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Sample records for 53r gene highly

  1. Modulation of gene expression in U251 glioblastoma cells by binding of mutant p53 R273H to intronic and intergenic sequences

    PubMed Central

    Brázdová, Marie; Quante, Timo; Tögel, Lars; Walter, Korden; Loscher, Christine; Tichý, Vlastimil; Činčárová, Lenka; Deppert, Wolfgang; Tolstonog, Genrich V.

    2009-01-01

    Missense point mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines derived thereof. Mutant p53 (mutp53) proteins have lost sequence-specific DNA binding, but have retained the ability to interact in a structure-selective manner with non-B DNA and to act as regulators of transcription. To identify functional binding sites of mutp53, we established a small library of genomic sequences bound by p53R273H in U251 human glioblastoma cells using chromatin immunoprecipitation (ChIP). Mutp53 binding to isolated DNA fragments confirmed the specificity of the ChIP. The mutp53 bound DNA sequences are rich in repetitive DNA elements, which are dispersed over non-coding DNA regions. Stable down-regulation of mutp53 expression strongly suggested that mutp53 binding to genomic DNA is functional. We identified the PPARGC1A and FRMD5 genes as p53R273H targets regulated by binding to intronic and intra-genic sequences. We propose a model that attributes the oncogenic functions of mutp53 to its ability to interact with intronic and intergenic non-B DNA sequences and modulate gene transcription via re-organization of chromatin. PMID:19139068

  2. Hot Spot Mutation in TP53 (R248Q) Causes Oncogenic Gain-of-Function Phenotypes in a Breast Cancer Cell Line Derived from an African American patient.

    PubMed

    Shtraizent, Nataly; Matsui, Hiroshi; Polotskaia, Alla; Bargonetti, Jill

    2016-01-01

    African American (AA) breast cancer patients often have triple negative breast cancer (TNBC) that contains mutations in the TP53 gene. The point mutations at amino acid residues R273 and R248 both result in oncogenic gain-of-function (GOF) phenotypes. Expression of mutant p53 (mtp53) R273H associates with increased cell elasticity, survival under serum deprivation conditions, and increased Poly (ADP ribose) polymerase 1 (PARP1) on the chromatin in the AA-derived TNBC breast cancer cell line MDA-MB-468. We hypothesized that GOF mtp53 R248Q expression could stimulate a similar phenotype in the AA-derived TNBC cell line HCC70. To test this hypothesis we depleted the R248Q protein in the HCC70 cell line using shRNA-mediated knockdown. Using impedance-based real-time analysis we correlated the expression of mtp53 R248Q with increased cell deformability. We also documented that depletion of mtp53 R248Q increased PARP1 in the cytoplasm and decreased PARP1 on the chromatin. We conclude that in the AA-derived TNBC HCC70 cells mtp53 R248Q expression results in a causative tumor associated phenotype. This study supports using the biological markers of high expression of mtp53 R273H or R248Q as additional diagnostics for TNBC resistant subtypes often found in the AA community. Each mtp53 protein must be considered separately and this work adds R248Q to the increasing list of p53 mutations that can be used for diagnostics and drug targeting. Here we report that when R248Q mtp53 proteins are expressed in TNBC, then targeting the gain-of-function pathways may improve treatment efficacy.

  3. Hot Spot Mutation in TP53 (R248Q) Causes Oncogenic Gain-of-Function Phenotypes in a Breast Cancer Cell Line Derived from an African American patient

    PubMed Central

    Shtraizent, Nataly; Matsui, Hiroshi; Polotskaia, Alla; Bargonetti, Jill

    2015-01-01

    African American (AA) breast cancer patients often have triple negative breast cancer (TNBC) that contains mutations in the TP53 gene. The point mutations at amino acid residues R273 and R248 both result in oncogenic gain-of-function (GOF) phenotypes. Expression of mutant p53 (mtp53) R273H associates with increased cell elasticity, survival under serum deprivation conditions, and increased Poly (ADP ribose) polymerase 1 (PARP1) on the chromatin in the AA-derived TNBC breast cancer cell line MDA-MB-468. We hypothesized that GOF mtp53 R248Q expression could stimulate a similar phenotype in the AA-derived TNBC cell line HCC70. To test this hypothesis we depleted the R248Q protein in the HCC70 cell line using shRNA-mediated knockdown. Using impedance-based real-time analysis we correlated the expression of mtp53 R248Q with increased cell deformability. We also documented that depletion of mtp53 R248Q increased PARP1 in the cytoplasm and decreased PARP1 on the chromatin. We conclude that in the AA-derived TNBC HCC70 cells mtp53 R248Q expression results in a causative tumor associated phenotype. This study supports using the biological markers of high expression of mtp53 R273H or R248Q as additional diagnostics for TNBC resistant subtypes often found in the AA community. Each mtp53 protein must be considered separately and this work adds R248Q to the increasing list of p53 mutations that can be used for diagnostics and drug targeting. Here we report that when R248Q mtp53 proteins are expressed in TNBC, then targeting the gain-of-function pathways may improve treatment efficacy. PMID:26703669

  4. Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis

    PubMed Central

    Lu, Tsung Chi; Zhao, Guan- Hao; Chen, Yao Yun; Chien, Chia-Ying; Huang, Chi-Hung; Lin, Kwang Hui; Chen, Shen Liang

    2016-01-01

    Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors. PMID:27390612

  5. Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF).

    PubMed

    Tan, B S; Tiong, K H; Choo, H L; Chung, F Fei-Lei; Hii, L-W; Tan, S H; Yap, I K S; Pani, S; Khor, N T W; Wong, S F; Rosli, R; Cheong, S-K; Leong, C-O

    2015-07-16

    p53 is the most frequently mutated tumor-suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor-suppressor function, but may also gain new oncogenic functions and promote tumorigenesis. Here, we showed that silencing of endogenous p53-R273H contact mutant, but not p53-R175H conformational mutant, reduced AKT phosphorylation, induced BCL2-modifying factor (BMF) expression, sensitized BIM dissociation from BCL-XL and induced mitochondria-dependent apoptosis in cancer cells. Importantly, cancer cells harboring endogenous p53-R273H mutant were also found to be inherently resistant to anoikis and lack BMF induction following culture in suspension. Underlying these activities is the ability of p53-R273H mutant to suppress BMF expression that is dependent on constitutively active PI3K/AKT signaling. Collectively, these findings suggest that p53-R273H can specifically drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. These findings open the possibility that blocking of PI3K/AKT will have therapeutic benefit in mutant p53-R273H expressing cancers.

  6. Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF)

    PubMed Central

    Tan, B S; Tiong, K H; Choo, H L; Fei-Lei Chung, F; Hii, L-W; Tan, S H; Yap, I KS; Pani, S; Khor, N TW; Wong, S F; Rosli, R; Cheong, S-K; Leong, C-O

    2015-01-01

    p53 is the most frequently mutated tumor-suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor-suppressor function, but may also gain new oncogenic functions and promote tumorigenesis. Here, we showed that silencing of endogenous p53-R273H contact mutant, but not p53-R175H conformational mutant, reduced AKT phosphorylation, induced BCL2-modifying factor (BMF) expression, sensitized BIM dissociation from BCL-XL and induced mitochondria-dependent apoptosis in cancer cells. Importantly, cancer cells harboring endogenous p53-R273H mutant were also found to be inherently resistant to anoikis and lack BMF induction following culture in suspension. Underlying these activities is the ability of p53-R273H mutant to suppress BMF expression that is dependent on constitutively active PI3K/AKT signaling. Collectively, these findings suggest that p53-R273H can specifically drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. These findings open the possibility that blocking of PI3K/AKT will have therapeutic benefit in mutant p53-R273H expressing cancers. PMID:26181206

  7. Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF).

    PubMed

    Tan, B S; Tiong, K H; Choo, H L; Chung, F Fei-Lei; Hii, L-W; Tan, S H; Yap, I K S; Pani, S; Khor, N T W; Wong, S F; Rosli, R; Cheong, S-K; Leong, C-O

    2015-01-01

    p53 is the most frequently mutated tumor-suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor-suppressor function, but may also gain new oncogenic functions and promote tumorigenesis. Here, we showed that silencing of endogenous p53-R273H contact mutant, but not p53-R175H conformational mutant, reduced AKT phosphorylation, induced BCL2-modifying factor (BMF) expression, sensitized BIM dissociation from BCL-XL and induced mitochondria-dependent apoptosis in cancer cells. Importantly, cancer cells harboring endogenous p53-R273H mutant were also found to be inherently resistant to anoikis and lack BMF induction following culture in suspension. Underlying these activities is the ability of p53-R273H mutant to suppress BMF expression that is dependent on constitutively active PI3K/AKT signaling. Collectively, these findings suggest that p53-R273H can specifically drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. These findings open the possibility that blocking of PI3K/AKT will have therapeutic benefit in mutant p53-R273H expressing cancers. PMID:26181206

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

    PubMed

    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.

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

    PubMed

    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. PMID:25736369

  10. Cdkn2a suppresses metastasis in squamous cell carcinomas induced by the gain-of-function mutant p53(R172H).

    PubMed

    Li, Zhongyou; Gonzalez, Cassandra L; Wang, Bingbing; Zhang, Yuanyuan; Mejia, Olga; Katsonis, Panagiotis; Lichtarge, Olivier; Myers, Jeffrey N; El-Naggar, Adel K; Caulin, Carlos

    2016-10-01

    p53 (TP53) is the most frequently mutated gene in squamous cell carcinomas (SCCs) of the skin and head and neck. Certain p53 mutations are oncogenic and promote invasion and metastasis in SCCs. However, it is unclear how the oncogenic function of mutant p53 is modulated by other molecular alterations that co-exist in SCCs. Here, we show that deletion of the p53 gene and activation of an endogenous p53(R172H) gain-of-function mutation in the skin induce carcinomas with similar kinetics and penetrance. Deletion of p53 induced primarily well-differentiated SCCs. However, most of the tumours induced by p53(R172H) were poorly differentiated SCCs, the only metastatic tumours in this model. These tumours expressed higher levels of cyclin D1 than the well-differentiated SCCs and spindle carcinomas that developed in these mice. Unexpectedly, metastasis was not observed in mice that developed spindle carcinomas, which expressed high levels of the tumour suppressors p16(Ink4a) and p19(Arf) , encoded by Cdkn2a, a gene frequently deleted in human SCCs. Remarkably, deletion of the Cdkn2a gene in p53(R172H) -induced SCCs promoted a dramatic increase in metastasis rates and a shorter survival in mice that developed these tumours, compared with those observed in mice with tumours in which Cdkn2a was deleted in the presence of a p53 loss-of-function mutation or wild-type p53. Accordingly, the survival of patients with head and neck SCCs bearing co-occurring high-risk p53 mutations and CDKN2A homozygous deletions was much shorter than that of patients with tumours in which high-risk p53 mutations did not contain CDKN2A homozygous deletions, or that of patients with tumours in which homozygous CDKN2A deletions co-existed with either low-risk p53 mutations or potential loss-of-function mutations in p53. These findings genetically identify a population of SCC patients with worst outcomes and will help to predict outcomes according to the p53 status and alterations in CDKN2A. Copyright

  11. Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

    SciTech Connect

    Whitley, Dexter S.; Yu, Kwang; Sample, Robert C.; Sinning, Allan; Henegar, Jeffrey; Norcross, Erin; Chinchar, V. Gregory

    2010-09-30

    Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.

  12. Mutant p53 interactome identifies nardilysin as a p53R273H-specific binding partner that promotes invasion.

    PubMed

    Coffill, Cynthia R; Muller, Patricia A J; Oh, Hue Kian; Neo, Suat Peng; Hogue, Kelly A; Cheok, Chit Fang; Vousden, Karen H; Lane, David P; Blackstock, Walter P; Gunaratne, Jayantha

    2012-07-01

    The invasiveness of tumour cells depends on changes in cell shape, polarity and migration. Mutant p53 induces enhanced tumour metastasis in mice, and human cells overexpressing p53R273H have aberrant polarity and increased invasiveness, demonstrating the 'gain of function' of mutant p53 in carcinogenesis. We hypothesize that p53R273H interacts with mutant p53-specific binding partners that control polarity, migration or invasion. Here we analyze the p53R273H interactome using stable isotope labelling by amino acids in cell culture and quantitative mass spectrometry, and identify at least 15 new potential mutant p53-specific binding partners. The interaction of p53R273H with one of them--nardilysin (NRD1)--promotes an invasive response to heparin binding-epidermal growth factor-like growth factor that is p53R273H-dependant but does not require Rab coupling protein or p63. Advanced proteomics has thus allowed the detection of a new mechanism of p53-driven invasion.

  13. Differential Expressions of p53, p53R2, hRRM2 and PBR in Chronic Lymphocytic Leukemia: A Correlation with Intracellular Cholesterol.

    PubMed

    Verma, Ankit; Chandra, N C

    2016-07-01

    Regulation of intracellular cholesterol homeostasis exists under balance between intracellular biosynthesis and uptake from extracellular origin by cell surface transport proteins. Expected role of cholesterol on either tumor suppressor gene and/or DNA synthesis has been aimed in the present study to explore intracellular cholesterol homeostasis in CLL subjects. Higher expressions of p53R2 (p53 dependent subunit of ribonucleotide reductase) and p53 were found in lymphocytes of chronic human lymphocytic leukemia as comparison to their normal counterparts. Inverse relation was found with p53 independent R2 subunit (in human hRRM2) of ribonucleotide reductase, which was found to be decreased from its control group. More expression of peripheral type benzodiazepine receptor, a cholesterol transporter, was noticed in isolated nuclear fraction with simultaneous increase of cholesterol concentration in cytoplasmic and nuclear compartments. A parallel increase of cholesterol in cell nucleus with increased p53R2 expression shows priority of the involvement of cholesterol in the process of cell replication.

