjacent loci. The number of markers within each linkage group ranged from 209 to 396. The new recessive dwarfing gene btwd1 in Huaai 11 was mapped onto the high density linkage maps. The result showed that the btwd1 is positioned between SNP marks 7HL_6335336 and 7_249275418 with a genetic distance of 0.9 cM and 0.7 cM on chromosome 7H, respectively. The SNP-based high-density genetic map developed and the dwarfing gene btwd1 mapped in this study provide critical information for position cloning of the btwd1 gene and molecular breeding of barley. PMID:27530597

  18. High-throughput Microarray Detection of Vomeronasal Receptor Gene Expression in Rodents

    OpenAIRE

    FlorenciaMarcucci

    2010-01-01

    We performed comprehensive data mining to explore the vomeronasal receptor (V1R & V2R) repertoires in mouse and rat using the mm5 and rn3 genome, respectively. This bioinformatic analysis was followed by investigation of gene expression using a custom designed high-density oligonucleotide array containing all of these receptors and other selected genes of interest. This array enabled us to detect the specific expression of V1R and V2Rs which were previously identified solely based on computa...

  19. High-Throughput Microarray Detection of Vomeronasal Receptor Gene Expression in Rodents

    OpenAIRE

    Zhang, Xiaohong; Marcucci, Florencia; Firestein, Stuart

    2010-01-01

    We performed comprehensive data mining to explore the vomeronasal receptor (V1R and V2R) repertoires in mouse and rat using the mm5 and rn3 genome, respectively. This bioinformatic analysis was followed by investigation of gene expression using a custom designed high-density oligonucleotide array containing all of these receptors and other selected genes of interest. This array enabled us to detect the specific expression of V1R and V2Rs which were previously identified solely based on comput...

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

    OpenAIRE

    Guo, Bingfu; Guo, Yong; Hong, Huilong; Jin, Longguo; Zhang, Lijuan; Chang, Ru-Zhen; Lu, Wei; Lin, Min; Qiu, Li-Juan

    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-...

  1. 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...... have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting...... knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer....

  2. 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

    2014-01-01

    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. PMID:25034029

  3. 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.

  4. 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.

  5. Evolutionary conservation of essential and highly expressed genes in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Scharfe Maren

    2010-04-01

    Full Text Available Abstract Background The constant increase in development and spread of bacterial resistance to antibiotics poses a serious threat to human health. New sequencing technologies are now on the horizon that will yield massive increases in our capacity for DNA sequencing and will revolutionize the drug discovery process. Since essential genes are promising novel antibiotic targets, the prediction of gene essentiality based on genomic information has become a major focus. Results In this study we demonstrate that pooled sequencing is applicable for the analysis of sequence variations of strain collections with more than 10 individual isolates. Pooled sequencing of 36 clinical Pseudomonas aeruginosa isolates revealed that essential and highly expressed proteins evolve at lower rates, whereas extracellular proteins evolve at higher rates. We furthermore refined the list of experimentally essential P. aeruginosa genes, and identified 980 genes that show no sequence variation at all. Among the conserved nonessential genes we found several that are involved in regulation, motility and virulence, indicating that they represent factors of evolutionary importance for the lifestyle of a successful environmental bacterium and opportunistic pathogen. Conclusion The detailed analysis of a comprehensive set of P. aeruginosa genomes in this study clearly disclosed detailed information of the genomic makeup and revealed a large set of highly conserved genes that play an important role for the lifestyle of this microorganism. Sequencing strain collections enables for a detailed and extensive identification of sequence variations as potential bacterial adaptation processes, e.g., during the development of antibiotic resistance in the clinical setting and thus may be the basis to uncover putative targets for novel treatment strategies.

  6. Morphological Profiles of RNAi-Induced Gene Knockdown Are Highly Reproducible but Dominated by Seed Effects.

    Directory of Open Access Journals (Sweden)

    Shantanu Singh

    Full Text Available RNA interference and morphological profiling-the measurement of thousands of phenotypes from individual cells by microscopy and image analysis-are a potentially powerful combination. We show that morphological profiles of RNAi-induced knockdown using the Cell Painting assay are in fact highly sensitive and reproducible. However, we find that the magnitude and prevalence of off-target effects via the RNAi seed-based mechanism make morphological profiles of RNAi reagents targeting the same gene look no more similar than reagents targeting different genes. Pairs of RNAi reagents that share the same seed sequence produce image-based profiles that are much more similar to each other than profiles from pairs designed to target the same gene, a phenomenon previously observed in small-scale gene-expression profiling experiments. Various strategies have been used to enrich on-target versus off-target effects in the context of RNAi screening where a narrow set of phenotypes are measured, mostly based on comparing multiple sequences targeting the same gene; however, new approaches will be needed to make RNAi morphological profiling (that is, comparing multi-dimensional phenotypes viable. We have shared our raw data and computational pipelines to facilitate research.

  7. 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.

  8. dNTP Supply Gene Expression Patterns after P53 Loss

    Energy Technology Data Exchange (ETDEWEB)

    Radivoyevitch, Tomas, E-mail: txr24@case.edu [Departments of Epidemiology and Biostatistics, General Medical Sciences (Oncology), and Pathology, Case Western Reserve School of Medicine, Cleveland, OH 44106 (United States); Saunthararajah, Yogen [Department of Translational Hematology & Oncology Research, Taussig Cancer Institute, Cleveland Clinic, 9500 Euclid Ave. R40, Cleveland, OH 44195 (United States); Pink, John [Departments of Epidemiology and Biostatistics, General Medical Sciences (Oncology), and Pathology, Case Western Reserve School of Medicine, Cleveland, OH 44106 (United States); Ferris, Gina [Department of Radiation Oncology, University Hospitals Case Medical Center and Case Western Reserve School of Medicine, Cleveland, OH 44106 (United States); Lent, Ian; Jackson, Mark; Junk, Damian [Departments of Epidemiology and Biostatistics, General Medical Sciences (Oncology), and Pathology, Case Western Reserve School of Medicine, Cleveland, OH 44106 (United States); Kunos, Charles A. [Department of Radiation Oncology, University Hospitals Case Medical Center and Case Western Reserve School of Medicine, Cleveland, OH 44106 (United States)

    2012-11-20

    Loss of the transcription factor p53 implies mRNA losses of target genes such as the p53R2 subunit of human ribonucleotide reductase (RNR). We hypothesized that other genes in the dNTP supply system would compensate for such p53R2 losses and looked for this in our own data and in data of the Gene Expression Omnibus (GEO). We found that the de novo dNTP supply system compensates for p53R2 losses with increases in RNR subunit R1, R2, or both. We also found compensatory increases in cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) and in mitochondrial deoxyguanosine kinase (dGK), all of the salvage dNTP supply system; in contrast, the remaining mitochondrial salvage enzyme thymidine kinase 2 (TK2) decreased with p53 loss. Thus, TK2 may be more dedicated to meeting mitochondrial dNTP demands than dGK which may be more obligated to assist cytosolic dNTP supply in meeting nuclear DNA dNTP demands.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. The prion-related protein (testis-specific) gene (PRNT) is highly polymorphic in Portuguese sheep.

    Science.gov (United States)

    Mesquita, P; Garcia, V; Marques, M R; Santos Silva, F; Oliveira Sousa, M C; Carolino, I; Pimenta, J; Fontes, C M G A; Horta, A E M; Prates, J A M; Pereira, R M

    2016-02-01

    The objective of this study was to search for polymorphisms in the ovine prion-related protein (testis-specific) gene (PRNT). Sampling included 567 sheep from eight Portuguese breeds. The PRNT gene-coding region was analyzed by single-strand conformation polymorphism and sequencing, allowing the identification of the first ovine PRNT polymorphisms, in codons 6, 38, 43 and 48: c.17C>T (p.Ser6Phe, which disrupts a consensus arginine-X-X-serine/threonine motif); c.112G>C (p.Gly38>Arg); c.129T>C and c.144A>G (synonymous) respectively. Polymorphisms in codons 6, 38 and 48 occur simultaneously in 50.6% of the animals, 38.8% presenting as heterozygous. To study the distribution of the polymorphism in codon 43, a restriction fragment length polymorphism analysis was performed. Polymorphic variant c.129C, identified in 89.8% of the animals with 32.8% presented as heterozygous, was considered the wild genotype in Portuguese sheep. Eight different haplotypes which have comparable distribution in all breeds were identified for the PRNT gene. In conclusion, the PRNT coding region is highly polymorphic in sheep, unlike the prion protein 2 dublet gene (PRND), in which we previously found only one synonymous substitution (c.78G>A), in codon 26. The absence or reduced number of PRND heterozygotes (c.78G>A) was significantly associated with three PRNT haplotypes (17C-112G-129T-144A,17CT-112GC-129CT-144AG and 17T-112C-129C-144G), and the only three animals found homozygous at c.78A had the 17C-112G-129C-144A PRNT haplotype. These results constitute evidence of an association between polymorphic variation in PRND and PRNT genes, as has already been observed for PRND and prion protein gene (PRNP). PMID:26538093

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

    Directory of Open Access Journals (Sweden)

    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.

  15. High Frequency of Large Intragenic Deletions in the Fanconi Anemia Group A Gene

    OpenAIRE

    Morgan, Neil V.; Tipping, Alex J.; Joenje, Hans; Mathew, Christopher G.

    1999-01-01

    Fanconi anemia (FA) is an autosomal recessive disorder exhibiting chromosomal fragility, bone-marrow failure, congenital abnormalities, and cancer. At least eight complementation groups have been described, with group A accounting for 60%–65% of FA patients. Mutation screening of the group A gene (FANCA) is complicated by its highly interrupted genomic structure and heterogeneous mutation spectrum. Recent reports of several large deletions of FANCA, coupled with modest mutation-detection rate...

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. Identifying gene-gene interactions that are highly associated with Body Mass Index using Quantitative Multifactor Dimensionality Reduction (QMDR)

    NARCIS (Netherlands)

    De, Rishika; Verma, Shefali S; Drenos, Fotios; Holzinger, Emily R; Holmes, Michael V; Hall, Molly A; Crosslin, David R; Carrell, David S; Hakonarson, Hakon; Jarvik, Gail; Larson, Eric; Pacheco, Jennifer A; Rasmussen-Torvik, Laura J; Moore, Carrie B; Asselbergs, Folkert W; Moore, Jason H; Ritchie, Marylyn D; Keating, Brendan J; Gilbert-Diamond, Diane

    2015-01-01

    BACKGROUND: Despite heritability estimates of 40-70 % for obesity, less than 2 % of its variation is explained by Body Mass Index (BMI) associated loci that have been identified so far. Epistasis, or gene-gene interactions are a plausible source to explain portions of the missing heritability of BMI

  1. 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.

  2. 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

  3. A simple but highly effective approach to evaluate the prognostic performance of gene expression signatures.

    Directory of Open Access Journals (Sweden)

    Maud H W Starmans

    Full Text Available BACKGROUND: Highly parallel analysis of gene expression has recently been used to identify gene sets or 'signatures' to improve patient diagnosis and risk stratification. Once a signature is generated, traditional statistical testing is used to evaluate its prognostic performance. However, due to the dimensionality of microarrays, this can lead to false interpretation of these signatures. PRINCIPAL FINDINGS: A method was developed to test batches of a user-specified number of randomly chosen signatures in patient microarray datasets. The percentage of random generated signatures yielding prognostic value was assessed using ROC analysis by calculating the area under the curve (AUC in six public available cancer patient microarray datasets. We found that a signature consisting of randomly selected genes has an average 10% chance of reaching significance when assessed in a single dataset, but can range from 1% to ∼40% depending on the dataset in question. Increasing the number of validation datasets markedly reduces this number. CONCLUSIONS: We have shown that the use of an arbitrary cut-off value for evaluation of signature significance is not suitable for this type of research, but should be defined for each dataset separately. Our method can be used to establish and evaluate signature performance of any derived gene signature in a dataset by comparing its performance to thousands of randomly generated signatures. It will be of most interest for cases where few data are available and testing in multiple datasets is limited.

  4. Male-specific region of the bovine Y chromosome is gene rich with a high transcriptomic activity in testis development.

    Science.gov (United States)

    Chang, Ti-Cheng; Yang, Yang; Retzel, Ernest F; Liu, Wan-Sheng

    2013-07-23

    The male-specific region of the mammalian Y chromosome (MSY) contains clusters of genes essential for male reproduction. The highly repetitive and degenerative nature of the Y chromosome impedes genomic and transcriptomic characterization. Although the Y chromosome sequence is available for the human, chimpanzee, and macaque, little is known about the annotation and transcriptome of nonprimate MSY. Here, we investigated the transcriptome of the MSY in cattle by direct testis cDNA selection and RNA-seq approaches. The bovine MSY differs radically from the primate Y chromosomes with respect to its structure, gene content, and density. Among the 28 protein-coding genes/families identified on the bovine MSY (12 single- and 16 multicopy genes), 16 are bovid specific. The 1,274 genes identified in this study made the bovine MSY gene density the highest in the genome; in comparison, primate MSYs have only 31-78 genes. Our results, along with the highly transcriptional activities observed from these Y-chromosome genes and 375 additional noncoding RNAs, challenge the widely accepted hypothesis that the MSY is gene poor and transcriptionally inert. The bovine MSY genes are predominantly expressed and are differentially regulated during the testicular development. Synonymous substitution rate analyses of the multicopy MSY genes indicated that two major periods of expansion occurred during the Miocene and Pliocene, contributing to the adaptive radiation of bovids. The massive amplification and vigorous transcription suggest that the MSY serves as a genomic niche regulating male reproduction during bovid expansion. PMID:23842086

  5. 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.

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

    Science.gov (United States)

    Wang, Yang; Chen, Zhi-Hao; Yin, Chun; Ma, Jian-Hua; Li, Di-Jie; Zhao, Fan; Sun, Yu-Long; Hu, Li-Fang; Shang, Peng; Qian, Ai-Rong

    2015-01-01

    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. PMID:25635858

  7. 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.

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    Jinming Luo

    Full Text Available 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.

  10. 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.

  11. 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.

  12. Characterization of upregulated genes associated with high phosphorus accumulation in cucumber.

    Science.gov (United States)

    Padmanabhan, Priya; Venkatachalam, Perumal; Sahi, Shivendra V

    2011-12-01

    Excessive application of phosphorus (P)-rich manures to agricultural lands often results in P-accumulation in soils leading to water pollution through runoffs and leaching. Use of suitable plant species that can extract and sequester excess P from soil into their biomass is an effective method of remediation of P-contaminated soils. Knowledge on the molecular responses of plants to high P-accumulation and tolerance is lacking. Therefore, a suppression subtractive hybridization (SSH) strategy was employed to identify and elucidate the pattern of gene expression related to P-tolerance and accumulation in cucumber (Cucumis sativus L.), a P-accumulator plant. RNA isolated from cucumber grown in high P was used for 'tester' cDNA synthesis and SSH library preparation. A total of 63 cDNAs were identified as showing upregulated expression in this plant in response to high P. No putative function could be assigned to 7 (11%) of the 63 upregulated high P-modulated genes and 11 expressed sequence tags (ESTs) (17%) did not match database entries. The remaining 45 ESTs were grouped into five functional classes. The majority of these ESTs belonged to three groups: 'metabolism', 'protein synthesis/degradation and signaling' and 'cell structure/cell wall'. Only six 'stress/defense'-related ESTs were identified from this library. The results of reverse northern blot analysis was further confirmed and validated through semi-quantitative RT-PCR carried out with representative ESTs identified in this study. The research reported here may contribute to a preliminary understanding of the high P-related gene expression in this P-accumulating plant. PMID:21883253

  13. 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.

  14. 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

  15. 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. PMID:25683503

  16. Engineering high-level aluminum tolerance in barley with the ALMT1 gene.

    Science.gov (United States)

    Delhaize, Emmanuel; Ryan, Peter R; Hebb, Diane M; Yamamoto, Yoko; Sasaki, Takayuki; Matsumoto, Hideaki

    2004-10-19

    Acidity is a serious limitation to plant production on many of the world's agricultural soils. Toxic aluminium (Al) cations solubilized by the acidity rapidly inhibit root growth and limit subsequent uptake of water and nutrients. Recent work has shown that the ALMT1 gene of wheat (Triticum aestivum) encodes a malate transporter that is associated with malate efflux and Al tolerance. We generated transgenic barley (Hordeum vulgare) plants expressing ALMT1 and assessed their ability to exude malate and withstand Al stress. ALMT1 expression in barley conferred an Al-activated efflux of malate with properties similar to those of Al-tolerant wheat. The transgenic barley showed a high level of Al tolerance when grown in both hydroponic culture and on acid soils. These findings provide additional evidence that ALMT1 is a major Al-tolerance gene and demonstrate its ability to confer effective tolerance to acid soils through a transgenic approach in an important crop species. PMID:15471989

  17. 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...

  18. 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.

  19. Genes with high penetrance for syndromic and non-syndromic autism typically function within the nucleus and regulate gene expression

    OpenAIRE

    Casanova, Emily L.; Sharp, Julia L.; Chakraborty, Hrishikesh; Sumi, Nahid Sultana; Casanova, Manuel F.

    2016-01-01

    Background Intellectual disability (ID), autism, and epilepsy share frequent yet variable comorbidities with one another. In order to better understand potential genetic divergence underlying this variable risk, we studied genes responsible for monogenic IDs, grouped according to their autism and epilepsy comorbidities. Methods Utilizing 465 different forms of ID with known molecular origins, we accessed available genetic databases in conjunction with gene ontology (GO) to determine whether t...

  20. 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

  1. 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 a...

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Self-assembled supramolecular nano vesicles for safe and highly efficient gene delivery to solid tumors

    Directory of Open Access Journals (Sweden)

    Li W

    2012-08-01

    Full Text Available Wei Li,1,2,* Huafei Li,1,* Jinfeng Li,1,* Huajing Wang,1,* He Zhao,1 Li Zhang,1 Yu Xia,1 Zengwei Ye,1 Jie Gao,1,2 Jianxin Dai,1–3 Hao Wang,1–3 Yajun Guo1–31International Joint Cancer Institute, The Second Military Medical University, Shanghai, 2National Engineering Research Center for Antibody Medicine, State Key Laboratory of Antibody Medicine and Targeting Therapy and Shanghai Key Laboratory of Cell Engineering, Shanghai, 3PLA General Hospital Cancer Center, PLA Graduate School of Medicine, Beijing, People's Republic of China*These authors contributed equally to this workAbstract: The main obstacles for cationic polyplexes in gene delivery are in vivo instability and low solid-tumor accumulation. Safe vectors with high transfection efficiency and in vivo tumor accumulation are therefore highly desirable. In this study, the amphiphilic block copolymer poly(n-butyl methacrylate-b-poly(N-acryloylmorpholine was synthesized by reversible addition–fragmentation chain-transfer (RAFT radical polymerization. The corresponding well-defined vesicles with narrow size distribution were tailored by finely regulating the packing parameter (β of copolymer (1/2 < β < 1. Compared with traditional "gold-standard" polycation (polyethylenimine, 25 kDa, plasmid DNA condensing efficiency, DNase I degradation protection, and cellular uptake were improved by the supramolecular nano vesicles. In addition, the plasmid DNA transferring efficiency in 10% fetal bovine serum medium was enlarged five times to that of polyethylenimine in renal tubular epithelial and human hepatocellular carcinoma cell lines. This improved in vitro transfection was mainly attributed to the densely packed bilayer. This stealth polyplex showed high serum stability via entropic repulsion, which further protected the polyplex from being destroyed during sterilization. As indicated by the IVIS® Lumina II Imaging System (Caliper Life Sciences, Hopkinton, MA 24 hours post

  7. 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

  8. A novel, highly efficient gene-cloning system for Micromonospora strains.

    OpenAIRE

    Hasegawa, M.; Dairi, T; T. Ohta; Hashimoto, E.

    1991-01-01

    A highly efficient gene-cloning system for Micromonospora olivasterospora, a producer of the antibiotic fortimicin A (astromicin), suited to shotgun cloning has been developed. The system is supported by two new advancements accomplished in this study. One is the construction of novel plasmid vectors pMO116, pMO126, pMO133, pMO136, and pMO217, all consisting of replicons from newly found Micromonospora plasmids and selectable markers cloned from a neomycin-producing Micromonospora strain. The...

  9. Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product.

    OpenAIRE

    Nakagama, H; Heinrich, G.; Pelletier, J; Housman, D E

    1995-01-01

    The Wilms' tumor suppressor gene, WT1, encodes a zinc finger polypeptide which plays a key role regulating cell growth and differentiation in the urogenital system. Using the whole-genome PCR approach, we searched murine genomic DNA for high-affinity WT1 binding sites and identified a 10-bp motif 5'GCGTGGGAGT3' which we term WTE). The WTE motif is similar to the consensus binding sequence 5'GCG(G/T)GGGCG3' recognized by EGR-1 and is also suggested to function as a binding site for WT1, settin...

  10. Berry intake changes hepatic gene expression and DNA methylation patterns associated with high-fat diet.

    Science.gov (United States)

    Heyman-Lindén, Lovisa; Seki, Yoshinori; Storm, Petter; Jones, Helena A; Charron, Maureen J; Berger, Karin; Holm, Cecilia

    2016-01-01

    The liver is a critical organ for regulation of energy homeostasis and fatty liver disease is closely associated with obesity and insulin resistance. We have previously found that lingonberries, blackcurrants and bilberries prevent, whereas açai berries exacerbate, the development of hepatic steatosis and obesity in the high-fat (HF)-fed C57BL/6J mouse model. In this follow-up study, we investigated the mechanisms behind these effects. Genome-wide hepatic gene expression profiling indicates that the protective effects of lingonberries and bilberries are accounted for by several-fold downregulation of genes involved in acute-phase and inflammatory pathways (e.g. Saa1, Cxcl1, Lcn2). In contrast, açai-fed mice exhibit marked upregulation of genes associated with steatosis (e.g. Cfd, Cidea, Crat) and lipid and cholesterol biosynthesis, which is in line with the exacerbation of HF-induced hepatic steatosis in these mice. In silico transcription factor analysis together with immunoblot analysis identified NF-κB, STAT3 and mTOR as upstream regulators involved in mediating the observed transcriptional effects. To gain further insight into mechanisms involved in the gene expression changes, the HELP-tagging assay was used to identify differentially methylated CpG sites. Compared to the HF control group, lingonberries induced genome-wide hypermethylation and specific hypermethylation of Ncor2, encoding the corepressor NCoR/SMRT implicated in the regulation of pathways of metabolic homeostasis and inflammation. We conclude that the beneficial metabolic effects of lingonberries and bilberries are associated with downregulation of inflammatory pathways, whereas for blackcurrants, exerting similar metabolic effects, different mechanisms of action appear to dominate. NF-κB, STAT3 and mTOR are potential targets of the health-promoting effects of berries. PMID:26423886

  11. 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.

  12. 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

  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. 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.

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

    Directory of Open Access Journals (Sweden)

    Catherine eRAVEL

    2014-11-01

    Full Text Available 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 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.

  16. Generation of novel high quality HMW-GS genes in two introgression lines of Triticum aestivum/Agropyron elongatum

    Directory of Open Access Journals (Sweden)

    Chen Fanguo

    2007-05-01

    Full Text Available Abstract Background High molecular weight glutenin subunits (HMW-GS have been proved to be mostly correlated with the processing quality of common wheat (Triticum aestivum. But wheat cultivars have limited number of high quality HMW-GS. However, novel HMW-GS were found to be present in many wheat asymmetric somatic hybrid introgression lines of common wheat/Agropyron elongatum. Results To exploit how these new subunits were generated, we isolated HMW-GS genes from two sib hybrid lines (II-12 and 11-4-6 and compared them with those from their parents. The result shows that two genes of hybrid (H11-3-3 and H11-4-3 are directly introgressed from the donor parent Agropyron elongatum; one hybrid gene (H1Dx5 comes from point mutation of a parental wheat gene (1Dx2.1; two other hybrid genes (H1By8 and H1By16 are likely resulting from unequal crossover or slippage of a parental wheat gene (1By9.1; and the sixth novel hybrid gene (H1Dy12 may come from recombination between two parental genes. Conclusion Therefore, we demonstrate that novel HMW-GS genes can be rapidly created through asymmetric somatic hybridization in a manner similar with the evolution mechanism of these genes supposed before. We also described gene shuffling as a new mechanism of novel HMW-GS gene formation in hybrids. The results suggest that asymmetric somatic hybridization is an important approach for widening HMW-GS genebank of wheat quality improvement.

  17. Application of high molecular weight DNA cloning in legume nodulation gene analysis

    International Nuclear Information System (INIS)

    High molecular weight (HMW) DNA was isolated from Glycine max (soybean) and the model legume Lotus japonicus for the purpose of legume genome analysis. The primary objectives were the gene regions that control nodulation, early plant-microbe interaction and cell division responses. HMW DNA was separated by pulse field gel electrophoresis (CHEF-PFGE) and analyzed with closely linked restriction fragment length polymorphism (RFLP) markers co-hybridized with clones, permitting estimation of the regional physical distances as they relate to recombination frequency. In the distal region of molecular linkage group H containing one of the genes controlling nodule number autoregulation and symbiotic nitrate tolerance (i.e. the nts gene), 1 cM was equivalent to less than 500 kb. Partially digested EcoRI soybean and L. japonicus HMW DNA were cloned into pYAC4. Stable yeast artificial chromosomes (YACs) carrying up to 960 kb DNA were generated. The average insert size was 200 kb. Hybridization with total genomic soybean DNA revealed YACs with different amounts of repeated DNA sequences. Mapping of the end clones demonstrated whether the YACs were chimeric. YACs of different complexity were used for chromosome identification using degenerate primer polymerase chain reaction and fluorescent in situ hybridization. This approach is a fast alternative to testing for YAC chimerism. Single arbitrary and structured mini-hairpin primers were used to amplify and DNA fingerprint the YACs, providing a means of identifying the additional markers needed for contig construction. HMW DNA was cloned into the F plasmid bacterial artificial chromosome (BAC) vector. The YACs and BACs were also constructed with DNA from the small genome/highly transformable legume L. japonicus. Mapping of the YAC and BAC clones with molecular markers will help to ascertain the degree of chimerism and stability in the different cloning systems. YACs, molecular markers and cDNA clones will be useful for chromosome

  18. A High Soy Diet Enhances Neurotropin Receptor and Bcl-XL Gene Expression in the Brains of Ovariectomized Female Rats

    OpenAIRE

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

    2007-01-01

    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 expre...

  19. A novel gene, lstC, of Listeria monocytogenes is implicated in high salt tolerance.

    Science.gov (United States)

    Burall, Laurel S; Simpson, Alexandra C; Chou, Luoth; Laksanalamai, Pongpan; Datta, Atin R

    2015-06-01

    Listeria monocytogenes, causative agent of human listeriosis, has been isolated from a wide variety of foods including deli meats, soft cheeses, cantaloupes, sprouts and canned mushrooms. Standard control measures for restricting microbial growth such as refrigeration and high salt are often inadequate as L. monocytogenes grows quite well in these environments. In an effort to better understand the genetic and physiological basis by which L. monocytogenes circumvents these controls, a transposon library of L. monocytogenes was screened for changes in their ability to grow in 7% NaCl and/ or at 5 °C. This work identified a transposon insertion upstream of an operon, here named lstABC, that led to a reduction in growth in 7% NaCl. In-frame deletion studies identified lstC which codes for a GNAT-acetyltransferase being responsible for the phenotype. Transcriptomic and RT-PCR analyses identified nine genes that were upregulated in the presence of high salt in the ΔlstC mutant. Further analysis of lstC and the genes affected by ΔlstC is needed to understand LstC's role in salt tolerance. PMID:25790994

  20. Screening of Highly-expressed-HMGB1-Gene Human Lung Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yi LIU

    2009-09-01

    Full Text Available Background and objective Lung cancer is a type of malignant tumor which threats human health and life. Its morbidity might increase dramatically in a long period. Lung cancer is the leading cause of cancer-related death all over the world. HMGB1 (high mobility group box B 1 is a non-histone chromosome binding protein in the cells. It takes part in many biological processes including genes transcription and DNA repair. HMGB1 overexpression can result in cell apoptosis, differentiation, migration and proliferation. The main purpose of this study was to detect the HMGB1 expression of 4 lung cancer cell lines in order to select the most suitable cell line to do the work next step. Methods Four lung cancer cell lines were cultured by normal method, Western blot and real-time quantitative PCR were used to verify the levels of expression of HMGB1. The cell line which HMGB1 over-expressed was selected. Results HMGB1 expressed in all 4 lung cancer cell lines, the cell line L9981 was the most highly expressed cell line (P < 0.001. Conclusion All 4 lung cancer cell lines expree HMGB1 gene. As the HMGB1 overexpression cell line, L9981 is an ideal material for follow-up research.