  14. Impact of Neonatal Screening and Surveillance for the TP53 R337H Mutation on Early Detection of Childhood Adrenocortical Tumors

    PubMed Central

    Custódio, Gislaine; Parise, Guilherme A.; Kiesel Filho, Nilton; Komechen, Heloisa; Sabbaga, Cesar C.; Rosati, Roberto; Grisa, Leila; Parise, Ivy Z.S.; Pianovski, Mara A.D.; Fiori, Carmem M.C.M.; Ledesma, Jorge A.; Barbosa, José Renato S.; Figueiredo, Francisco R.O.; Sade, Elis R.; Ibañez, Humberto; Arram, Sohaila B.I.; Stinghen, Sérvio T.; Mengarelli, Luciano R.; Figueiredo, Mirna M.O.; Carvalho, Danilo C.; Avilla, Sylvio G.A.; Woiski, Thiago D.; Poncio, Lisiane C.; Lima, Geneci F.R.; Pontarolo, Roberto; Lalli, Enzo; Zhou, Yinmei; Zambetti, Gerard P.; Ribeiro, Raul C.; Figueiredo, Bonald C.

    2013-01-01

    Purpose The incidence of pediatric adrenocortical tumors (ACTs) is remarkably high in southern Brazil, where more than 90% of patients carry the germline TP53 mutation R337H. We assessed the impact of early detection of this mutation and of surveillance of carriers. Patients and Methods Free newborn screening was offered at all hospitals in the state of Paraná. Parents of positive newborns were tested, and relatives in the carrier line were offered screening. Positive newborns and their relatives age < 15 years were offered surveillance (periodic clinical, laboratory, and ultrasound evaluations). ACTs detected by imaging were surgically resected. Results Of 180,000 newborns offered screening, 171,649 were screened, and 461 (0.27%) were carriers. As of April 2012, ACTs had been diagnosed in 11 of these carriers but in only two neonatally screened noncarriers (P < .001); six patient cases were identified among 228 carrier relatives age < 15 years (total, 19 ACTs). Surveillance participants included 347 (49.6%) of 699 carriers. Tumors were smaller in surveillance participants (P < .001) and more advanced in nonparticipants (four with stage III disease; two deaths). Neonatally screened carriers also had neuroblastoma (n = 1), glioblastoma multiforme (n = 1), choroid plexus carcinoma (n = 2), and Burkitt lymphoma (n = 1). Cancer histories and pedigrees were obtained for 353 families that included 1,704 identified carriers. ACTs were the most frequent cancer among carrier children (n = 48). Conclusion These findings establish the prevalence of the TP53 R337H mutation in Paraná state and the penetrance of ACTs among carriers. Importantly, screening and surveillance of heterozygous carriers are effective in detecting ACTs when readily curable. PMID:23733769

  15. Gene therapy for high-grade glioma

    PubMed Central

    Natsume, Atsushi

    2008-01-01

    The treatment of high-grade gliomas remains difficult despite recent advances in surgery, radiotherapy and chemotherapy. True advances may emerge from the increasing understanding in molecular biology and discovery of novel mechanisms for the delivery of tumoricidal agents. In an attempt to overcome this formidable neoplasm, molecular approaches using gene therapy have been investigated clinically since 1992. The clinical trials have mainly been classified into three approaches: suicide gene therapy, immune gene therapy and oncolytic viral therapy. In this article, we review these approaches, which have been studied in previous and ongoing clinical trials. PMID:19262115

  16. Clustering of High Throughput Gene Expression Data

    PubMed Central

    Pirim, Harun; Ekşioğlu, Burak; Perkins, Andy; Yüceer, Çetin

    2012-01-01

    High throughput biological data need to be processed, analyzed, and interpreted to address problems in life sciences. Bioinformatics, computational biology, and systems biology deal with biological problems using computational methods. Clustering is one of the methods used to gain insight into biological processes, particularly at the genomics level. Clearly, clustering can be used in many areas of biological data analysis. However, this paper presents a review of the current clustering algorithms designed especially for analyzing gene expression data. It is also intended to introduce one of the main problems in bioinformatics - clustering gene expression data - to the operations research community. PMID:23144527

  17. Partial genetic deficiency in tissue kallikrein impairs adaptation to high potassium intake in humans.

    PubMed

    Monteiro, Joana S; Blanchard, Anne; Curis, Emmanuel; Chambrey, Régine; Jeunemaitre, Xavier; Azizi, Michel

    2013-12-01

    Inactivation of the tissue kallikrein gene in mice impairs renal handling of potassium due to enhanced H, K-ATPase activity, and induces hyperkalemia. We investigated whether the R53H loss-of-function polymorphism of the human tissue kallikrein gene affects renal potassium handling. In a crossover study, 30 R53R homozygous and 10 R53H heterozygous healthy males were randomly assigned to a low-sodium/high-potassium or a high-sodium/low-potassium diet to modulate tissue kallikrein synthesis. On the seventh day of each diet, participants were studied before and during a 2-h infusion of furosemide to stimulate distal potassium secretion. Urinary kallikrein activity was significantly lower in R53H than in R53R subjects on the low-sodium/high-potassium diet and was similarly reduced in both genotypes on high-sodium/low-potassium. Plasma potassium and renal potassium reabsorption were similar in both genotypes on an ad libitum sodium/potassium diet or after 7 days of a high-sodium/low-potassium diet. However, the median plasma potassium was significantly higher after 7 days of low-sodium/high-potassium diet in R53H than in R53R individuals. Urine potassium excretion and plasma aldosterone concentrations were similar. On the low-sodium/high-potassium diet, furosemide-induced decrease in plasma potassium was significantly larger in R53H than in R53R subjects. Thus, impaired tissue kallikrein stimulation by a low-sodium/high-potassium diet in R53H subjects with partial tissue kallikrein deficiency highlights an inappropriate renal adaptation to potassium load, consistent with experimental data in mice.

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

    NASA Astrophysics Data System (ADS)

    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

  19. High diversity of polyketide synthase genes and the melanin biosynthesis gene cluster in Penicillium marneffei.

    PubMed

    Woo, Patrick C Y; Tam, Emily W T; Chong, Ken T K; Cai, James J; Tung, Edward T K; Ngan, Antonio H Y; Lau, Susanna K P; Yuen, Kwok-Yung

    2010-09-01

    Despite the unique phenotypic properties and clinical importance of Penicillium marneffei, the polyketide synthase genes in its genome have never been characterized. Twenty-three putative polyketide synthase genes and two putative polyketide synthase nonribosomal peptide-synthase hybrid genes were identified in the P. marneffei genome, a diversity much higher than found in other pathogenic thermal dimorphic fungi, such as Histoplasma capsulatum (one polyketide synthase gene) and Coccidioides immitis (10 polyketide synthase genes). These genes were evenly distributed on the phylogenetic tree with polyketide synthase genes of Aspergillus and other fungi, indicating that the high diversity was not a result of lineage-specific gene expansion through recent gene duplication. The melanin-biosynthesis gene cluster had gene order and orientations identical to those in the Talaromyces stipitatus (a teleomorph of Penicillium emmonsii) genome. Phylogenetically, all six genes of the melanin-biosynthesis gene cluster in P. marneffei were also most closely related to those in T. stipitatus, with high bootstrap supports. The polyketide synthase gene of the melanin-biosynthesis gene cluster (alb1) in P. marneffei was knocked down, which was accompanied by loss of melanin pigment production and reduced ornamentation in conidia. The survival of mice challenged with the alb1 knockdown mutant was significantly better than those challenged with wild-type P. marneffei (P < 0.005). The sterilizing doses of hydrogen peroxide, leading to a 50% reduction in survival of conidia, were 11 min for wild-type P. marneffei and 6 min for the alb1 knockdown mutant of P. marneffei, implying that the melanin-biosynthesis gene cluster contributed to virulence through decreased susceptibility to killing by hydrogen peroxide. PMID:20718860

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

    PubMed

    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.

  1. A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

    PubMed

    Xie, Wensheng; Silvers, Robert; Ouellette, Michael; Wu, Zining; Lu, Quinn; Li, Hu; Gallagher, Kathleen; Johnson, Kathy; Montoute, Monica

    2016-01-01

    Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic β-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase. PMID:27316998

  2. High magnetic field induced changes of gene expression in arabidopsis

    PubMed Central

    Paul, Anna-Lisa; Ferl, Robert J; Meisel, Mark W

    2006-01-01

    Background High magnetic fields are becoming increasingly prevalent components of non-invasive, biomedical imaging tools (such as MRI), thus, an understanding of the molecular impacts associated with these field strengths in biological systems is of central importance. The biological impact of magnetic field strengths up to 30 Tesla were investigated in this study through the use of transgenic Arabidopsis plants engineered with a stress response gene consisting of the alcohol dehydrogenase (Adh) gene promoter driving the β-glucuronidase (GUS) gene reporter. Methods Magnetic field induced Adh/GUS activity was evaluated with histochemical staining to assess tissue specific expression and distribution, and with quantitative, spectrofluometric assays to measure degree of activation. The evaluation of global changes in the Arabidopsis genome in response to exposure to high magnetic fields was facilitated with Affymetrix Gene Chip microarrays. Quantitative analyses of gene expression were performed with quantitative real-time polymerase-chain-reaction (qRT-PCR). Results Field strengths in excess of about 15 Tesla induce expression of the Adh/GUS transgene in the roots and leaves. From the microarray analyses that surveyed 8000 genes, 114 genes were differentially expressed to a degree greater than 2.5 fold over the control. These results were quantitatively corroborated by qRT-PCR examination of 4 of the 114 genes. Conclusion The data suggest that magnetic fields in excess of 15 Tesla have far-reaching effect on the genome. The wide-spread induction of stress-related genes and transcription factors, and a depression of genes associated with cell wall metabolism, are prominent examples. The roles of magnetic field orientation of macromolecules and magnetophoretic effects are discussed as possible factors that contribute to the mounting of this response. PMID:17187667

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

    PubMed Central

    Payton, Paxton; Kottapalli, Kameswara Rao; Rowland, Diane; Faircloth, Wilson; Guo, Baozhu; Burow, Mark; Puppala, Naveen; Gallo, Maria

    2009-01-01

    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. PMID:19523230

  4. Gene expression in papillary thyroid carcinoma reveals highly consistent profiles

    PubMed Central

    Huang, Ying; Prasad, Manju; Lemon, William J.; Hampel, Heather; Wright, Fred A.; Kornacker, Karl; LiVolsi, Virginia; Frankel, Wendy; Kloos, Richard T.; Eng, Charis; Pellegata, Natalia S.; de la Chapelle, Albert

    2001-01-01

    Papillary thyroid carcinoma (PTC) is clinically heterogeneous. Apart from an association with ionizing radiation, the etiology and molecular biology of PTC is poorly understood. We used oligo-based DNA arrays to study the expression profiles of eight matched pairs of normal thyroid and PTC tissues. Additional PTC tumors and other tissues were studied by reverse transcriptase–PCR and immunohistochemistry. The PTCs showed concordant expression of many genes and distinct clustered profiles. Genes with increased expression in PTC included many encoding adhesion and extracellular matrix proteins. Expression was increased in 8/8 tumors for 24 genes and in 7/8 tumors for 22 genes. Among these genes were several previously known to be overexpressed in PTC, such as MET, LGALS3, KRT19, DPP4, MDK, TIMP1, and FN1. The numerous additional genes include CITED1, CHI3L1, ODZ1, N33, SFTPB, and SCEL. Reverse transcriptase–PCR showed high expression of CITED1, CHI3L1, ODZ1, and SCEL in 6/6 additional PTCs. Immunohistochemical analysis detected CITED1 and SFTPB in 49/52 and 39/52 PTCs, respectively, but not in follicular thyroid carcinoma and normal thyroid tissue. Genes underexpressed in PTC included tumor suppressors, thyroid function-related proteins, and fatty acid binding proteins. Expression was decreased in 7/8 tumors for eight genes and decreased in 6/8 tumors for 19 genes. We conclude that, despite its clinical heterogeneity, PTC is characterized by consistent and specific molecular changes. These findings reveal clues to the molecular pathways involved in PTC and may provide biomarkers for clinical use. PMID:11752453

  5. High Intensity Focused Ultrasound induced Gene Activation in Solid Tumors

    NASA Astrophysics Data System (ADS)

    Liu, Yunbo; Kon, Takashi; Li, Chuanyuan; Zhong, Pei

    2006-05-01

    In this work, the feasibility of using high intensity focused ultrasound (HIFU) to activate trans-gene expression in a mouse tumor model was investigated. 4T1 cancer cells were implanted subcutaneously in the hind limbs of Balb/C mice and adenovirus luciferase gene vectors under the control of heat shock protein 70B promoter (Adeno-hsp70B-Luc) were injected intratumoraly for gene transfection. One day following the virus injection, the transfected tumors were heated to a peak temperature of 55, 65, 75, and 85°C, respectively, in 10s at multiple sites around the center of the tumor using a HIFU transducer operated at either 1.1-MHz (fundamental) or 3.3-MHz (3rd harmonic) frequency. Inducible luciferase gene expression was found to vary from 15-fold to 120-fold of the control group following 1.1-MHz HIFU exposure. The maximum gene activation was produced at a peak temperature of 65˜75°C one day following HIFU exposure and decayed gradually to baseline level within 7 days. The inducible gene activation produced by 3.3-MHz HIFU exposure (75°C-10s) was found to be comparable to that produced by hyperthermia (42°C-30min). Altogether, these results demonstrate the feasibility of using HIFU as a simple and versatile physical means to regulate trans-gene expression in vivo. This unique feature may be explored in the future for a synergistic combination of HIFU-induced thermal ablation with heat-induced gene therapy for improved cancer therapy.

  6. High pressure-sensitive gene expression in Lactobacillus sanfranciscensis.

    PubMed

    Vogel, R F; Pavlovic, M; Hörmann, S; Ehrmann, M A

    2005-08-01

    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. PMID:16082466

  7. MYRIAD VOICES AGAINST GENE PATENTS IN THE HIGH COURT.

    PubMed

    McCallum, Lucas; Faunce, Thomas

    2015-12-01

    The Australian High Court's recent landmark decision in D'Arcy v Myriad Genetics Inc overturned the decision by the Federal Court in Cancer Voices Australia v Myriad Genetics Inc regarding patenting of genetic material. The Federal Court had found that isolated DNA and RNA can constitute a patentable invention under s 18(1)(a) of the Patents Act 1990 (Cth). The decision by the High Court unanimously reversed this and declared it was appropriate to look to the policy implications at the heart of the legal question: are genes a category of things that can be patented? This column critically examines the implications of the High Court decision for both research and public health in Australia.

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

    PubMed

    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.

  9. Statistically invalid classification of high throughput gene expression data.

    PubMed

    Barbash, Shahar; Soreq, Hermona

    2013-01-01

    Classification analysis based on high throughput data is a common feature in neuroscience and other fields of science, with a rapidly increasing impact on both basic biology and disease-related studies. The outcome of such classifications often serves to delineate novel biochemical mechanisms in health and disease states, identify new targets for therapeutic interference, and develop innovative diagnostic approaches. Given the importance of this type of studies, we screened 111 recently-published high-impact manuscripts involving classification analysis of gene expression, and found that 58 of them (53%) based their conclusions on a statistically invalid method which can lead to bias in a statistical sense (lower true classification accuracy then the reported classification accuracy). In this report we characterize the potential methodological error and its scope, investigate how it is influenced by different experimental parameters, and describe statistically valid methods for avoiding such classification mistakes.

  10. High-Dimensional Gene Expression Profiling Studies in High and Low Responders to Primary Smallpox Vaccination

    PubMed Central

    Haralambieva, Iana H.; Oberg, Ann L.; Dhiman, Neelam; Ovsyannikova, Inna G.; Kennedy, Richard B.; Grill, Diane E.; Jacobson, Robert M.; Poland, Gregory A.

    2012-01-01

    Background. The mechanisms underlying smallpox vaccine-induced variations in immune responses are not well understood, but are of considerable interest to a deeper understanding of poxvirus immunity and correlates of protection. Methods. We assessed transcriptional messenger RNA expression changes in 197 recipients of primary smallpox vaccination representing the extremes of humoral and cellular immune responses. Results. The 20 most significant differentially expressed genes include a tumor necrosis factor–receptor superfamily member, an interferon (IFN) gene, a chemokine gene, zinc finger protein genes, nuclear factors, and histones (P ≤ 1.06E−20, q ≤ 2.64E−17). A pathway analysis identified 4 enriched pathways with cytokine production by the T-helper 17 subset of CD4+ T cells being the most significant pathway (P = 3.42E−05). Two pathways (antiviral actions of IFNs, P = 8.95E−05; and IFN-α/β signaling pathway, P = 2.92E−04), integral to innate immunity, were enriched when comparing high with low antibody responders (false discovery rate, < 0.05). Genes related to immune function and transcription (TLR8, P = .0002; DAPP1, P = .0003; LAMP3, P = 9.96E−05; NR4A2, P ≤ .0002; EGR3, P = 4.52E−05), and other genes with a possible impact on immunity (LNPEP, P = 3.72E−05; CAPRIN1, P = .0001; XRN1, P = .0001), were found to be expressed differentially in high versus low antibody responders. Conclusion. We identified novel and known immunity-related genes and pathways that may account for differences in immune response to smallpox vaccination. PMID:22949304

  11. Deciphering the onychophoran 'segmentation gene cascade': Gene expression reveals limited involvement of pair rule gene orthologs in segmentation, but a highly conserved segment polarity gene network.

    PubMed

    Janssen, Ralf; Budd, Graham E

    2013-10-01

    The hallmark of the arthropods is their segmented body, although origin of segmentation, however, is unresolved. In order to shed light on the origin of segmentation we investigated orthologs of pair rule genes (PRGs) and segment polarity genes (SPGs) in a member of the closest related sister-group to the arthropods, the onychophorans. Our gene expression data analysis suggests that most of the onychophoran PRGs do not play a role in segmentation. One possible exception is the even-skipped (eve) gene that is expressed in the posterior end of the onychophoran where new segments are likely patterned, and is also expressed in segmentation-gene typical transverse stripes in at least a number of newly formed segments. Other onychophoran PRGs such as runt (run), hairy/Hes (h/Hes) and odd-skipped (odd) do not appear to have a function in segmentation at all. Onychophoran PRGs that act low in the segmentation gene cascade in insects, however, are potentially involved in segment-patterning. Most obvious is that from the expression of the pairberry (pby) gene ortholog that is expressed in a typical SPG-pattern. Since this result suggested possible conservation of the SPG-network we further investigated SPGs (and associated factors) such as Notum in the onychophoran. We find that the expression patterns of SPGs in arthropods and the onychophoran are highly conserved, suggesting a conserved SPG-network in these two clades, and indeed also in an annelid. This may suggest that the common ancestor of lophotrochozoans and ecdysozoans was already segmented utilising the same SPG-network, or that the SPG-network was recruited independently in annelids and onychophorans/arthropods. PMID:23880430

  12. Deciphering the onychophoran 'segmentation gene cascade': Gene expression reveals limited involvement of pair rule gene orthologs in segmentation, but a highly conserved segment polarity gene network.

    PubMed

    Janssen, Ralf; Budd, Graham E

    2013-10-01

    The hallmark of the arthropods is their segmented body, although origin of segmentation, however, is unresolved. In order to shed light on the origin of segmentation we investigated orthologs of pair rule genes (PRGs) and segment polarity genes (SPGs) in a member of the closest related sister-group to the arthropods, the onychophorans. Our gene expression data analysis suggests that most of the onychophoran PRGs do not play a role in segmentation. One possible exception is the even-skipped (eve) gene that is expressed in the posterior end of the onychophoran where new segments are likely patterned, and is also expressed in segmentation-gene typical transverse stripes in at least a number of newly formed segments. Other onychophoran PRGs such as runt (run), hairy/Hes (h/Hes) and odd-skipped (odd) do not appear to have a function in segmentation at all. Onychophoran PRGs that act low in the segmentation gene cascade in insects, however, are potentially involved in segment-patterning. Most obvious is that from the expression of the pairberry (pby) gene ortholog that is expressed in a typical SPG-pattern. Since this result suggested possible conservation of the SPG-network we further investigated SPGs (and associated factors) such as Notum in the onychophoran. We find that the expression patterns of SPGs in arthropods and the onychophoran are highly conserved, suggesting a conserved SPG-network in these two clades, and indeed also in an annelid. This may suggest that the common ancestor of lophotrochozoans and ecdysozoans was already segmented utilising the same SPG-network, or that the SPG-network was recruited independently in annelids and onychophorans/arthropods.

  13. Gene Duplicability of Core Genes Is Highly Consistent across All Angiosperms.

    PubMed

    Li, Zhen; Defoort, Jonas; Tasdighian, Setareh; Maere, Steven; Van de Peer, Yves; De Smet, Riet

    2016-02-01

    Gene duplication is an important mechanism for adding to genomic novelty. Hence, which genes undergo duplication and are preserved following duplication is an important question. It has been observed that gene duplicability, or the ability of genes to be retained following duplication, is a nonrandom process, with certain genes being more amenable to survive duplication events than others. Primarily, gene essentiality and the type of duplication (small-scale versus large-scale) have been shown in different species to influence the (long-term) survival of novel genes. However, an overarching view of "gene duplicability" is lacking, mainly due to the fact that previous studies usually focused on individual species and did not account for the influence of genomic context and the time of duplication. Here, we present a large-scale study in which we investigated duplicate retention for 9178 gene families shared between 37 flowering plant species, referred to as angiosperm core gene families. For most gene families, we observe a strikingly consistent pattern of gene duplicability across species, with gene families being either primarily single-copy or multicopy in all species. An intermediate class contains gene families that are often retained in duplicate for periods extending to tens of millions of years after whole-genome duplication, but ultimately appear to be largely restored to singleton status, suggesting that these genes may be dosage balance sensitive. The distinction between single-copy and multicopy gene families is reflected in their functional annotation, with single-copy genes being mainly involved in the maintenance of genome stability and organelle function and multicopy genes in signaling, transport, and metabolism. The intermediate class was overrepresented in regulatory genes, further suggesting that these represent putative dosage-balance-sensitive genes.

  14. Gene Duplicability of Core Genes Is Highly Consistent across All Angiosperms[OPEN

    PubMed Central

    Li, Zhen; Van de Peer, Yves; De Smet, Riet

    2016-01-01

    Gene duplication is an important mechanism for adding to genomic novelty. Hence, which genes undergo duplication and are preserved following duplication is an important question. It has been observed that gene duplicability, or the ability of genes to be retained following duplication, is a nonrandom process, with certain genes being more amenable to survive duplication events than others. Primarily, gene essentiality and the type of duplication (small-scale versus large-scale) have been shown in different species to influence the (long-term) survival of novel genes. However, an overarching view of “gene duplicability” is lacking, mainly due to the fact that previous studies usually focused on individual species and did not account for the influence of genomic context and the time of duplication. Here, we present a large-scale study in which we investigated duplicate retention for 9178 gene families shared between 37 flowering plant species, referred to as angiosperm core gene families. For most gene families, we observe a strikingly consistent pattern of gene duplicability across species, with gene families being either primarily single-copy or multicopy in all species. An intermediate class contains gene families that are often retained in duplicate for periods extending to tens of millions of years after whole-genome duplication, but ultimately appear to be largely restored to singleton status, suggesting that these genes may be dosage balance sensitive. The distinction between single-copy and multicopy gene families is reflected in their functional annotation, with single-copy genes being mainly involved in the maintenance of genome stability and organelle function and multicopy genes in signaling, transport, and metabolism. The intermediate class was overrepresented in regulatory genes, further suggesting that these represent putative dosage-balance-sensitive genes. PMID:26744215

  15. High intensity ultrasound transducer used in gene transfection

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    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

  17. α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras(G12D), and Kras(G12D)/tp53R270H) mice.

    PubMed

    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 (Pdx(Cre);LSL-Kras(G12D)) pancreatic CSC characteristics in vitro, and pancreatic carcinogenesis in KPC (Pdx(Cre);LSLKras(G12D);LSL-Trp53(R172H)) mice. Mang-NPs inhibited human and Kras(G12D) 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

  18. Vitamin E δ-tocotrienol prolongs survival in the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) transgenic mouse model of pancreatic cancer.

    PubMed

    Husain, Kazim; Centeno, Barbara A; Chen, Dung-Tsa; Hingorani, Sunil R; Sebti, Said M; Malafa, Mokenge P

    2013-10-01

    Previous work has shown that vitamin E δ-tocotrienol (VEDT) prolongs survival and delays progression of pancreatic cancer in the LSL-Kras(G12D)(/+);Pdx-1-Cre mouse model of pancreatic cancer. However, the effect of VEDT alone or in combination with gemcitabine in the more aggressive LSL-Kras(G12D)(/+);LSL-Trp53(R172H)(/+);Pdx-1-Cre (KPC) mouse model is unknown. Here, we studied the effects of VEDT and the combination of VEDT and gemcitabine in the KPC mice. KPC mice were randomized into four groups: (i) vehicle [olive oil, 1.0 mL/kg per os twice a day and PBS 1.0 mL/kg intrapertoneally (i.p.) twice a week], (ii) gemcitabine (100 mg/kg i.p. twice a week), (iii) VEDT (200 mg/kg per os twice a day), and (iv) gemcitabine + VEDT. Mice received treatment until they displayed symptoms of impending death from pancreatic cancer, at which point animals were euthanized. At 16 weeks, survival was 10% in the vehicle group, 30% in the gemcitabine group, 70% in the VEDT group (P < 0.01), and 90% in the VEDT combined with gemcitabine group (P < 0.05). VEDT alone and combined with gemcitabine resulted in reversal of epithelial-to-mesenchymal transition in tumors. Biomarkers of apoptosis (plasma CK18), PARP1 cleavage, and Bax expression were more greatly induced in tumors subjected to combined treatment versus individual treatment. Combined treatment induced cell-cycle inhibitors (p27(Kip1) and p21(Cip1)) and inhibited VEGF, vascularity (CD31), and oncogenic signaling (pAKT, pMEK, and pERK) greater than individual drugs. No significant differences in body weight gain between drug treatment and control mice were observed. These results strongly support further investigation of VEDT alone and in combination with gemcitabine for pancreatic cancer prevention and treatment.

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

    PubMed

    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

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

    PubMed

    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.