  1. 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.

  2. High-Throughput, Motility-Based Sorter for Microswimmers and Gene Discovery Platform

    Science.gov (United States)

    Yuan, Jinzhou; Raizen, David; Bau, Haim

    2015-11-01

    Animal motility varies with genotype, disease progression, aging, and environmental conditions. In many studies, it is desirable to carry out high throughput motility-based sorting to isolate rare animals for, among other things, forward genetic screens to identify genetic pathways that regulate phenotypes of interest. Many commonly used screening processes are labor-intensive, lack sensitivity, and require extensive investigator training. Here, we describe a sensitive, high throughput, automated, motility-based method for sorting nematodes. Our method was implemented in a simple microfluidic device capable of sorting many thousands of animals per hour per module, and is amenable to parallelism. The device successfully enriched for known C. elegans motility mutants. Furthermore, using this device, we isolated low-abundance mutants capable of suppressing the somnogenic effects of the flp-13 gene, which regulates sleep-like quiescence in C. elegans. Subsequent genomic sequencing led to the identification of a flp-13-suppressor gene. This research was supported, in part, by NIH NIA Grant 5R03AG042690-02.

  3. 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.

  4. High-Resolution Analysis of Gene Copy Number Alterations in Human Prostate Cancer Using CGH on cDNA Microarrays: Impact of Copy Number on Gene Expression

    Directory of Open Access Journals (Sweden)

    Maija Wolf

    2004-05-01

    Full Text Available Identification of target genes for genetic rearrangements in prostate cancer and the impact of copy number changes on gene expression are currently not well understood. Here, we applied high-resolution comparative genomic hybridization (CGH on cDNA microarrays for analysis of prostate cancer cell lines. CGH microarrays identified most of the alterations detected by classical chromosomal CGH, as well as a number of previously unreported alterations. Specific recurrent regions of gain (28 and loss (18 were found, their boundaries defined with sub-megabasepair accuracy. The most common changes included copy number decreases at 13% and gains at iq and 5p. Refined mapping identified several sites, such as at 13q (33-44, 49-51, 74-76 Mbp from the p-telomere, which matched with minimal regions of loss seen in extensive loss of heterozygosity mapping studies of large numbers of tumors. Previously unreported recurrent changes were found at 2p, 2q, 3p, 17q (losses, at 3q, 5p, 6p (gains. Integration of genomic and transcriptomic data revealed the role of individual candidate target genes for genomic alterations as well as a highly significant (P < .0001 overall association between copy number levels and the percentage of differentially expressed genes. Across the genome, the overall impact of copy number on gene expression levels was, to a large extent, attributable to low-level gains and losses of copy number, corresponding to common deletions and gains of often large chromosomal regions.

  5. 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.

  6. Lognormality and oscillations in the coverage of high-throughput transcriptomic data towards gene ends

    International Nuclear Information System (INIS)

    High-throughput transcriptomics experiments have reached the stage where the count of the number of reads alignable to a given position can be treated as an almost-continuous signal. This allows us to ask questions of biophysical/biotechnical nature, but which may still have biological implications. Here we show that when sequencing RNA fragments from one end, as is the case on most platforms, an oscillation in the read count is observed at the other end. We further show that these oscillations can be well described by Kolmogorov’s 1941 broken stick model. We investigate how the model can be used to improve predictions of gene ends (3′ transcript ends), but conclude that with present data the improvement is only marginal. The results highlight subtle effects in high-throughput transcriptomics experiments which do not have a biological origin, but which may still be used to obtain biological information. (paper)

  7. 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....

  8. A high-throughput transient gene expression system for switchgrass (Panicum virgatum L. seedlings

    Directory of Open Access Journals (Sweden)

    Agarwal Sujata

    2010-05-01

    Full Text Available Abstract Background Grasses are relatively recalcitrant to genetic transformation in comparison to certain dicotyledons, yet they constitute some of the most important biofuel crops. Genetic transformation of switchgrass (Panicum virgatum L. has previously been reported after cocultivation of explants with Agrobacterium and biolistics of embryogenic calli. Experiments to increase transient gene expression in planta may lead to stable transformation methods with increased efficiency. Results A high-throughput Agrobacterium-mediated transient gene expression system has been developed for in planta inoculation of germinating switchgrass seedlings. Four different Agrobacterium strains were compared for their ability to infect switchgrass seedlings, and strain AGL1 was found to be the most infective. Wounding pretreatments such as sonication, mixing by vortex with carborundum, separation by centrifugation, vacuum infiltration, and high temperature shock significantly increased transient expression of a reporter gene (GUSPlus, a variation of the β-glucuronidase (GUS gene. The addition of L-cysteine and dithiothreitol in the presence of acetosyringone significantly increased GUS expression compared with control treatments, whereas the addition of 0.1% surfactants such as Silwet L77 or Li700 decreased GUS expression. 4-Methylumbelliferyl beta-D-galactopyranoside (MUG assays showed a peak of β-glucuronidase (GUS enzyme activity 3 days after cocultivation with Agrobacterium harboring pCambia1305.2, whereas MUG assays showed a peak of enzyme activity 5 days after cocultivation with Agrobacterium harboring pCambia1305.1. Conclusion Agrobacterium strains C58, GV3101 and EHA105 are less able to deliver transfer DNA to switchgrass seedlings (cultivar Alamo compared with strain AGL1. Transient expression was increased by double or triple wounding treatments such as mixing by vortex with carborundum, sonication, separation by centrifugation, and heat shock

  9. Aberrant host immune response induced by highly virulent PRRSV identified by digital gene expression tag profiling

    Directory of Open Access Journals (Sweden)

    Zhao Xiao

    2010-10-01

    Full Text Available Abstract Background There was a large scale outbreak of the highly pathogenic porcine reproductive and respiratory syndrome (PRRS in China and Vietnam during 2006 and 2007 that resulted in unusually high morbidity and mortality among pigs of all ages. The mechanisms underlying the molecular pathogenesis of the highly virulent PRRS virus (H-PRRSV remains unknown. Therefore, the relationship between pulmonary gene expression profiles after H-PRRSV infection and infection pathology were analyzed in this study using high-throughput deep sequencing and histopathology. Results H-PRRSV infection resulted in severe lung pathology. The results indicate that aberrant host innate immune responses to H-PRRSV and induction of an anti-apoptotic state could be responsible for the aggressive replication and dissemination of H-PRRSV. Prolific rapid replication of H-PRRSV could have triggered aberrant sustained expression of pro-inflammatory cytokines and chemokines leading to a markedly robust inflammatory response compounded by significant cell death and increased oxidative damage. The end result was severe tissue damage and high pathogenicity. Conclusions The systems analysis utilized in this study provides a comprehensive basis for better understanding the pathogenesis of H-PRRSV. Furthermore, it allows the genetic components involved in H-PRRSV resistance/susceptibility in swine populations to be identified.

  10. 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.

  11. 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

  12. Phylogenetic analysis of bacterial isolates from man-made high-pH, high-salt environments and identification of gene-cassette-associated open reading frames.

    Science.gov (United States)

    Ghauri, Muhammad A; Khalid, Ahmad M; Grant, Susan; Grant, William D; Heaphy, Shaun

    2006-06-01

    Environmental samples were collected from high-pH sites in Pakistan, including a uranium heap set up for carbonate leaching, the lime unit of a tannery, and the Khewra salt mine. Another sample was collected from a hot spring on the shore of the soda lake, Magadi, in Kenya. Microbial cultures were enriched from Pakistani samples. Phylogenetic analysis of isolates was carried out by sequencing 16S rRNA genes. Genomic DNA was amplified by polymerase chain reaction using integron gene-cassette-specific primers. Different gene-cassette-linked genes were recovered from the cultured strains related to Halomonas magadiensis, Virgibacillus halodenitrificans, and Yania flava and from the uncultured environmental DNA sample. The usefulness of this technique as a tool for gene mining is indicated. PMID:16732461

  13. High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes.

    Science.gov (United States)

    Alout, H; Labbé, P; Berthomieu, A; Makoundou, P; Fort, P; Pasteur, N; Weill, M

    2016-02-01

    We investigated the genetic determinism of high chlorpyrifos resistance (HCR), a phenotype first described in 1999 in Culex pipiens mosquitoes surviving chlorpyrifos doses ⩾1 mg l(-1) and more recently found in field samples from Tunisia, Israel or Indian Ocean islands. Through chlorpyrifos selection, we selected several HCR strains that displayed over 10 000-fold resistance. All strains were homozygous for resistant alleles at two main loci: the ace-1 gene, with the resistant ace-1(R) allele expressing the insensitive G119S acetylcholinesterase, and a resistant allele of an unknown gene (named T) linked to the sex and ace-2 genes. We constructed a strain carrying only the T-resistant allele and studied its resistance characteristics. By crossing this strain with strains harboring different alleles at the ace-1 locus, we showed that the resistant ace-1(R) and the T alleles act in strong synergy, as they elicited a resistance 100 times higher than expected from a simple multiplicative effect. This effect was specific to chlorpyrifos and parathion and was not affected by synergists. We also examined how HCR was expressed in strains carrying other ace-1-resistant alleles, such as ace-1(V) or the duplicated ace-1(D) allele, currently spreading worldwide. We identified two major parameters that influenced the level of resistance: the number and the nature of the ace-1-resistant alleles and the number of T alleles. Our data fit a model that predicts that the T allele acts by decreasing chlorpyrifos concentration in the compartment targeted in insects. PMID:26463842

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. Analysis of Canis mitochondrial DNA demonstrates high concordance between the control region and ATPase genes

    Directory of Open Access Journals (Sweden)

    White Bradley N

    2010-07-01

    Full Text Available Abstract Background Phylogenetic studies of wild Canis species have relied heavily on the mitochondrial DNA control region (mtDNA CR to infer species relationships and evolutionary lineages. Previous analyses of the CR provided evidence for a North American evolved eastern wolf (C. lycaon, that is more closely related to red wolves (C. rufus and coyotes (C. latrans than grey wolves (C. lupus. Eastern wolf origins, however, continue to be questioned. Therefore, we analyzed mtDNA from 89 wolves and coyotes across North America and Eurasia at 347 base pairs (bp of the CR and 1067 bp that included the ATPase6 and ATPase8 genes. Phylogenies and divergence estimates were used to clarify the evolutionary history of eastern wolves, and regional comparisons of nonsynonomous to synonomous substitutions (dN/dS at the ATPase6 and ATPase8 genes were used to elucidate the potential role of selection in shaping mtDNA geographic distribution. Results We found high concordance across analyses between the mtDNA regions studied. Both had a high percentage of variable sites (CR = 14.6%; ATP = 9.7% and both phylogenies clustered eastern wolf haplotypes monophyletically within a North American evolved lineage apart from coyotes. Divergence estimates suggest the putative red wolf sequence is more closely related to coyotes (DxyCR = 0.01982 ± 0.00494 SD; DxyATP = 0.00332 ± 0.00097 SD than the eastern wolf sequences (DxyCR = 0.03047 ± 0.00664 SD; DxyATP = 0.00931 ± 0.00205 SD. Neutrality tests on both genes were indicative of the population expansion of coyotes across eastern North America, and dN/dS ratios suggest a possible role for purifying selection in the evolution of North American lineages. dN/dS ratios were higher in European evolved lineages from northern climates compared to North American evolved lineages from temperate regions, but these differences were not statistically significant. Conclusions These results demonstrate high concordance between coding

  19. A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes

    Directory of Open Access Journals (Sweden)

    Ke Tao

    2012-03-01

    Full Text Available Abstract Background To facilitate the screening of large quantities of new antimicrobial peptides (AMPs, we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity. Results Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia. Conclusions The method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a

  20. 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.

  1. A high frequency of distinct ATM gene mutations in ataxia-telangiectasia

    Energy Technology Data Exchange (ETDEWEB)

    Wright, J.; Teraoka, S.; Concannon, P. [Univ. of Washington School of Medicine, Seattle, WA (United States)] [and others

    1996-10-01

    The clinical features of the autosomal recessive disorder ataxia-telangiectasia (AT) include a progressive cerebellar ataxia, hypersensitivity to ionizing radiation, and an increased susceptibility to malignancies. Epidemiological studies have suggested that AT heterozygotes may also be at increased risk for malignancy, possibly as a consequence of radiation exposure. A gene mutated in AT patients (ATM) has recently been isolated, making mutation screening in both patients and the general population possible. Because of the relatively large size of the ATM gene, the design of screening programs will depend on the types and distribution of mutations in the general population. In this report, we describe 30 mutations identified in a panel of unrelated AT patients and controls. Twenty-five of the 30 were distinct, and most patients were compound heterozygotes. The most frequently detected mutation was found in three different families and had previously been reported in five others. This corresponds to a frequency of 8% of all reported ATM mutations. Twenty-two of the alterations observed would be predicted to lead to protein truncation at sites scattered throughout the molecule. Two fibroblast cell lines, which displayed normal responses to ionizing radiation, also proved to be heterozygous for truncation mutations of ATM. These observations suggest that the carrier frequency of ATM mutations may be sufficiently high to make population screening practical. However, such screening may need to be done prospectively, that is, by searching for new mutations rather than by screening for just those already identified in AT families. 33 refs., 1 fig., 1 tab.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

    2015-12-01

    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. PMID:26598698

  8. 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.

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

    Science.gov (United States)

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

    2009-07-01

    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 functions. Cytoskeleton, as an intracellular load-bearing structure, 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), WIPF1 (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 cytoskeleton-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.

  10. 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.

  11. Molecular deregulation induced by silencing of the high mobility group protein A2 gene in retinoblastoma cells

    OpenAIRE

    Venkatesan, Nalini; Krishnakumar, Subramanian; Deepa, Perinkulam Ravi; Deepa, Murali; Khetan, Vikas; Reddy, M. Ashwin

    2012-01-01

    Aim To explore the molecular mechanisms deregulated by high mobility group protein A2 (HMGA2) gene silencing in retinoblastoma (RB) cells. Methods Synthetic anti-HMGA2 short interfering RNA (siRNA) was used to silence the HMGA2 gene in cultured Y79 RB cells that were subjected to whole genome microarray analysis. The expression of differentially regulated key genes was confirmed with quantitative reverse-transcriptase polymerase chain reaction (qRT–PCR) in post-silenced RB cell lines (Y79 and...

  12. Effects of dietary high fructose corn syrup on regulation of energy intake and leptin gene expression in rats

    OpenAIRE

    Guadalupe López-Rodríguez; Silke Kotasek Osuna; Marcos Galván García; Teodoro Suárez Dieguez

    2015-01-01

    OBJECTIVE: To evaluate in Wistar rats the effect of chronic use of high fructose corn syrup on serum lipids, body weight, energy intake regulation, and expression of associated genes. METHODS: For 11 weeks, male rats were fed a standard diet with either water (control) or 15% high fructose corn syrup solution, or fed a high-fat diet. The rats' food intake and body weight were measured weekly. Expression of leptin and fatty acid synthase genes was quantified in their brain and adipose tissue ...

  13. Implications of using whole genome sequencing to test unselected populations for high risk breast cancer genes: a modelling study

    OpenAIRE

    Warren-Gash, Charlotte; Kroese, Mark; Burton, Hilary; Pharoah, Paul

    2016-01-01

    Background The decision to test for high risk breast cancer gene mutations is traditionally based on risk scores derived from age, family and personal cancer history. Next generation sequencing technologies such as whole genome sequencing (WGS) make wider population testing more feasible. In the UK’s 100,000 Genomes Project, mutations in 16 genes including BRCA1 and BRCA2 are to be actively sought regardless of clinical presentation. The implications of deploying this approach at scale for pa...

  14. DAG Expression: High-Throughput Gene Expression Analysis of Real-Time PCR Data Using Standard Curves for Relative Quantification

    OpenAIRE

    Ballester, María; Cordón, Rubén; Folch, Josep M.

    2013-01-01

    Background Real-time quantitative PCR (qPCR) is still the gold-standard technique for gene-expression quantification. Recent technological advances of this method allow for the high-throughput gene-expression analysis, without the limitations of sample space and reagent used. However, non-commercial and user-friendly software for the management and analysis of these data is not available. Results The recently developed commercial microarrays allow for the drawing of standard curves of multipl...

  15. Highly luminescent and cytocompatible cationic Ag2S NIR-emitting quantum dots for optical imaging and gene transfection

    OpenAIRE

    Duman, Fatma Demir; Hocaoğlu, Ibrahim; Kiraz, Alper; Acar, Havva Yagci; Ozturk, Deniz Gulfem; Gozuacik, Devrim

    2015-01-01

    The development of non-toxic theranostic nanoparticles capable of delivering a therapeutic cargo and providing a means for diagnosis is one of the most challenging tasks in nano-biotechnology. Gene therapy is a very important mode of therapy and polyethyleneimine (PEI) is one of the most successful vehicles for gene transfection, yet poses significant toxicity. Optical imaging utilizing quantum dots is one of the newer but fast growing diagnostic modalities, which requires non-toxic, highly l...

  16. DAG expression: high-throughput gene expression analysis of real-time PCR data using standard curves for relative quantification.

    Directory of Open Access Journals (Sweden)

    María Ballester

    Full Text Available BACKGROUND: Real-time quantitative PCR (qPCR is still the gold-standard technique for gene-expression quantification. Recent technological advances of this method allow for the high-throughput gene-expression analysis, without the limitations of sample space and reagent used. However, non-commercial and user-friendly software for the management and analysis of these data is not available. RESULTS: The recently developed commercial microarrays allow for the drawing of standard curves of multiple assays using the same n-fold diluted samples. Data Analysis Gene (DAG Expression software has been developed to perform high-throughput gene-expression data analysis using standard curves for relative quantification and one or multiple reference genes for sample normalization. We discuss the application of DAG Expression in the analysis of data from an experiment performed with Fluidigm technology, in which 48 genes and 115 samples were measured. Furthermore, the quality of our analysis was tested and compared with other available methods. CONCLUSIONS: DAG Expression is a freely available software that permits the automated analysis and visualization of high-throughput qPCR. A detailed manual and a demo-experiment are provided within the DAG Expression software at http://www.dagexpression.com/dage.zip.

  17. Manifestation in F2 of pleiotropic gene in the cross between mother variety and its high protein mutant pt. 1

    International Nuclear Information System (INIS)

    High protein rice mutant (M12 plant generation in 1976) was obtained from the progeny of X-rayed Hokwang variety. Besides high protein, the mutant accompanied two other mutated characters of reduced culm length and earlier heading date. Crossing between the mutant and its mother variety was made (1) to examine the gene(s) controlling high protein, (2) to verify the pleiotropic (or linkage) relation among the three changed characters and (3) to test the possibility of introducing the high protein gene(s) into mother variety. The assumption that a single pleiotropic recessive gene might be responsible for the three mutated characters was evidenced in F2 by the segregation of mother and mutant types in the ratio of 3:1. Besides these parental types there were numbers of segregant groups in F2. Among them were types which were mother-like phenotypically but had as high protein content as the mutant. These segregants were placed under further experiment for the confirmation of the character manifestation or fixation in later generations. (author)

  18. Pathways-driven sparse regression identifies pathways and genes associated with high-density lipoprotein cholesterol in two Asian cohorts.

    Directory of Open Access Journals (Sweden)

    Matt Silver

    2013-11-01

    Full Text Available Standard approaches to data analysis in genome-wide association studies (GWAS ignore any potential functional relationships between gene variants. In contrast gene pathways analysis uses prior information on functional structure within the genome to identify pathways associated with a trait of interest. In a second step, important single nucleotide polymorphisms (SNPs or genes may be identified within associated pathways. The pathways approach is motivated by the fact that genes do not act alone, but instead have effects that are likely to be mediated through their interaction in gene pathways. Where this is the case, pathways approaches may reveal aspects of a trait's genetic architecture that would otherwise be missed when considering SNPs in isolation. Most pathways methods begin by testing SNPs one at a time, and so fail to capitalise on the potential advantages inherent in a multi-SNP, joint modelling approach. Here, we describe a dual-level, sparse regression model for the simultaneous identification of pathways and genes associated with a quantitative trait. Our method takes account of various factors specific to the joint modelling of pathways with genome-wide data, including widespread correlation between genetic predictors, and the fact that variants may overlap multiple pathways. We use a resampling strategy that exploits finite sample variability to provide robust rankings for pathways and genes. We test our method through simulation, and use it to perform pathways-driven gene selection in a search for pathways and genes associated with variation in serum high-density lipoprotein cholesterol levels in two separate GWAS cohorts of Asian adults. By comparing results from both cohorts we identify a number of candidate pathways including those associated with cardiomyopathy, and T cell receptor and PPAR signalling. Highlighted genes include those associated with the L-type calcium channel, adenylate cyclase, integrin, laminin, MAPK

  19. 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.

  20. High accuracy mass spectrometry analysis as a tool to verify and improve gene annotation using Mycobacterium tuberculosis as an example

    Directory of Open Access Journals (Sweden)

    Prasad Swati

    2008-07-01

    Full Text Available Abstract Background While the genomic annotations of diverse lineages of the Mycobacterium tuberculosis complex are available, divergences between gene prediction methods are still a challenge for unbiased protein dataset generation. M. tuberculosis gene annotation is an example, where the most used datasets from two independent institutions (Sanger Institute and Institute of Genomic Research-TIGR differ up to 12% in the number of annotated open reading frames, and 46% of the genes contained in both annotations have different start codons. Such differences emphasize the importance of the identification of the sequence of protein products to validate each gene annotation including its sequence coding area. Results With this objective, we submitted a culture filtrate sample from M. tuberculosis to a high-accuracy LTQ-Orbitrap mass spectrometer analysis and applied refined N-terminal prediction to perform comparison of two gene annotations. From a total of 449 proteins identified from the MS data, we validated 35 tryptic peptides that were specific to one of the two datasets, representing 24 different proteins. From those, 5 proteins were only annotated in the Sanger database. In the remaining proteins, the observed differences were due to differences in annotation of transcriptional start sites. Conclusion Our results indicate that, even in a less complex sample likely to represent only 10% of the bacterial proteome, we were still able to detect major differences between different gene annotation approaches. This gives hope that high-throughput proteomics techniques can be used to improve and validate gene annotations, and in particular for verification of high-throughput, automatic gene annotations.

  1. Interleukin-6 gene knockout antagonizes high-fat-induced trabecular bone loss.

    Science.gov (United States)

    Wang, Chunyu; Tian, Li; Zhang, Kun; Chen, Yaxi; Chen, Xiang; Xie, Ying; Zhao, Qian; Yu, Xijie

    2016-10-01

    The purpose of the study was to determine the roles of interleukin-6 (IL6) in fat and bone communication. Male wild-type (WT) mice and IL6 knockout (IL6(-/-)) mice were fed with either regular diet (RD) or high-fat diet (HFD) for 12 weeks. Bone mass and bone microstructure were evaluated by micro-computed tomography. Gene expression related to lipid and bone metabolisms was assayed with real-time quantitative polymerase chain reaction. Bone marrow cells from both genotypes were induced to differentiate into osteoblasts or osteoclasts, and treated with palmitic acid (PA). HFD increased the body weight and fat pad weight, and impaired lipid metabolism in both WT and IL6(-/-) mice. The dysregulation of lipid metabolism was more serious in IL6(-/-) mice. Trabecular bone volume fraction, trabecular bone number and trabecular bone thickness were significantly downregulated in WT mice after HFD than those in the RD (P alkaline phosphatase (ALP) activity and higher mRNA levels of Runx2 and Colla1 than those in WT osteoblasts both in the control and PA treatment group (P < 0.05). IL6(-/-) mice showed significantly lower mRNA levels of PPARγ and leptin and higher mRNA levels of adiponectin in comparison with WT mice on HFD. In conclusion, these findings suggested that IL6 gene deficiency antagonized HFD-induced bone loss. IL6 might bridge lipid and bone metabolisms and could be a new potential therapeutic target for lipid metabolism disturbance-related bone loss. PMID:27493246

  2. 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.

  3. Biocathodes reducing oxygen at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes.

    Science.gov (United States)

    Desmond-Le Quéméner, Elie; Rimboud, Mickaël; Bridier, Arnaud; Madigou, Céline; Erable, Benjamin; Bergel, Alain; Bouchez, Théodore

    2016-08-01

    Biocathodes polarized at high potential are promising for enhancing Microbial Fuel Cell performances but the microbes and genes involved remain poorly documented. Here, two sets of five oxygen-reducing biocathodes were formed at two potentials (-0.4V and +0.1V vs. saturated calomel electrode) and analyzed combining electrochemical and metagenomic approaches. Slower start-up but higher current densities were observed at high potential and a distinctive peak increasing over time was recorded on cyclic voltamogramms, suggesting the growth of oxygen reducing microbes. 16S pyrotag sequencing showed the enrichment of two operational taxonomic units (OTUs) affiliated to Ectothiorodospiraceae on high potential electrodes with the best performances. Shotgun metagenome sequencing and a newly developed method for the identification of Taxon Specific Gene Annotations (TSGA) revealed Ectothiorhodospiraceae specific genes possibly involved in electron transfer and in autotrophic growth. These results give interesting insights into the genetic features underlying the selection of efficient oxygen reducing microbes on biocathodes. PMID:27126080

  4. Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell-derived medium spiny neurons.

    Science.gov (United States)

    Straccia, Marco; Garcia-Diaz Barriga, Gerardo; Sanders, Phil; Bombau, Georgina; Carrere, Jordi; Mairal, Pedro Belio; Vinh, Ngoc-Nga; Yung, Sun; Kelly, Claire M; Svendsen, Clive N; Kemp, Paul J; Arjomand, Jamshid; Schoenfeld, Ryan C; Alberch, Jordi; Allen, Nicholas D; Rosser, Anne E; Canals, Josep M

    2015-01-01

    A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC) into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE) and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening. PMID:26417608

  5. Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell–derived medium spiny neurons

    Science.gov (United States)

    Straccia, Marco; Garcia-Diaz Barriga, Gerardo; Sanders, Phil; Bombau, Georgina; Carrere, Jordi; Mairal, Pedro Belio; Vinh, Ngoc-Nga; Yung, Sun; Kelly, Claire M; Svendsen, Clive N; Kemp, Paul J; Arjomand, Jamshid; Schoenfeld, Ryan C; Alberch, Jordi; Allen, Nicholas D; Rosser, Anne E; Canals, Josep M

    2015-01-01

    A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC) into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE) and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening. PMID:26417608

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots.

    Directory of Open Access Journals (Sweden)

    Cheng Yuan

    Full Text Available Barley stripe mosaic virus (BSMV is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS, magnesium chelatase subunit H (ChlH, and plastid transketolase (TK gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5 also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

  12. 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

  13. Gene transfection in high serum levels: case studies with new cholesterol based cationic gemini lipids.

    Directory of Open Access Journals (Sweden)

    Santosh K Misra

    Full Text Available BACKGROUND: Six new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH2CH2OH and oligo-oxyethylene -(CH2CH2On- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media. METHODOLOGY/PRINCIPAL FINDINGS: To understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM, dynamic light scattering (DLS, zeta potential measurements and X-ray diffraction (XRD. We studied the lipid/DNA complex (lipoplex formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH2-CH2-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50% greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features. CONCLUSIONS/SIGNIFICANCE: -OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible in vivo studies.

  14. 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.

  15. Identification of candidate genes for yeast engineering to improve bioethanol production in very high gravity and lignocellulosic biomass industrial fermentations

    Directory of Open Access Journals (Sweden)

    Pereira Francisco B

    2011-12-01

    Full Text Available Abstract Background The optimization of industrial bioethanol production will depend on the rational design and manipulation of industrial strains to improve their robustness against the many stress factors affecting their performance during very high gravity (VHG or lignocellulosic fermentations. In this study, a set of Saccharomyces cerevisiae genes found, through genome-wide screenings, to confer resistance to the simultaneous presence of different relevant stresses were identified as required for maximal fermentation performance under industrial conditions. Results Chemogenomics data were used to identify eight genes whose expression confers simultaneous resistance to high concentrations of glucose, acetic acid and ethanol, chemical stresses relevant for VHG fermentations; and eleven genes conferring simultaneous resistance to stresses relevant during lignocellulosic fermentations. These eleven genes were identified based on two different sets: one with five genes granting simultaneous resistance to ethanol, acetic acid and furfural, and the other with six genes providing simultaneous resistance to ethanol, acetic acid and vanillin. The expression of Bud31 and Hpr1 was found to lead to the increase of both ethanol yield and fermentation rate, while Pho85, Vrp1 and Ygl024w expression is required for maximal ethanol production in VHG fermentations. Five genes, Erg2, Prs3, Rav1, Rpb4 and Vma8, were found to contribute to the maintenance of cell viability in wheat straw hydrolysate and/or the maximal fermentation rate of this substrate. Conclusions The identified genes stand as preferential targets for genetic engineering manipulation in order to generate more robust industrial strains, able to cope with the most significant fermentation stresses and, thus, to increase ethanol production rate and final ethanol titers.