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

    PubMed

    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

  2. Cell types differ in global coordination of splicing and proportion of highly expressed genes.

    PubMed

    Trakhtenberg, Ephraim F; Pho, Nam; Holton, Kristina M; Chittenden, Thomas W; Goldberg, Jeffrey L; Dong, Lingsheng

    2016-01-01

    Balance in the transcriptome is regulated by coordinated synthesis and degradation of RNA molecules. Here we investigated whether mammalian cell types intrinsically differ in global coordination of gene splicing and expression levels. We analyzed RNA-seq transcriptome profiles of 8 different purified mouse cell types. We found that different cell types vary in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, and that the cell types that express more variants of alternatively spliced transcripts per gene are those that have higher proportion of highly expressed genes. Cell types segregated into two clusters based on high or low proportion of highly expressed genes. Biological functions involved in negative regulation of gene expression were enriched in the group of cell types with low proportion of highly expressed genes, and biological functions involved in regulation of transcription and RNA splicing were enriched in the group of cell types with high proportion of highly expressed genes. Our findings show that cell types differ in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, which represent distinct properties of the transcriptome and may reflect intrinsic differences in global coordination of synthesis, splicing, and degradation of RNA molecules. PMID:27577089

  3. Cell types differ in global coordination of splicing and proportion of highly expressed genes

    PubMed Central

    Trakhtenberg, Ephraim F.; Pho, Nam; Holton, Kristina M.; Chittenden, Thomas W.; Goldberg, Jeffrey L.; Dong, Lingsheng

    2016-01-01

    Balance in the transcriptome is regulated by coordinated synthesis and degradation of RNA molecules. Here we investigated whether mammalian cell types intrinsically differ in global coordination of gene splicing and expression levels. We analyzed RNA-seq transcriptome profiles of 8 different purified mouse cell types. We found that different cell types vary in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, and that the cell types that express more variants of alternatively spliced transcripts per gene are those that have higher proportion of highly expressed genes. Cell types segregated into two clusters based on high or low proportion of highly expressed genes. Biological functions involved in negative regulation of gene expression were enriched in the group of cell types with low proportion of highly expressed genes, and biological functions involved in regulation of transcription and RNA splicing were enriched in the group of cell types with high proportion of highly expressed genes. Our findings show that cell types differ in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, which represent distinct properties of the transcriptome and may reflect intrinsic differences in global coordination of synthesis, splicing, and degradation of RNA molecules. PMID:27577089

  4. Mitochondrial Toxicity Studied with the PBMC of Children from the Chinese National Pediatric Highly Active Antiretroviral Therapy Cohort

    PubMed Central

    Liu, Daojie; Yin, Jiming; Qiao, Luxin; Shi, Ying; Dong, Yaowu; Li, Ning; Zhang, Fujie; Chen, Dexi

    2013-01-01

    As the backbone of highly active antiretroviral therapy (HAART), nucleoside reverse transcriptase inhibitors (NRTIs) have effectively improved outcomes for HIV-infected patients. However, long-term treatment with NRTIs can cause a series of pathologies associated with mitochondrial toxicity. To date, the status and mechanism of mitochondrial toxicity induced by NRTIs are still not clear, especially in HIV-infected children. As part of the national pediatric HAART program in China, our study focused on mitochondrial toxicity and its potential mechanism in HIV-1-infected children who were divided into two groups based on their duration of treatment with NRTIs: one group received treatment for less than 36 months and one group was treated for 36 to 72 months. The control group comprised age-matched non-HIV-infected children. Blood lactic acid and ATP levels in peripheral blood mononuclear cells (PBMCs) were measured to evaluate mitochondrial function, and mtDNA copies and mutations in PBMCs were determined for detecting mtDNA lesions. Simultaneously, TK2 and P53R2 gene expression in PBMC was measured. As compared with the control group, blood lactic acid levels in both NRTI treatment groups were significantly higher, whereas ATP levels and mtDNA mutation rates in PBMCs did not differ between the control and the two NRTI treatment groups. Both NRTI treatment groups exhibited significant mtDNA loss. N Moreover, we found that P53R2 mRNA expression and protein levels were significantly reduced in both treatment groups and that TK2 mRNA expression and protein levels were induced in the long-term NRTI treatment group. These results suggest that mitochondrial toxicity occurs in long-term HAART patients and that P53R2 and TK2 levels in PBMCs are useful biomarkers for detecting mitochondrial toxicity in patients on long-term treatment with NRTIs. PMID:23468942

  5. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

    PubMed Central

    Nishina, Kazutaka; Piao, Wenying; Yoshida-Tanaka, Kie; Sujino, Yumiko; Nishina, Tomoko; Yamamoto, Tsuyoshi; Nitta, Keiko; Yoshioka, Kotaro; Kuwahara, Hiroya; Yasuhara, Hidenori; Baba, Takeshi; Ono, Fumiko; Miyata, Kanjiro; Miyake, Koichi; Seth, Punit P.; Low, Audrey; Yoshida, Masayuki; Bennett, C. Frank; Kataoka, Kazunori; Mizusawa, Hidehiro; Obika, Satoshi; Yokota, Takanori

    2015-01-01

    Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field. PMID:26258894

  6. Rates of Lateral Gene Transfer in Prokaryotes: High but Why?

    PubMed

    Vos, Michiel; Hesselman, Matthijn C; te Beek, Tim A; van Passel, Mark W J; Eyre-Walker, Adam

    2015-10-01

    Lateral gene transfer is of fundamental importance to the evolution of prokaryote genomes and has important practical consequences, as evidenced by the rapid dissemination of antibiotic resistance and virulence determinants. Relatively little effort has so far been devoted to explicitly quantifying the rate at which accessory genes are taken up and lost, but it is possible that the combined rate of lateral gene transfer and gene loss is higher than that of point mutation. What evolutionary forces underlie the rate of lateral gene transfer are not well understood. We here use theory developed to explain the evolution of mutation rates to address this question and explore its consequences for the study of prokaryote evolution.

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

    PubMed

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

    2013-09-01

    Sulindac has been identified as a competitive inhibitor of aldo-keto reductase 1B10 (AKR1B10), an enzyme that plays a key role in carcinogenesis. AKR1B10 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and exhibits lipid substrate specificity, especially for farnesyl and geranylgeranyl. There have been no studies though showing that the inhibition of PDAC by sulindac is via inhibition of AKR1B10, particularly the metabolism of farnesyl/geranylgeranyl and Kras protein prenylation. To determine the chemopreventive effects of sulindac on pancreatic carcinogenesis, 5-week-old LSL-Kras(G12D)-LSL-Trp53(R172H)-Pdx-1-Cre mice (Pan(kras/p53) mice) were fed an AIN93M diet with or without 200 p.p.m. sulindac (n = 20/group). Kaplan-Meier survival analysis showed that average animal survival in Pan(kras/p53) mice was 143.7 ± 8.8 days, and average survival with sulindac was increased to 168.0 ± 8.8 days (P < 0.005). Histopathological analyses revealed that 90% of mice developed PDAC, 10% with metastasis to the liver and lymph nodes. With sulindac, the incidence of PDAC was reduced to 56% (P < 0.01) and only one mouse had lymph node metastasis. Immunochemical analysis showed that sulindac significantly decreased Ki-67-labeled cell proliferation and markedly reduced the expression of phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Raf and mitogen-activated protein kinase kinase 1 and 2. In in vitro experiments with PDAC cells from Pan(kras/p53) mice, sulindac exhibited dose-dependent inhibition of AKR1B10 activity. By silencing AKR1B10 expression through small interfering RNA or by sulindac treatment, these in vitro models showed a reduction in Kras and human DNA-J homolog 2 protein prenylation, and downregulation of phosphorylated C-raf, ERK1/2 and MEK1/2 expression. Our results demonstrate that sulindac inhibits pancreatic carcinogenesis by the inhibition of Kras protein prenylation by targeting AKR1B10.

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

    PubMed Central

    2010-01-01

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

  9. The ovary is an alternative site of origin for high-grade serous ovarian cancer in mice.

    PubMed

    Kim, Jaeyeon; Coffey, Donna M; Ma, Lang; Matzuk, Martin M

    2015-06-01

    Although named "ovarian cancer," it has been unclear whether the cancer actually arises from the ovary, especially for high-grade serous carcinoma (HGSC), also known as high-grade serous ovarian cancer, the most common and deadliest ovarian cancer. In addition, the tumor suppressor p53 is the most frequently mutated gene in HGSC. However, whether mutated p53 can cause HGSC remains unknown. In this study, we bred a p53 mutation, p53(R172H), into conditional Dicer-Pten double-knockout (DKO) mice, a mouse model duplicating human HGSC, to generate triple-mutant (TKO) mice. Like DKO mice, these TKO mice develop metastatic HGSCs originating from the fallopian tube. Unlike DKO mice, however, even after fallopian tubes are removed in TKO mice, ovaries alone can develop metastatic HGSCs, indicating that a p53 mutation can drive HGSC arising from the ovary. To confirm this, we generated p53(R172H)-Pten double-mutant mice, one of the genetic control lines for TKO mice. As anticipated, these double-mutant mice also develop metastatic HGSCs from the ovary, verifying the HGSC-forming ability of ovaries with a p53 mutation. Our study therefore shows that ovaries harboring a p53 mutation, as well as fallopian tubes, can be a distinct tissue source of high-grade serous ovarian cancer in mice.

  10. Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi

    PubMed Central

    2009-01-01

    Background Trypanosoma cruzi, a kinetoplastid protozoan parasite that causes Chagas disease, infects approximately 15 million people in Central and South America. In contrast to the substantial in silico studies of the T. cruzi genome, transcriptome, and proteome, only a few genes have been experimentally characterized and validated, mainly due to the lack of facile methods for gene manipulation needed for reverse genetic studies. Current strategies for gene disruption in T. cruzi are tedious and time consuming. In this study we have compared the conventional multi-step cloning technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway-based systems. Results While the one-step-PCR strategy was found to be the fastest method for production of knockout constructs, it does not efficiently target genes of interest using gene-specific sequences of less than 80 nucleotides. Alternatively, the Multisite Gateway based approach is less time-consuming than conventional methods and is able to efficiently and reproducibly delete target genes. Conclusion Using the Multisite Gateway strategy, we have rapidly produced constructs that successfully produce specific gene deletions in epimastigotes of T. cruzi. This methodology should greatly facilitate reverse genetic studies in T. cruzi. PMID:19432966

  11. Cell type-selective disease-association of genes under high regulatory load

    PubMed Central

    Galhardo, Mafalda; Berninger, Philipp; Nguyen, Thanh-Phuong; Sauter, Thomas; Sinkkonen, Lasse

    2015-01-01

    We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3′ UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner. PMID:26338775

  12. Cell type-selective disease-association of genes under high regulatory load.

    PubMed

    Galhardo, Mafalda; Berninger, Philipp; Nguyen, Thanh-Phuong; Sauter, Thomas; Sinkkonen, Lasse

    2015-10-15

    We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3' UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner.

  13. Tay-Sachs disease: high gene frequency in a non-Jewish population.

    PubMed

    Kelly, T E; Chase, G A; Kaback, M M; Kumor, K; McKusick, V A

    1975-05-01

    A non-Amish "Pennsylvania Dutch" semi-isolate was found to have a high frequency of Tay-Sachs gene. This high frequency could be ascribed to founder effect and may represent, in microcosm, how this mechanism could have produced the high gene frequency among Ashkenazi Jews. PMID:803011

  14. High presence/absence gene variability in defense-related gene clusters of Cucumis melo

    PubMed Central

    2013-01-01

    Background Changes in the copy number of DNA sequences are one of the main mechanisms generating genome variability in eukaryotes. These changes are often related to phenotypic effects such as genetic disorders or novel pathogen resistance. The increasing availability of genome sequences through the application of next-generation massive sequencing technologies has allowed the study of genomic polymorphisms at both the interspecific and intraspecific levels, thus helping to understand how species adapt to changing environments through genome variability. Results Data on gene presence/absence variation (PAV) in melon was obtained by resequencing a cultivated accession and an old-relative melon variety, and using previously obtained resequencing data from three other melon cultivars, among them DHL92, on which the current draft melon genome sequence is based. A total of 1,697 PAV events were detected, involving 4.4% of the predicted melon gene complement. In all, an average 1.5% of genes were absent from each analyzed cultivar as compared to the DHL92 reference genome. The most populated functional category among the 304 PAV genes of known function was that of stress response proteins (30% of all classified PAVs). Our results suggest that genes from multi-copy families are five times more likely to be affected by PAV than singleton genes. Also, the chance of genes present in the genome in tandem arrays being affected by PAV is double that of isolated genes, with PAV genes tending to be in longer clusters. The highest concentration of PAV events detected in the melon genome was found in a 1.1 Mb region of linkage group V, which also shows the highest density of melon stress-response genes. In particular, this region contains the longest continuous gene-containing PAV sequence so far identified in melon. Conclusions The first genome-wide report of PAV variation among several melon cultivars is presented here. Multi-copy and clustered genes, especially those with

  15. Highly Fluorescent Gene Carrier Based on Ag-Au Alloy Nanoclusters.

    PubMed

    Wang, Ping; Lin, Lin; Guo, Zhaopei; Chen, Jie; Tian, Huayu; Chen, Xuesi; Yang, Hua

    2016-01-01

    For systemic delivery of gene, gold nanoparticles (GNPs) have been exploited as novel gene carriers because of the excellent characteristics for "visible" in intracellular trafficking. Herein, a highly fluorescent gene carrier was prepared by conjugating polyethylenimines on Ag-Au alloy nanoclusters. This carrier exhibited remarkable high gene transfection efficiencies and relatively low cytotoxicity toward B16F10, HeLa, and CHO cells. More interestingly, the high fluorescent Ag-Au-PEI conjugates showed high quantum yield of 14.56%, which is much higher than most of the reported gold nanocluster-based quantum dots and Ag-Au-PEI possessed bioimaging capacity both in vitro and in vivo.

  16. New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

    DOE PAGESBeta

    Guss, Adam M.; Rother, Michael; Zhang, Jun Kai; Kulkkarni, Gargi; Metcalf, William W.

    2008-01-01

    A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri P mcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline inmore » strains that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR -regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.« less

  17. New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

    PubMed Central

    Guss, Adam M.; Rother, Michael; Zhang, Jun Kai; Kulkkarni, Gargi; Metcalf, William W.

    2008-01-01

    A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion. PMID:19054746

  18. High levels of gene expression explain the strong evolutionary constraint of mitochondrial protein-coding genes.

    PubMed

    Nabholz, Benoit; Ellegren, Hans; Wolf, Jochen B W

    2013-02-01

    The nearly neutral theory of molecular evolution has been widely accepted as the guiding principle for understanding how selection affects gene sequence evolution. One of its central predictions is that the rate at which proteins evolve should negatively scale with effective population size (N(e)). In contrast to the expectation of reduced selective constraint in the mitochondrial genome following from its lower N(e), we observe what can be interpreted as the opposite: for a taxonomically diverse set of organisms (birds, mammals, insects, and nematodes), mitochondrially encoded protein-coding genes from the oxidative phosphorylation pathway (mtOXPHOS; n = 12-13) show markedly stronger signatures of purifying selection (illustrated by low d(N)/d(S)) than their nuclear counterparts interacting in the same pathway (nuOXPHOS; n: ∼75). To understand these unexpected evolutionary dynamics, we consider a number of structural and functional parameters including gene expression, hydrophobicity, transmembrane position, gene ontology, GC content, substitution rate, proportion of amino acids in transmembrane helices, and protein-protein interaction. Across all taxa, unexpectedly large differences in gene expression levels (RNA-seq) between nuclear and mitochondrially encoded genes, and to a lower extent hydrophobicity, explained most of the variation in d(N)/d(S). Similarly, differences in d(N)/d(S) between functional OXPHOS protein complexes could largely be explained by gene expression differences. Overall, by including gene expression and other functional parameters, the unexpected mitochondrial evolutionary dynamics can be understood. Our results not only reaffirm the link between gene expression and protein evolution but also open new questions about the functional role of expression level variation between mitochondrial genes. PMID:23071102

  19. High levels of gene expression explain the strong evolutionary constraint of mitochondrial protein-coding genes.

    PubMed

    Nabholz, Benoit; Ellegren, Hans; Wolf, Jochen B W

    2013-02-01

    The nearly neutral theory of molecular evolution has been widely accepted as the guiding principle for understanding how selection affects gene sequence evolution. One of its central predictions is that the rate at which proteins evolve should negatively scale with effective population size (N(e)). In contrast to the expectation of reduced selective constraint in the mitochondrial genome following from its lower N(e), we observe what can be interpreted as the opposite: for a taxonomically diverse set of organisms (birds, mammals, insects, and nematodes), mitochondrially encoded protein-coding genes from the oxidative phosphorylation pathway (mtOXPHOS; n = 12-13) show markedly stronger signatures of purifying selection (illustrated by low d(N)/d(S)) than their nuclear counterparts interacting in the same pathway (nuOXPHOS; n: ∼75). To understand these unexpected evolutionary dynamics, we consider a number of structural and functional parameters including gene expression, hydrophobicity, transmembrane position, gene ontology, GC content, substitution rate, proportion of amino acids in transmembrane helices, and protein-protein interaction. Across all taxa, unexpectedly large differences in gene expression levels (RNA-seq) between nuclear and mitochondrially encoded genes, and to a lower extent hydrophobicity, explained most of the variation in d(N)/d(S). Similarly, differences in d(N)/d(S) between functional OXPHOS protein complexes could largely be explained by gene expression differences. Overall, by including gene expression and other functional parameters, the unexpected mitochondrial evolutionary dynamics can be understood. Our results not only reaffirm the link between gene expression and protein evolution but also open new questions about the functional role of expression level variation between mitochondrial genes.

  20. Altered Gene Expression in Mice Selected for High Maternal Aggression

    PubMed Central

    Gammie, Stephen C.; Auger, Anthony P.; Jessen, Heather M.; Vanzo, Rena J.; Awad, Tarif A.; Stevenson, Sharon A.

    2007-01-01

    We previously applied selective breeding on outbred mice to increase maternal aggression (maternal defense). In this study, we compared gene expression within a continuous region of the CNS involved in maternal aggression (hypothalamus and preoptic regions) between lactating selected (S) and non-selected control (C) mice (n = 6 per group). Using microarrays representing over 40,000 genes or expressed sequence tags, two statistical algorithms were used to identify significant differences in gene expression: robust multi array and the probe logarithmic intensity error method. ∼ 200 genes were identified as significant using an intersection from both techniques. A subset of genes were examined for confirmation by real-time PCR. Significant decreases were found in S mice for neurotensin and neuropeptide Y receptor Y2 (both confirmed by PCR). Significant increases were found in S mice for neuronal nitric oxide synthase (confirmed by PCR), the K+ channel subunit, Kcna1 (confirmed by PCR), corticotrophin releasing factor binding protein (just above significance using PCR; p = 0.051), and GABA A receptor subunit 1A (not confirmed by PCR, but similar direction). S mice also exhibited significantly higher levels of the neurotransmitter receptor, adenosine A1 receptor, and the transcription factors, c-Fos, and Egr-1. Interestingly, for 24 genes related to metabolism, all were significantly elevated in S mice, suggesting altered metabolism in these mice. Together, this study provides a list of candidate genes (some previously implicated in maternal aggression and some novel) that may play an important role in the production of this behavior. PMID:16939635

  1. Locally adapted traits maintained in the face of high gene flow.

    PubMed

    Fitzpatrick, S W; Gerberich, J C; Kronenberger, J A; Angeloni, L M; Funk, W C

    2015-01-01

    Gene flow between phenotypically divergent populations can disrupt local adaptation or, alternatively, may stimulate adaptive evolution by increasing genetic variation. We capitalised on historical Trinidadian guppy transplant experiments to test the phenotypic effects of increased gene flow caused by replicated introductions of adaptively divergent guppies, which were translocated from high- to low-predation environments. We sampled two native populations prior to the onset of gene flow, six historic introduction sites, introduction sources and multiple downstream points in each basin. Extensive gene flow from introductions occurred in all streams, yet adaptive phenotypic divergence across a gradient in predation level was maintained. Descendants of guppies from a high-predation source site showed high phenotypic similarity with native low-predation guppies in as few as ~12 generations after gene flow, likely through a combination of adaptive evolution and phenotypic plasticity. Our results demonstrate that locally adapted phenotypes can be maintained despite extensive gene flow from divergent populations. PMID:25363522

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is ‘open’, with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order. PMID:26484663

  4. High-throughput Gene Tagging in Trypanosoma brucei.

    PubMed

    Dyer, Philip; Dean, Samuel; Sunter, Jack

    2016-01-01

    Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible. PMID:27584862

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

    PubMed

    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.

  6. Polymorphism in two genes for B2 high sulfur proteins of wool.

    PubMed

    Rogers, G R; Hickford, J G; Bickerstaffe, R

    1994-12-01

    Variation in the nucleotide sequence of the B2 high-sulfur protein genes has not been reported previously. This paper reports 15 nucleotide substitutions in each of the genes for the B2A and B2C proteins and a length of polymorphism in the B2A gene which translates to the insertion/deletion of one 30-nucleotide repeat sequence. Evidence is presented for gene conversion occurring within the B2 high-sulfur multigene family. These DNA polymorphisms may account for some of the microheterogeneity observed in the B2 high-sulfur proteins and may also be useful genetic markers of the B2 high-sulfur protein gene loci for future use in analysing wool fibre characteristics.

  7. Gene interaction network analysis suggests differences between high and low doses of acetaminophen

    SciTech Connect

    Toyoshiba, Hiroyoshi . E-mail: toyoshiba.hiroyoshi@nies.go.jp; Sone, Hideko; Yamanaka, Takeharu; Parham, Frederick M.; Irwin, Richard D.; Boorman, Gary A.; Portier, Christopher J.

    2006-09-15

    Bayesian networks for quantifying linkages between genes were applied to detect differences in gene expression interaction networks between multiple doses of acetaminophen at multiple time points. Seventeen (17) genes were selected from the gene expression profiles from livers of rats orally exposed to 50, 150 and 1500 mg/kg acetaminophen (APAP) at 6, 24 and 48 h after exposure using a variety of statistical and bioinformatics approaches. The selected genes are related to three biological categories: apoptosis, oxidative stress and other. Gene interaction networks between all 17 genes were identified for the nine dose-time observation points by the TAO-Gen algorithm. Using k-means clustering analysis, the estimated nine networks could be clustered into two consensus networks, the first consisting of the low and middle dose groups, and the second consisting of the high dose. The analysis suggests that the networks could be segregated by doses and were consistent in structure over time of observation within grouped doses. The consensus networks were quantified to calculate the probability distribution for the strength of the linkage between genes connected in the networks. The quantifying analysis showed that, at lower doses, the genes related to the oxidative stress signaling pathway did not interact with the apoptosis-related genes. In contrast, the high-dose network demonstrated significant interactions between the oxidative stress genes and the apoptosis genes and also demonstrated a different network between genes in the oxidative stress pathway. The approaches shown here could provide predictive information to understand high- versus low-dose mechanisms of toxicity.

  8. Gene Model Annotations for Drosophila melanogaster: Impact of High-Throughput Data

    PubMed Central

    Matthews, Beverley B.; dos Santos, Gilberto; Crosby, Madeline A.; Emmert, David B.; St. Pierre, Susan E.; Gramates, L. Sian; Zhou, Pinglei; Schroeder, Andrew J.; Falls, Kathleen; Strelets, Victor; Russo, Susan M.; Gelbart, William M.

    2015-01-01

    We report the current status of the FlyBase annotated gene set for Drosophila melanogaster and highlight improvements based on high-throughput data. The FlyBase annotated gene set consists entirely of manually annotated gene models, with the exception of some classes of small non-coding RNAs. All gene models have been reviewed using evidence from high-throughput datasets, primarily from the modENCODE project. These datasets include RNA-Seq coverage data, RNA-Seq junction data, transcription start site profiles, and translation stop-codon read-through predictions. New annotation guidelines were developed to take into account the use of the high-throughput data. We describe how this flood of new data was incorporated into thousands of new and revised annotations. FlyBase has adopted a philosophy of excluding low-confidence and low-frequency data from gene model annotations; we also do not attempt to represent all possible permutations for complex and modularly organized genes. This has allowed us to produce a high-confidence, manageable gene annotation dataset that is available at FlyBase (http://flybase.org). Interesting aspects of new annotations include new genes (coding, non-coding, and antisense), many genes with alternative transcripts with very long 3′ UTRs (up to 15–18 kb), and a stunning mismatch in the number of male-specific genes (approximately 13% of all annotated gene models) vs. female-specific genes (less than 1%). The number of identified pseudogenes and mutations in the sequenced strain also increased significantly. We discuss remaining challenges, for instance, identification of functional small polypeptides and detection of alternative translation starts. PMID:26109357

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

    PubMed

    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

  10. Evaluating evolutionary history in the face of high gene tree discordance in Australian Gehyra (Reptilia: Gekkonidae).

    PubMed

    Sistrom, M; Hutchinson, M; Bertozzi, T; Donnellan, S

    2014-07-01

    Species tree methods have provided improvements for estimating species relationships and the timing of diversification in recent radiations by allowing for gene tree discordance. Although gene tree discordance is often observed, most discordance is attributed to incomplete lineage sorting rather than other biological phenomena, and the causes of discordance are rarely investigated. We use species trees from multi-locus data to estimate the species relationships, evolutionary history and timing of diversification among Australian Gehyra-a group renowned for taxonomic uncertainty and showing a large degree of gene tree discordance. We find support for a recent Asian origin and two major clades: a tropically adapted clade and an arid adapted clade, with some exceptions, but no support for allopatric speciation driven by chromosomal rearrangement in the group. Bayesian concordance analysis revealed high gene tree discordance and comparisons of Robinson-Foulds distances showed that discordance between gene trees was significantly higher than that generated by topological uncertainty within each gene. Analysis of gene tree discordance and incomplete taxon sampling revealed that gene tree discordance was high whether terminal taxon or gene sampling was maximized, indicating discordance is due to biological processes, which may be important in contributing to gene tree discordance in many recently diversified organisms.

  11. Evaluating evolutionary history in the face of high gene tree discordance in Australian Gehyra (Reptilia: Gekkonidae)

    PubMed Central

    Sistrom, M; Hutchinson, M; Bertozzi, T; Donnellan, S

    2014-01-01

    Species tree methods have provided improvements for estimating species relationships and the timing of diversification in recent radiations by allowing for gene tree discordance. Although gene tree discordance is often observed, most discordance is attributed to incomplete lineage sorting rather than other biological phenomena, and the causes of discordance are rarely investigated. We use species trees from multi-locus data to estimate the species relationships, evolutionary history and timing of diversification among Australian Gehyra—a group renowned for taxonomic uncertainty and showing a large degree of gene tree discordance. We find support for a recent Asian origin and two major clades: a tropically adapted clade and an arid adapted clade, with some exceptions, but no support for allopatric speciation driven by chromosomal rearrangement in the group. Bayesian concordance analysis revealed high gene tree discordance and comparisons of Robinson–Foulds distances showed that discordance between gene trees was significantly higher than that generated by topological uncertainty within each gene. Analysis of gene tree discordance and incomplete taxon sampling revealed that gene tree discordance was high whether terminal taxon or gene sampling was maximized, indicating discordance is due to biological processes, which may be important in contributing to gene tree discordance in many recently diversified organisms. PMID:24642886

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

    PubMed

    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.

  13. Gene expression profiling in undifferentiated thyroid carcinoma induced by high-dose radiation

    PubMed Central

    Bang, Hyun Soon; Choi, Moo Hyun; Kim, Cha Soon; Choi, Seung Jin

    2016-01-01

    Published gene expression studies for radiation-induced thyroid carcinogenesis have used various methodologies. In this study, we identified differential gene expression in a human thyroid epithelial cell line after exposure to high-dose γ-radiation. HTori-3 cells were exposed to 5 or 10 Gy of ionizing radiation using two dose rates (high-dose rate: 4.68 Gy/min, and low-dose rate: 40 mGy/h) and then implanted into the backs of BALB/c nude mice after 4 (10 Gy) or 5 weeks (5 Gy). Decreases in cell viability, increases in giant cell frequency, anchorage-independent growth in vitro, and tumorigenicity in vivo were observed. Particularly, the cells irradiated with 5 Gy at the high-dose rate or 10 Gy at the low-dose rate demonstrated more prominent tumorigenicity. Gene expression profiling was analyzed via microarray. Numerous genes that were significantly altered by a fold-change of >50% following irradiation were identified in each group. Gene expression analysis identified six commonly misregulated genes, including CRYAB, IL-18, ZNF845, CYP24A1, OR4N4 and VN1R4, at all doses. These genes involve apoptosis, the immune response, regulation of transcription, and receptor signaling pathways. Overall, the altered genes in high-dose rate (HDR) 5 Gy and low-dose rate (LDR) 10 Gy were more than those of LDR 5 Gy and HDR 10 Gy. Thus, we investigated genes associated with aggressive tumor development using the two dosage treatments. In this study, the identified gene expression profiles reflect the molecular response following high doses of external radiation exposure and may provide helpful information about radiation-induced thyroid tumors in the high-dose range. PMID:27006382

  14. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

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

    1998-01-01

    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.