  16. Constitutive gene expression profile segregates toxicity in locally advanced breast cancer patients treated with high-dose hyperfractionated radical radiotherapy

    International Nuclear Information System (INIS)

    Breast cancer patients show a wide variation in normal tissue reactions after radiotherapy. The individual sensitivity to x-rays limits the efficiency of the therapy. Prediction of individual sensitivity to radiotherapy could help to select the radiation protocol and to improve treatment results. The aim of this study was to assess the relationship between gene expression profiles of ex vivo un-irradiated and irradiated lymphocytes and the development of toxicity due to high-dose hyperfractionated radiotherapy in patients with locally advanced breast cancer. Raw data from microarray experiments were uploaded to the Gene Expression Omnibus Database http://www.ncbi.nlm.nih.gov/geo/ (GEO accession GSE15341). We obtained a small group of 81 genes significantly regulated by radiotherapy, lumped in 50 relevant pathways. Using ANOVA and t-test statistical tools we found 20 and 26 constitutive genes (0 Gy) that segregate patients with and without acute and late toxicity, respectively. Non-supervised hierarchical clustering was used for the visualization of results. Six and 9 pathways were significantly regulated respectively. Concerning to irradiated lymphocytes (2 Gy), we founded 29 genes that separate patients with acute toxicity and without it. Those genes were gathered in 4 significant pathways. We could not identify a set of genes that segregates patients with and without late toxicity. In conclusion, we have found an association between the constitutive gene expression profile of peripheral blood lymphocytes and the development of acute and late toxicity in consecutive, unselected patients. These observations suggest the possibility of predicting normal tissue response to irradiation in high-dose non-conventional radiation therapy regimens. Prospective studies with higher number of patients are needed to validate these preliminary results

  17. Genomics and relative expression analysis identifies key genes associated with high female to male flower ratio in Jatropha curcas L.

    Science.gov (United States)

    Gangwar, Manali; Sood, Hemant; Chauhan, Rajinder Singh

    2016-04-01

    Jatropha curcas, has been projected as a major source of biodiesel due to high seed oil content (42 %). A major roadblock for commercialization of Jatropha-based biodiesel is low seed yield per inflorescence, which is affected by low female to male flower ratio (1:25-30). Molecular dissection of female flower development by analyzing genes involved in phase transitions and floral organ development is, therefore, crucial for increasing seed yield. Expression analysis of 42 genes implicated in floral organ development and sex determination was done at six floral developmental stages of a J. curcas genotype (IC561235) with inherently higher female to male flower ratio (1:8-10). Relative expression analysis of these genes was done on low ratio genotype. Genes TFL1, SUP, AP1, CRY2, CUC2, CKX1, TAA1 and PIN1 were associated with reproductive phase transition. Further, genes CUC2, TAA1, CKX1 and PIN1 were associated with female flowering while SUP and CRY2 in female flower transition. Relative expression of these genes with respect to low female flower ratio genotype showed up to ~7 folds increase in transcript abundance of SUP, TAA1, CRY2 and CKX1 genes in intermediate buds but not a significant increase (~1.25 folds) in female flowers, thereby suggesting that these genes possibly play a significant role in increased transition towards female flowering by promoting abortion of male flower primordia. The outcome of study has implications in feedstock improvement of J. curcas through functional validation and eventual utilization of key genes associated with female flowering. PMID:26878857

  18. HIGH FREQUENCY GENETIC TRANSFORMATION OF CICHORIUM INTYBUS L. USING nptII GENE AS A SELECTIVE MARKER.

    Science.gov (United States)

    Matvieieva, N; Shakhovsky, A; Kvasko, O; Kuchuk, N

    2015-01-01

    Cichorium intybus L. is an important vegetable crop used as salad (leaf form) and for the production of coffee substitutes (root form). At the same time these plants can also be used in biotechnologies for synthesis of pharmaceutical proteins. Here we report the possibility of high frequency Agrobacterium rhizogenes- or A. tumefaciens-mediated transformation of C. intybus L. for construction of transgenic "hairy" roots and plants. The used plasmids contained target human interferonifn-α2b gene, Mycobacterium tuberculosis ESAT6:Ag85B antigene esxA::fbpB(ΔTMD) fused gene and human telomerase reverse transcriptase h Tert gene. Using of nptII gene as a selective one was preferable to the bar gene for chicory. In this case the frequency of transgenic plants or "hairy" roots formation was significantly higher. Cultivation of explants on the medium with Basta in concentration 1-2 mg/l have led to plants death or to significant reduction of number of shoots formed. Frequency of "hairy" roots formation varied from 5.9 to 42.3% after A. rhizogenes-mediated transformation. Frequency of regeneration of transgenic plants varied from 10 to 86% after A. tumefaciens-mediated transformation. Both A. rhizogenes- and A. tumefaciens-mediated transformation frequency depended on the type of explants, roots or cotyledons, and vector used. Usage of A. tumefaciens carrying pCB064 plasmid (target esxA:fbpB(ΔTMD) fused gene and nptII selective gene) resulted in the most effective regeneration of transgenic plants with regeneration frequency up to 86%. In the case of chicory A. rhizogenes-mediated transformation the highest regeneration frequency up to 42.3% was demonstrated using p CB161 vector with ifn-α2b target gene and nptII selective gene. PMID:26419064

  19. Effects of dietary high fructose corn syrup on regulation of energy intake and leptin gene expression in rats

    Directory of Open Access Journals (Sweden)

    Guadalupe López-Rodríguez

    2015-12-01

    Full Text Available OBJECTIVE: To evaluate in Wistar rats the effect of chronic use of high fructose corn syrup on serum lipids, body weight, energy intake regulation, and expression of associated genes. METHODS: For 11 weeks, male rats were fed a standard diet with either water (control or 15% high fructose corn syrup solution, or fed a high-fat diet. The rats' food intake and body weight were measured weekly. Expression of leptin and fatty acid synthase genes was quantified in their brain and adipose tissue upon sacrifice at age 119 days using real-time polymerase chain reaction. RESULTS: The intake of 15% high fructose corn syrup did not affect the rats' weight, only the rats on the high-fat diet gained significant weight. The rats in both diets had lower levels of leptin expression and high levels of fatty acid synthase in the brain, which were associated with high serum triglycerides. CONCLUSION: Fifteen percent high fructose corn syrup intake and the high-fat diet reduced leptin gene expression in the brain of Wistar rats, with differential effects on weight gain.

  20. 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.

  1. High Expression of the RECK Gene in Breast Cancer Cells is Related to Low Invasive Capacity

    Institute of Scientific and Technical Information of China (English)

    Tao Sun; Daqing Jiang; Jinming Li; Dongyun Han; Zhiguo Song

    2006-01-01

    OBJECTIVE To investigate the expression of the RECK gene in human breast (cancer) cell lines, and to determine the relationship between RECK gene expression and the invasive capacity of the breast cancer cell lines.METHODS The invasive capacity of breast (cancer) cell lines including HBL-100, MCF-7 and MDA-MB-435S were determined by the Transwell method. The protein expression levels of RECK, MMP-2 and MMP- 9 genes in these three cell lines were measured by immunocytochemical methods. The expressions of the RECK gene and protein level were measured by RT-PCR and Western blots in the cell lines respectively.RESULTS The order of the invasive capacity of the breast (cancer) cell lines was MDA-MB-435S, being the highest, and HBL-100, being the lowest. The invasive capacity difference between any two groups among the three groups was significant (P<0.01). The protein expression level of the RECK gene in the HBL-100 cell line was highest, and no expression was detected in MDA-MB-435S cells. Moreover, the expression of the RECK gene was negatively correlated with the expression of the MMP-2 and MMP-9 genes. The mRNA level of the RECK gene in HBL-100 cells was the highest, but no expression was found in the MDA-MB-435S cells (P<0.001).CONCLUSION There was a significant negative correlation between the expression level of the RECK gene and invasive capacity in vitro, and the RECK gene expression showed an inverse proportion to that of the MMP-2, MMP-9 genes.

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

    International Nuclear Information System (INIS)

    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. 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. 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 male patient, expressed the mutated WTX

  3. 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

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

    Institute of Scientific and Technical Information of China (English)

    Chen Hualan; Yu Kangzhen; Bu Zhigao

    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.

  5. Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map.

    Science.gov (United States)

    Wicks, S R; Yeh, R T; Gish, W R; Waterston, R H; Plasterk, R H

    2001-06-01

    Single nucleotide polymorphisms (SNPs) are valuable genetic markers of human disease. They also comprise the highest potential density marker set available for mapping experimentally derived mutations in model organisms such as Caenorhabditis elegans. To facilitate the positional cloning of mutations we have identified polymorphisms in CB4856, an isolate from a Hawaiian island that shows a uniformly high density of polymorphisms compared with the reference Bristol N2 strain. Based on 5.4 Mbp of aligned sequences, we predicted 6,222 polymorphisms. Furthermore, 3,457 of these markers modify restriction enzyme recognition sites ('snip-SNPs') and are therefore easily detected as RFLPs. Of these, 493 were experimentally confirmed by restriction digest to produce a snip-SNP map of the worm genome. A mapping strategy using snip-SNPs and bulked segregant analysis (BSA) is outlined. CB4856 is crossed into a mutant strain, and exclusion of CB4856 alleles of a subset of snip-SNPs in mutant progeny is assessed with BSA. The proximity of a linked marker to the mutation is estimated by the relative proportion of each form of the biallelic marker in populations of wildtype and mutant genomes. The usefulness of this approach is illustrated by the rapid mapping of the dyf-5 gene. PMID:11381264

  6. 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.

  7. High gene flow between alternative morphs and the evolutionary persistence of facultative paedomorphosis.

    Science.gov (United States)

    Oromi, Neus; Michaux, Johan; Denoël, Mathieu

    2016-01-01

    Paedomorphosis and metamorphosis are two major developmental processes that characterize the evolution of complex life cycles in many lineages. Whereas these processes were fixed in some taxa, they remained facultative in others, with alternative phenotypes expressed in the same populations. From a genetic perspective, it is still unknown whether such phenotypes form a single population or whether they show some patterns of isolation in syntopy. This has deep implications for understanding the evolution of the phenotypes, i.e. towards their persistence or their fixation and speciation. Newts and salamanders are excellent models to test this hypothesis because they exhibit both developmental processes in their populations: the aquatic paedomorphs retain gills, whereas the metamorphs are able to colonize land. Using microsatellite data of coexisting paedomorphic and metamorphic palmate newts (Lissotriton helveticus), we found that they formed a panmictic population, which evidences sexual compatibility between the two phenotypes. The high gene flow could be understood as an adaptation to unstable habitats in which phenotypic plasticity is favored over the fixation of developmental alternatives. This makes then possible the persistence of a polyphenism: only metamorphosis could be maintained in case of occasional drying whereas paedomorphosis could offer specific advantages in organisms remaining in water. PMID:27534370

  8. 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.

  9. High-frequency conjugative transfer of antibiotic resistance genes to Yersinia pestis in the flea midgut.

    Science.gov (United States)

    Hinnebusch, B Joseph; Rosso, Marie-Laure; Schwan, Tom G; Carniel, Elisabeth

    2002-10-01

    The acquisition of foreign DNA by horizontal transfer from unrelated organisms is a major source of variation leading to new strains of bacterial pathogens. The extent to which this occurs varies widely, due in part to lifestyle factors that determine exposure to potential donors. Yersinia pestis, the plague bacillus, infects normally sterile sites in its mammalian host, but forms dense aggregates in the non-sterile digestive tract of its flea vector to produce a transmissible infection. Here we show that unrelated co-infecting bacteria in the flea midgut are readily incorporated into these aggregates, and that this close physical contact leads to high-frequency conjugative genetic exchange. Transfer of an antibiotic resistance plasmid from an Escherichia coli donor to Y. pestis occurred in the flea midgut at a frequency of 10-3 after only 3 days of co-infection, and after 4 weeks 95% of co-infected fleas contained an average of 103 antibiotic-resistant Y. pestis transconjugants. Thus, transit in its arthropod vector exposes Y. pestis to favourable conditions for efficient genetic exchange with microbial flora of the flea gut. Horizontal gene transfer in the flea may be the source of antibiotic-resistant Y. pestis strains recently isolated from plague patients in Madagascar. PMID:12406213

  10. 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

    and affective disorders. Recently, copy number variants (CNVs) in SLC6A3 have been identified in healthy subjects but so far, the implication of CNVs affecting this gene in psychiatric diseases has not been addressed. AIMS: In the present study, we aimed to investigate whether CNVs affecting SLC6A3 represent...... 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....

  11. 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...

  12. 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...

  13. Quality Control Usage in High-Density Microarrays Reveals Differential Gene Expression Profiles in Ovarian Cancer.

    Science.gov (United States)

    Villegas-Ruiz, Vanessa; Moreno, Jose; Jacome-Lopez, Karina; Zentella-Dehesa, Alejandro; Juarez-Mendez, Sergio

    2016-01-01

    There are several existing reports of microarray chip use for assessment of altered gene expression in different diseases. In fact, there have been over 1.5 million assays of this kind performed over the last twenty years, which have influenced clinical and translational research studies. The most commonly used DNA microarray platforms are Affymetrix GeneChip and Quality Control Software along with their GeneChip Probe Arrays. These chips are created using several quality controls to confirm the success of each assay, but their actual impact on gene expression profiles had not been previously analyzed until the appearance of several bioinformatics tools for this purpose. We here performed a data mining analysis, in this case specifically focused on ovarian cancer, as well as healthy ovarian tissue and ovarian cell lines, in order to confirm quality control results and associated variation in gene expression profiles. The microarray data used in our research were downloaded from ArrayExpress and Gene Expression Omnibus (GEO) and analyzed with Expression Console Software using RMA, MAS5 and Plier algorithms. The gene expression profiles were obtained using Partek Genomics Suite v6.6 and data were visualized using principal component analysis, heat map, and Venn diagrams. Microarray quality control analysis showed that roughly 40% of the microarray files were false negative, demonstrating over- and under-estimation of expressed genes. Additionally, we confirmed the results performing second analysis using independent samples. About 70% of the significant expressed genes were correlated in both analyses. These results demonstrate the importance of appropriate microarray processing to obtain a reliable gene expression profile. PMID:27268623

  14. 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.

  15. 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.

  16. Changes in Expression of Genes Regulating Airway Inflammation Following a High-Fat Mixed Meal in Asthmatics.

    Science.gov (United States)

    Li, Qian; Baines, Katherine J; Gibson, Peter G; Wood, Lisa G

    2016-01-01

    Consumption of a high fat meal can increase neutrophilic airway inflammation in asthma subjects. This study investigates the molecular mechanisms driving airway neutrophilia following a high fat meal in asthmatics. Subjects with asthma (n = 11) and healthy controls (n = 8) consumed a high-fat/energy meal, containing total energy (TE) of 3846 kJ and 48 g of total fat (20.5 g saturated). Sputum was induced at 0 and 4 h, and gene expression was examined by microarray and quantitative real-time PCR (qPCR). Following the high fat dietary challenge, 168 entities were significantly differentially expressed greater than >1.5 fold in subjects with asthma, whereas, in healthy controls, only 14 entities were differentially expressed. Of the 168 genes that were changed in asthma, several biological processes were overrepresented, with 25 genes involved in "immune system processes". qPCR confirmed that S100P, S100A16, MAL and MUC1 were significantly increased in the asthma group post-meal. We also observed a strong correlation and a moderate correlation between the change in NLRP12 and S100A16 gene expression at 4 h compared to baseline, and the change in total and saturated non-esterified plasma fatty acid levels at 2 h compared to baseline. In summary, our data identifies differences in inflammatory gene expression that may contribute to increased airway neutrophilia following a high fat meal in subjects with asthma and may provide useful therapeutic targets for immunomodulation. This may be particularly relevant to obese asthmatics, who are habitually consuming diets with a high fat content. PMID:26751474

  17. Changes in Expression of Genes Regulating Airway Inflammation Following a High-Fat Mixed Meal in Asthmatics

    Directory of Open Access Journals (Sweden)

    Qian Li

    2016-01-01

    Full Text Available Consumption of a high fat meal can increase neutrophilic airway inflammation in asthma subjects. This study investigates the molecular mechanisms driving airway neutrophilia following a high fat meal in asthmatics. Subjects with asthma (n = 11 and healthy controls (n = 8 consumed a high-fat/energy meal, containing total energy (TE of 3846 kJ and 48 g of total fat (20.5 g saturated. Sputum was induced at 0 and 4 h, and gene expression was examined by microarray and quantitative real-time PCR (qPCR. Following the high fat dietary challenge, 168 entities were significantly differentially expressed greater than >1.5 fold in subjects with asthma, whereas, in healthy controls, only 14 entities were differentially expressed. Of the 168 genes that were changed in asthma, several biological processes were overrepresented, with 25 genes involved in “immune system processes”. qPCR confirmed that S100P, S100A16, MAL and MUC1 were significantly increased in the asthma group post-meal. We also observed a strong correlation and a moderate correlation between the change in NLRP12 and S100A16 gene expression at 4 h compared to baseline, and the change in total and saturated non-esterified plasma fatty acid levels at 2 h compared to baseline. In summary, our data identifies differences in inflammatory gene expression that may contribute to increased airway neutrophilia following a high fat meal in subjects with asthma and may provide useful therapeutic targets for immunomodulation. This may be particularly relevant to obese asthmatics, who are habitually consuming diets with a high fat content.

  18. 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 gene...... low pathogenic avian influenza A viruses. (c) 2006 Elsevier Ltd. All rights reserved....

  19. The Genome Sequence of Alcaligenes faecalis NBIB-017 Contains Genes with Potentially High Activities against Erwinia carotovora

    Science.gov (United States)

    Liu, Xiaoyan; Huang, Daye; Wu, Jinping; Yu, Cui; Zhou, Ronghua; Liu, Cuijun; Zhang, Wei; Yao, Jingwu; Cheng, Meng

    2016-01-01

    Alcaligenes faecalis NBIB-017, a Gram-negative bacterium, was isolated from soil in China. Here, we provide the complete genome sequence of this bacterium, which possesses a high number of genes encoding antibacterial factors, including proteins and small molecular peptides. PMID:27056227

  20. 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

  1. 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

  2. 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.

  3. High-throughput sequence analysis of turbot (Scophthalmus maximus transcriptome using 454-pyrosequencing for the discovery of antiviral immune genes.

    Directory of Open Access Journals (Sweden)

    Patricia Pereiro

    Full Text Available BACKGROUND: Turbot (Scophthalmus maximus L. is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations. METHODOLOGY/PRINCIPAL FINDINGS: Turbot were injected with viral stimuli to increase the expression level of immune-related genes. High-throughput deep sequencing using 454-pyrosequencing technology yielded 915,256 high-quality reads. These sequences were assembled into 55,404 contigs that were subjected to annotation steps. Intriguingly, 55.16% of the deduced protein was not significantly similar to any sequences in the databases used for the annotation and only 0.85% of the BLASTx top-hits matched S. maximus protein sequences. This relatively low level of annotation is possibly due to the limited information for this specie and other flatfish in the database. These results suggest the identification of a large number of new genes in turbot and in fish in general. A more detailed analysis showed the presence of putative members of several innate and specific immune pathways. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this study is the first transcriptome analysis using 454-pyrosequencing for turbot. Previously, there were only 12,471 EST and less of 1,500 nucleotide sequences for S. maximus in NCBI database. Our results provide a rich source of data (55,404 contigs and 181,845 singletons for discovering and identifying new genes, which will serve as a basis for microarray construction, gene expression characterization and for identification of genetic markers to be used in several applications. Immune stimulation in turbot was very

  4. 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.

  5. 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.

  6. 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. PMID:26746430

  7. High preservation of CpG cytosine methylation patterns at imprinted gene loci in liver and brain of aged mice.

    Directory of Open Access Journals (Sweden)

    Silvia Gravina

    Full Text Available A gradual loss of the correct patterning of 5-methyl cytosine marks in gene promoter regions has been implicated in aging and age-related diseases, most notably cancer. While a number of studies have examined DNA methylation in aging, there is no consensus on the magnitude of the effects, particularly at imprinted loci. Imprinted genes are likely candidate to undergo age-related changes because of their demonstrated plasticity in utero, for example, in response to environmental cues. Here we quantitatively analyzed a total of 100 individual CpG sites in promoter regions of 11 imprinted and non-imprinted genes in liver and cerebral cortex of young and old mice using mass spectrometry. The results indicate a remarkably high preservation of methylation marks during the aging process in both organs. To test if increased genotoxic stress associated with premature aging would destabilize DNA methylation we analyzed two DNA repair defective mouse models showing a host of premature aging symptoms in liver and brain. However, also in these animals, at the end of their life span, we found a similarly high preservation of DNA methylation marks. We conclude that patterns of DNA methylation in gene promoters of imprinted genes are surprisingly stable over time in normal, postmitotic tissues and that the multiple documented changes with age are likely to involve exceptions to this pattern, possibly associated with specific cellular responses to age-related changes other than genotoxic stress.

  8. 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.

  9. 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.

  10. 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

  11. PCR primers to study the diversity of expressed fungal genes encoding lignocellulolytic enzymes in soils using high-throughput sequencing.

    Science.gov (United States)

    Barbi, Florian; Bragalini, Claudia; Vallon, Laurent; Prudent, Elsa; Dubost, Audrey; Fraissinet-Tachet, Laurence; Marmeisse, Roland; Luis, Patricia

    2014-01-01

    Plant biomass degradation in soil is one of the key steps of carbon cycling in terrestrial ecosystems. Fungal saprotrophic communities play an essential role in this process by producing hydrolytic enzymes active on the main components of plant organic matter. Open questions in this field regard the diversity of the species involved, the major biochemical pathways implicated and how these are affected by external factors such as litter quality or climate changes. This can be tackled by environmental genomic approaches involving the systematic sequencing of key enzyme-coding gene families using soil-extracted RNA as material. Such an approach necessitates the design and evaluation of gene family-specific PCR primers producing sequence fragments compatible with high-throughput sequencing approaches. In the present study, we developed and evaluated PCR primers for the specific amplification of fungal CAZy Glycoside Hydrolase gene families GH5 (subfamily 5) and GH11 encoding endo-β-1,4-glucanases and endo-β-1,4-xylanases respectively as well as Basidiomycota class II peroxidases, corresponding to the CAZy Auxiliary Activity family 2 (AA2), active on lignin. These primers were experimentally validated using DNA extracted from a wide range of Ascomycota and Basidiomycota species including 27 with sequenced genomes. Along with the published primers for Glycoside Hydrolase GH7 encoding enzymes active on cellulose, the newly design primers were shown to be compatible with the Illumina MiSeq sequencing technology. Sequences obtained from RNA extracted from beech or spruce forest soils showed a high diversity and were uniformly distributed in gene trees featuring the global diversity of these gene families. This high-throughput sequencing approach using several degenerate primers constitutes a robust method, which allows the simultaneous characterization of the diversity of different fungal transcripts involved in plant organic matter degradation and may lead to the

  12. PCR primers to study the diversity of expressed fungal genes encoding lignocellulolytic enzymes in soils using high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Florian Barbi

    Full Text Available Plant biomass degradation in soil is one of the key steps of carbon cycling in terrestrial ecosystems. Fungal saprotrophic communities play an essential role in this process by producing hydrolytic enzymes active on the main components of plant organic matter. Open questions in this field regard the diversity of the species involved, the major biochemical pathways implicated and how these are affected by external factors such as litter quality or climate changes. This can be tackled by environmental genomic approaches involving the systematic sequencing of key enzyme-coding gene families using soil-extracted RNA as material. Such an approach necessitates the design and evaluation of gene family-specific PCR primers producing sequence fragments compatible with high-throughput sequencing approaches. In the present study, we developed and evaluated PCR primers for the specific amplification of fungal CAZy Glycoside Hydrolase gene families GH5 (subfamily 5 and GH11 encoding endo-β-1,4-glucanases and endo-β-1,4-xylanases respectively as well as Basidiomycota class II peroxidases, corresponding to the CAZy Auxiliary Activity family 2 (AA2, active on lignin. These primers were experimentally validated using DNA extracted from a wide range of Ascomycota and Basidiomycota species including 27 with sequenced genomes. Along with the published primers for Glycoside Hydrolase GH7 encoding enzymes active on cellulose, the newly design primers were shown to be compatible with the Illumina MiSeq sequencing technology. Sequences obtained from RNA extracted from beech or spruce forest soils showed a high diversity and were uniformly distributed in gene trees featuring the global diversity of these gene families. This high-throughput sequencing approach using several degenerate primers constitutes a robust method, which allows the simultaneous characterization of the diversity of different fungal transcripts involved in plant organic matter degradation and may

  13. 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.

  14. Response of antibiotics and resistance genes to high-intensity and low-intensity manure management.

    Science.gov (United States)

    Storteboom, Heather N; Kim, Sung-Chul; Doesken, Kathy C; Carlson, Kenneth H; Davis, Jessica G; Pruden, Amy

    2007-01-01

    The purpose of this study was to determine the response of antibiotics and antibiotic resistance genes (ARG) to manure management. A pilot field study was conducted using horse manure containing no antibiotics, into which chlortetracycline (CTC), tylosin (TYL), and monensin (MON) were spiked and compared to unspiked controls. Subsequently, a large-scale field study was conducted comparing manure from a dairy with minimal use of antibiotics and a feedlot with regular subtherapeutic use of antibiotics. The manures were subjected to high-intensity management (HIM) (amending, watering, and turning) and low-intensity management (LIM) (no amending, watering, or turning) and were monitored for antibiotic concentrations and levels of tetracycline ARG [tet(W) and tet(O)] using quantitative real-time polymerase chain reaction. All three antibiotics in the pilot study dissipated more rapidly in HIM manure, with half-lives ranging from 4 to 15 d, compared to LIM manure, with half-lives ranging from 8 to 30 d. Levels of tet(W) were significantly higher after 141 d of treatment, but levels of tet(O) were significantly lower in all treatments. In the large-scale study, the feedlot manure had higher initial concentrations than the dairy manure of tetracycline (TC), oxytetracycline (OTC), and CTC as well as tet(W) and tet(O). Tetracycline and OTC dissipated more rapidly in HIM manure, with half-lives ranging from 6 to 15 d, compared to LIM manure, with half-lives ranging from 7 to 31 d. After 6 mo of treatment, tet(W) and tet(O) decreased significantly in feedlot manure, whereas dairy manure required only 4 mo of treatment for similar results.

  15. A multi-layer microchip for high-throughput single-cell gene expression profiling.

    Science.gov (United States)

    Sun, Hao

    2016-09-01

    Microfluidics or Bio-MEMS technology offers significant advantages for performing high-throughput screens and sensitive assays. The ability to correlate single-cell genetic information with cellular phenotypes is of great importance to biology and medicine because it holds the potential to gain insight into disease pathways that is unavailable from ensemble measurements. Previously, we reported two kinds of prototypes for integrated on-chip gene expression profiling at the single-cell level, and the throughput was designed to be 6. In this work, we present a five-layer microfluidic system for parallelized, rapid, quantitative analysis of RNA templates with low abundance at the single-cell level. The microchip contains two multiplexors and one partitioning valve group, and it leverages a matrix (6 × 8) of quantitative reverse transcription polymerase chain reaction (RT-qPCR) units formed by a set of parallel microchannels concurrently controlled by elastomeric pneumatic valves, thereby enabling parallelized handling and processing of biomolecules in a simplified operation procedure. A comprehensive metallic nanofilm with passivation layer is used to run polymerase chain reaction (PCR) temperature cycles. To demonstrate the utility of the approach, artificial synthesized RNA templates (XenoRNA) and mRNA templates from single cells are employed to perform the 48-readout RT-qPCRs. The PCR products are imaged on a fluorescence microscope using a hydrolysis probe/primer set (TaqMan). Fluorescent intensities of passive reference dye and a fluorescein amidite reporter dye are acquired and measured at the end of PCR cycles. PMID:27255567

  16. Discovering biclusters in gene expression data based on high-dimensional linear geometries

    OpenAIRE

    Liew Alan; Gan Xiangchao; Yan Hong

    2008-01-01

    Abstract Background In DNA microarray experiments, discovering groups of genes that share similar transcriptional characteristics is instrumental in functional annotation, tissue classification and motif identification. However, in many situations a subset of genes only exhibits consistent pattern over a subset of conditions. Conventional clustering algorithms that deal with the entire row or column in an expression matrix would therefore fail to detect these useful patterns in the data. Rece...