  15. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

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

    2000-08-22

    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.

  16. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

    1998-10-13

    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. 13 figs.

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

    DOEpatents

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

  20. Genetic control of eosinophilia in mice: gene(s) expressed in bone marrow-derived cells control high responsiveness

    SciTech Connect

    Vadas, M.A.

    1982-02-01

    A heterogeneity in the capacity of strains of mice to mount eosinophilia is described. BALB/c and C3H are eosinophil high responder strains (EO-HR) and CBA and A/J are eosinophil low responder strains (EO-LR), judged by the response of blood eosinophils to Ascaris suum, and the response of blood, bone marrow, and spleen eosinophils to keyhole limpet hemocyanin given 2 days after 150 mg/kg cyclophosphamide. Some of the gene(s) for high responsiveness appear to be dominant because (EO-HR x EO-LR)F/sub 1/ mice were intermediate to high responders. This gene is expressed in bone marrow-derived cells because radiation chimeras of the type EO-HR..-->..F/sub 1/ were high responders and EO-LR..-->..F/sub 1/ were low responders. This description of a genetic control of eosinophilia in mice may be useful in understanding the role of this cell in parasite immunity and allergy.

  1. High-Copy Overexpression Screening Reveals PDR5 as the Main Doxorubicin Resistance Gene in Yeast

    PubMed Central

    Demir, Ayse Banu; Koc, Ahmet

    2015-01-01

    Doxorubicin is one of the most potent anticancer drugs used in the treatment of various cancer types. The efficacy of doxorubicin is influenced by the drug resistance mechanisms and its cytotoxicity. In this study, we performed a high-copy screening analysis to find genes that play a role in doxorubicin resistance and found several genes (CUE5, AKL1, CAN1, YHR177W and PDR5) that provide resistance. Among these genes, overexpression of PDR5 provided a remarkable resistance, and deletion of it significantly rendered the tolerance level for the drug. Q-PCR analyses suggested that transcriptional regulation of these genes was not dependent on doxorubicin treatment. Additionally, we profiled the global expression pattern of cells in response to doxorubicin treatment and highlighted the genes and pathways that are important in doxorubicin tolerance/toxicity. Our results suggest that many efflux pumps and DNA metabolism genes are upregulated by the drug and required for doxorubicin tolerance. PMID:26690737

  2. High-resolution chromosome ideogram representation of currently recognized genes for autism spectrum disorders.

    PubMed

    Butler, Merlin G; Rafi, Syed K; Manzardo, Ann M

    2015-03-20

    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.

  3. High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides

    PubMed Central

    Borovkov, Alex Y.; Loskutov, Andrey V.; Robida, Mark D.; Day, Kristen M.; Cano, Jose A.; Le Olson, Tien; Patel, Hetal; Brown, Kevin; Hunter, Preston D.; Sykes, Kathryn F.

    2010-01-01

    To meet the growing demand for synthetic genes more robust, scalable and inexpensive gene assembly technologies must be developed. Here, we present a protocol for high-quality gene assembly directly from low-cost marginal-quality microarray-synthesized oligonucleotides. Significantly, we eliminated the time- and money-consuming oligonucleotide purification steps through the use of hybridization-based selection embedded in the assembly process. The protocol was tested on mixtures of up to 2000 oligonucleotides eluted directly from microarrays obtained from three different chip manufacturers. These mixtures containing <5% perfect oligos, and were used directly for assembly of 27 test genes of different sizes. Gene quality was assessed by sequencing, and their activity was tested in coupled in vitro transcription/translation reactions. Genes assembled from the microarray-eluted material using the new protocol matched the quality of the genes assembled from >95% pure column-synthesized oligonucleotides by the standard protocol. Both averaged only 2.7 errors/kb, and genes assembled from microarray-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones. This report represents the first demonstration of cost-efficient gene assembly from microarray-synthesized oligonucleotides. The overall cost of assembly by this method approaches 5¢ per base, making gene synthesis more affordable than traditional cloning. PMID:20693531

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

    PubMed Central

    Butler, Merlin G.; Rafi, Syed K.; Manzardo, Ann M.

    2015-01-01

    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. PMID:25803107

  5. Searching for the Genes of Unconventional High Temperature Superconductors

    NASA Astrophysics Data System (ADS)

    Hu, Jiangping

    In the past, both curates and iron-based superconductors were discovered accidentally. Lacking of successful predictions on new high Tc materials is one of major obstacles to reach a consensus on the high Tc mechanism. In this talk, we discuss two emergent principles, which are called as the correspondence principle and the selective magnetic pairing rule, to unify the understanding of both cuprates and iron-based superconductors. These two principles provide an unified explanation why the d-wave pairing symmetry and the s-wave pairing symmetry are robust respectively in cuprates and iron-based superconductors. In the meanwhile, the above two principles explain the rareness of unconventional high Tc superconductivity, identify necessary electronic environments required for high Tc superconductivity and finally serve as direct guiding rules to search new high Tc materials. We predict that the third family of unconventional high Tc superconductors exist in the compounds which carry two dimensional hexagonal lattices formed by cation-anion trigonal bipyramidal complexes with a d filling configuration on the cation ions. Their superconducting states are expected to be dominated by the d+id pairing symmetry and their maximum Tc should be higher than those of iron-based superconductors. Verifying the prediction can convincingly establish the high Tc superconducting mechanism and pave a way to design new high Tc superconductors

  6. Identifying reproducible cancer-associated highly expressed genes with important functional significances using multiple datasets

    PubMed Central

    Huang, Haiyan; Li, Xiangyu; Guo, You; Zhang, Yuncong; Deng, Xusheng; Chen, Lufei; Zhang, Jiahui; Guo, Zheng; Ao, Lu

    2016-01-01

    Identifying differentially expressed (DE) genes between cancer and normal tissues is of basic importance for studying cancer mechanisms. However, current methods, such as the commonly used Significance Analysis of Microarrays (SAM), are biased to genes with low expression levels. Recently, we proposed an algorithm, named the pairwise difference (PD) algorithm, to identify highly expressed DE genes based on reproducibility evaluation of top-ranked expression differences between paired technical replicates of cells under two experimental conditions. In this study, we extended the application of the algorithm to the identification of DE genes between two types of tissue samples (biological replicates) based on several independent datasets or sub-datasets of a dataset, by constructing multiple paired average gene expression profiles for the two types of samples. Using multiple datasets for lung and esophageal cancers, we demonstrated that PD could identify many DE genes highly expressed in both cancer and normal tissues that tended to be missed by the commonly used SAM. These highly expressed DE genes, including many housekeeping genes, were significantly enriched in many conservative pathways, such as ribosome, proteasome, phagosome and TNF signaling pathways with important functional significances in oncogenesis. PMID:27796338

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

    PubMed

    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

  8. Highly Fluorescent Gene Carrier Based on Ag-Au Alloy Nanoclusters.

    PubMed

    Wang, Ping; Lin, Lin; Guo, Zhaopei; Chen, Jie; Tian, Huayu; Chen, Xuesi; Yang, Hua

    2016-01-01

    For systemic delivery of gene, gold nanoparticles (GNPs) have been exploited as novel gene carriers because of the excellent characteristics for "visible" in intracellular trafficking. Herein, a highly fluorescent gene carrier was prepared by conjugating polyethylenimines on Ag-Au alloy nanoclusters. This carrier exhibited remarkable high gene transfection efficiencies and relatively low cytotoxicity toward B16F10, HeLa, and CHO cells. More interestingly, the high fluorescent Ag-Au-PEI conjugates showed high quantum yield of 14.56%, which is much higher than most of the reported gold nanocluster-based quantum dots and Ag-Au-PEI possessed bioimaging capacity both in vitro and in vivo. PMID:26287567

  9. An integrated probabilistic approach for gene function prediction using multiple sources of high-throughput data.

    PubMed

    Zhang, Chao; Joshi, Trupti; Lin, Guan Ning; Xu, Dong

    2008-01-01

    Characterising gene function is one of the major challenging tasks in the post-genomic era. Various approaches have been developed to integrate multiple sources of high-throughput data to predict gene function. Most of those approaches are just used for research purpose and have not been implemented as publicly available tools. Even for those implemented applications, almost all of them are still web-based 'prediction servers' that have to be managed by specialists. This paper introduces a systematic method for integrating various sources of high-throughput data to predict gene function and analyse our prediction results and evaluates its performances based on the competition for mouse gene function prediction (MouseFunc). A stand-alone Java-based software package 'GeneFAS' is freely available at http://digbio. missouri.eduigenefas.

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

    PubMed Central

    van den Broek, Evert; Dijkstra, Maurits J. J.; Krijgsman, Oscar; Sie, Daoud; Haan, Josien C.; Traets, Joleen J. H.; van de Wiel, Mark A.; Nagtegaal, Iris D.; Punt, Cornelis J. A.; Carvalho, Beatriz; Ylstra, Bauke; Abeln, Sanne; Meijer, Gerrit A.; Fijneman, Remond J. A.

    2015-01-01

    Background Cancer is caused by somatic DNA alterations such as gene point mutations, DNA copy number aberrations (CNA) and structural variants (SVs). Genome-wide analyses of SVs in large sample series with well-documented clinical information are still scarce. Consequently, the impact of SVs on carcinogenesis and patient outcome remains poorly understood. This study aimed to perform a systematic analysis of genes that are affected by CNA-associated chromosomal breaks in colorectal cancer (CRC) and to determine the clinical relevance of recurrent breakpoint genes. Methods Primary CRC samples of patients with metastatic disease from CAIRO and CAIRO2 clinical trials were previously characterized by array-comparative genomic hybridization. These data were now used to determine the prevalence of CNA-associated chromosomal breaks within genes across 352 CRC samples. In addition, mutation status of the commonly affected APC, TP53, KRAS, PIK3CA, FBXW7, SMAD4, BRAF and NRAS genes was determined for 204 CRC samples by targeted massive parallel sequencing. Clinical relevance was assessed upon stratification of patients based on gene mutations and gene breakpoints that were observed in >3% of CRC cases. Results In total, 748 genes were identified that were recurrently affected by chromosomal breaks (FDR <0.1). MACROD2 was affected in 41% of CRC samples and another 169 genes showed breakpoints in >3% of cases, indicating that prevalence of gene breakpoints is comparable to the prevalence of well-known gene point mutations. Patient stratification based on gene breakpoints and point mutations revealed one CRC subtype with very poor prognosis. Conclusions We conclude that CNA-associated chromosomal breaks within genes represent a highly prevalent and clinically relevant subset of SVs in CRC. PMID:26375816

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

    PubMed Central

    2012-01-01

    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. Conclusions Defense-related genes

  12. 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

    PubMed Central

    2010-01-01

    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 orientation within the gene

  13. Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae

    PubMed Central

    Azvolinsky, Anna; Giresi, Paul G.; Lieb, Jason D.; Zakian, Virginia A.

    2009-01-01

    SUMMARY Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. PMID:19560424

  14. High prevalence of DUOX2 gene mutations among children with congenital hypothyroidism in central China.

    PubMed

    Jiang, Hong; Wu, Jinhua; Ke, Shengzhong; Hu, Yue; Fei, Anxing; Zhen, Yan; Yu, Jin; Zhu, Kuichun

    2016-10-01

    Congenial hypothyroidism (CH) is the most common congenital endocrine disease and is treatable when recognized early enough. We investigated the genetic variants in 12 children diagnosed with CH by newborn screening in Huangshi area central China. Twelve genes commonly involved in CH development were studied. Genomic DNA from peripheral blood was used to amplify all exons of the selected genes, and the constructed sequencing libraries were subjected to next generation high throughput DNA sequencing (NGS). Analysis of the sequencing results identified rare genetic variants in 11 of the 12 patients (91.7%), and two novel rare variants were found in DUOX2 gene and two in TPO gene. Mutations in DUOX2 gene were identified in 10 patients (83.3%), and all these patients were found to carry bi-allelic, tri-allelic mutations or compound mutations with other genes. Recurrent DUOX2 mutations include K530X, R683L, R1110Q, and L1343F. Truncating, splicing, and proven deleterious DUOX2 missense mutations were detected in 50% of the patients. Mutations in TG gene were identified in four patients, and mutations in TPO, THSR, SLC26A4 genes were identified, one in each patient, respectively. The high prevalence of DUOX2 mutations in this cohort of children with CH appears striking and surprising. The clinical implications were discussed. PMID:27498126

  15. High prevalence of DUOX2 gene mutations among children with congenital hypothyroidism in central China.

    PubMed

    Jiang, Hong; Wu, Jinhua; Ke, Shengzhong; Hu, Yue; Fei, Anxing; Zhen, Yan; Yu, Jin; Zhu, Kuichun

    2016-10-01

    Congenial hypothyroidism (CH) is the most common congenital endocrine disease and is treatable when recognized early enough. We investigated the genetic variants in 12 children diagnosed with CH by newborn screening in Huangshi area central China. Twelve genes commonly involved in CH development were studied. Genomic DNA from peripheral blood was used to amplify all exons of the selected genes, and the constructed sequencing libraries were subjected to next generation high throughput DNA sequencing (NGS). Analysis of the sequencing results identified rare genetic variants in 11 of the 12 patients (91.7%), and two novel rare variants were found in DUOX2 gene and two in TPO gene. Mutations in DUOX2 gene were identified in 10 patients (83.3%), and all these patients were found to carry bi-allelic, tri-allelic mutations or compound mutations with other genes. Recurrent DUOX2 mutations include K530X, R683L, R1110Q, and L1343F. Truncating, splicing, and proven deleterious DUOX2 missense mutations were detected in 50% of the patients. Mutations in TG gene were identified in four patients, and mutations in TPO, THSR, SLC26A4 genes were identified, one in each patient, respectively. The high prevalence of DUOX2 mutations in this cohort of children with CH appears striking and surprising. The clinical implications were discussed.