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

    OpenAIRE

    Lingli eYu; Florie eReynaud; Julien eFalk; Ambre eSpencer; Yin-Di eDing; Véronique eBaumlé; Ruisheng eLu; Valérie eCastellani; Chonggang eYuan; Rudkin, Brian B.

    2015-01-01

    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...

  18. High-frequency intrachromosomal gene conversion induced by triplex-forming oligonucleotides microinjected into mouse cells

    OpenAIRE

    Luo, Zhongjun; Macris, Margaret A.; Faruqi, A. Fawad; Glazer, Peter M.

    2000-01-01

    To test the ability of triple helix-forming oligonucleotides (TFOs) to promote recombination within chromosomal sites in mammalian cells, a mouse LTK− cell line was established carrying two mutant copies of the herpes simplex virus thymidine kinase (TK) gene as direct repeats in a single chromosomal locus. Recombination between these repeats can produce a functional TK gene and occurs at a spontaneous frequency of 4 × 10−6 under standard culture conditions. When cells were microinjected with ...

  19. Mutation detection in the human HSP70B′ gene by denaturing high-performance liquid chromatography

    OpenAIRE

    Hecker, Karl H.; Asea, Alexzander; Kobayashi, Kaoru; Green, Stacy; Tang, Dan; Calderwood, Stuart K

    2000-01-01

    Variances, particularly single nucleotide polymorphisms (SNP), in the genomic sequence of individuals are the primary key to understanding gene function as it relates to differences in the susceptibility to disease, environmental influences, and therapy. In this report, the HSP70B′ gene is the target sequence for mutation detection in biopsy samples from human prostate cancer patients undergoing combined hyperthermia and radiation therapy at the Dana-Farber Cancer Institute, using temperature...

  20. 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.

  1. Synthetic cryIIIA gene from Bacillus thuringiensis improved for high expression in plants.

    Science.gov (United States)

    Sutton, D W; Havstad, P K; Kemp, J D

    1992-09-01

    A 1974 bp synthetic gene was constructed from chemically synthesized oligonucleotides in order to improve transgenic protein expression of the cryIIIA gene from Bacillus thuringiensis var. tenebrionis in transgenic tobacco. The crystal toxin genes (cry) from B. thuringiensis are difficult to express in plants even when under the control of efficient plant regulatory sequences. We identified and eliminated five classes of sequence found throughout the cryIIIA gene that mimic eukaryotic processing signals and which may be responsible for the low levels of transcription and translation. Furthermore, the GC content of the gene was raised from 36% to 49% and the codon usage was changed to be more plant-like. When the synthetic gene was placed behind the cauliflower mosaic virus 35S promoter and the alfalfa mosaic virus translational enhancer, up to 0.6% of the total protein in transgenic tobacco plants was cryIIIA as measured from immunoblot analysis. Bioassay data using potato beetle larvae confirmed this estimate. PMID:1301214

  2. Bioinformatic analysis reveals high diversity of bacterial genes for laccase-like enzymes.

    Directory of Open Access Journals (Sweden)

    Luka Ausec

    Full Text Available Fungal laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and three-domain laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76% of the genes, signal peptides were predicted, indicating that these bacterial laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications.

  3. Bioinformatic Analysis Reveals High Diversity of Bacterial Genes for Laccase-Like Enzymes

    Science.gov (United States)

    Ausec, Luka; Zakrzewski, Martha; Goesmann, Alexander; Schlüter, Andreas; Mandic-Mulec, Ines

    2011-01-01

    Fungal laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and three- domain laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76% of the genes, signal peptides were predicted, indicating that these bacterial laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications. PMID:22022440

  4. 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.

  5. 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. PMID:26919178

  6. 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.

  7. Manifestation in F3 of pleiotropic gene in the cross between mother variety and its high protein mutant pt. 2

    International Nuclear Information System (INIS)

    Rice variety Hokwang was X-rayed in 1965 and a high protein line No. 398 was selected. This mutant, besides high protein, accompanied two visible mutated characters, short culm and early heading. In 1973 crossing was made between mutant 398 and its mother variety and in 1976 F3 generations were grown to examine the manifestation of the mutated gene. It was confirmed that the above-mentioned mutated characters were controlled by a single recessive major gene of pleiotropic nature. Recessive deficit was observed in the segregation ratio of mutated characters. Besides the parental types, there were deviants which were assumed to be caused by the changed genetic background. It was considered possible to make a selection, from among the deviants, of mother-like type with high protein content. (author)

  8. 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.

  9. 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.

  10. 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.

  11. A high-density association screen of 155 ion transport genes for involvement with common migraine

    Science.gov (United States)

    Nyholt, Dale R.; LaForge, K. Steven; Kallela, Mikko; Alakurtti, Kirsi; Anttila, Verneri; Färkkilä, Markus; Hämaläinen, Eija; Kaprio, Jaakko; Kaunisto, Mari A.; Heath, Andrew C.; Montgomery, Grant W.; Göbel, Hartmut; Todt, Unda; Ferrari, Michel D.; Launer, Lenore J.; Frants, Rune R.; Terwindt, Gisela M.; de Vries, Boukje; Verschuren, W.M. Monique; Brand, Jan; Freilinger, Tobias; Pfaffenrath, Volker; Straube, Andreas; Ballinger, Dennis G.; Zhan, Yiping; Daly, Mark J.; Cox, David R.; Dichgans, Martin; van den Maagdenberg, Arn M.J.M.; Kubisch, Christian; Martin, Nicholas G.; Wessman, Maija; Peltonen, Leena; Palotie, Aarno

    2008-01-01

    The clinical overlap between monogenic Familial Hemiplegic Migraine (FHM) and common migraine subtypes, and the fact that all three FHM genes are involved in the transport of ions, suggest that ion transport genes may underlie susceptibility to common forms of migraine. To test this leading hypothesis, we examined common variation in 155 ion transport genes using 5257 single nucleotide polymorphisms (SNPs) in a Finnish sample of 841 unrelated migraine with aura cases and 884 unrelated non-migraine controls. The top signals were then tested for replication in four independent migraine case–control samples from the Netherlands, Germany and Australia, totalling 2835 unrelated migraine cases and 2740 unrelated controls. SNPs within 12 genes (KCNB2, KCNQ3, CLIC5, ATP2C2, CACNA1E, CACNB2, KCNE2, KCNK12, KCNK2, KCNS3, SCN5A and SCN9A) with promising nominal association (0.00041 < P < 0.005) in the Finnish sample were selected for replication. Although no variant remained significant after adjusting for multiple testing nor produced consistent evidence for association across all cohorts, a significant epistatic interaction between KCNB2 SNP rs1431656 (chromosome 8q13.3) and CACNB2 SNP rs7076100 (chromosome 10p12.33) (pointwise P = 0.00002; global P = 0.02) was observed in the Finnish case–control sample. We conclude that common variants of moderate effect size in ion transport genes do not play a major role in susceptibility to common migraine within these European populations, although there is some evidence for epistatic interaction between potassium and calcium channel genes, KCNB2 and CACNB2. Multiple rare variants or trans-regulatory elements of these genes are not ruled out. PMID:18676988

  12. 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.

  13. Liver Fatty Acid Binding Protein Gene-ablation Exacerbates Weight Gain in High-Fat Fed Female Mice

    OpenAIRE

    McIntosh, Avery L.; Atshaves, Barbara P.; Landrock, Danilo; Landrock, Kerstin K.; Martin, Gregory G.; Storey, Stephen M.; Kier, Ann B.; Schroeder, Friedhelm

    2013-01-01

    Loss of liver fatty acid binding protein (L-FABP) decreases long chain fatty acid uptake and oxidation in primary hepatocytes and in vivo. On this basis, L-FABP gene ablation would potentiate high-fat diet-induced weight gain and weight gain/energy intake. While this was indeed the case when L-FABP null (−/−) mice on the C57BL/6NCr background were pair-fed high fat diet, whether this would also be observed under high-fat diet fed ad libitum was not known. Therefore, this possibility was exami...

  14. 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.

  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. Identification of novel highly expressed genes in pancreatic ductal adenocarcinomas through a bioinformatics analysis of expressed sequence tags.

    Science.gov (United States)

    Cao, Dengfeng; Hustinx, Steven R; Sui, Guoping; Bala, P; Sato, Norihiro; Martin, Sean; Maitra, Anirban; Murphy, Kathleen M; Cameron, John L; Yeo, Charles J; Kern, Scott E; Goggins, Michael; Pandey, Akhilesh; Hruban, Ralph H

    2004-11-01

    In most microarray experiments, a significant fraction of the differentially expressed mRNAs identified correspond to expressed sequence tags (ESTs) and are generally discarded from further analyses. We used careful bioinformatics analyses to characterize those ESTs that were found to be highly overexpressed in a series of pancreatic adenocarcinomas. cDNA was prepared from 60 non-neoplastic samples (normal pancreas [n = 20], normal colon [n = 10], or normal duodenal mucosal [n = 30]) and from 64 pancreatic cancers (resected cancers [n = 50] or cancer cell lines [n = 14]) and hybridized to the complete Affymetrix Human Genome U133 GeneChip(R) set (arrays U133A and B) for simultaneous analysis of 45,000 fragments corresponding to 33,000 known genes and 6,000 ESTs. The GeneExpress(R) software system Fold Change Analysis Tool was used and 60 ESTs were identified that were expressed at levels at least 3-fold greater in the pancreatic cancers as compared to normal tissues. Searches against the human genomic sequence and comparative genomic analysis of human and mouse genomes was carried out using basic local alignment search tools (BLAST), BLASTN, and BLASTX, for identifying protein coding genes corresponding to the ESTs. Subsequently, in order to pick the most relevant candidate genes for a more detailed analysis, we looked for domains/motifs in the open reading frames using SMART and Pfam programs. We were able to definitively map 43 of the 60 ESTs to known or novel genes, and 15 of the ESTs could be localized in close proximity to a gene in the human genome although we were unable to establish that the EST was indeed derived from those genes. The differential expression of a subset of genes was confirmed at the protein level by immunohistochemical labeling of tissue microarrays (inhibin beta A [INHBA] and CD29) and/or at the transcript level by RT-PCR (INHBA, AKAP12, ELK3, FOXQ1, EIF5A2, and EFNA5). We conclude that bioinformatics tools can be used to characterize

  17. A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression

    Directory of Open Access Journals (Sweden)

    Amsterdam Adam

    2006-06-01

    identification of approximately 25% of the essential genes required for craniofacial development. The identification of zebrafish models for two human disease syndromes indicates that homologs to the other genes are likely to also be relevant for human craniofacial development. The initial characterization of wdr68 suggests an important role in craniofacial development for the highly conserved Wdr68-Dyrk1 protein complexes.

  18. High Rate of Chimeric Gene Origination by Retroposition in Plant Genomes

    DEFF Research Database (Denmark)

    Wang, Wen; Zheng, Hongkung; Fan, Chuanzhu;

    2006-01-01

    Retroposition is widely found to play essential roles in origination of new mammalian and other animal genes. However, the scarcity of retrogenes in plants has led to the assumption that plant genomes rarely evolve new gene duplicates by retroposition, despite abundant retrotransposons in plants...... retrotransposons, confirming a previously observed role of retroelements in generating plant retrogenes. Substitution analyses revealed that the vast majority are subject to negative selection, suggesting, along with expression data and evidence of age, that they are likely functional retrogenes. In addition, 42...

  19. High-level chromate resistance in Arthrobacter sp. strain FB24 requires previously uncharacterized accessory genes

    Directory of Open Access Journals (Sweden)

    Thompson Dorothea K

    2009-09-01

    Full Text Available Abstract Background The genome of Arthrobacter sp. strain FB24 contains a chromate resistance determinant (CRD, consisting of a cluster of 8 genes located on a 10.6 kb fragment of a 96 kb plasmid. The CRD includes chrA, which encodes a putative chromate efflux protein, and three genes with amino acid similarities to the amino and carboxy termini of ChrB, a putative regulatory protein. There are also three novel genes that have not been previously associated with chromate resistance in other bacteria; they encode an oxidoreductase (most similar to malate:quinone oxidoreductase, a functionally unknown protein with a WD40 repeat domain and a lipoprotein. To delineate the contribution of the CRD genes to the FB24 chromate [Cr(VI] response, we evaluated the growth of mutant strains bearing regions of the CRD and transcript expression levels in response to Cr(VI challenge. Results A chromate-sensitive mutant (strain D11 was generated by curing FB24 of its 96-kb plasmid. Elemental analysis indicated that chromate-exposed cells of strain D11 accumulated three times more chromium than strain FB24. Introduction of the CRD into strain D11 conferred chromate resistance comparable to wild-type levels, whereas deletion of specific regions of the CRD led to decreased resistance. Using real-time reverse transcriptase PCR, we show that expression of each gene within the CRD is specifically induced in response to chromate but not by lead, hydrogen peroxide or arsenate. Higher levels of chrA expression were achieved when the chrB orthologs and the WD40 repeat domain genes were present, suggesting their possible regulatory roles. Conclusion Our findings indicate that chromate resistance in Arthrobacter sp. strain FB24 is due to chromate efflux through the ChrA transport protein. More importantly, new genes have been identified as having significant roles in chromate resistance. Collectively, the functional predictions of these additional genes suggest the

  20. [Construction of the flavinogenic yeast Candida famata strains with high riboflavin kinase activity using gene engineering].

    Science.gov (United States)

    Ishchuk, O P; Iatsyshyn, V Iu; Dmytruk, K V; Voronovs'kyĭ, A Ia; Fedorovych, D V; Sybirnyĭ, A A

    2006-01-01

    The recombinant strains of the flavinogenic yeast Candida famata, which contain the DNA fragment consisting of the FMN1 gene (encoding the riboflavin kinase, enzyme that converts riboflavin to flavinmononucleotide) driven by the strong promoters (the regulated RIB1 or constitutive TEF1 promoter) were isolated. Riboflavin kinase activity in the isolated transformants was tested. The 6-8-fold increase of the riboflavin kinase activity was shown in the recombinant strains containing the integrated Debaryomyces hansenii FMN1 gene under the strong constitutive TEF1 promoter. The recombinant strains can be used for the following construction of flavinmononucleotide overproducers. PMID:17290783

  1. Systematic analysis of Zn2Cys6 transcription factors required for development and pathogenicity by high-throughput gene knockout in the rice blast fungus.

    Directory of Open Access Journals (Sweden)

    Jianping Lu

    2014-10-01

    Full Text Available Because of great challenges and workload in deleting genes on a large scale, the functions of most genes in pathogenic fungi are still unclear. In this study, we developed a high-throughput gene knockout system using a novel yeast-Escherichia-Agrobacterium shuttle vector, pKO1B, in the rice blast fungus Magnaporthe oryzae. Using this method, we deleted 104 fungal-specific Zn(2Cys(6 transcription factor (TF genes in M. oryzae. We then analyzed the phenotypes of these mutants with regard to growth, asexual and infection-related development, pathogenesis, and 9 abiotic stresses. The resulting data provide new insights into how this rice pathogen of global significance regulates important traits in the infection cycle through Zn(2Cys(6TF genes. A large variation in biological functions of Zn(2Cys(6TF genes was observed under the conditions tested. Sixty-one of 104 Zn(2Cys(6 TF genes were found to be required for fungal development. In-depth analysis of TF genes revealed that TF genes involved in pathogenicity frequently tend to function in multiple development stages, and disclosed many highly conserved but unidentified functional TF genes of importance in the fungal kingdom. We further found that the virulence-required TF genes GPF1 and CNF2 have similar regulation mechanisms in the gene expression involved in pathogenicity. These experimental validations clearly demonstrated the value of a high-throughput gene knockout system in understanding the biological functions of genes on a genome scale in fungi, and provided a solid foundation for elucidating the gene expression network that regulates the development and pathogenicity of M. oryzae.

  2. 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.

  3. 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.

  4. Correlation between single nucleotide polymorphisms in hypoxia-related genes and susceptibility to acute high-altitude pulmonary edema.

    Science.gov (United States)

    Wu, A L; Xiong, Y S; Li, Z Q; Liu, Y G; Quan, Q; Wu, L J

    2015-01-01

    This study aimed to explore the relationship between genetic changes and high-altitude pulmonary edema (HAPE) susceptibility, and to screen for the key single nucleotide polymorphism (SNP) loci in the HAPE-susceptibility gene, by investigating the SNPs occurring in hypoxia-related genes in HAPE-susceptible and control (non-susceptible) populations. This research was conducted on Han recruits, who travelled to the Lhasa plateau (altitude, 3658 m). Ten loci located on ten genes extracted from the HAPE and healthy populations were amplified by polymerase chain reaction, and subsequently sequenced. The investigated genes included those coding for aldosterone synthase 2 (CYP11B2), angiotensin-converting enzyme (ACE), heat-shock protein 70 (HSP70), nuclear factor kappa B (NF-κB), surfactant protein A2 (SP-A2), plasminogen activator inhibitor-1 (PAI-1), nitric oxide synthetase (NOS), vascular endothelial growth factor (VEGF), prolyl hydroxylase (EGLN1), and zinc finger protein A20. The gene distribution of each SNP loci and its correlation with HAPE was analyzed. Statistical analyses of the genotype frequencies of the SNPs revealed significant differences in the ACE (rs4309), EGLN1 (rs480902), SP-A2 (rs1965708), HSP70 (rs1008438), PAI-1 (rs1799889), and NOS (rs199983) expressions between the HAPE and healthy control groups (P polymorphism and HAPE susceptibility revealed that 6 hypoxia-related genes were key sites accounting for HAPE. These findings could help assess the risk of HAPE in populations expressing different genotypes, in order to reduce the occurrence of HAPE. PMID:26436397

  5. Effect of high-intensity training on exercise-induced gene expression specific to ion homeostasis and metabolism

    DEFF Research Database (Denmark)

    Nordsborg, Nikolai; Bangsbo, Jens; Pilegaard, Henriette

    2003-01-01

    Changes in gene expression during recovery from high-intensity, intermittent, one-legged exercise were studied before and after 5.5 wk of training. Genes related to metabolism, as well as Na+, K+, and pH homeostasis, were selected for analyses. After the same work was performed before and after the...... training period, several muscle biopsies were obtained from vastus lateralis muscle. In the untrained state, the Na+-K+-ATPase alpha1-subunit mRNA level was approximately threefold higher (P < 0.01) at 0, 1, and 3 h after exercise, relative to the preexercise resting level. After 3-5 h of recovery in the...... resting mRNA levels were observed as a result of training. In conclusion, cellular adaptations to high-intensity exercise training may, in part, be induced by transcriptional regulation. After training, the transcriptional response to an exercise bout at a given workload is diminished....

  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. 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 start...

  8. 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

  9. Identification of rare and frequent variants of the CASR gene by high-resolution melting

    DEFF Research Database (Denmark)

    Nissen, Peter H; Christensen, Signe E; Ladefoged, Søren A;

    2012-01-01

    BACKGROUND: Calcium metabolic disorders like familial hypocalciuric hypercalcemia (FHH) and autosomal dominant familial isolated hypoparathyroidism (FIH) can be caused by rare variants of the calcium sensing receptor gene (CASR). Molecular genetic screening of the CASR is often based on DNA seque...

  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 rep...

  11. Identification of candidate genes for yeast engineering to improve bioethanol production in very high gravity and lignocellulosic biomass industrial fermentations

    OpenAIRE

    Pereira Francisco B; Guimarães Pedro; Gomes Daniel G; Mira Nuno P; Teixeira Miguel C; Sá-Correia Isabel; Domingues Lucília

    2011-01-01

    Abstract Background The optimization of industrial bioethanol production will depend on the rational design and manipulation of industrial strains to improve their robustness against the many stress factors affecting their performance during very high gravity (VHG) or lignocellulosic fermentations. In this study, a set of Saccharomyces cerevisiae genes found, through genome-wide screenings, to confer resistance to the simultaneous presence of different relevant stresses were identified as req...

  12. Identification of candidate genes for yeast engineering to improve bioethanol production in very high gravity and lignocellulosic biomass industrial fermentations

    OpenAIRE

    Pereira, Francisco B; Guimarães, Pedro M. R.; Gomes, Daniel Gonçalves; Mira, Nuno P.; Teixeira, Miguel C

    2011-01-01

    Background: The optimization of industrial bioethanol production will depend on the rational design and manipulation of industrial strains to improve their robustness against the many stress factors affecting their performance during very high gravity (VHG) or lignocellulosic fermentations. In this study, a set of Saccharomyces cerevisiae genes found, through genome-wide screenings, to confer resistance to the simultaneous presence of different relevant stresses were identified as...

  13. E2 represses the late gene promoter of human papillomavirus type 8 at high concentrations by interfering with cellular factors.

    OpenAIRE

    Stubenrauch, F.; Leigh, I M; Pfister, H

    1996-01-01

    The late gene promoter P7535 of the epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV8) is regulated by the viral E2 protein. Transfection experiments performed with the human skin keratinocyte cell line RTS3b and P7535 reporter plasmids revealed transactivation at low amounts and a repression of basal promoter activity at high amounts of E2 expression vector. This repression was promoter specific and correlated with the amount of transiently expressed E2 protein. Mu...

  14. High-throughput knockout screen in Schizosaccharomyces pombe identifies a novel gene required for efficient homolog disjunction during meiosis I

    OpenAIRE

    Rumpf, Cornelia; Cipak, Lubos; Novatchkova, Maria; LI, ZHANG; Polakova, Silvia; Dudas, Andrej; Kovacikova, Ines; Miadokova, Eva; Ammerer, Gustav; Gregan, Juraj

    2010-01-01

    Meiosis is the process which produces haploid gametes from diploid precursor cells. This reduction of chromosome number is achieved by two successive divisions. Whereas homologs segregate during meiosis I, sister chromatids segregate during meiosis II. To identify novel proteins required for proper segregation of chromosomes during meiosis, we applied a high-throughput knockout technique to delete 87 S. pombe genes whose expression is upregulated during meiosis and analyzed the mutant phenoty...

  15. Moderate exercise training provides modest protection against adipose tissue inflammatory gene expression in response to high-fat feeding.

    Science.gov (United States)

    Linden, Melissa A; Pincu, Yair; Martin, Stephen A; Woods, Jeffrey A; Baynard, Tracy

    2014-01-01

    As white adipose tissue (WAT) expands under obesogenic conditions, local WAT hypoxia may contribute to the chronic low-grade inflammation observed in obesity. Aerobic exercise training is beneficial in treating WAT inflammation after obesity is established, but it remains unknown whether exercise training, while on a concomitant high-fat (HF) diet, influences WAT inflammation during the development of obesity. We sought to determine the effects of 4, 8, and 12 weeks of HF feeding and/or moderate intensity treadmill exercise training (EX) on the relationship between inflammatory and hypoxic gene expression within mouse WAT. Male C57Bl6/J mice (n = 113) were randomized into low-fat (LF)/sedentary (SED), LF/EX, HF/SED, or HF/EX groups. The low-fat and high-fat diets contained 10% and 60% energy from fat, respectively. Exercise training consisted of treadmill running 5 days/week at 12 m/min, 8% incline, 40 min/day. Quantitative real-time PCR was used to assess gene expression. HF diet impaired glucose regulation, and upregulated WAT gene expression of inflammation (IL-1β, IL-1ra, TNFα), macrophage recruitment and infiltration (F4/80 and monocyte chemoattractant protein), and M1 (CD11c) and M2 (CD206 and Arginase-1) macrophage polarization markers. Treadmill training resulted in a modest reduction of WAT macrophage and inflammatory gene expression. HF diet had little effect on hypoxia-inducible factor-1α and vascular endothelial growth factor, suggesting that WAT inflammatory gene expression may not be driven by hypoxia within the adipocytes. Treadmill training may provide protection by preventing WAT expansion and macrophage recruitment. PMID:25347855

  16. Effects of Persian leek (Allium ampeloprasum) on hepatic lipids and the expression of proinflammatory gene in hamsters fed a high-fat/ high-cholesterol diet

    Science.gov (United States)

    Fatoorechi, Vahideh; Rismanchi, Marjan; Nasrollahzadeh, Javad

    2016-01-01

    Objective: Persian leek is one of the most widely used herbal foods among Iranians. In this study, effects of oral administration of Persian leek on plasma and liver lipids were examined in hamster. Materials and Methods: Male Syrian hamsters were randomly divided into three groups: control (standard diet), high fat control (high-fat/high-cholesterol diet), Persian leek (high-fat/high-cholesterol diet + 1% per weight of diet from dried powdered Persian leek) for 14 weeks. Results: High fat diet increased plasma and liver lipids as compared to standard diet. Adding Persian leek to the high-fat/high-cholesterol diet resulted in no significant changes in the concentration of the plasma lipids or liver cholesterol. However, liver triglycerides (TG), plasma Alanine aminotransferase and gene expression of tumor necrosis factor- α were decreased in hamsters fed high-fat diet containing Persian leek as compared to high-fat diet only. Conclusion: Persian leek might be considered as a herbal food that can reduce liver TG accumulation induced by high fat diets.

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

    Science.gov (United States)

    Lawrenson, Kate; Li, Qiyuan; Kar, Siddhartha; Seo, Ji-Heui; Tyrer, Jonathan; Spindler, Tassja J.; Lee, Janet; Chen, Yibu; Karst, Alison; Drapkin, Ronny; Aben, Katja K. H.; Anton-Culver, Hoda; Antonenkova, Natalia; Bowtell, David; Webb, Penelope M.; deFazio, Anna; Baker, Helen; Bandera, Elisa V.; Bean, Yukie; Beckmann, Matthias W.; Berchuck, Andrew; Bisogna, Maria; Bjorge, Line; Bogdanova, Natalia; Brinton, Louise A.; Brooks-Wilson, Angela; Bruinsma, Fiona; Butzow, Ralf; Campbell, Ian G.; Carty, Karen; Chang-Claude, Jenny; Chenevix-Trench, Georgia; Chen, Anne; Chen, Zhihua; Cook, Linda S.; Cramer, Daniel W.; Cunningham, Julie M.; Cybulski, Cezary; Dansonka-Mieszkowska, Agnieszka; Dennis, Joe; Dicks, Ed; Doherty, Jennifer A.; Dörk, Thilo; du Bois, Andreas; Dürst, Matthias; Eccles, Diana; Easton, Douglas T.; Edwards, Robert P.; Eilber, Ursula; Ekici, Arif B.; Fasching, Peter A.; Fridley, Brooke L.; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G.; Glasspool, Rosalind; Goode, Ellen L.; Goodman, Marc T.; Grownwald, Jacek; Harrington, Patricia; Harter, Philipp; Hasmad, Hanis Nazihah; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A. T.; Hillemanns, Peter; Hogdall, Estrid; Hogdall, Claus; Hosono, Satoyo; Iversen, Edwin S.; Jakubowska, Anna; James, Paul; Jensen, Allan; Ji, Bu-Tian; Karlan, Beth Y.; Kruger Kjaer, Susanne; Kelemen, Linda E.; Kellar, Melissa; Kelley, Joseph L.; Kiemeney, Lambertus A.; Krakstad, Camilla; Kupryjanczyk, Jolanta; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D.; Lee, Alice W.; Lele, Shashi; Leminen, Arto; Lester, Jenny; Levine, Douglas A.; Liang, Dong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon F. A. G.; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R.; Nevanlinna, Heli; McNeish, Ian; Menon, Usha; Modugno, Francesmary; Moysich, Kirsten B.; Narod, Steven A.; Nedergaard, Lotte; Ness, Roberta B.; Azmi, Mat Adenan Noor; Odunsi, Kunle; Olson, Sara H.; Orlow, Irene; Orsulic, Sandra; Weber, Rachel Palmieri; Pearce, Celeste L.; Pejovic, Tanja; Pelttari, Liisa M.; Permuth-Wey, Jennifer; Phelan, Catherine M.; Pike, Malcolm C.; Poole, Elizabeth M.; Ramus, Susan J.; Risch, Harvey A.; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H.; Rudolph, Anja; Runnebaum, Ingo B.; Rzepecka, Iwona K.; Salvesen, Helga B.; Schildkraut, Joellen M.; Schwaab, Ira; Sellers, Thomas A.; Shu, Xiao-Ou; Shvetsov, Yurii B.; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa C.; Sucheston, Lara; Tangen, Ingvild L.; Teo, Soo-Hwang; Terry, Kathryn L.; Thompson, Pamela J.; Timorek, Agnieszka; Tsai, Ya-Yu; Tworoger, Shelley S.; van Altena, Anne M.; Van Nieuwenhuysen, Els; Vergote, Ignace; Vierkant, Robert A.; Wang-Gohrke, Shan; Walsh, Christine; Wentzensen, Nicolas; Whittemore, Alice S.; Wicklund, Kristine G.; Wilkens, Lynne R.; Woo, Yin-Ling; Wu, Xifeng; Wu, Anna H.; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Monteiro, Alvaro; Pharoah, Paul D.; Gayther, Simon A.; Freedman, Matthew L.

    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 associated with HGSOC risk (P≤10−5). For three cis-eQTL associations (P<1.4 × 10−3, FDR<0.05) at 1p36 (CDC42), 1p34 (CDCA8) and 2q31 (HOXD9), we evaluate the functional role of each candidate by perturbing expression of each gene in HGSOC precursor cells. Overexpression of HOXD9 increases anchorage-independent growth, shortens population-doubling time and reduces contact inhibition. Chromosome conformation capture identifies an interaction between rs2857532 and the HOXD9 promoter, suggesting this SNP is a leading causal variant. Transcriptomic profiling after HOXD9 overexpression reveals enrichment of HGSOC risk variants within HOXD9 target genes (P=6 × 10−10 for risk variants (P<10−4) within 10 kb of a HOXD9 target gene in ovarian cells), suggesting a broader role for this network in genetic susceptibility to HGSOC. PMID:26391404

  18. 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.

  19. Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction.

    Science.gov (United States)

    Li, Chao; Ding, Lei; Sun, Chiao-Wang; Wu, Li-Chen; Zhou, Dewang; Pawlik, Kevin M; Khodadadi-Jamayran, Alireza; Westin, Erik; Goldman, Frederick D; Townes, Tim M

    2016-01-01

    CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical, iPSC generation must be rapid and efficient. Therefore, we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector, rCLAE-R6, that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently, the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% (β(A)/[β(S)+β(A)]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected, patient-specific iPSCs for therapeutic applications. PMID:27460639

  20. 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.

  1. A high affinity kidney targeting by chitobionic acid-conjugated polysorbitol gene transporter alleviates unilateral ureteral obstruction in rats.

    Science.gov (United States)

    Islam, Mohammad Ariful; Kim, Sanghwa; Firdous, Jannatul; Lee, Ah-Young; Hong, Seong-Ho; Seo, Min Kyeong; Park, Tae-Eun; Yun, Cheol-Heui; Choi, Yun-Jaie; Chae, Chanhee; Cho, Chong-Su; Cho, Myung-Haing

    2016-09-01

    Aside from kidney transplantation - a procedure which is exceedingly dependent on donor-match and availability leading to excessive costs - there are currently no permanent treatments available which reverse kidney injury and failure. However, kidney-specific targeted gene therapy has outstanding potential to treat kidney-related dysfunction. Herein we report a novel kidney-specific targeted gene delivery system developed through the conjugation of chitobionic acid (CBA) to a polysorbitol gene transporter (PSGT) synthesized from sorbitol diacrylate and low molecular weight polyethylenimine (PEI) carrying hepatocyte growth factor (HGF) gene to alleviate unilateral ureteral obstruction (UUO) in rats. CBA-PSGT performed exceptionally well for targeted delivery of HGF to kidney tissues compared to its non-targeted counterparts (P type I and II), blood urea nitrogen (BUN), creatinine, and the expressions of ICAM-1, TIMP-1 and α-SMA which play a critical role in obstructive kidney functions. Therefore, CBA-PSGT should be further investigated because of its potential to alleviate UUO and kidney-related diseases using high affinity kidney targeting. PMID:27318934

  2. 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.

  3. Closing the gaps on human chromosome 19 revealed genes with a high density of repetitive tandemly arrayed elements.

    Energy Technology Data Exchange (ETDEWEB)

    Leem, Sun-Hee; Kouprina, Natalay; Grimwood, Jane; Kim, Jung-Hyun; Mullokandov, Michael; Yoon, Young-Ho; Chae, Ji-Youn; Morgan, Jenna; Lucas, Susan; Richardson, Paul; Detter, Chris; Glavina, Tijana; Rubin, Eddy; Barrett, J. Carl; Larionov, Vladimir

    2003-09-01

    The reported human genome sequence includes about 400 gaps of unknown sequence that were not found in the bacterial artificial chromosome (BAC) and cosmid libraries used for sequencing of the genome. These missing sequences correspond to {approx} 1 percent of euchromatic regions of the human genome. Gap filling is a laborious process because it relies on analysis of random clones of numerous genomic BAC or cosmid libraries. In this work we demonstrate that closing the gaps can be accelerated by a selective recombinational capture of missing chromosomal segments in yeast. The use of both methodologies allowed us to close the four remaining gaps on the human chromosome 19. Analysis of the gap sequences revealed that they contain several abnormalities that could result in instability of the sequences in microbe hosts, including large blocks of micro- and minisatellites and a high density of Alu repeats. Sequencing of the gap regions, in both BAC and YAC forms, allowed us to generate a complete sequence of four genes, including the neuronal cell signaling gene SCK1/SLI. The SCK1/SLI gene contains a record number of minisatellites, most of which are polymorphic and transmitted through meiosis following a Mendelian inheritance. In conclusion, the use of the alternative recombinational cloning system in yeast may greatly accelerate work on closing the remaining gaps in the human genome (as well as in other complex genomes) to achieve the goal of annotation of all human genes.

  4. 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.

  5. Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction

    Science.gov (United States)

    Li, Chao; Ding, Lei; Sun, Chiao-Wang; Wu, Li-Chen; Zhou, Dewang; Pawlik, Kevin M.; Khodadadi-Jamayran, Alireza; Westin, Erik; Goldman, Frederick D.; Townes, Tim M.

    2016-01-01

    CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical, iPSC generation must be rapid and efficient. Therefore, we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector, rCLAE-R6, that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently, the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% (βA/[βS+βA]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected, patient-specific iPSCs for therapeutic applications. PMID:27460639

  6. High Temperature in Combination with UV Irradiation Enhances Horizontal Transfer of stx2 Gene from E. coli O157:H7 to Non-Pathogenic E. coli

    OpenAIRE

    Wan-Fu Yue; Min Du; Mei-Jun Zhu

    2012-01-01

    BACKGROUND: Shiga toxin (stx) genes have been transferred to numerous bacteria, one of which is E. coli O157:H7. It is a common belief that stx gene is transferred by bacteriophages, because stx genes are located on lambdoid prophages in the E. coli O157:H7 genome. Both E. coli O157:H7 and non-pathogenic E. coli are highly enriched in cattle feedlots. We hypothesized that strong UV radiation in combination with high temperature accelerates stx gene transfer into non-pathogenic E. coli in feed...

  7. High Temperature in Combination with UV Irradiation Enhances Horizontal Transfer of stx2 Gene from E. coli O157:H7 to Non-Pathogenic E. coli

    OpenAIRE

    Yue, Wan-Fu; Du, Min; Zhu, Mei-Jun

    2012-01-01

    Background Shiga toxin (stx) genes have been transferred to numerous bacteria, one of which is E. coli O157:H7. It is a common belief that stx gene is transferred by bacteriophages, because stx genes are located on lambdoid prophages in the E. coli O157:H7 genome. Both E. coli O157:H7 and non-pathogenic E. coli are highly enriched in cattle feedlots. We hypothesized that strong UV radiation in combination with high temperature accelerates stx gene transfer into non-pathogenic E. coli in feedl...

  8. High Dietary Fat Exacerbates Weight Gain and Obesity in Female Liver Fatty Acid Binding Protein Gene-Ablated Mice

    OpenAIRE

    Atshaves, Barbara P.; McIntosh, Avery L.; Storey, Stephen M.; Landrock, Kerstin K.; Kier, Ann B.; Schroeder, Friedhelm

    2009-01-01

    Since liver fatty acid binding protein (L-FABP) facilitates uptake/oxidation of long-chain fatty acids in cultured transfected cells and primary hepatocytes, loss of L-FABP was expected to exacerbate weight gain and/or obesity in response to high dietary fat. Male and female wild-type (WT) and L-FABP gene-ablated mice, pair-fed a defined isocaloric control or high fat diet for 12 weeks, consumed equal amounts of food by weight and kcal. Male WT mice gained weight faster than their female WT c...

  9. High dietary zinc supplementation increases the occurrence of tetracycline and sulfonamide resistance genes in the intestine of weaned pigs

    OpenAIRE

    Vahjen, Wilfried; Pietruszyńska, Dominika; Starke, Ingo C.; Zentek, Jürgen

    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...

  10. 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.

  11. 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.

  12. 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

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

    OpenAIRE

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

    2013-01-01

    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 ea...

  14. Chickpea rhizobia symbiosis genes are highly conserved across multiple Mesorhizobium species

    OpenAIRE

    Laranjo, Marta; Alexandre, Ana; Rivas, Raul; Velázquez, Encarna; Young, J. Peter W.; Oliveira, Solange

    2008-01-01

    ABSTRACT Chickpea has been considered as a restrictive host for nodulation by rhizobia. However, recent studies have reported that several Mesorhizobium species may effectively nodulate chickpea. With the purpose of investigating the evolutionary relationships between these different species with the ability of nodulating the same host, we analysed 21 Portuguese chickpea rhizobial isolates. Symbiosis genes nifH and nodC were sequenced and used for phylogenetic studies. Symbiotic effectiven...

  15. Highly modular bow-tie gene circuits with programmable dynamic behavior

    OpenAIRE

    Prochazka, Laura; Angelici, Bartolomeo; Haefliger, Benjamin; Benenson, Yaakov

    2014-01-01

    Synthetic gene circuits often require extensive mutual optimization of their components for successful operation, while modular and programmable design platforms are rare. A possible solution lies in the “bow-tie” architecture, which stipulates a focal component - a “knot” - uncoupling circuits’ inputs and outputs, simplifying component swapping, and introducing additional layer of control. Here we construct, in cultured human cells, synthetic bow-tie circuits that transduce microRNA inputs i...

  16. Engineering high-level aluminum tolerance in barley with the ALMT1 gene

    OpenAIRE

    Delhaize, Emmanuel; Ryan, Peter R.; Hebb, Diane M.; Yamamoto, Yoko; Sasaki, Takayuki; Matsumoto, Hideaki

    2004-01-01

    Acidity is a serious limitation to plant production on many of the world's agricultural soils. Toxic aluminium (Al) cations solubilized by the acidity rapidly inhibit root growth and limit subsequent uptake of water and nutrients. Recent work has shown that the ALMT1 gene of wheat (Triticum aestivum) encodes a malate transporter that is associated with malate efflux and Al tolerance. We generated transgenic barley (Hordeum vulgare) plants expressing ALMT1 and assessed their ability to exude m...

  17. A Highly Polymorphic Copy Number Variant in the NSF Gene is Associated with Cocaine Dependence.

    Science.gov (United States)

    Cabana-Domínguez, Judit; Roncero, Carlos; Grau-López, Lara; Rodríguez-Cintas, Laia; Barral, Carmen; Abad, Alfonso C; Erikson, Galina; Wineinger, Nathan E; Torrico, Bàrbara; Arenas, Concepció; Casas, Miquel; Ribasés, Marta; Cormand, Bru; Fernàndez-Castillo, Noèlia

    2016-01-01

    Cocaine dependence is a complex psychiatric disorder involving both genetic and environmental factors. Several neurotransmitter systems mediate cocaine's effects, dependence and relapse, being the components of the neurotransmitter release machinery good candidates for the disorder. Previously, we identified a risk haplotype for cocaine dependence in the NSF gene, encoding the protein N-Ethylmaleimide-Sensitive Factor essential for synaptic vesicle turnover. Here we examined the possible contribution to cocaine dependence of a large copy number variant (CNV) that encompasses part of the NSF gene. We performed a case-control association study in a discovery sample (359 cases and 356 controls) and identified an association between cocaine dependence and the CNV (P = 0.013), that was confirmed in the replication sample (508 cases and 569 controls, P = 7.1e-03) and in a pooled analysis (P = 1.8e-04), with an over-representation of low number of copies in cases. Subsequently, we studied the functional impact of the CNV on gene expression and found that the levels of two NSF transcripts were significantly increased in peripheral blood mononuclear cells (PBMC) along with the number of copies of the CNV. These results, together with a previous study from our group, support the role of NSF in the susceptibility to cocaine dependence. PMID:27498889

  18. 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

  19. 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 × 10(7) 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

  20. High-throughput phenotypic profiling of gene-environment interactions by quantitative growth curve analysis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Weiss, Andrew; Delproposto, James; Giroux, Craig N

    2004-04-01

    Cell-based assays are widely used in high-throughput screening to determine the effects of toxicants and drugs on their biological targets. To enable a functional genomics modeling of gene-environment interactions, quantitative assays are required both for gene expression and for the phenotypic responses to environmental challenge. To address this need, we describe an automated high-throughput methodology that provides phenotypic profiling of the cellular responses to environmental stress in Saccharomyces cerevisiae. Standardized assay conditions enable the use of a single metric value to quantify yeast microculture growth curves. This assay format allows precise control of both genetic and environmental determinants of the cellular responses to oxidative stress, a common mechanism of environmental insult. These yeast-cell-based assays are validated with hydrogen peroxide, a simple direct-acting oxidant. Phenotypic profiling of the oxidative stress response of a yap1 mutant strain demonstrates the mechanistic analysis of genetic susceptibility to oxidative stress. As a proof of concept for analysis of more complex gene-environment interactions, we describe a combinatorial assay design for phenotypic profiling of the cellular responses to tert-butyl hydroperoxide, a complex oxidant that is actively metabolized by its target cells. Thus, the yeast microculture assay format supports comprehensive applications in toxicogenomics. PMID:15033507

  1. 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.

  2. 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.

  3. 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.

  4. Coevolution of genes and languages and high levels of population structure among the highland populations of Daghestan.

    Science.gov (United States)

    Karafet, Tatiana M; Bulayeva, Kazima B; Nichols, Johanna; Bulayev, Oleg A; Gurgenova, Farida; Omarova, Jamilia; Yepiskoposyan, Levon; Savina, Olga V; Rodrigue, Barry H; Hammer, Michael F

    2016-03-01

    As a result of the combination of great linguistic and cultural diversity, the highland populations of Daghestan present an excellent opportunity to test the hypothesis of language-gene coevolution at a fine geographic scale. However, previous genetic studies generally have been restricted to uniparental markers and have not included many of the key populations of the region. To improve our understanding of the genetic structure of Daghestani populations and to investigate possible correlations between genetic and linguistic variation, we analyzed ~550,000 autosomal single nucleotide polymorphisms, phylogenetically informative Y chromosome markers and mtDNA haplotypes in 21 ethnic Daghestani groups. We found high levels of population structure in Daghestan consistent with the hypothesis of long-term isolation among populations of the highland Caucasus. Highland Daghestani populations exhibit extremely high levels of between-population diversity for all genetic systems tested, leading to some of the highest FST values observed for any region of the world. In addition, we find a significant positive correlation between gene and language diversity, suggesting that these two aspects of human diversity have coevolved as a result of historical patterns of social interaction among highland farmers at the community level. Finally, our data are consistent with the hypothesis that most Daghestanian-speaking groups descend from a common ancestral population (~6000-6500 years ago) that spread to the Caucasus by demic diffusion followed by population fragmentation and low levels of gene flow. PMID:26607180

  5. 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.

  6. 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.

  7. 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.

  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. 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.

  10. 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...

  11. 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

  12. Highly conserved gene order and numerous novel repetitive elements in genomic regions linked to wing pattern variation in Heliconius butterflies

    Directory of Open Access Journals (Sweden)

    Halder Georg

    2008-07-01

    Full Text Available Abstract Background With over 20 parapatric races differing in their warningly colored wing patterns, the butterfly Heliconius erato provides a fascinating example of an adaptive radiation. Together with matching races of its co-mimic Heliconius melpomene, H. erato also represents a textbook case of Müllerian mimicry, a phenomenon where common warning signals are shared amongst noxious organisms. It is of great interest to identify the specific genes that control the mimetic wing patterns of H. erato and H. melpomene. To this end we have undertaken comparative mapping and targeted genomic sequencing in both species. This paper reports on a comparative analysis of genomic sequences linked to color pattern mimicry genes in Heliconius. Results Scoring AFLP polymorphisms in H. erato broods allowed us to survey loci at approximately 362 kb intervals across the genome. With this strategy we were able to identify markers tightly linked to two color pattern genes: D and Cr, which were then used to screen H. erato BAC libraries in order to identify clones for sequencing. Gene density across 600 kb of BAC sequences appeared relatively low, although the number of predicted open reading frames was typical for an insect. We focused analyses on the D- and Cr-linked H. erato BAC sequences and on the Yb-linked H. melpomene BAC sequence. A comparative analysis between homologous regions of H. erato (Cr-linked BAC and H. melpomene (Yb-linked BAC revealed high levels of sequence conservation and microsynteny between the two species. We found that repeated elements constitute 26% and 20% of BAC sequences from H. erato and H. melpomene respectively. The majority of these repetitive sequences appear to be novel, as they showed no significant similarity to any other available insect sequences. We also observed signs of fine scale conservation of gene order between Heliconius and the moth Bombyx mori, suggesting that lepidopteran genome architecture may be conserved

  13. Medullary Epithelial Cells of the Human Thymus Express a Highly Diverse Selection of Tissue-specific Genes Colocalized in Chromosomal Clusters

    OpenAIRE

    Gotter, Jörn; Brors, Benedikt; Hergenhahn, Manfred; Kyewski, Bruno

    2004-01-01

    Promiscuous expression of tissue-specific self-antigens in the thymus imposes T cell tolerance and protects from autoimmune diseases, as shown in animal studies. Analysis of promiscuous gene expression in purified stromal cells of the human thymus at the single and global gene level documents the species conservation of this phenomenon. Medullary thymic epithelial cells overexpress a highly diverse set of genes (>400) including many tissue-specific antigens, disease-associated autoantigens, a...

  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. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. Polymorphism profiling of nine high altitude relevant candidate gene loci in acclimatized sojourners and adapted natives

    OpenAIRE

    Tomar, Arvind; Malhotra, Seema; Sarkar, Soma

    2015-01-01

    Background Sea level sojourners, on ascent to high altitude, undergo acclimatization through integrated physiological processes for defending the body against oxygen deprivation while the high altitude natives (resident population) are adapted to the prevailing hypobaric hypoxic condition through natural selection. Separating the acclimatization processes from adaptive changes and identifying genetic markers in lowlanders that may be beneficial for offsetting the high altitude hypoxic stress,...

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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...

  11. 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; M. Vidal; J. Kaeda; L. Luzzatto; D.R. Greaves; 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

  12. 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

  13. 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; Del Moral-Chávez, Víctor; 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

  14. 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.

  15. 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.

  16. HFE gene mutation (C282Y) and phenotypic expression among a hospitalised population in a high prevalence area of haemochromatosis

    OpenAIRE

    DISTANTE, S; Berg, J.; Lande, K.; Haug, E.; Bell, H.

    2000-01-01

    BACKGROUND—Previous studies have shown that up to 0.5% of the Caucasian population is homozygous for the HFE gene C282Y mutation. High prevalence values have been reported in Northern Europe. To what extent the presence of this mutation is associated with overt clinical haemochromatosis is unclear.
AIM—To determine the prevalence of the C282Y allele in a hospitalised population of an acute medical department, and study the phenotypic expression in the homozygotes.
METHODS—Blood samples were o...

  17. 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...

  18. Cloning and Expression of Highly Pathogenic Avian Influenza Virus Full-Length Nonstructural Gene in Pichia pastoris

    OpenAIRE

    Abubakar, M. B.; I. Aini; Omar, A. R.; Hair-Bejo, M

    2011-01-01

    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...

  19. Highly variable clinical phenotype of carbamylphosphate synthetase 1 deficiency in one family: an effect of allelic variation in gene expression?

    DEFF Research Database (Denmark)

    Klaus, V; Vermeulen, T; Minassian, B;

    2009-01-01

    Deficiency of the urea cycle enzyme carbamylphosphate synthetase 1 (CPS1) causes hyperammonemia with a vast range of clinical severity from neonatal onset with early lethality to onset after age 40 with rare episodes of hyperammonemic confusion. The cause for this variability is not understood. We...... report two patients from one family with highly divergent clinical course, one presenting neonatally with a fatal form and the other at age 45 with benign diet-responsive disease. The patients are compound heterozygous for two mutations of the CPS1 gene, c.3558 + 1G > C and c.4101 + 2T > C...

  20. 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.

  1. 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.

  2. Pre-amplification in the context of high-throughput qPCR gene expression experiment

    OpenAIRE

    Korenková, Vlasta; Scott, Justin; Novosadová, Vendula; Jindřichová, Marie; Langerová, Lucie; Švec, David; Šídová, Monika; Sjöback, Robert

    2015-01-01

    Background With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-a...

  3. Highly modular bow-tie gene circuits with programmable dynamic behaviour.

    Science.gov (United States)

    Prochazka, Laura; Angelici, Bartolomeo; Haefliger, Benjamin; Benenson, Yaakov

    2014-01-01

    Synthetic gene circuits often require extensive mutual optimization of their components for successful operation, while modular and programmable design platforms are rare. A possible solution lies in the 'bow-tie' architecture, which stipulates a focal component-a 'knot'-uncoupling circuits' inputs and outputs, simplifying component swapping, and introducing additional layer of control. Here we construct, in cultured human cells, synthetic bow-tie circuits that transduce microRNA inputs into protein outputs with independently programmable logical and dynamic behaviour. The latter is adjusted via two different knot configurations: a transcriptional activator causing the outputs to track input changes reversibly, and a recombinase-based cascade, converting transient inputs into permanent actuation. We characterize the circuits in HEK293 cells, confirming their modularity and scalability, and validate them using endogenous microRNA inputs in additional cell lines. This platform can be used for biotechnological and biomedical applications in vitro, in vivo and potentially in human therapy. PMID:25311543

  4. High Resolution Melting Analysis for Detecting p53 Gene Mutations in Patients with Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Zhihong CHEN

    2011-10-01

    Full Text Available Background and objective It has been proven that p53 gene was related to many human cancers. The mutations in p53 gene play an important role in carcinogensis and mostly happened in exon 5-8. The aim of this study is to establish a high resolution melting (HRM assay to detect p53 mutations from patients with non-small cell lung cancer (NSCLC, to investigate the characteristics of p53 gene mutations, and to analyze the relationship between p53 mutations and evolution regularity of pathogenesis. Methods p53 mutations in exon 5-8 were detected by HRM assay on DNA insolated from 264 NSCLC samples derived from tumor tissues and 54 control samples from pericancerous pulmonary tissues. The mutation samples by the HRM assay were confirmed by sequencing technique. Samples which were positive by HRM but wild type by sequencing were further confirmed by sub-clone and sequencing. Results No mutation was found in 54 pericancerous pulmonary samples by HRM assay. 104 of the 264 tumor tissues demonstrated mutation curves by HRM assay, 102 samples were confirmed by sequencing, including 95 point mutations and 7 frame shift mutations by insertion or deletion. The mutation rate of p53 gene was 39.4%. The mutation rate from exon 5-8 were 11.7%, 8%, 12.5% and 10.6%, respectively and there was no statistically significant difference between them (P=0.35. p53 mutations were significantly more frequent in males than that in females, but not related to the other clinicopathologic characteristics. Conclusion The results indicate that HRM is a sensitive in-tube methodology to detect for mutations in clinical samples. The results suggest that the arising p53 mutations in NSCLC may be due to spontaneous error in DNA synthesis and repair.

  5. High temperature inhibits ascorbate recycling and light stimulation of the ascorbate pool in tomato despite increased expression of biosynthesis genes.

    Directory of Open Access Journals (Sweden)

    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.

  6. 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.

  7. Extreme positive selection on a new highly-expressed larval glycoprotein (LGP) gene in Galaxias fishes (Osmeriformes: Galaxiidae).

    Science.gov (United States)

    Wallis, Lise J; Wallis, Graham P

    2011-01-01

    We describe the intron-exon structure and DNA/protein sequences of a new larval glycoprotein (LGP) gene from nine species of galaxiid fish. The gene has a distant similarity to Danio THP (Tamm-Horsfall urinary glycoprotein; uromodulin) and cichlid SPP120 (seminal plasma glycoprotein) due to conserved features of its zona pellucida (ZP) domain, including eight highly conserved cysteines and a consensus furin cleavage site. Using a combination of 454 sequencing of cDNA and exon-primed intron-spanning sequencing of genomic DNA, we obtained full sequences of the coding region (996 bp) and its intervening sequences (1,459 bp). LGP shows an exceptionally strong signal of positive selection over the entire coding region, as evidenced by d(N)/d(S) values >1. Across nine species of Galaxias, 87/332 (26%) amino acid residues are variable, compared with 9/386 (2%) for mitochondrial cytochrome b (cytb) in the same group of species. Across 36 interspecific pairwise comparisons, genetic distances are in all cases larger for coding region than for introns, by a factor of 2.4-fold on average. Reading frame, gene structure, splice sites, and many ZP motifs are conserved across all species. Together with the fact that the gene is expressed in all species, these results argue clearly against the possibility of a pseudogene. We show by 454 sequencing and quantitative polymerase chain reaction that the transcript is abundant (ca. 0.5%) in newly hatched larvae and appears to be almost absent from a range of adult tissues. We postulate that the strong Darwinian evolution exhibited by this protein may reflect some type of immunoprotection at this vulnerable larval stage. PMID:20696791

  8. 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.

  9. 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.

  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. 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.

  12. 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.

  13. High throughput gene complementation screening permits identification of a mammalian mitochondrial protein synthesis (ρ(-)) mutant.

    Science.gov (United States)

    Potluri, Prasanth; Procaccio, Vincent; Scheffler, Immo E; Wallace, Douglas C

    2016-08-01

    To identify nuclear DNA (nDNA) oxidative phosphorylation (OXPHOS) gene mutations using cultured cells, we have developed a complementation system based on retroviral transduction with a full length cDNA expression library and selection for OXHOS function by growth in galactose. We have used this system to transduce the Chinese hamster V79-G7 OXPHOS mutant cell line with a defect in mitochondrial protein synthesis. The complemented cells were found to have acquired the cDNA for the bS6m polypeptide of the small subunit of the mitochondrial ribosome. bS6m is a 14 kDa polypeptide located on the outside of the mitochondrial 28S ribosomal subunit and interacts with the rRNA. The V79-G7 mutant protein was found to harbor a methionine to threonine missense mutation at codon 13. The hamster bS6m null mutant could also be complemented by its orthologs from either mouse or human. bS6m protein tagged at its C-terminus by HA, His or GFP localized to the mitochondrion and was fully functional. Through site-directed mutagenesis we identified the probable RNA interacting residues of the bS6m peptide and tested the functional significance of mammalian specific C-terminal region. The N-terminus of the bS6m polypeptide functionally corresponds to that of the prokaryotic small ribosomal subunit, but deletion of C-terminal residues along with the zinc ion coordinating cysteine had no functional effect. Since mitochondrial diseases can result from hundreds to thousands of different nDNA gene mutations, this one step viral complementation cloning may facilitate the molecular diagnosis of a range of nDNA mitochondrial disease mutations. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26946086

  14. 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.

  15. 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.

  16. 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

  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. 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

    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...

  19. 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/.

  20. 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.

  1. High-resolution linkage analyses to identify genes that influence Varroa sensitive hygiene behavior in honey bees.

    Directory of Open Access Journals (Sweden)

    Jennifer M Tsuruda

    Full Text Available 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.

  2. Associations between organohalogen concentrations and transcription of thyroid-related genes in a highly contaminated gull population.

    Science.gov (United States)

    Técher, Romy; Houde, Magali; Verreault, Jonathan

    2016-03-01

    A number of studies have reported altered circulating thyroid hormone levels in birds exposed either in controlled settings or in their natural habitat to ubiquitous organohalogen compounds including organochlorines (OCs) and polybrominated diphenyl ether (PBDE) flame retardants. However, limited attention has been paid to underlying homeostatic mechanisms in wild birds such as changes in the expression of genes in the hypothalamic-pituitary-thyroid (HPT) axis. The objective of the present study was to investigate the relationships between hepatic concentrations of major organohalogens (PBDEs and OCs), and circulating thyroid hormone (free and total thyroxine (T4) and triiodothyronine (T3)) levels and transcription of 14 thyroid-related genes in three tissues (thyroid, brain, and liver) of an urban-adapted bird exposed to high organohalogen concentrations in the Montreal area (QC, Canada), the ring-billed gull (Larus delawarensis). Positive correlations were found between liver concentrations of several polychlorinated biphenyls (PCBs), PBDEs as well as chlordanes and total plasma T4 levels. Hepatic concentrations of several PBDEs were negatively correlated with mRNA levels of deiodinase type 3, thyroid peroxidase, and thyroid hormone receptor β (TRβ) in the thyroid gland. Liver PCB (deca-CB) correlated positively with mRNA levels of sodium-iodide symporter and TRα. In brain, concentrations of most PBDEs were positively correlated with mRNA levels of organic anion transporter protein 1C1 and transthyretin, while PCBs positively correlated with expression of TRα and TRβ as well as deiodinase type 2. These multiple correlative linkages suggest that organohalogens operate through several mechanisms (direct or compensatory) involving gene transcription, thus potentially perturbing the HPT axis of this highly organohalogen-contaminated ring-billed gull population. PMID:26747993

  3. 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.