  16. High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

    PubMed Central

    Brommage, Robert; Liu, Jeff; Hansen, Gwenn M; Kirkpatrick, Laura L; Potter, David G; Sands, Arthur T; Zambrowicz, Brian; Powell, David R; Vogel, Peter

    2014-01-01

    Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening (HTS) data for 3 762 distinct global gene knockout (KO) mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type (WT) cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass: 23 previously reported genes (Calcr, Cebpb, Crtap, Dcstamp, Dkk1, Duoxa2, Enpp1, Fgf23, Kiss1/Kiss1r, Kl (Klotho), Lrp5, Mstn, Neo1, Npr2, Ostm1, Postn, Sfrp4, Slc30a5, Slc39a13, Sost, Sumf1, Src, Wnt10b), five novel genes extensively characterized (Cldn18, Fam20c, Lrrk1, Sgpl1, Wnt16), five novel genes with preliminary characterization (Agpat2, Rassf5, Slc10a7, Slc26a7, Slc30a10) and three novel undisclosed genes coding for potential osteoporosis drug targets. PMID:26273529

  17. A novel highly differentially expressed gene in wheat endosperm associated with bread quality

    PubMed Central

    Furtado, A.; Bundock, P. C.; Banks, P. M.; Fox, G.; Yin, X.; Henry, R. J.

    2015-01-01

    Analysis of gene expression in developing wheat seeds was used to identify a gene, wheat bread making (wbm), with highly differential expression (~1000 fold) in the starchy endosperm of genotypes varying in bread making quality. Several alleles differing in the 5’-upstream region (promoter) of this gene were identified, with one present only in genotypes with high levels of wbm expression. RNA-Seq analysis revealed low or no wbm expression in most genotypes but high expression (0.2-0.4% of total gene expression) in genotypes that had good bread loaf volume. The wbm gene is predicted to encode a mature protein of 48 amino acids (including four cysteine residues) not previously identified in association with wheat quality, possibly because of its small size and low frequency in the wheat gene pool. Genotypes with high wbm expression all had good bread making quality but not always good physical dough qualities. The predicted protein was sulphur rich suggesting the possibility of a contribution to bread loaf volume by supporting the crossing linking of proteins in gluten. Improved understanding of the molecular basis of differences in bread making quality may allow more rapid development of high performing genotypes with acceptable end-use properties and facilitate increased wheat production. PMID:26011437

  18. Biocleavable Polycationic Micelles as Highly Efficient Gene Delivery Vectors

    NASA Astrophysics Data System (ADS)

    Zhang, Min; Xue, Ya-Nan; Liu, Min; Zhuo, Ren-Xi; Huang, Shi-Wen

    2010-11-01

    An amphiphilic disulfide-containing polyamidoamine was synthesized by Michael-type polyaddition reaction of piperazine to equimolar N, N'-bis(acryloyl)cystamine with 90% yield. The polycationic micelles (198 nm, 32.5 mV), prepared from the amphiphilic polyamidoamine by dialysis method, can condense foreign plasmid DNA to form nanosized polycationic micelles/DNA polyelectrolyte complexes with positive charges, which transfected 293T cells with high efficiency. Under optimized conditions, the transfection efficiencies of polycationic micelles/DNA complexes are comparable to, or even higher than that of commercially available branched PEI (Mw 25 kDa).

  19. Sclera-related gene polymorphisms in high myopia

    PubMed Central

    Lin, Hui-Ju; Tsai, Yuhsin; Liu, Su-Ching; Chen, Wen-Chi; Tsai, Shih-Wei; Tsai, Fuu-Jen

    2009-01-01

    Purpose Transforming growth factor-β2 (TGF-β2), basic fibroblast growth factor (bFGF), and fibromodulin (FMOD) are important extracellular matrix components of the sclera and have been shown to be associated with the development of high myopia. Our aim was to examine the association between myopia and the polymorphisms within TGF-β2, bFGF, and FMOD. Methods The study group comprised of patients (n=195; age range: 17−24 years) with a spherical equivalent of −6.5 diopters (D) or a more negative refractive error. The control group comprised of individuals (n=94; age range: 17−25 years) with a spherical equivalent ranging from −0.5 D to +1.0 D. The subjects with astigmatism over –0.75 D were excluded from the study. High resolution melting (HRM) genotyping and restriction fragment length polymorphism (RFLP) genotyping were used to detect single nucleotide polymorphisms (SNPs). The polymorphisms detected were TGF-β2 (rs7550232 and rs991967), bFGF (rs308395 and rs41348645), and FMOD (rs7543418). Moreover, a stepwise logistic regression procedure was used to detect which of the significant SNPs contributed to the main effects of myopia development. Results There were significant differences in the frequency of the A allele and A/A genotype in TGF-β2 (rs7550232; p=0.0178 and 0.03, respectively). Moreover, the haplotype distribution of haplotype 2 (Ht2; A/A) of TGF-β2 differed significantly between the two groups (p=0.014). The results of the stepwise logistic regression procedure revealed that TGF-β2 (rs7550232) contributed significantly to the development of high myopia. Conclusions TGF-β2 is an important structure of sclera and might contribute to the formation of myopia. TGF-β2 (rs7550232) polymorphisms, A allele and A/A genotype, had a protective role against the development of high myopia. PMID:19710942

  20. Identification of candidate genes related to rice grain weight under high-temperature stress.

    PubMed

    Liao, Jiang-Lin; Zhang, Hong-Yu; Liu, Jun-Bao; Zhong, Ping-An; Huang, Ying-Jin

    2012-11-01

    The rise of global warming presents a problem for all living organisms, including rice and other staple plants. High temperatures impair rice grain weight by inhibiting the filling of the caryopses during the milky stage. The molecular mechanism behind this process, however, is poorly understood. Identifying candidate genes involved in responses to high-temperature stress may provide a basis for the improvement of heat tolerance in rice. Using paired, genetically similar heat-tolerant and heat-sensitive rice lines as plant materials, cDNA-AFLP analysis revealed a total of 54 transcript derived fragments (TDFs), mainly from the heat-tolerant lines. This clearly indicated variations in gene expression between the two rice lines. BLAST results showed that 28 of the 54 TDFs were homologous sequences. These homologous genes were found to encode proteins involved in signal transduction, oxidation, transcriptional regulation, transport, and metabolism. The functions and differential expression patterns of some important genes are further discussed. High temperature stress may trigger a wide range of changes in gene expression in rice caryopses, in turn affecting functions ranging from signal transduction to cellular metabolism. Forty-five of the 54 TDFs were mapped to rice chromosomes. The genes identified in the present study would make good candidates for further study into the molecular mechanisms underlying rice adaptation to high-temperature stress.

  1. Epigenetic priming of inflammatory response genes by high glucose in adipose progenitor cells.

    PubMed

    Rønningen, Torunn; Shah, Akshay; Reiner, Andrew H; Collas, Philippe; Moskaug, Jan Øivind

    2015-11-27

    Cellular metabolism confers wide-spread epigenetic modifications required for regulation of transcriptional networks that determine cellular states. Mesenchymal stromal cells are responsive to metabolic cues including circulating glucose levels and modulate inflammatory responses. We show here that long term exposure of undifferentiated human adipose tissue stromal cells (ASCs) to high glucose upregulates a subset of inflammation response (IR) genes and alters their promoter histone methylation patterns in a manner consistent with transcriptional de-repression. Modeling of chromatin states from combinations of histone modifications in nearly 500 IR genes unveil three overarching chromatin configurations reflecting repressive, active, and potentially active states in promoter and enhancer elements. Accordingly, we show that adipogenic differentiation in high glucose predominantly upregulates IR genes. Our results indicate that elevated extracellular glucose levels sensitize in ASCs an IR gene expression program which is exacerbated during adipocyte differentiation. We propose that high glucose exposure conveys an epigenetic 'priming' of IR genes, favoring a transcriptional inflammatory response upon adipogenic stimulation. Chromatin alterations at IR genes by high glucose exposure may play a role in the etiology of metabolic diseases.

  2. Contribution of nonohnologous duplicated genes to high habitat variability in mammals.

    PubMed

    Tamate, Satoshi C; Kawata, Masakado; Makino, Takashi

    2014-07-01

    The mechanism by which genetic systems affect environmental adaptation is a focus of considerable attention in the fields of ecology, evolution, and conservation. However, the genomic characteristics that constrain adaptive evolution have remained unknown. A recent study showed that the proportion of duplicated genes in whole Drosophila genomes correlated with environmental variability within habitat, but it remains unclear whether the correlation is observed even in vertebrates whose genomes including a large number of duplicated genes generated by whole-genome duplication (WGD). Here, we focus on fully sequenced mammalian genomes that experienced WGD in early vertebrate lineages and show that the proportion of small-scale duplication (SSD) genes in the genome, but not that of WGD genes, is significantly correlated with habitat variability. Moreover, species with low habitat variability have a higher proportion of lost duplicated genes, particularly SSD genes, than those with high habitat variability. These results indicate that species that inhabit variable environments may maintain more SSD genes in their genomes and suggest that SSD genes are important for adapting to novel environments and surviving environmental changes. These insights may be applied to predicting invasive and endangered species. PMID:24714078

  3. Phylogenetic Resolution of Deep Eukaryotic and Fungal Relationships Using Highly Conserved Low-Copy Nuclear Genes.

    PubMed

    Ren, Ren; Sun, Yazhou; Zhao, Yue; Geiser, David; Ma, Hong; Zhou, Xiaofan

    2016-09-11

    A comprehensive and reliable eukaryotic tree of life is important for many aspects of biological studies from comparative developmental and physiological analyses to translational medicine and agriculture. Both gene-rich and taxon-rich approaches are effective strategies to improve phylogenetic accuracy and are greatly facilitated by marker genes that are universally distributed, well conserved, and orthologous among divergent eukaryotes. In this article, we report the identification of 943 low-copy eukaryotic genes and we show that many of these genes are promising tools in resolving eukaryotic phylogenies, despite the challenges of determining deep eukaryotic relationships. As a case study, we demonstrate that smaller subsets of ∼20 and 52 genes could resolve controversial relationships among widely divergent taxa and provide strong support for deep relationships such as the monophyly and branching order of several eukaryotic supergroups. In addition, the use of these genes resulted in fungal phylogenies that are congruent with previous phylogenomic studies that used much larger datasets, and successfully resolved several difficult relationships (e.g., forming a highly supported clade with Microsporidia, Mitosporidium and Rozella sister to other fungi). We propose that these genes are excellent for both gene-rich and taxon-rich analyses and can be applied at multiple taxonomic levels and facilitate a more complete understanding of the eukaryotic tree of life.

  4. Phylogenetic Resolution of Deep Eukaryotic and Fungal Relationships Using Highly Conserved Low-Copy Nuclear Genes.

    PubMed

    Ren, Ren; Sun, Yazhou; Zhao, Yue; Geiser, David; Ma, Hong; Zhou, Xiaofan

    2016-01-01

    A comprehensive and reliable eukaryotic tree of life is important for many aspects of biological studies from comparative developmental and physiological analyses to translational medicine and agriculture. Both gene-rich and taxon-rich approaches are effective strategies to improve phylogenetic accuracy and are greatly facilitated by marker genes that are universally distributed, well conserved, and orthologous among divergent eukaryotes. In this article, we report the identification of 943 low-copy eukaryotic genes and we show that many of these genes are promising tools in resolving eukaryotic phylogenies, despite the challenges of determining deep eukaryotic relationships. As a case study, we demonstrate that smaller subsets of ∼20 and 52 genes could resolve controversial relationships among widely divergent taxa and provide strong support for deep relationships such as the monophyly and branching order of several eukaryotic supergroups. In addition, the use of these genes resulted in fungal phylogenies that are congruent with previous phylogenomic studies that used much larger datasets, and successfully resolved several difficult relationships (e.g., forming a highly supported clade with Microsporidia, Mitosporidium and Rozella sister to other fungi). We propose that these genes are excellent for both gene-rich and taxon-rich analyses and can be applied at multiple taxonomic levels and facilitate a more complete understanding of the eukaryotic tree of life. PMID:27604879

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    Summary 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. PMID:26771356

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

    PubMed

    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. High-resolution prediction of mouse brain connectivity using gene expression patterns.

    PubMed

    Fakhry, Ahmed; Ji, Shuiwang

    2015-02-01

    The brain is a multi-level system in which the high-level functions are generated by low-level genetic mechanisms. Thus, elucidating the relationship among multiple brain levels via correlative and predictive analytics is an important area in brain research. Currently, studies in multiple species have indicated that the spatiotemporal gene expression patterns are predictive of brain wiring. Specifically, results on the worm Caenorhabditis elegans have shown that the prediction of neuronal connectivity using gene expression signatures yielded statistically significant results. Recent studies on the mammalian brain produced similar results at the coarse regional level. In this study, we provide the first high-resolution, large-scale integrative analysis of the transcriptome and connectome in a single mammalian brain at a fine voxel level. By using the Allen Brain Atlas data, we predict voxel-level brain connectivity based on the gene expressions in the adult mouse brain. We employ regularized models to show that gene expression is predictive of connectivity at the voxel-level with an accuracy of 93%. We also identify a set of genes playing the most important role in connectivity prediction. We use only this small number of genes to predict the brain wiring with an accuracy over 80%. We discover that these important genes are enriched in neurons as compared to glia, and they perform connectivity-related functions. We perform several interesting correlative studies to further elucidate the transcriptome-connectome relationship.

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

    PubMed

    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.