  4. 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

  5. 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.

  6. [Gene expression of the key enzymes controlling starch synthesis and metabolism in rice grain endosperm under effects of high temperature after anthesis].

    Science.gov (United States)

    Zhong, Lian-Jin; Dong, Hu; Cai, Xiao-Bo; Feng, Yan-Ning; Ren, Ping; Cheng, Fang-Min

    2012-03-01

    Taking an early-season indica cultivar 'Jiazao 935' whose grain quality was sensitive to temperature as test material, and by using artificial climatic chamber and real-time fluorescence quantitative PCR (FQ-PCR), this paper studied the relative expression amount and its dynamic changes of ten isoform genes of the key enzymes controlling starch synthesis and metabolism in rice grain endosperm, including sbe1, sbe3, and sbe4 of starch branching enzyme (SBE), isal, isa2, isa3, and pul of starch debranching enzyme (DBE), and Wx, sss1, and sss2a of starch synthase (SS), at the mean daily temperature 22 and 32 degrees C after anthesis. There existed obvious differences in the expression patterns of these genes under the high temperature stress, and the expression patterns were isoform-dependent. The relative expression amount of sbe1 and sbe3 under high temperature decreased significantly, and both of the genes were the sensitive isoform genes of SBE to high temperature stress. Among the DBE genes, pul was the isoform gene with high expression level, being more sensitive to high temperature stress than isa1, isa2, and isa3. Among the SS genes, sss2a had a significantly lower relative expression amount than sss1 and Wx, but sss2a and sss1 were more sensitive to high temperature than Wx, suggesting that sss2a and sss1 could be the important genes that adjusted the starch structure in rice endosperm under high temperature stress, especially at the middle and late grain filling stages. PMID:22720620

  7. 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)

  8. Cloning, Purification and Characterization of a Highly Thermostable Amylase Gene of Thermotoga petrophila into Escherichia coli.

    Science.gov (United States)

    Zafar, Asma; Aftab, Muhammad Nauman; ud Din, Zia; Aftab, Saima; Iqbal, Irfana; ul Haq, Ikram

    2016-02-01

    A putative α-amylase gene of Thermotoga petrophila was cloned and expressed in Escherichia coli BL21 (DE3) using pET-21a (+), as an expression vector. The growth conditions were optimized for maximal expression of the α-amylase using various parameters, such as pH, temperature, time of induction and addition of an inducer. The optimum temperature and pH for the maximum expression of α-amylase were 22 °C and 7.0 pH units, respectively. Purification of the recombinant enzyme was carried out by heat treatment method, followed by ion exchange chromatography with 34.6-fold purification having specific activity of 126.31 U mg(-1) and a recovery of 56.25%. Molecular weight of the purified α-amylase, 70 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 100 °C temperature and at pH of 7.0. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA indicating that the α-amylase was a metalloenzyme. However, addition of 1% Tween 20, Tween 80 and β-mercaptoethanol constrained the enzyme activity to 87, 96 and 89%, respectively. No considerable effect of organic solvents (ethanol, methanol, isopropanol, acetone and n-butanol) was observed on enzyme activity. With soluble starch as a substrate, the enzyme activity under optimized conditions was 73.8 U mg(-1). The α-amylase enzyme was active to hydrolyse starch forming maltose. PMID:26526464

  9. 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.

  10. 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.

  11. 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.

  12. A high-resolution gene expression atlas of epistasis between gene-specific transcription factors exposes potential mechanisms for genetic interactions

    OpenAIRE

    Sameith, Katrin; Amini, Saman; Groot Koerkamp, Marian J. A.; van Leenen, Dik; Brok, Mariel; Brabers, Nathalie; Lijnzaad, Philip; van Hooff, Sander R; Joris J Benschop; Lenstra, Tineke L.; Apweiler, Eva; van Wageningen, Sake; Snel, Berend; Holstege, Frank C. P.; Kemmeren, Patrick

    2015-01-01

    Background Genetic interactions, or non-additive effects between genes, play a crucial role in many cellular processes and disease. Which mechanisms underlie these genetic interactions has hardly been characterized. Understanding the molecular basis of genetic interactions is crucial in deciphering pathway organization and understanding the relationship between genotype, phenotype and disease. Results To investigate the nature of genetic interactions between gene-specific transcription factor...

  13. 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

  14. 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.

  15. Liver gene transfer of interkeukin-15 constructs that become part of circulating high density lipoproteins for immunotherapy.

    Directory of Open Access Journals (Sweden)

    Maria C Ochoa

    Full Text Available Apolipoprotein A-I (Apo A-I is a major component of high density lipoproteins (HDL that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15 and Apo A-I (pApo-hIL15 that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα(-/- mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.

  16. Highly variable individual donor cell fates characterize robust horizontal gene transfer of an integrative and conjugative element.

    Science.gov (United States)

    Delavat, François; Mitri, Sara; Pelet, Serge; van der Meer, Jan Roelof

    2016-06-14

    Horizontal gene transfer is an important evolutionary mechanism for bacterial adaptation. However, given the typical low transfer frequencies in a bacterial population, little is known about the fate and interplay of donor cells and the mobilized DNA during transfer. Here we study transfer of an integrative and conjugative element (ICE) among individual live bacterial cells. ICEs are widely distributed mobile DNA elements that are different than plasmids because they reside silent in the host chromosome and are maintained through vertical descent. Occasionally, ICEs become active, excise, and transmit their DNA to a new recipient, where it is reintegrated. We develop a fluorescent tool to differentiate excision, transfer, and reintegration of a model ICE named ICEclc (for carrying the clc genes for chlorocatechol metabolism) among single Pseudomonas cells by using time-lapse microscopy. We find that ICEclc activation is initiated in stationary phase cells, but excision and transfer predominantly occur only when such cells have been presented with new nutrients. Donors with activated ICE develop a number of different states, characterized by reduced cell division rates or growth arrest, persistence, or lysis, concomitant with ICE excision, and likely, ICE loss or replication. The donor cell state transitions can be described by using a stochastic model, which predicts that ICE fitness is optimal at low initiation rates in stationary phase. Despite highly variable donor cell fates, ICE transfer is remarkably robust overall, with 75% success after excision. Our results help to better understand ICE behavior and shed a new light on bacterial cellular differentiation during horizontal gene transfer. PMID:27247406

  17. 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.

  18. 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.

  19. Interplay between path and speed in decision making by high-dimensional stochastic gene regulatory networks.

    Directory of Open Access Journals (Sweden)

    Nuno R Nené

    Full Text Available Induction of a specific transcriptional program by external signaling inputs is a crucial aspect of intracellular network functioning. The theoretical concept of coexisting attractors representing particular genetic programs is reasonably adapted to experimental observations of "genome-wide" expression profiles or phenotypes. Attractors can be associated either with developmental outcomes such as differentiation into specific types of cells, or maintenance of cell functioning such as proliferation or apoptosis. Here we review a mechanism known as speed-dependent cellular decision making (SdCDM in a small epigenetic switch and generalize the concept to high-dimensional space. We demonstrate that high-dimensional network clustering capacity is dependent on the level of intrinsic noise and the speed at which external signals operate on the transcriptional landscape.

  20. Algorithms for Determining Differentially Expressed Genes and Chromosome Structures From High-Throughput Sequencing Data

    OpenAIRE

    Yang, Yi-Wen

    2015-01-01

    Next-generation sequencing (NGS) technologies are able to sequence DNA or RNA molecules at unprecedented speed and with high accuracy. Recently, NGS technologies have been applied in a variety of contexts, e.g., whole genome sequencing, transcript expression profiling, chromatin immunoprecipitation sequencing, and small RNA sequencing, to accelerate genomic researches. The size of NGS data is usually gigantic such that the data analysis in these applications of NGS largely relies on efficient...

  1. 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.

  2. Genes and Gene Therapy

    Science.gov (United States)

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  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. The use of high-frequency ultrasound imaging and biofluorescence for in vivo evaluation of gene therapy vectors

    International Nuclear Information System (INIS)

    Non-invasive imaging of the biodistribution of novel therapeutics including gene therapy vectors in animal models is essential. 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. HF-US imaging of xenografts resulted in an accurate and informative xenograft volume in a longitudinal study. The volumes correlated better with final ex vivo volume than mechanical callipers (R2 = 0.7993, p = 0.0002 vs. R2 = 0.7867, p = 0.0014). HF-US showed that the amplicon caused lobe formation. BFI demonstrated retention and expression of the amplicon in the xenografts and quantitation of the fluorescence levels also correlated with tumour volumes. The use of multi-modal imaging provided useful and enhanced insights into the behaviour of gene therapy vectors in vivo in real-time. These relatively inexpensive technologies are easy to incorporate into pre-clinical studies

  5. 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.

  6. 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.

  7. The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

    International Nuclear Information System (INIS)

    Chondrosarcoma is virtually resistant to chemotherapy and radiation therapy. Survivin, the smallest member of the inhibitor of apoptosis protein family, is a critical factor for tumor progression and resistance to conventional therapeutic approaches in a wide range of malignancies. However, the role of survivin in chondrosarcoma has not been well studied. We examined the importance of survivin gene expression in chondrosarcoma and analysed its influences on proliferation, apoptosis and resistance to chemotherapy in vitro. Resected chondrosarcoma specimens from which paraffin-embedded tissues could be extracted were available from 12 patients. In vitro experiments were performed in human chondrosarcoma cell lines SW1353 and Hs819.T. Immunohistochemistry, immunoblot, quantitative PCR, RNA interference, gene-overexpression and analyses of cell proliferation and apoptosis were performed. Expression of survivin protein was detected in all chondrosarcoma specimens analyzed, while undetectable in adult human cartilage. RNA interference targeting survivin resulted in a G2/M-arrest of the cell cycle and led to increased rates of apoptosis in chondrosarcoma cells in vitro. Overexpression of survivin resulted in pronounced resistance to doxorubicin treatment. These findings indicate that survivin plays a role in the pathogenesis and pronounced chemoresistance of high grade chondrosarcoma. Survivin antagonizing therapeutic strategies may lead to new treatment options in unresectable and metastasized chondrosarcoma

  8. 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.

  9. 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. PMID:26039904

  10. 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.

  11. Feeding a high dosage of zinc oxide affects suppressor of cytokine gene expression in Salmonella Typhimurium infected piglets.

    Science.gov (United States)

    Schulte, Jasper N; Brockmann, Gudrun A; Kreuzer-Redmer, Susanne

    2016-10-01

    Suppressor of cytokine signaling (SOCS) proteins play an important role in the regulation of the immune response by inhibiting cytokines. Here we investigated the effects of zinc oxide fed at three different dosages (LZN=57ppm, MZN=167ppm, HZN=2425ppm) to weaned piglets that were or were not orally infected with Salmonella enterica serovar Typhimurium DT 104. We detected higher expression of SOCS3 six days after weaning for all analyzed piglets, regardless of the infection or the zinc feeding, suggesting a stress induced immune response. Whereas, SOCS1 showed only higher transcript amounts in S. Typhimurium infected piglets, especially the LZN group. This might indicate an infection regulating effect of zinc oxide in the infection model. After 42days of infection, the expression of SOCS2, SOCS4, and SOCS7 was increased only in animals fed the highest concentrations of zinc oxide, while non-infected piglets at the age of 56days showed no regulation for these genes. The up-regulation of SOCS genes in the mesenteric lymph nodes of piglets fed a diet with a very high concentration of zinc over 6 weeks suggests that such treatments may impair the immune response. PMID:27496737

  12. 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.

  13. Genome-Wide High-Throughput Screening to Investigate Essential Genes Involved in Methicillin-Resistant Staphylococcus aureus Sequence Type 398 Survival

    DEFF Research Database (Denmark)

    Christiansen, Mette Theilgaard; Kaas, Rolf Sommer; Chaudhuri, Roy R.;

    2014-01-01

    Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) Sequence Type 398 (ST398) is an opportunistic pathogen that is able to colonize and cause disease in several animal species including humans. To better understand the adaptation, evolution, transmission and pathogenic....... The transposon mutant library consisted of approximately 1 million mutants with around 140,000 unique insertion sites and an average number of unique inserts per gene of 44.8. We identified LA-MRSA ST398 essential genes comparable to other high-throughput S. aureus essential gene studies. As ST398 is the most...... capacity, further investigations into the importance of the different genes harboured by LA-MRSA ST398 are required. In this study we generated a genome-wide transposon mutant library in an LA-MRSA ST398 isolate to evaluate genes important for bacterial survival in laboratory and host-specific environments...

  14. 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

  15. High-Resolution Comparative Genomic Hybridization of Inflammatory Breast Cancer and Identification of Candidate Genes

    OpenAIRE

    Bekhouche, Ismahane; Finetti, Pascal; Adelaïde, José; Ferrari, Anthony; Tarpin, Carole; Charafe-Jauffret, Emmanuelle; Charpin, Colette; Houvenaeghel, Gilles; Jacquemier, Jocelyne; Bidaut, Ghislain; Birnbaum, Daniel; Viens, Patrice; Chaffanet, Max; Bertucci, François

    2011-01-01

    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 ...

  16. 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.

  17. 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. PMID:21540065

  18. Analysis of image-based phenotypic parameters for high throughput gene perturbation assays.

    Science.gov (United States)

    Song, Mee; Jeong, Euna; Lee, Tae-Kyu; Tsoy, Yury; Kwon, Yong-Jun; Yoon, Sukjoon

    2015-10-01

    Although image-based phenotypic assays are considered a powerful tool for siRNA library screening, the reproducibility and biological implications of various image-based assays are not well-characterized in a systematic manner. Here, we compared the resolution of high throughput assays of image-based cell count and typical cell viability measures for cancer samples. It was found that the optimal plating density of cells was important to obtain maximal resolution in both types of assays. In general, cell counting provided better resolution than the cell viability measure in diverse batches of siRNAs. In addition to cell count, diverse image-based measures were simultaneously collected from a single screening and showed good reproducibility in repetitions. They were classified into a few functional categories according to biological process, based on the differential patterns of hit (i.e., siRNAs) prioritization from the same screening data. The presented systematic analyses of image-based parameters provide new insight to a multitude of applications and better biological interpretation of high content cell-based assays. PMID:26256799

  19. Finding Important Genes from High-Dimensional Data: An Appraisal of Statistical Tests and Machine-Learning Approaches

    CERN Document Server

    Wang, Chamont; Chen, Chaur-Chin; Auslender, Leonardo

    2012-01-01

    Over the past decades, statisticians and machine-learning researchers have developed literally thousands of new tools for the reduction of high-dimensional data in order to identify the variables most responsible for a particular trait. These tools have applications in a plethora of settings, including data analysis in the fields of business, education, forensics, and biology (such as microarray, proteomics, brain imaging), to name a few. In the present work, we focus our investigation on the limitations and potential misuses of certain tools in the analysis of the benchmark colon cancer data (2,000 variables; Alon et al., 1999) and the prostate cancer data (6,033 variables; Efron, 2010, 2008). Our analysis demonstrates that models that produce 100% accuracy measures often select different sets of genes and cannot stand the scrutiny of parameter estimates and model stability. Furthermore, we created a host of simulation datasets and "artificial diseases" to evaluate the reliability of commonly used statistica...

  20. Shallow gene pools in the high intertidal: extreme loss of genetic diversity in viviparous sea stars (Parvulastra).

    Science.gov (United States)

    Keever, Carson C; Puritz, Jonathan B; Addison, Jason A; Byrne, Maria; Grosberg, Richard K; Toonen, Robert J; Hart, Michael W

    2013-10-23

    We document an extreme example of reproductive trait evolution that affects population genetic structure in sister species of Parvulastra cushion stars from Australia. Self-fertilization by hermaphroditic adults and brood protection of benthic larvae causes strong inbreeding and range-wide genetic poverty. Most samples were fixed for a single allele at nearly all nuclear loci; heterozygotes were extremely rare (0.18%); mitochondrial DNA sequences were more variable, but few populations shared haplotypes in common. Isolation-with-migration models suggest that these patterns are caused by population bottlenecks (relative to ancestral population size) and low gene flow. Loss of genetic diversity and low potential for dispersal between high-intertidal habitats may have dire consequences for extinction risk and potential for future adaptive evolution in response to climate and other selective agents.

  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...... that gut-expressed human celiac epitope homologous peptides are unlikely to represent non-HLA risk factors in the development of celiac disease in Caucasians....

  2. Determination of gene expression patterns using high-throughput RNA in situ hybridizaion to whole-mount Drosophila embryos

    Energy Technology Data Exchange (ETDEWEB)

    Weiszmann, R.; Hammonds, A.S.; Celniker, S.E.

    2009-04-09

    We describe a high-throughput protocol for RNA in situ hybridization (ISH) to Drosophila embryos in a 96-well format. cDNA or genomic DNA templates are amplified by PCR and then digoxigenin-labeled ribonucleotides are incorporated into antisense RNA probes by in vitro transcription. The quality of each probe is evaluated before ISH using a RNA probe quantification (dot blot) assay. RNA probes are hybridized to fixed, mixed-staged Drosophila embryos in 96-well plates. The resulting stained embryos can be examined and photographed immediately or stored at 4oC for later analysis. Starting with fixed, staged embryos, the protocol takes 6 d from probe template production through hybridization. Preparation of fixed embryos requires a minimum of 2 weeks to collect embryos representing all stages. The method has been used to determine the expression patterns of over 6,000 genes throughout embryogenesis.

  3. Genetics and Molecular Mapping of a High-Temperature Resistance Gene to Stripe Rust in Seeding-Stage in Winter Wheat Cultivar Lantian 1

    Institute of Scientific and Technical Information of China (English)

    MA Dong-fang; JING Jin-xue; HOU Dong-yuan; LI Qiang; ZHOU Xin-li; DU Jiu-yuan; and LU Qing-lin

    2013-01-01

    Stripe rust, caused by Puccinia striiformis Westend. f. sp. tritici (Pst), is a severe foliar disease of common wheat (Triticum aestivum L.) in the world. Resistance is the best approach to control the disease. The winter wheat cultivar Lantian 1 has high-temperature resistance to stripe rust. To determing the gene(s) for the stripe rust resistance, Lantian 1 was crossed with Mingxian 169 (M169). Seedlings of the parents, and F1, F2 and F2-3 progenies were tested with races CYR32 of Pst under controlled greenhouse conditions. Lantian 1 has a single partially dominant gene conferred resistance to race CYR32, designated as YrLT1. Simple sequence repeat (SSR) techniques were used to identify molecular markers linked to YrLT1. A linkage group of five SSR markers was constructed for YrLT1 using 166 F2 plants. Based on the SSR marker consensus map and the position on wheat chromosome, the resistance gene was assigned on chromosome 2DL. Amplification of a set of nulli-tetrasomic Chinese Spring lines with SSR marker Xwmc797 confirmed that the resistance gene was located on the long arm of chromosome 2D. Because of its chromosomal location and the high-temperature resistance, this gene is different from previously described genes. The molecular map spanned 29.9 cM, and the genetic distance of two close markers Xbarc228 and Xcfd16 to resistance gene locus was 4.0 and 5.7 cM, respectively. The polymorphism rates of the flanking markers in 46 wheat lines were 2.1 and 2.1%, respectively;and the two markers in combination could distinguish the alleles at the resistance locus in 97.9%of tested genotypes. This new gene and flanking markers should be useful in developing wheat cultivars with high level and possible durable resistance to stripe rust.

  4. Effects of abscisic acid and high osmoticum on storage protein gene expression in microspore embryos of Brassica napus

    International Nuclear Information System (INIS)

    Storage protein gene expression, characteristic of mid- to late embryogenesis, was investigated in microspore embryos of rapeseed (Brassica napus). These embryos, derived from the immature male gametophyte, accumulate little or no detectable napin or cruciferin mRNA when cultured on hormone-free medium containing 13% sucrose. The addition of abscisic acid (ABA) to the medium results in an increase in detectable transcripts encoding both these polypeptides. Storage protein mRNA is induced at 1 micromolar ABA with maximum stimulation occurring between 5 and 50 micromolar. This hormone induction results in a level of storage protein mRNA that is comparable to that observed in zygotic embryos of an equivalent morphological stage. Effects similar to that of ABA are noted when 12.5% sorbitol is added to the microspore embryo medium (osmotic potential = 25.5 bars). Time course experiments, to study the induction of napin and cruciferin gene expression demonstrated that the ABA effect occurred much more rapidly than the high osmoticum effect, although after 48 hours, the levels of napin or cruciferin mRNA detected were similar in both treatments. This difference in the rates of induction is consistent with the idea that the osmotic effect may be mediated by ABA which is synthesized in response to the reduced water potential. Measurements of ABA (by gas chromatography-mass spectrometry using [2H6]ABA as an internal standard) present in microspore embryos during sorbitol treatment and in embryos treated with 10 micromolar ABA were performed to investigate this possibility. Within 2 hours of culture on high osmoticum the level of ABA increased substantially and significantly above control and reached a maximum concentration within 24 hours. This elevated concentration was maintained for 48 hours after culturing and represents a sixfold increase over control embryos

  5. Complete Sequence Construction of the Highly Repetitive Ribosomal RNA Gene Repeats in Eukaryotes Using Whole Genome Sequence Data.

    Science.gov (United States)

    Agrawal, Saumya; Ganley, Austen R D

    2016-01-01

    The ribosomal RNA genes (rDNA) encode the major rRNA species of the ribosome, and thus are essential across life. These genes are highly repetitive in most eukaryotes, forming blocks of tandem repeats that form the core of nucleoli. The primary role of the rDNA in encoding rRNA has been long understood, but more recently the rDNA has been implicated in a number of other important biological phenomena, including genome stability, cell cycle, and epigenetic silencing. Noncoding elements, primarily located in the intergenic spacer region, appear to mediate many of these phenomena. Although sequence information is available for the genomes of many organisms, in almost all cases rDNA repeat sequences are lacking, primarily due to problems in assembling these intriguing regions during whole genome assemblies. Here, we present a method to obtain complete rDNA repeat unit sequences from whole genome assemblies. Limitations of next generation sequencing (NGS) data make them unsuitable for assembling complete rDNA unit sequences; therefore, the method we present relies on the use of Sanger whole genome sequence data. Our method makes use of the Arachne assembler, which can assemble highly repetitive regions such as the rDNA in a memory-efficient way. We provide a detailed step-by-step protocol for generating rDNA sequences from whole genome Sanger sequence data using Arachne, for refining complete rDNA unit sequences, and for validating the sequences obtained. In principle, our method will work for any species where the rDNA is organized into tandem repeats. This will help researchers working on species without a complete rDNA sequence, those working on evolutionary aspects of the rDNA, and those interested in conducting phylogenetic footprinting studies with the rDNA. PMID:27576718

  6. HLA-A Gene Polymorphism Defined by High-Resolution Sequence Based Typing in 161 Northern Chinese Han People

    Institute of Scientific and Technical Information of China (English)

    Chunxia Yan; Haiyan Sun; Xiuqing Zhang; Jian Wang; Huanming Yang; Shengbin Li; Ruilin Wang; Jingxiang Li; Yajun Deng; Dongying Wu; Hongbo Zhang; Hongxing Zhang; Lidong Wang; Chunrong Zhang

    2003-01-01

    Human leukocyte antigen (HLA) system is the most polymorphic region known in the human genome. In the present study, we analyzed for the first time the HLA-A gene polymorphisms defined by the high-resolution typing methods--sequence-based typing (SBT) in 161 Northern Chinese Han people. A total of 74 different HLA-A gene types and 36 alleles were detected. The most frequent alleles were A*110101 (GF=0.2360), A*24020101 (GF=0.1646), and A*020101 (GF=0.1553); followed by A*3303 (GF=0.1180), A*3001 (GF=0.0590),and A*310102 (GF=0.0404). The frequencies of following alleles, A*0203, A*0205,A*0206, A*0207, A*030101, A*2423, A*2601, A*3201, and A*3301, are all higher than 0.0093. The homozygous alleles include A*020101, A*110101, A*24020101 and A*310102. Heterozygosity (H), polymorphism information content (PIC), discrimination power (DP) and probability of paternity exclusion (PPE) of HLA-A in the samples were calculated and their values were 0.8705, 0.8491, 0.6014, and 0.9475, respectively. These results by SBT analysis of HLA-A polymorphism in Northern Chinese Han population, especially the allele subtypes character, will be of great interest for clinical transplantation, disease-associated study and forensic identification. Implementation of high-resolution typing methods allows a significantly wider spectrum of HLA variation including rare alleles. This spectrum will further be extensively utilized in many fields.

  7. High-throughput Gene Expression Analysis In Pigs As Model For Respiratory Infections

    DEFF Research Database (Denmark)

    Skovgaard, Kerstin; Brogaard, Louise; Schou, Kirstine Klitgaard;

    perform highly controlled experimental infections and to study changes of symptoms, viral titer, and expression of microRNAs/mRNAs as the influenza infection progresses in time, generating information that would be difficult to obtain from human patients. The Gram-negative bacterium Actinobacillus......Influenza A virus infections have great impact on human health and welfare and significant resources are linked to influenza epidemics due to excess hospitalizations and lost productivity. Up to 15% of the human population is affected when Influenza spreads around the world in seasonal epidemics...... (WHO). Animal models are essential in understanding the mechanisms involved in human infectious disease and for the development of effective prevention and treatment strategies. It is increasingly realized that large animal models like the pig are exceptionally human like and serve as an excellent...

  8. High-quality reference genes for quantifying the transcriptional responses of Oryza sativa L.(ssp.indica and japonica) to abiotic stress conditions

    Institute of Scientific and Technical Information of China (English)

    MAKSUP Sarunyaporn; SUPAIBULWATANA Kanyaratt; SELVARAJ Gopalan

    2013-01-01

    Rice (Oryza sativa L.) is important to food security and is also an excellent model plant for numerous cereal crops.A functional genomics study in rice includes characterization of the expression dynamics of genes by quantitative real-time PCR (qPCR) analysis; this is a significant key for developing rice varieties that perform well in the face of adverse climate change.The qPCR analysis requires the use of appropriate reference genes in order to make any quantitative interpretations meaningful.Here,the new potential reference genes were selected from a huge public database of rice microarray experiments.The expression stability of 14 candidates and 4 conventional reference genes was validated by geNormPLUs and NormFinder software.Seven candidates are superior to the conventionally used reference genes in qPCR and three genes can be used reliably for quantitating the expression of genes involved in abiotic stress responses.These high-quality references EP (LOC_Os05g08980),HNR (LOC_Os01g71770),and TBC (LOC_Os09g34040) worked very well in three indica genotypes and one japonica genotype.One of indica genotypes including the Jasmine rice,KDML105 developed in Thailand for which no reference genes have been reported until now.

  9. Mechanistic understanding of gene delivery mediated by highly efficient multicomponent envelope-type nanoparticle systems.

    Science.gov (United States)

    Pozzi, D; Marchini, C; Cardarelli, F; Rossetta, A; Colapicchioni, V; Amici, A; Montani, M; Motta, S; Brocca, P; Cantù, L; Caracciolo, G

    2013-12-01

    We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability. PMID:24188138

  10. Virulence Factors and Antibiotic Susceptibility of Staphylococcus aureus Isolates in Ready-to-Eat Foods: Detection of S. aureus Contamination and a High Prevalence of Virulence Genes

    Directory of Open Access Journals (Sweden)

    Suat Moi Puah

    2016-02-01

    Full Text Available Staphylococcus aureus is one of the leading causes of food poisoning. Its pathogenicity results from the possession of virulence genes that produce different toxins which result in self-limiting to severe illness often requiring hospitalization. In this study of 200 sushi and sashimi samples, S. aureus contamination was confirmed in 26% of the food samples. The S. aureus isolates were further characterized for virulence genes and antibiotic susceptibility. A high incidence of virulence genes was identified in 96.2% of the isolates and 20 different virulence gene profiles were confirmed. DNA amplification showed that 30.8% (16/52 of the S. aureus carried at least one SE gene which causes staphylococcal food poisoning. The most common enterotoxin gene was seg (11.5% and the egc cluster was detected in 5.8% of the isolates. A combination of hla and hld was the most prevalent coexistence virulence genes and accounted for 59.6% of all isolates. Antibiotic resistance studies showed tetracycline resistance to be the most common at 28.8% while multi-drug resistance was found to be low at 3.8%. In conclusion, the high rate of S. aureus in the sampled sushi and sashimi indicates the need for food safety guidelines.

  11. Isolation of a new butanol-producing Clostridium strain: high level of hemicellulosic activity and structure of solventogenesis genes of a new Clostridium saccharobutylicum isolate.

    Science.gov (United States)

    Berezina, Oksana V; Brandt, Agnieszka; Yarotsky, Sergey; Schwarz, Wolfgang H; Zverlov, Vladimir V

    2009-10-01

    New isolates of solventogenic bacteria exhibited high hemicellulolytic activity. They produced butanol and acetone with high selectivity for butanol (about 80% of butanol from the total solvent yield). Their 16S rDNA sequence was 99% identical to that of Clostridium saccharobutylicum. The genes responsible for the last steps of solventogenesis and encoding crotonase, butyryl-CoA dehydrogenase, electron-transport protein subunits A and B, 3-hydroxybutyryl-CoA dehydrogenase, alcohol dehydrogenase, CoA-transferase (subunits A and B), acetoacetate decarboxylase, and aldehyde dehydrogenase were identified in the new C. saccharobutylicum strain Ox29 and cloned into Escherichia coli. The genes for crotonase, butyryl-CoA dehydrogenase, electron-transport protein subunits A and B, and 3-hydroxybutyryl-CoA dehydrogenase composed the bcs-operon. A monocistronic operon containing the alcohol dehydrogenase gene was located downstream of the bcs-operon. Genes for aldehyde dehydrogenase, CoA-transferase (subunits A and B), and acetoacetate decarboxylase composed the sol-operon. The gene sequences and the gene order within the sol- and bcs-operons of C. saccharobutylicum Ox29 were most similar to those of Clostridium beijerinckii. The activity of some of the bcs-operon genes, expressed in heterologous E. coli, was determined. PMID:19674858

  12. Virulence Factors and Antibiotic Susceptibility of Staphylococcus aureus Isolates in Ready-to-Eat Foods: Detection of S. aureus Contamination and a High Prevalence of Virulence Genes.

    Science.gov (United States)

    Puah, Suat Moi; Chua, Kek Heng; Tan, Jin Ai Mary Anne

    2016-02-01

    Staphylococcus aureus is one of the leading causes of food poisoning. Its pathogenicity results from the possession of virulence genes that produce different toxins which result in self-limiting to severe illness often requiring hospitalization. In this study of 200 sushi and sashimi samples, S. aureus contamination was confirmed in 26% of the food samples. The S. aureus isolates were further characterized for virulence genes and antibiotic susceptibility. A high incidence of virulence genes was identified in 96.2% of the isolates and 20 different virulence gene profiles were confirmed. DNA amplification showed that 30.8% (16/52) of the S. aureus carried at least one SE gene which causes staphylococcal food poisoning. The most common enterotoxin gene was seg (11.5%) and the egc cluster was detected in 5.8% of the isolates. A combination of hla and hld was the most prevalent coexistence virulence genes and accounted for 59.6% of all isolates. Antibiotic resistance studies showed tetracycline resistance to be the most common at 28.8% while multi-drug resistance was found to be low at 3.8%. In conclusion, the high rate of S. aureus in the sampled sushi and sashimi indicates the need for food safety guidelines. PMID:26861367

  13. Identification of Novel Pepper Genes Involved in Bax- or INF1-Mediated Cell Death Responses by High-Throughput Virus-Induced Gene Silencing

    Directory of Open Access Journals (Sweden)

    Jeong Hee Lee

    2013-11-01

    Full Text Available Hot pepper is one of the economically important crops in Asia. A large number of gene sequences, including expressed sequence tag (EST and genomic sequences are publicly available. However, it is still a daunting task to determine gene function due to difficulties in genetic modification of a pepper plants. Here, we show the application of the virus-induced gene silencing (VIGS repression for the study of 459 pepper ESTs selected as non-host pathogen-induced cell death responsive genes from pepper microarray experiments in Nicotiana benthamiana. Developmental abnormalities in N. benthamiana plants are observed in the 32 (7% pepper ESTs-silenced plants. Aberrant morphological phenotypes largely comprised of three groups: stunted, abnormal leaf, and dead. In addition, by employing the combination of VIGS and Agrobacterium-mediated transient assays, we identified novel pepper ESTs that involved in Bax or INF1-mediated cell death responses. Silencing of seven pepper ESTs homologs suppressed Bax or INF1-induced cell death, five of which suppressed both cell death responses in N. benthamiana. The genes represented by these five ESTs encode putative proteins with functions in endoplasmic reticulum (ER stress and lipid signaling. The genes represented by the other two pepper ESTs showing only Bax-mediated cell death inhibition encode a CCCH-type zinc finger protein containing an ankyrin-repeat domain and a probable calcium-binding protein, CML30-like. Taken together, we effectively isolated novel pepper clones that are involved in hypersensitive response (HR-like cell death using VIGS, and identified silenced clones that have different responses to Bax and INF1 exposure, indicating separate signaling pathways for Bax- and INF1-mediated cell death.

  14. High-throughput analysis of ammonia oxidiser community composition via a novel, amoA-based functional gene array.

    Directory of Open Access Journals (Sweden)

    Guy C J Abell

    Full Text Available Advances in microbial ecology research are more often than not limited by the capabilities of available methodologies. Aerobic autotrophic nitrification is one of the most important and well studied microbiological processes in terrestrial and aquatic ecosystems. We have developed and validated a microbial diagnostic microarray based on the ammonia-monooxygenase subunit A (amoA gene, enabling the in-depth analysis of the community structure of bacterial and archaeal ammonia oxidisers. The amoA microarray has been successfully applied to analyse nitrifier diversity in marine, estuarine, soil and wastewater treatment plant environments. The microarray has moderate costs for labour and consumables and enables the analysis of hundreds of environmental DNA or RNA samples per week per person. The array has been thoroughly validated with a range of individual and complex targets (amoA clones and environmental samples, respectively, combined with parallel analysis using traditional sequencing methods. The moderate cost and high throughput of the microarray makes it possible to adequately address broader questions of the ecology of microbial ammonia oxidation requiring high sample numbers and high resolution of the community composition.

  15. High production of plant type levan in sugar beet transformed with timothy (Phleum pratense) 6-SFT genes.

    Science.gov (United States)

    Matsuhira, Hiroaki; Tamura, Ken-ichi; Tamagake, Hideto; Sato, Yutaka; Anzai, Hiroyuki; Yoshida, Midori

    2014-12-20

    Levan, a type of fructan, is an oligomer or polymer with mainly a β(2,6)-linked fructose chain attached to sucrose. We introduced two timothy genes, PpFT1 and PpFT2, coding for two homologous sucrose:fructan 6-fructosyltransferases into sugar beet. Sugar beet produces a high concentration of sucrose, a starting substrate in fructan synthesis, in the root. Among transgenic T1 lines, we obtained sugar beet transformants that accumulated large amounts of β(2,6)-linked levans (about 20 to 75mgg(-1) FW) in the roots. The transformed sugar beet plants possessing PpFT1 or PpFT2 produced linear levans with different degrees of polymerization (DP). Namely, the PpFT1 transformants accumulated mainly high DP levans including those with DP>40, while the PpFT2 transformants accumulated levans with DP between 3 and 40. Chromatograms showed that PpFT2 produces pure β(2,6)-linked linear levans compared with fructans synthesized by PpFT1. These levans belong to the high DP class of plant fructans, but have much shorter DP than that of levans generally produced by microorganisms. PMID:25305472

  16. High-Throughput Analysis of Ammonia Oxidiser Community Composition via a Novel, amoA-Based Functional Gene Array

    Science.gov (United States)

    Abell, Guy C. J.; Robert, Stan S.; Frampton, Dion M. F.; Volkman, John K.; Rizwi, Farhan; Csontos, József; Bodrossy, Levente

    2012-01-01

    Advances in microbial ecology research are more often than not limited by the capabilities of available methodologies. Aerobic autotrophic nitrification is one of the most important and well studied microbiological processes in terrestrial and aquatic ecosystems. We have developed and validated a microbial diagnostic microarray based on the ammonia-monooxygenase subunit A (amoA) gene, enabling the in-depth analysis of the community structure of bacterial and archaeal ammonia oxidisers. The amoA microarray has been successfully applied to analyse nitrifier diversity in marine, estuarine, soil and wastewater treatment plant environments. The microarray has moderate costs for labour and consumables and enables the analysis of hundreds of environmental DNA or RNA samples per week per person. The array has been thoroughly validated with a range of individual and complex targets (amoA clones and environmental samples, respectively), combined with parallel analysis using traditional sequencing methods. The moderate cost and high throughput of the microarray makes it possible to adequately address broader questions of the ecology of microbial ammonia oxidation requiring high sample numbers and high resolution of the community composition. PMID:23284709

  17. High gene flow and outcrossing within populations of two cryptic fungal pathogens on a native and non-native host in Cameroon.

    Science.gov (United States)

    Begoude Boyogueno, Aime Didier; Slippers, Bernard; Perez, Guillermo; Wingfield, Michael J; Roux, Jolanda

    2012-03-01

    In this study, we determined the genetic diversity of 126 isolates representing both Lasiodiplodia theobromae and Lasiodiplodia pseudotheobromae, collected from Theobroma cacao and Terminalia spp. in Cameroon, using simple sequence repeat (SSR) markers. SSR alleles showed clear genetic distinction between L. theobromae and L. pseudotheobromae, supporting their earlier separation as sister species. Both L. theobromae and L. pseudotheobromae populations from Cameroon had high levels of gene diversity, moderate degrees of genotypic diversity, and high levels of gene flow between isolates from T. cacao and Terminalia spp. There was no evidence for geographic substructure in these populations across the region studied, and the SSR alleles were randomly associated in both species, suggesting outcrossing. The significant levels of aggressiveness, evolutionary potential represented by high levels of diversity, outcrossing and gene flow between geographically and host defined populations, identify these fungi as high-risk pathogens for their native and non-native hosts in Cameroon. PMID:22385617

  18. The TIS11 primary response gene is a member of a gene family that encodes proteins with a highly conserved sequence containing an unusual Cys-His repeat.

    OpenAIRE

    Varnum, B C; Ma, Q F; T. H. Chi; Fletcher, B.; Herschman, H.R.

    1991-01-01

    The TIS11 primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors. The predicted TIS11 protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TIS11b and TIS11d, that contain the YKTELC sequence. TIS11, TIS11b, and TIS11d proteins share a 67-amino-acid region of sequence similarity that includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS11 gene family members are not coordinately...

  19. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

    Directory of Open Access Journals (Sweden)

    Nerea Irigoyen

    2016-02-01

    Full Text Available Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV, are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59, a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the

  20. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling

    Science.gov (United States)

    Jones, Joshua D.; Chung, Betty Y.-W.; Siddell, Stuart G.; Brierley, Ian

    2016-01-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  1. A genetic combination of silent beta-thalassaemia, high Hb A2 beta-thalassaemia, and single alpha globin gene deletion causing mild thalassaemia intermedia.

    OpenAIRE

    R. Galanello; Maccioni, L; ROSATELLI, M. C.; Ibba, P; Nurchi, A M; Cao, A

    1984-01-01

    This paper reports a Sardinian patient, who was a compound heterozygote for silent beta-thalassaemia and high Hb A2 beta o-thalassaemia with the clinical phenotype of mild thalassaemia intermedia; alpha globin gene mapping showed a single alpha globin gene deletion. The reduced alpha globin chain output resulted in more balanced globin chain synthesis, which in turn accounted for the mild clinical phenotype.

  2. Prolonged high fat diet reduces dopamine reuptake without altering DAT gene expression.

    Directory of Open Access Journals (Sweden)

    Jackson J Cone

    Full Text Available The development of diet-induced obesity (DIO can potently alter multiple aspects of dopamine signaling, including dopamine transporter (DAT expression and dopamine reuptake. However, the time-course of diet-induced changes in DAT expression and function and whether such changes are dependent upon the development of DIO remains unresolved. Here, we fed rats a high (HFD or low (LFD fat diet for 2 or 6 weeks. Following diet exposure, rats were anesthetized with urethane and striatal DAT function was assessed by electrically stimulating the dopamine cell bodies in the ventral tegmental area (VTA and recording resultant changes in dopamine concentration in the ventral striatum using fast-scan cyclic voltammetry. We also quantified the effect of HFD on membrane associated DAT in striatal cell fractions from a separate group of rats following exposure to the same diet protocol. Notably, none of our treatment groups differed in body weight. We found a deficit in the rate of dopamine reuptake in HFD rats relative to LFD rats after 6 but not 2 weeks of diet exposure. Additionally, the increase in evoked dopamine following a pharmacological challenge of cocaine was significantly attenuated in HFD relative to LFD rats. Western blot analysis revealed that there was no effect of diet on total DAT protein. However, 6 weeks of HFD exposure significantly reduced the 50 kDa DAT isoform in a synaptosomal membrane-associated fraction, but not in a fraction associated with recycling endosomes. Our data provide further evidence for diet-induced alterations in dopamine reuptake independent of changes in DAT production and demonstrates that such changes can manifest without the development of DIO.

  3. Rice Bran Protein Hydrolysates Improve Insulin Resistance and Decrease Pro-inflammatory Cytokine Gene Expression in Rats Fed a High Carbohydrate-High Fat Diet

    Directory of Open Access Journals (Sweden)

    Kampeebhorn Boonloh

    2015-08-01

    Full Text Available A high carbohydrate-high fat (HCHF diet causes insulin resistance (IR and metabolic syndrome (MS. Rice bran has been demonstrated to have anti-dyslipidemic and anti-atherogenic properties in an obese mouse model. In the present study, we investigated the beneficial effects of rice bran protein hydrolysates (RBP in HCHF-induced MS rats. After 12 weeks on this diet, the HCHF-fed group was divided into four subgroups, which were orally administered RBP 100 or 500 mg/kg, pioglitazone 10 mg/kg, or tap water for a further 6 weeks. Compared with normal diet control group, the MS rats had elevated levels of blood glucose, lipid, insulin, and HOMA-IR. Treatment with RBP significantly alleviated all those changes and restored insulin sensitivity. Additionally, RBP treatment increased adiponectin and suppressed leptin levels. Expression of Ppar-γ mRNA in adipose tissues was significantly increased whereas expression of lipogenic genes Srebf1 and Fasn was significantly decreased. Levels of mRNA of proinflammatory cytokines, Il-6, Tnf-α, Nos-2 and Mcp-1 were significantly decreased. In conclusion, the present findings support the consumption of RBP as a functional food to improve insulin resistance and to prevent the development of metabolic syndrome.

  4. Rice Bran Protein Hydrolysates Improve Insulin Resistance and Decrease Pro-inflammatory Cytokine Gene Expression in Rats Fed a High Carbohydrate-High Fat Diet.

    Science.gov (United States)

    Boonloh, Kampeebhorn; Kukongviriyapan, Veerapol; Kongyingyoes, Bunkerd; Kukongviriyapan, Upa; Thawornchinsombut, Supawan; Pannangpetch, Patchareewan

    2015-08-01

    A high carbohydrate-high fat (HCHF) diet causes insulin resistance (IR) and metabolic syndrome (MS). Rice bran has been demonstrated to have anti-dyslipidemic and anti-atherogenic properties in an obese mouse model. In the present study, we investigated the beneficial effects of rice bran protein hydrolysates (RBP) in HCHF-induced MS rats. After 12 weeks on this diet, the HCHF-fed group was divided into four subgroups, which were orally administered RBP 100 or 500 mg/kg, pioglitazone 10 mg/kg, or tap water for a further 6 weeks. Compared with normal diet control group, the MS rats had elevated levels of blood glucose, lipid, insulin, and HOMA-IR. Treatment with RBP significantly alleviated all those changes and restored insulin sensitivity. Additionally, RBP treatment increased adiponectin and suppressed leptin levels. Expression of Ppar-γ mRNA in adipose tissues was significantly increased whereas expression of lipogenic genes Srebf1 and Fasn was significantly decreased. Levels of mRNA of proinflammatory cytokines, Il-6, Tnf-α, Nos-2 and Mcp-1 were significantly decreased. In conclusion, the present findings support the consumption of RBP as a functional food to improve insulin resistance and to prevent the development of metabolic syndrome. PMID:26247962

  5. High expression of Sonic Hedgehog signaling pathway genes indicates a risk of recurrence of breast carcinoma

    Directory of Open Access Journals (Sweden)

    Jeng KS

    2013-12-01

    Full Text Available Kuo-Shyang Jeng,1 I-Shyan Sheen,2 Wen-Juei Jeng,2 Ming-Che Yu,3 Hsin-I Hsiau,3 Fang-Yu Chang31Department of Surgery, Far Eastern Memorial Hospital, Taipei, 2Department of Internal Medicine, Chang-Gung Memorial Hospital, Linkou Medical Center, Chang-Gung University, Tao-Yuan, 3Department of Medical Research, Far Eastern Memorial Hospital, Taipei, TaiwanBackground: Abnormal activation of the Sonic Hedgehog (SHH signaling pathway contributing to carcinogenesis of some organs has been reported in the literature. We hypothesize that activation of the SHH pathway contributes to the recurrence of breast carcinoma.Methods: Fifty consecutive patients with invasive breast carcinoma following curative resection were enrolled in this prospective study. The ratios of messenger RNA (mRNA expression for Sonic Hedgehog (SHH, patched homolog-1 (PTCH-1, glioma-associated oncogene-1 (GLI-1, and smoothened (SMOH were measured between breast carcinoma tissue and paired noncancerous breast tissue. These ratios were compared with their clinicopathologic characteristics. These factors and the mRNA ratios were compared between patients with recurrence and those without recurrence.Results: The size of the invasive cancer correlated significantly with the ratio of SHH mRNA (P=0.001, that of PTCH-1 mRNA (P=0.005, and that of SMOH mRNA (P=0.021. Lymph node involvement correlated significantly with the ratio of SMOH mRNA (P=0.041. The correlation between Her-2 neu and the ratio of GLI-1 mRNA was statistically significant (P=0.012. Each ratio of mRNA of SHH, PTCH-1, GLI-1, and SMOH correlated significantly with cancer recurrence (P<0.001 for each.Conclusion: We suggest that high expression of SHH mRNA, PTCH-1 mRNA, GLI-1 mRNA, and SMOH mRNA in breast cancer tissue correlates with invasiveness and is a potential biomarker to predict postoperative recurrence.Keywords: SHH pathway, breast carcinoma, prediction, recurrence

  6. High-resolution mapping, cloning and molecular characterization of the Pi-k ( h ) gene of rice, which confers resistance to Magnaporthe grisea.

    Science.gov (United States)

    Sharma, T R; Madhav, M S; Singh, B K; Shanker, P; Jana, T K; Dalal, V; Pandit, A; Singh, A; Gaikwad, K; Upreti, H C; Singh, N K

    2005-12-01

    In order to understand the molecular mechanisms involved in the gene-for-gene type of pathogen resistance, high-resolution genetic and physical mapping of resistance loci is required to facilitate map-based cloning of resistance genes. Here, we report the molecular mapping and cloning of a dominant gene (Pi-k ( h )) present in the rice line Tetep, which is associated with resistance to rice blast disease caused by Magnaporthe grisea. This gene is effective against M. grisea populations prevalent in the Northwestern Himalayan region of India. Using 178 sequence tagged microsatellite, sequence-tagged site, expressed sequence tag and simple sequence repeat (SSR) markers to genotype a population of 208 F(2) individuals, we mapped the Pi-k ( h ) gene between two SSR markers (TRS26 and TRS33) which are 0.7 and 0.5 cM away, respectively, and can be used in marker-assisted-selection for blast-resistant rice cultivars. We used the markers to identify the homologous region in the genomic sequence of Oryza sativa cv. Nipponbare, and a physical map consisting of two overlapping bacterial artificial chromosome and P1 artificial chromosome clones was assembled, spanning a region of 143,537 bp on the long arm of chromosome 11. Using bioinformatic analyses, we then identified a candidate blast-resistance gene in the region, and cloned the homologous sequence from Tetep. The putative Pi-k ( h ) gene cloned from Tetep is 1.5 kbp long with a single ORF, and belongs to the nucleotide binding site-leucine rich repeat class of disease resistance genes. Structural and expression analysis of the Pi-k ( h ) gene revealed that its expression is pathogen inducible. PMID:16228246

  7. The use of Gene Ontology terms for predicting highly-connected 'hub' nodes in protein-protein interaction networks

    Directory of Open Access Journals (Sweden)

    Cherkasov Artem

    2008-09-01

    Full Text Available Abstract Background Protein-protein interactions mediate a wide range of cellular functions and responses and have been studied rigorously through recent large-scale proteomics experiments and bioinformatics analyses. One of the most important findings of those endeavours was the observation that 'hub' proteins participate in significant numbers of protein interactions and play critical roles in the organization and function of cellular protein interaction networks (PINs 12. It has also been demonstrated that such hub proteins may constitute an important pool of attractive drug targets. Thus, it is crucial to be able to identify hub proteins based not only on experimental data but also by means of bioinformatics predictions. Results A hub protein classifier has been developed based on the available interaction data and Gene Ontology (GO annotations for proteins in the Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster and Homo sapiens genomes. In particular, by utilizing the machine learning method of boosting trees we were able to create a predictive bioinformatics tool for the identification of proteins that are likely to play the role of a hub in protein interaction networks. Testing the developed hub classifier on external sets of experimental protein interaction data in Methicillin-resistant Staphylococcus aureus (MRSA 252 and Caenorhabditis elegans demonstrated that our approach can predict hub proteins with a high degree of accuracy. A practical application of the developed bioinformatics method has been illustrated by the effective protein bait selection for large-scale pull-down experiments that aim to map complete protein-protein interaction networks for several species. Conclusion The successful development of an accurate hub classifier demonstrated that highly-connected proteins tend to share certain relevant functional properties reflected in their Gene Ontology annotations. It is anticipated that the developed

  8. Development of a Computational Steering Framework for High Performance Computing Environments on Blue Gene/P Systems

    KAUST Repository

    Danani, Bob K.

    2012-07-01

    Computational steering has revolutionized the traditional workflow in high performance computing (HPC) applications. The standard workflow that consists of preparation of an application’s input, running of a simulation, and visualization of simulation results in a post-processing step is now transformed into a real-time interactive workflow that significantly reduces development and testing time. Computational steering provides the capability to direct or re-direct the progress of a simulation application at run-time. It allows modification of application-defined control parameters at run-time using various user-steering applications. In this project, we propose a computational steering framework for HPC environments that provides an innovative solution and easy-to-use platform, which allows users to connect and interact with running application(s) in real-time. This framework uses RealityGrid as the underlying steering library and adds several enhancements to the library to enable steering support for Blue Gene systems. Included in the scope of this project is the development of a scalable and efficient steering relay server that supports many-to-many connectivity between multiple steered applications and multiple steering clients. Steered applications can range from intermediate simulation and physical modeling applications to complex computational fluid dynamics (CFD) applications or advanced visualization applications. The Blue Gene supercomputer presents special challenges for remote access because the compute nodes reside on private networks. This thesis presents an implemented solution and demonstrates it on representative applications. Thorough implementation details and application enablement steps are also presented in this thesis to encourage direct usage of this framework.

  9. Influences of graphene on microbial community and antibiotic resistance genes in mouse gut as determined by high-throughput sequencing.

    Science.gov (United States)

    Xie, Yongchao; Wu, Bing; Zhang, Xu-Xiang; Yin, Jinbao; Mao, Liang; Hu, Maojie

    2016-02-01

    Graphene is a promising candidate as an antibacterial material owning to its bacterial toxicity. However, little information on influence of graphene on gut microbiota is available. In this study, mice were exposed to graphene for 4 weeks, and high-throughput sequencing was applied to characterize the changes in microbial community and antibiotic resistance genes (ARGs) in mouse gut. The results showed that graphene exposure increased biodiversity of gut microbiota, and changed their community. The 1 μg/d graphene exposure had higher influences on the gut microbiota than 10 μg/d and 100 μg/d graphene exposures, which might be due to higher aggregation of high-level graphene. The influence of graphene on gut microbiota might attribute to that graphene could induce oxidative stress and damage of cell membrane integrity. The results were verified by the increase of ratio of Gram-negative bacteria. Outer membrane of Gram-negative bacteria could reduce the membrane damage induced by graphene and make them more tolerance to graphene. Further, we found that graphene exposure significantly increased the abundance and types of ARGs, indicating a potential health risk of graphene. This study firstly provides new insight to the health effects of graphene on gut microbiota.

  10. N-acetylcysteine enhances cystic fibrosis sputum penetration and airway gene transfer by highly compacted DNA nanoparticles.

    Science.gov (United States)

    Suk, Jung Soo; Boylan, Nicholas J; Trehan, Kanika; Tang, Benjamin C; Schneider, Craig S; Lin, Jung-Ming G; Boyle, Michael P; Zeitlin, Pamela L; Lai, Samuel K; Cooper, Mark J; Hanes, Justin

    2011-11-01

    For effective airway gene therapy of cystic fibrosis (CF), inhaled gene carriers must first penetrate the hyperviscoelastic sputum covering the epithelium. Whether clinically studied gene carriers can penetrate CF sputum remains unknown. Here, we measured the diffusion of a clinically tested nonviral gene carrier, composed of poly-l-lysine conjugated with a 10 kDa polyethylene glycol segment (CK(30)PEG(10k)). We found that CK(30)PEG(10k)/DNA nanoparticles were trapped in CF sputum. To improve gene carrier diffusion across sputum, we tested adjuvant regimens consisting of N-acetylcysteine (NAC), recombinant human DNase (rhDNase) or NAC together with rhDNase. While rhDNase alone did not enhance gene carrier diffusion, NAC and NAC + rhDNase increased average effective diffusivities by 6-fold and 13-fold, respectively, leading to markedly greater fractions of gene carriers that may penetrate sputum layers. We further tested the adjuvant effects of NAC in the airways of mice with Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced mucus hypersecretion. Intranasal dosing of NAC prior to CK(30)PEG(10k)/DNA nanoparticles enhanced gene expression by up to ~12-fold compared to saline control, reaching levels observed in the lungs of mice without LPS challenge. Our findings suggest that a promising synthetic nanoparticle gene carrier may transfer genes substantially more effectively to lungs of CF patients if administered following adjuvant mucolytic therapy with NAC or NAC + rhDNase.

  11. N-acetylcysteine enhances cystic fibrosis sputum penetration and airway gene transfer by highly compacted DNA nanoparticles.

    Science.gov (United States)

    Suk, Jung Soo; Boylan, Nicholas J; Trehan, Kanika; Tang, Benjamin C; Schneider, Craig S; Lin, Jung-Ming G; Boyle, Michael P; Zeitlin, Pamela L; Lai, Samuel K; Cooper, Mark J; Hanes, Justin

    2011-11-01

    For effective airway gene therapy of cystic fibrosis (CF), inhaled gene carriers must first penetrate the hyperviscoelastic sputum covering the epithelium. Whether clinically studied gene carriers can penetrate CF sputum remains unknown. Here, we measured the diffusion of a clinically tested nonviral gene carrier, composed of poly-l-lysine conjugated with a 10 kDa polyethylene glycol segment (CK(30)PEG(10k)). We found that CK(30)PEG(10k)/DNA nanoparticles were trapped in CF sputum. To improve gene carrier diffusion across sputum, we tested adjuvant regimens consisting of N-acetylcysteine (NAC), recombinant human DNase (rhDNase) or NAC together with rhDNase. While rhDNase alone did not enhance gene carrier diffusion, NAC and NAC + rhDNase increased average effective diffusivities by 6-fold and 13-fold, respectively, leading to markedly greater fractions of gene carriers that may penetrate sputum layers. We further tested the adjuvant effects of NAC in the airways of mice with Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced mucus hypersecretion. Intranasal dosing of NAC prior to CK(30)PEG(10k)/DNA nanoparticles enhanced gene expression by up to ~12-fold compared to saline control, reaching levels observed in the lungs of mice without LPS challenge. Our findings suggest that a promising synthetic nanoparticle gene carrier may transfer genes substantially more effectively to lungs of CF patients if administered following adjuvant mucolytic therapy with NAC or NAC + rhDNase. PMID:21829177

  12. Detecting population structure in a high gene-flow species, Atlantic herring (Clupea harengus): direct, simultaneous evaluation of neutral vs putatively selected loci

    DEFF Research Database (Denmark)

    André, C.; Larsson, L. C.; Laikre, L.;

    2010-01-01

    DNA, with one microsatellite locus, Cpa112, previously shown to be influenced by divergent selection associated with salinity, and one locus located in the major histocompatibility complex class IIA (MHC-IIA) gene, using the same individuals across analyses. Samples were collected in 2002 and 2003......In many marine fish species, genetic population structure is typically weak because populations are large, evolutionarily young and have a high potential for gene flow. We tested whether genetic markers influenced by natural selection are more efficient than the presumed neutral genetic markers...... presumed neutral microsatellite loci, sample sizes could be reduced by up to a tenth while still retaining high statistical power. Our results show that the loci influenced by selection can serve as powerful markers for detecting population structure in high gene-flow marine fish species....

  13. Visual demonstration of the organization of the human complement C4 and 21-hydroxylase genes by high-resolution fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Suto, Yumiko; Tokunaga, Katsushi; Watanabe, Yoshihisa; Hirai, Momoki [Univ. of Tokyo (Japan)

    1996-04-15

    We analyzed the gene organization in the complement component C4 and 21-hydroxylase (21OH) gene region of the human major histocompatibility complex using visual mapping of stretched DNA by multicolor fluorescence in situ hybridization (FISH). Normally, this region contains a duplicated 21OH-C4 gene unit are known to occur frequently. Biotin-labeled cDNA of the C4 gene and digoxigenin-labeled cDNA of the 21OH gene were hybridized to decondensed nuclei of peripheral blood lymphocytes obtained from individuals with various 21OH-C4 haplotypes. Hybridization signals of the C4 and 21OH probes were detected with fluorescein isothiocyanate (green) and rhodamine (red), respectively. Two linear green and red signal clusters were observed in each nucleus showing the normal haplotype. Gene duplication and deletion were visualized as addition and deletion of the signal cluster, respectively. The DNA types of the 21OH-C4 region determined by FISH were concordant with the results previously obtained by conventional molecular studies. Our high-resolution FISH technique is found to be useful for screening gene duplications and deletions. 14 refs., 1 fig.

  14. Highly efficient EIAV-mediated in utero gene transfer and expression in the major muscle groups affected by Duchenne muscular dystrophy.

    Science.gov (United States)

    Gregory, L G; Waddington, S N; Holder, M V; Mitrophanous, K A; Buckley, S M K; Mosley, K L; Bigger, B W; Ellard, F M; Walmsley, L E; Lawrence, L; Al-Allaf, F; Kingsman, S; Coutelle, C; Themis, M

    2004-07-01

    Gene therapy for Duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. In addition, the prenatal onset of disease complicates postnatal gene therapy. We have therefore proposed a fetal approach to overcome these barriers. We have applied beta-galactosidase expressing equine infectious anaemia virus (EIAV) lentiviruses pseudotyped with VSV-G by single or combined injection via different routes to the MF1 mouse fetus on day 15 of gestation and describe substantial gene delivery to the musculature. Highly efficient gene transfer to skeletal muscles, including the diaphragm and intercostal muscles, as well as to cardiac myocytes was observed and gene expression persisted for at least 15 months after administration of this integrating vector. These findings support the concept of in utero gene delivery for therapeutic and long-term prevention/correction of muscular dystrophies and pave the way for a future application in the clinic. PMID:15141156

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1998-08-01

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

  16. High voltage discharge gene gun used in plant gene engineering%在植物基因工程中应用的高压脉冲放电基因枪

    Institute of Scientific and Technical Information of China (English)

    孙鹞鸿; 秦曾衍

    2001-01-01

    本文介绍了一种用于植物基因工程领域的外源基因导入技术和高压放电基因枪的工作原 理 及关键结构,采用高速摄影法获得了射弹的运动速度。当储能为500J时,测得第一放电电流 峰值为19.3kA,电流上升时间为9μs。生物学试验结果表明,高压放电基因枪能有效地将 外源基因导入植物细胞并得到表达。%This paper describes a technique of introducing foreign gene into pla nt cells and the principles of the high-voltage discharge gene gun and its key s tructures. The velocity of the macroprojectile are measured with high speed phot ography. The main specifications are: when storing energy is 500J,the first max discharge current is 19.3kA and the rise time is 9μs. The biological tests pro v e that the foreign gene can be introduced into plant cells and can be expressed effectively using the high voltage discharge gene gun.

  17. Bioinformatics analysis of potential essential genes that response to the high intraocular pressure on astrocyte due to glaucoma

    Institute of Scientific and Technical Information of China (English)

    Yang; Yang; Jing-Zhu; Duan; Yu; Di; Dong-Mei; Gui; Dian-Wen; Gao

    2015-01-01

    AIM: To study the gene expression response and predict the network in cell due to pressure effects on optic nerve injury of glaucoma.METHODS: We used glaucoma related microarray data in public database [Gene Expression Omnibus(GEO)] to explore the potential gene expression changes as well as correspondent biological process alterations due to increased pressure in astrocytes during glaucoma development.RESULTS: A total of six genes were identified to be related with pressure increasing. Through the annotation and network analysis, we found these genes might be involved in cell morphological remodeling, angiogenesis,mismatch repair.CONCLUSION: Increasing pressure in glaucoma on astrocytes might cause gene expression alterations,which might induce some cellular responses changes.

  18. Reactive oxygen species modulate the differential expression of methionine sulfoxide reductase genes in Chlamydomonas reinhardtii under high light illumination.

    Science.gov (United States)

    Chang, Hsueh-Ling; Tseng, Yu-Lu; Ho, Kuan-Lin; Shie, Shu-Chiu; Wu, Pei-Shan; Hsu, Yuan-Ting; Lee, Tse-Min

    2014-04-01

    Illumination of Chlamydomonas reinhardtii cells at 1000 (high light, HL) or 3000 (very high light, VHL) µmol photons m(-2)  s(-1) intensity increased superoxide anion radical (O(2)(•-)) and hydrogen peroxide (H(2)O(2)) production, and VHL illumination also increased the singlet oxygen ((1)O(2)) level. HL and VHL illumination decreased methionine sulfoxide reductase A4 (CrMSRA4) transcript levels but increased CrMSRA3, CrMSRA5 and CrMSRB2.1 transcripts levels. CrMSRB2.2 transcript levels increased only under VHL conditions. The role of reactive oxygen species (ROS) on CrMSR expression was studied using ROS scavengers and generators. Treatment with dimethylthiourea (DMTU), a H(2)O(2) scavenger, suppressed HL- and VHL-induced CrMSRA3, CrMSRA5 and CrMSRB2.1 expression, whereas H(2)O(2) treatment stimulated the expression of these genes under 50 µmol photons m(-2)  s(-1) conditions (low light, LL). Treatment with diphenylamine (DPA), a (1)O(2) quencher, reduced VHL-induced CrMSRA3, CrMSRA5 and CrMSRB2.2 expression and deuterium oxide, which delays (1)O(2) decay, enhanced these gene expression, whereas treatment with (1)O(2) (rose bengal, methylene blue and neutral red) or O(2)(•-) (menadione and methyl viologen) generators under LL conditions induced their expression. DPA treatment inhibited the VHL-induced decrease in CrMSRA4 expression, but other ROS scavengers and ROS generators did not affect its expression under LL or HL conditions. These results demonstrate that the differential expression of CrMSRs under HL illumination can be attributed to different types of ROS. H(2)O(2), O(2) (•-) and (1)O(2) modulate CrMSRA3 and CrMSRA5 expression, whereas H(2)O(2) and O(2)(•-) regulate CrMSRB2.1 and CrMSRB2.2 expression, respectively. (1)O(2) mediates the decrease of CrMSRA4 expression by VHL illumination, but ROS do not modulate its decrease under HL conditions. PMID:24102363

  19. Nme gene family evolutionary history reveals pre-metazoan origins and high conservation between humans and the sea anemone, Nematostella vectensis.

    Directory of Open Access Journals (Sweden)

    Thomas Desvignes

    Full Text Available BACKGROUND: The Nme gene family is involved in multiple physiological and pathological processes such as cellular differentiation, development, metastatic dissemination, and cilia functions. Despite the known importance of Nme genes and their use as clinical markers of tumor aggressiveness, the associated cellular mechanisms remain poorly understood. Over the last 20 years, several non-vertebrate model species have been used to investigate Nme functions. However, the evolutionary history of the family remains poorly understood outside the vertebrate lineage. The aim of the study was thus to elucidate the evolutionary history of the Nme gene family in Metazoans. METHODOLOGY/PRINCIPAL FINDINGS: Using a total of 21 eukaryote species including 14 metazoans, the evolutionary history of Nme genes was reconstructed in the metazoan lineage. We demonstrated that the complexity of the Nme gene family, initially thought to be restricted to chordates, was also shared by the metazoan ancestor. We also provide evidence suggesting that the complexity of the family is mainly a eukaryotic innovation, with the exception of Nme8 that is likely to be a choanoflagellate/metazoan innovation. Highly conserved gene structure, genomic linkage, and protein domains were identified among metazoans, some features being also conserved in eukaryotes. When considering the entire Nme family, the starlet sea anemone is the studied metazoan species exhibiting the most conserved gene and protein sequence features with humans. In addition, we were able to show that most of the proteins known to interact with human NME proteins were also found in starlet sea anemone. CONCLUSION/SIGNIFICANCE: Together, our observations further support the association of Nme genes with key cellular functions that have been conserved throughout metazoan evolution. Future investigations of evolutionarily conserved Nme gene functions using the starlet sea anemone could shed new light on a wide variety of

  20. A 1. 8-Mb YAC contig in Xp11. 23: Identification of Cpg islands and physical mapping of CA repeats in a region of high gene density

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, M.P.; Nemeth, A.H.; Campbell, L.; Raut, C.P.; Davies, K.E. (Institute of Molecular Medicine, Oxford (United Kingdom)); Weissenbach, J. (Unite de Genetique Moleculaire Humaine, Paris (France))

    1994-05-15

    The genes ARAF1, SYN1, TIMP, and PFC are clustered within 70 kb of one another, and, as reported in the accompanying paper, at least four more genes map within 400 kb: a cluster of Krueppel-type zinc finger genes (including ZNF21, ZNF41, and ZNF81) and ELK-1, a member of the ets oncogene superfamily. This gene-rich region is of particular interest because of the large number of disease genes mapping to Xp11.23: At least three eye diseases (retinitis pigmentosa type 2, congenital stationary night blindness CSNB1, and Aland Island eye disease), Wiskott-Aldrich syndrome, X-linked nephrolithiasis, and a translocation breakpoint associated with synovial sarcoma. The authors have constructed a 1.8-Mb YAC contig in this region, confirming the link between TIMP and OATL1 reported by Knight et al. (1994) and extending the map in the distal direction. To investigate the likelihood that more genes are located within this region, they have carried out detailed mapping of rare-cutter restriction sites in these YACs and identified seven CpG islands. At least six of these islands are located over 50 kb from any known gene locations, suggesting that the region contains at least this many as yet unidentified genes. They have also mapped the physical locations of six highly polymorphic CA repeats within the contig, thus integrating the physical, genetic, and transcriptional maps of the region and facilitating the mapping and identification of disease genes. Together with the report by Knight et al., these data indicate the following order of loci: Xpter-DXS1264-DXS1055-DXS1003-DXS1146-DXS1266-(ZNF41, ARAF1)-SYN1 CA repeat-SYN1 (3[prime] end)-TIMP-SYN1 (5[prime] end)-PFC CA repeat-PFC-(DXS426, ELK1)-(DXS1265, ZNF81)-ZNF21-DXS1267-OATL1-Xcen. 40 refs., 4 figs., 3 tabs.

  1. Abundance and diversity of bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants revealed by high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Zhu Wang

    Full Text Available Biological nitrification/denitrification is frequently used to remove nitrogen from tannery wastewater containing high concentrations of ammonia. However, information is limited about the bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants (WWTPs due to the low-throughput of the previously used methods. In this study, 454 pyrosequencing and Illumina high-throughput sequencing, combined with molecular methods, were used to comprehensively characterize structures and functions of nitrification and denitrification bacterial communities in aerobic and anaerobic sludge of two full-scale tannery WWTPs. Pyrosequencing of 16S rRNA genes showed that Proteobacteria and Synergistetes dominated in the aerobic and anaerobic sludge, respectively. Ammonia-oxidizing bacteria (AOB amoA gene cloning revealed that Nitrosomonas europaea dominated the ammonia-oxidizing community in the WWTPs. Metagenomic analysis showed that the denitrifiers mainly included the genera of Thauera, Paracoccus, Hyphomicrobium, Comamonas and Azoarcus, which may greatly contribute to the nitrogen removal in the two WWTPs. It is interesting that AOB and ammonia-oxidizing archaea had low abundance although both WWTPs demonstrated high ammonium removal efficiency. Good correlation between the qPCR and metagenomic analysis is observed for the quantification of functional genes amoA, nirK, nirS and nosZ, indicating that the metagenomic approach may be a promising method used to comprehensively investigate the abundance of functional genes of nitrifiers and denitrifiers in the environment.

  2. Operator dependent choice of prostate cancer biopsy has limited impact on a gene signature analysis for the highly expressed genes IGFBP3 and F3 in prostate cancer epithelial cells.

    Directory of Open Access Journals (Sweden)

    Zhuochun Peng

    Full Text Available BACKGROUND: Predicting the prognosis of prostate cancer disease through gene expression analysis is receiving increasing interest. In many cases, such analyses are based on formalin-fixed, paraffin embedded (FFPE core needle biopsy material on which Gleason grading for diagnosis has been conducted. Since each patient typically has multiple biopsy samples, and since Gleason grading is an operator dependent procedure known to be difficult, the impact of the operator's choice of biopsy was evaluated. METHODS: Multiple biopsy samples from 43 patients were evaluated using a previously reported gene signature of IGFBP3, F3 and VGLL3 with potential prognostic value in estimating overall survival at diagnosis of prostate cancer. A four multiplex one-step qRT-PCR test kit, designed and optimized for measuring the signature in FFPE core needle biopsy samples was used. Concordance of gene expression levels between primary and secondary Gleason tumor patterns, as well as benign tissue specimens, was analyzed. RESULTS: The gene expression levels of IGFBP3 and F3 in prostate cancer epithelial cell-containing tissue representing the primary and secondary Gleason patterns were high and consistent, while the low expressed VGLL3 showed more variation in its expression levels. CONCLUSION: The assessment of IGFBP3 and F3 gene expression levels in prostate cancer tissue is independent of Gleason patterns, meaning that the impact of operator's choice of biopsy is low.

  3. Tackling heterogeneity: a leaf disc-based assay for the high-throughput screening of transient gene expression in tobacco.

    Directory of Open Access Journals (Sweden)

    Natalia Piotrzkowski

    Full Text Available Transient Agrobacterium-mediated gene expression assays for Nicotiana tabacum (N. tabacum are frequently used because they facilitate the comparison of multiple expression constructs regarding their capacity for maximum recombinant protein production. However, for three model proteins, we found that recombinant protein accumulation (rpa was significantly influenced by leaf age and leaf position effects. The ratio between the highest and lowest amount of protein accumulation (max/min ratio was found to be as high as 11. Therefore, construct-based impacts on the rpa level that are less than 11-fold will be masked by background noise. To address this problem, we developed a leaf disc-based screening assay and infiltration device that allows the rpa level in a whole tobacco plant to be reliably and reproducibly determined. The prototype of the leaf disc infiltration device allows 14 Agrobacterium-mediated infiltration events to be conducted in parallel. As shown for three model proteins, the average max/min rpa ratio was reduced to 1.4 using this method, which allows for a sensitive comparison of different genetic elements affecting recombinant protein expression.

  4. Genome editing of BmFib-H gene provides an empty Bombyx mori silk gland for a highly efficient bioreactor.

    Science.gov (United States)

    Ma, Sanyuan; Shi, Run; Wang, Xiaogang; Liu, Yuanyuan; Chang, Jiasong; Gao, Jie; Lu, Wei; Zhang, Jianduo; Zhao, Ping; Xia, Qingyou

    2014-01-01

    Evolution has produced some remarkable creatures, of which silk gland is a fascinating organ that exists in a variety of insects and almost half of the 34,000 spider species. The impressive ability to secrete huge amount of pure silk protein, and to store proteins at an extremely high concentration (up to 25%) make the silk gland of Bombyx mori hold great promise to be a cost-effective platform for production of recombinant proteins. However, the extremely low production yields of the numerous reported expression systems greatly hindered the exploration and application of silk gland bioreactors. Using customized zinc finger nucleases (ZFN), we successfully performed genome editing of Bmfib-H gene, which encodes the largest and most abundant silk protein, in B. mori with efficiency higher than any previously reported. The resulted Bmfib-H knocked-out B. mori showed a smaller and empty silk gland, abnormally developed posterior silk gland cells, an extremely thin cocoon that contain only sericin proteins, and a slightly heavier pupae. We also showed that removal of endogenous Bmfib-H protein could significantly increase the expression level of exogenous protein. Furthermore, we demonstrated that the bioreactor is suitable for large scale production of protein-based materials. PMID:25359576

  5. Analysis of Beta-Lactoglobuline Gene (LGB Polymorphism in Different Breeds of Bulls by High Resolution Melting

    Directory of Open Access Journals (Sweden)

    Martina Miluchová

    2012-05-01

    Full Text Available The goal of the paper was to identify  - lactoglobulin gene polymorphism in bulls. The  - lactoglobulin (LGB is expressed in milk and is important in the evaluation of milk production potential and butterfat and protein content. LGB is localized on bovine chromosome 11. The AA genotype of LGB is associated with higher milk yield, the BB genotype with higher fat and casein content and is more desirable for cheese making. The material involved 46 bulls (Slovak spotted breed – 41 bulls, Pinzgau breed – 3 bulls and Holstein breed – 2 bulls. Bovine genomic DNA was isolated from sperm using commercial kit NucleoSpin Tissue and used in order to estimate LGB genotypes by means of PCR RFLP method and high resolution melting analysis (HRMA. In the population of Slovak spotted breed we detected all genotypes AA, AB and BB with frequency 0.3415, 0.4390 and 0.2195, subsequently. In Pinzgau breed was detected homozygote genotypes AA and BB with frequency 0.3333 and 0.6667. In Holstein breed was observed only heterozygote genotype AB with frequency 1.

  6. Taxonomic analysis of the microbial community in stored sugar beets using high-throughput sequencing of different marker genes.

    Science.gov (United States)

    Liebe, Sebastian; Wibberg, Daniel; Winkler, Anika; Pühler, Alfred; Schlüter, Andreas; Varrelmann, Mark

    2016-02-01

    Post-harvest colonization of sugar beets accompanied by rot development is a serious problem due to sugar losses and negative impact on processing quality. Studies on the microbial community associated with rot development and factors shaping their structure are missing. Therefore, high-throughput sequencing was applied to describe the influence of environment, plant genotype and storage temperature (8°C and 20°C) on three different communities in stored sugar beets, namely fungi (internal transcribed spacers 1 and 2), Fusarium spp. (elongation factor-1α gene fragment) and oomycetes (internal transcribed spacers 1). The composition of the fungal community changed during storage mostly influenced by the storage temperature followed by a weak environmental effect. Botrytis cinerea was the prevalent species at 8°C whereas members of the fungal genera Fusarium and Penicillium became dominant at 20°C. This shift was independent of the plant genotype. Species richness within the genus Fusarium also increased during storage at both temperatures whereas the oomycetes community did not change. Moreover, oomycetes species were absent after storage at 20°C. The results of the present study clearly show that rot development during sugar beet storage is associated with pathogens well known as causal agents of post-harvest diseases in many other crops. PMID:26738557

  7. A new high phenyl lactic acid-yielding Lactobacillus plantarum IMAU10124 and a comparative analysis of lactate dehydrogenase gene.

    Science.gov (United States)

    Zhang, Xiqing; Zhang, Shuli; Shi, Yan; Shen, Fadi; Wang, Haikuan

    2014-07-01

    Phenyl lactic acid (PLA) has been widely reported as a new natural antimicrobial compound. In this study, 120 Lactobacillus plantarum strains were demonstrated to produce PLA using high-performance liquid chromatography. Lactobacillus plantarum IMAU10124 was screened with a PLA yield of 0.229 g L(-1) . Compared with all previous reports, this is the highest PLA-producing lactic acid bacteria (LAB) when grown in MRS broth without any optimizing conditions. When 3.0 g L(-1) phenyl pyruvic acid (PPA) was added to the medium as substrate, PLA production reached 2.90 g L(-1) , with the highest 96.05% conversion rate. A lowest PLA-yielding L. plantarum IMAU40105 (0.043 g L(-1) ) was also screened. It was shown that the conversion from PPA to PLA by lactic dehydrogenase (LDH) is the key factor in the improvement of PLA production by LAB. Comparing the LDH gene of two strains, four amino acid mutation sites were found in this study in the LDH of L. plantarum IMAU10124.

  8. Parallel high-throughput screening of polymer vectors for nonviral gene delivery: evaluation of structure-property relationships of transfection.

    Science.gov (United States)

    Rinkenauer, Alexandra C; Vollrath, Antje; Schallon, Anja; Tauhardt, Lutz; Kempe, Kristian; Schubert, Stephanie; Fischer, Dagmar; Schubert, Ulrich S

    2013-09-01

    In recent years, "high-throughput" (HT) has turned into a keyword in polymer research. In this study, we present a novel HT workflow for the investigation of cationic polymers for gene delivery applications. For this purpose, various poly(ethylene imine)s (PEI) were used as representative vectors and investigated via HT-assays in a 96-well plate format, starting from polyplex preparation up to the examination of the transfection process. In detail, automated polyplex preparation, complex size determination, DNA binding affinity, polyplex stability, cytotoxicity, and transfection efficiency were performed in the well plate format. With standard techniques, investigation of the biological properties of polymers is quite time-consuming, so only a limited number of materials and conditions (such as pH, buffer composition, and concentration) can be examined. The approach described here allows many different polymers and parameters to be tested for transfection properties and cytotoxicity, giving faster insights into structure-activity relationships for biological activity. PMID:23886244

  9. Taxonomic analysis of the microbial community in stored sugar beets using high-throughput sequencing of different marker genes.

    Science.gov (United States)

    Liebe, Sebastian; Wibberg, Daniel; Winkler, Anika; Pühler, Alfred; Schlüter, Andreas; Varrelmann, Mark

    2016-02-01

    Post-harvest colonization of sugar beets accompanied by rot development is a serious problem due to sugar losses and negative impact on processing quality. Studies on the microbial community associated with rot development and factors shaping their structure are missing. Therefore, high-throughput sequencing was applied to describe the influence of environment, plant genotype and storage temperature (8°C and 20°C) on three different communities in stored sugar beets, namely fungi (internal transcribed spacers 1 and 2), Fusarium spp. (elongation factor-1α gene fragment) and oomycetes (internal transcribed spacers 1). The composition of the fungal community changed during storage mostly influenced by the storage temperature followed by a weak environmental effect. Botrytis cinerea was the prevalent species at 8°C whereas members of the fungal genera Fusarium and Penicillium became dominant at 20°C. This shift was independent of the plant genotype. Species richness within the genus Fusarium also increased during storage at both temperatures whereas the oomycetes community did not change. Moreover, oomycetes species were absent after storage at 20°C. The results of the present study clearly show that rot development during sugar beet storage is associated with pathogens well known as causal agents of post-harvest diseases in many other crops.

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

    Science.gov (United States)

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

    2013-02-01

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

  11. Effects of Ethyl Pyruvate on High Mobility Group Box1 gene expression in septic lung of rat

    Institute of Scientific and Technical Information of China (English)

    Lian Zeng; Shanglong Yao; Dong Liu; Yuelan Wang

    2005-01-01

    Objective: Ethyl Pyruvate (EP) has been shown to be an effective anti-inflammatory agent in a variety of model systems. The aim of this study was to investigate the effects of EP on High Mobility Group Box1 (HMGB1) genes expression and the possible mechanisms of EP protecting against acute lung injury induced by sepsis. Methods: Forty Wistar rats were randomly divided into normal controls, sham operation, acute lung injury, and EP treatment (40 mg/kg intra-peritoneally every 6 hrs ) groups. At the time points of 24 hours the animals in each group were sacrificed, and the lungs were harvested. Wet/dry lung weight ratio, the protein in the bronchoalveolar lavage fluid(BALF), and pulmonary permeability index(PPI) were determined. The histological morphology of lung was observed under microscope. The expression of HMGB1 mRNA was measured using semi-quantitative RT-PCR. Results: EP treatment decreased wet/dry lung weight ratio, the protein in the BALF, and PPI ( P < 0.01 ). The histological morphology of lung injury was ameliorated. EP significantly inhibited the HMGB1 mRNA expression (P < 0.01 ). HMGB1 mRNA expression in lungs positivelycorrelation with wet/dry lung weight ratio, the protein in the BALF, and PPI. Conclusion: EP administered inhibits HMGB1 mRNAexpression, and protects the lungs against acute injury induced by sepsis.

  12. Depression of p53-independent Akt survival signals in human oral cancer cells bearing mutated p53 gene after exposure to high-LET radiation

    International Nuclear Information System (INIS)

    Highlights: ► High-LET radiation induces efficiently apoptosis regardless of p53 gene status. ► We examined whether high-LET radiation depresses the Akt-survival signals. ► High-LET radiation depresses of survival signals even in the mp53 cancer cells. ► High-LET radiation activates Caspase-9 through depression of survival signals. ► High-LET radiation suppresses cell growth through depression of survival signals. -- Abstract: Although mutations and deletions in the p53 tumor suppressor gene lead to resistance to low linear energy transfer (LET) radiation, high-LET radiation efficiently induces cell lethality and apoptosis regardless of the p53 gene status in cancer cells. Recently, it has been suggested that the induction of p53-independent apoptosis takes place through the activation of Caspase-9 which results in the cleavage of Caspase-3 and poly (ADP-ribose) polymerase (PARP). This study was designed to examine if high-LET radiation depresses serine/threonine protein kinase B (PKB, also known as Akt) and Akt-related proteins. Human gingival cancer cells (Ca9–22 cells) harboring a mutated p53 (mp53) gene were irradiated with 2 Gy of X-rays or Fe-ion beams. The cellular contents of Akt-related proteins participating in cell survival signaling were analyzed with Western Blotting 1, 2, 3 and 6 h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis. Akt-related protein levels decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G2/M phase arrests and suppressed the progression of the cell cycle much more efficiently when compared to low-LET radiation. These results suggest that high-LET radiation enhances apoptosis through the activation of Caspase-3 and Caspase-9, and suppresses cell growth by suppressing Akt-related signaling, even in mp53 bearing cancer cells.

  13. Impact of high-fat diet on liver genes expression profiles in mice model of nonalcoholic fatty liver disease.

    Science.gov (United States)

    Wang, Chunhua; Tao, Qimeng; Wang, Xinghe; Wang, Xiurong; Zhang, Xiuying

    2016-07-01

    Evidences have shown that NAFLD influences expression of some drug metabolic enzyme genes. This study aims to investigate the role of HFD-induced NAFLD in regulating the transcription of genes, particularly the drug metabolizing genes variation. Transcriptome analysis demonstrated that HFD feeding caused the 150 genes expression to change, most genes associated with lipid metabolism, inflammatory, oxidative stress and oxidoreductase activity up-regulated, whereas most genes involved in nucleic acid metabolism repressed. The genes involved in drug metabolism had 16 down-regulated and 21 up-regulated in NAFLD. The over-4-fold change genes included the down-regulation of Cyp8b1, Cyp7a1, Sult3a1, Sult1e1, Cyp17a1, Cyp3a41a, Gstt3, Cyp51, Cyp2c54 and Cyp4f14, and the up-regulation of Asns, Past1, Cyp2c55, Gstm2, Cyp2e1 and Gstaα1. In conclusion, significant alterations in the expression of drug metabolizing enzymes may affect the clearance of therapeutic drugs, with the potential to result in adverse drug reactions or drug toxicity in nonalcoholic fatty liver disease. PMID:27262986

  14. Regulation of Coding and Non-coding Genes : New insights obtained through analysis of high-throughput sequencing data

    NARCIS (Netherlands)

    K. Rooijers (Koos)

    2016-01-01

    markdownabstractThe genetic code of a cell is kept in its DNA. However, a vast number of functions of a cell are carried out by proteins. Through gene expression the genetic code can be expressed and give rise to proteins. The expression of genes into proteins follows two steps: transcription of DNA

  15. Efficiency of high- and low-voltage pulse combinations for gene electrotransfer in muscle, li