  10. High Throughput Gene Expression Measurement with Real Time PCR in a Microfluidic Dynamic Array

    PubMed Central

    Spurgeon, Sandra L.; Jones, Robert C.; Ramakrishnan, Ramesh

    2008-01-01

    We describe a high throughput gene expression platform based on microfluidic dynamic arrays. This system allows 2,304 simultaneous real time PCR gene expression measurements in a single chip, while requiring less pipetting than is required to set up a 96 well plate. We show that one can measure the expression of 45 different genes in 18 tissues with replicates in a single chip. The data have excellent concordance with conventional real time PCR and the microfluidic dynamic arrays show better reproducibility than commercial DNA microarrays. PMID:18301740

  11. Transfection microarrays for high-throughput phenotypic screening of genes involved in cell migration.

    PubMed

    Onuki-Nagasaki, Reiko; Nagasaki, Akira; Hakamada, Kazumi; Uyeda, Taro Q P; Fujita, Satoshi; Miyake, Masato; Miyake, Jun

    2010-01-01

    Cell migration is important in several biological phenomena, such as cancer metastasis. Therefore, the identification of genes involved in cell migration might facilitate the discovery of antimetastatic drugs. However, screening of genes by the current methods can be complicated by factors related to cell stimulation, for example, abolition of contact inhibition and the release inflammatory cytokines from wounded cells during examinations of wound healing in vitro. To overcome these problems and identify genes involved in cell migration, in this chapter we describe the use of transfection microarrays for high-throughput phenotypic screening. PMID:20387151

  12. High-Throughput Screening of Tyrosine Kinase Inhibitor Resistant Genes in CML.

    PubMed

    Ma, Leyuan; Roderick, Justine; Kelliher, Michelle A; Green, Michael R

    2016-01-01

    Genome-wide RNA interference (RNAi) screening in mammalian cells has proven to be a powerful tool for identifying new genes and molecular pathways relevant to many cellular processes and diseases. For example, screening for genes that, when inactivated, lead to resistance to cancer therapeutic drugs can reveal new mechanisms for how resistance develops and identify potential targetable strategies to overcome drug resistance. Here, we describe a detailed procedure for performing a high-throughput RNAi screen using a genome-wide human short hairpin RNA (shRNA) library for identifying tyrosine kinase inhibitor (TKI)-resistance genes in a human CML cell line model. PMID:27581147

  13. High- and low-threshold genes in the Spo0A regulon of Bacillus subtilis.

    PubMed

    Fujita, Masaya; González-Pastor, José Eduardo; Losick, Richard

    2005-02-01

    The master regulator for entry into sporulation in Bacillus subtilis is the response regulator Spo0A, which directly governs the expression of about 121 genes. Using cells in which the synthesis of Spo0A was under the control of an inducible promoter or in which production of the regulatory protein was impaired by a promoter mutation, we found that sporulation required a high (threshold) level of Spo0A and that many genes in the regulon differentially responded to high and low doses of the regulator. We distinguished four categories of genes, as follows: (i) those that required a high level of Spo0A to be activated, (ii) those that required a high level of Spo0A to be repressed, (iii) those that were activated at a low level of the regulator, and (iv) those that were repressed at a low dose of the regulator. Genes that required a high dose of Spo0A to be activated were found to have low binding constants for the DNA-binding protein. Some genes that were turned on at a low dose of Spo0A either had a high binding constant for the regulatory protein or were activated by an indirect mechanism involving Spo0A-mediated relief of repression by the repressor protein AbrB. We propose that progressive increases in the level of Spo0A leads to an early phase of transcription in which genes that play auxiliary roles in development, such as cannibalism and biofilm formation, are turned on and a later phase in which genes that play a direct role in sporulation are activated. PMID:15687200

  14. A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes.

    PubMed

    Herbold, Craig W; Pelikan, Claus; Kuzyk, Orest; Hausmann, Bela; Angel, Roey; Berry, David; Loy, Alexander

    2015-01-01

    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. PMID:26236305

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

    PubMed

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

    2015-07-14

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

  16. High divergence in primate-specific duplicated regions: Human and chimpanzee Chorionic Gonadotropin Beta genes

    PubMed Central

    2008-01-01

    Background Low nucleotide divergence between human and chimpanzee does not sufficiently explain the species-specific morphological, physiological and behavioral traits. As gene duplication is a major prerequisite for the emergence of new genes and novel biological processes, comparative studies of human and chimpanzee duplicated genes may assist in understanding the mechanisms behind primate evolution. We addressed the divergence between human and chimpanzee duplicated genomic regions by using Luteinizing Hormone Beta (LHB)/Chorionic Gonadotropin Beta (CGB) gene cluster as a model. The placental CGB genes that are essential for implantation have evolved from an ancestral pituitary LHB gene by duplications in the primate lineage. Results We shotgun sequenced and compared the human (45,165 bp) and chimpanzee (39,876 bp) LHB/CGB regions and hereby present evidence for structural variation resulting in discordant number of CGB genes (6 in human, 5 in chimpanzee). The scenario of species-specific parallel duplications was supported (i) as the most parsimonious solution requiring the least rearrangement events to explain the interspecies structural differences; (ii) by the phylogenetic trees constructed with fragments of intergenic regions; (iii) by the sequence similarity calculations. Across the orthologous regions of LHB/CGB cluster, substitutions and indels contributed approximately equally to the interspecies divergence and the distribution of nucleotide identity was correlated with the regional repeat content. Intraspecies gene conversion may have shaped the LHB/CGB gene cluster. The substitution divergence (1.8–2.59%) exceeded two-three fold the estimates for single-copy loci and the fraction of transversional mutations was increased compared to the unique sequences (43% versus ~30%). Despite the high sequence identity among LHB/CGB genes, there are signs of functional differentiation among the gene copies. Estimates for dn/ds rate ratio suggested a purifying

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

    PubMed Central

    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

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed

    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.

  1. High-resolution genome-wide scan of genes, gene-networks and cellular systems impacting the yeast ionome

    PubMed Central

    2012-01-01

    Background To balance the demand for uptake of essential elements with their potential toxicity living cells have complex regulatory mechanisms. Here, we describe a genome-wide screen to identify genes that impact the elemental composition (‘ionome’) of yeast Saccharomyces cerevisiae. Using inductively coupled plasma – mass spectrometry (ICP-MS) we quantify Ca, Cd, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, S and Zn in 11890 mutant strains, including 4940 haploid and 1127 diploid deletion strains, and 5798 over expression strains. Results We identified 1065 strains with an altered ionome, including 584 haploid and 35 diploid deletion strains, and 446 over expression strains. Disruption of protein metabolism or trafficking has the highest likelihood of causing large ionomic changes, with gene dosage also being important. Gene over expression produced more extreme ionomic changes, but over expression and loss of function phenotypes are generally not related. Ionomic clustering revealed the existence of only a small number of possible ionomic profiles suggesting fitness tradeoffs that constrain the ionome. Clustering also identified important roles for the mitochondria, vacuole and ESCRT pathway in regulation of the ionome. Network analysis identified hub genes such as PMR1 in Mn homeostasis, novel members of ionomic networks such as SMF3 in vacuolar retrieval of Mn, and cross-talk between the mitochondria and the vacuole. All yeast ionomic data can be searched and downloaded at http://www.ionomicshub.org. Conclusions Here, we demonstrate the power of high-throughput ICP-MS analysis to functionally dissect the ionome on a genome-wide scale. The information this reveals has the potential to benefit both human health and agriculture. PMID:23151179

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

    PubMed

    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.

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

    PubMed

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

    2015-05-01

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

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

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

    Gene expression variation within species is relatively common, however, the role of natural selection in the maintenance of this variation is poorly understood. Here we investigate low and high latitude populations of Drosophila melanogaster and its sister species, D. simulans, to determine whether the two species show similar patterns of population differentiation, consistent with a role for spatially varying selection in maintaining gene expression variation. We compared at two temperatures the whole male transcriptome of D. melanogaster and D. simulans sampled from Panama City (Panama) and Maine (USA). We observed a significant excess of genes exhibiting differential expression in both species, consistent with parallel adaptation to heterogeneous environments. Moreover, the majority of genes showing parallel expression differentiation showed the same direction of differential expression in the two species and the magnitudes of expression differences between high and low latitude populations were correlated across species, further bolstering the conclusion that parallelism for expression phenotypes results from spatially varying selection. However, the species also exhibited important differences in expression phenotypes. For example, the genomic extent of genotype × environment interaction was much more common in D. melanogaster. Highly differentiated SNPs between low and high latitudes were enriched in the 3’ UTRs and CDS of the geographically differently expressed genes in both species, consistent with an important role for cis-acting variants in driving local adaptation for expression-related phenotypes. PMID:25950438

  6. Comparative gene expression profiles for highly similar aggressive phenotypes in male and female cichlid fishes (Julidochromis)

    PubMed Central

    Schumer, Molly; Krishnakant, Kavita; Renn, Suzy C. P.

    2011-01-01

    SUMMARY Julidochromis marlieri and Julidochromis transcriptus are two closely related Tanganyikan cichlids that have evolved different behavior and mating strategies since they diverged from their common ancestor. While J. transcriptus follows the ancestral pattern of male dominance, male-biased sexual size dimorphism and territoriality, the pattern is reversed in J. marlieri. In J. marlieri, females show all of these behavioral and morphological characteristics. This raises the question of whether female J. marlieri achieve the dominant phenotype by expressing the same genes as J. transcriptus males or whether novel brain gene expression patterns have evolved to produce a similar behavioral phenotype in the females of J. marlieri. This study used cDNA microarrays to investigate whether female J. marlieri and male J. transcriptus show conserved or divergent patterns of brain gene expression. Analysis of microarray data in both species showed certain gene expression patterns associated with sex role independent of gonadal sex and, to a lesser extent, gene expression patterns associated with sex independent of sex role. In general, these data suggest that while there has been substantial divergence in gene expression patterns between J. transcriptus and J. marlieri, we can detect a highly significant overlap for a core set of genes related to aggression in both species. These results suggest that the proximate mechanisms regulating aggressive behavior in J. transcriptus and J. marlieri may be shared. PMID:21900474

  7. Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs

    PubMed Central

    Cade, Lindsay; Reyon, Deepak; Hwang, Woong Y.; Tsai, Shengdar Q.; Patel, Samir; Khayter, Cyd; Joung, J. Keith; Sander, Jeffry D.; Peterson, Randall T.; Yeh, Jing-Ruey Joanna

    2012-01-01

    Transcription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission. For one target gene sequence, we observed substantially reduced mutagenesis efficiency for a variant site bearing two mismatched nucleotides, raising the possibility that TALENs might be used to perform allele-specific gene disruption. Our results suggest that construction of one to two heterodimeric TALEN pairs for any given gene will, in most cases, enable researchers to rapidly generate knockout zebrafish. PMID:22684503

  8. 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.

    PubMed

    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.

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

    PubMed

    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.

  10. Evolution of high cellulolytic activity in symbiotic Streptomyces through selection of expanded gene content and coordinated gene expression

    DOE PAGESBeta

    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.

    2016-06-08

    In this study, 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 andmore » 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.« less

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

    PubMed

    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

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

    PubMed Central

    McDonald, Bradon R.; Takasuka, Taichi E.; Wendt-Pienkowski, Evelyn; Doering, Drew T.; Raffa, Kenneth F.; Fox, Brian G.; Currie, Cameron R.

    2016-01-01

    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

  13. High-throughput platform for the discovery of elicitors of silent bacterial gene clusters.

    PubMed

    Seyedsayamdost, Mohammad R

    2014-05-20

    Over the past decade, bacterial genome sequences have revealed an immense reservoir of biosynthetic gene clusters, sets of contiguous genes that have the potential to produce drugs or drug-like molecules. However, the majority of these gene clusters appear to be inactive for unknown reasons prompting terms such as "cryptic" or "silent" to describe them. Because natural products have been a major source of therapeutic molecules, methods that rationally activate these silent clusters would have a profound impact on drug discovery. Herein, a new strategy is outlined for awakening silent gene clusters using small molecule elicitors. In this method, a genetic reporter construct affords a facile read-out for activation of the silent cluster of interest, while high-throughput screening of small molecule libraries provides potential inducers. This approach was applied to two cryptic gene clusters in the pathogenic model Burkholderia thailandensis. The results not only demonstrate a prominent activation of these two clusters, but also reveal that the majority of elicitors are themselves antibiotics, most in common clinical use. Antibiotics, which kill B. thailandensis at high concentrations, act as inducers of secondary metabolism at low concentrations. One of these antibiotics, trimethoprim, served as a global activator of secondary metabolism by inducing at least five biosynthetic pathways. Further application of this strategy promises to uncover the regulatory networks that activate silent gene clusters while at the same time providing access to the vast array of cryptic molecules found in bacteria.

  14. A fast and high performance multiple data integration algorithm for identifying human disease genes

    PubMed Central

    2015-01-01

    Background Integrating multiple data sources is indispensable in improving disease gene identification. It is not only due to the fact that disease genes associated with similar genetic diseases tend to lie close with each other in various biological networks, but also due to the fact that gene-disease associations are complex. Although various algorithms have been proposed to identify disease genes, their prediction performances and the computational time still should be further improved. Results In this study, we propose a fast and high performance multiple data integration algorithm for identifying human disease genes. A posterior probability of each candidate gene associated with individual diseases is calculated by using a Bayesian analysis method and a binary logistic regression model. Two prior probability estimation strategies and two feature vector construction methods are developed to test the performance of the proposed algorithm. Conclusions The proposed algorithm is not only generated predictions with high AUC scores, but also runs very fast. When only a single PPI network is employed, the AUC score is 0.769 by using F2 as feature vectors. The average running time for each leave-one-out experiment is only around 1.5 seconds. When three biological networks are integrated, the AUC score using F3 as feature vectors increases to 0.830, and the average running time for each leave-one-out experiment takes only about 12.54 seconds. It is better than many existing algorithms. PMID:26399620

  15. Suppression of α-amylase genes improves quality of rice grain ripened under high temperature.

    PubMed

    Hakata, Makoto; Kuroda, Masaharu; Miyashita, Tomomi; Yamaguchi, Takeshi; Kojima, Mikiko; Sakakibara, Hitoshi; Mitsui, Toshiaki; Yamakawa, Hiromoto

    2012-12-01

    High temperature impairs rice (Oryza sativa) grain filling by inhibiting the deposition of storage materials such as starch, resulting in mature grains with a chalky appearance, currently a major problem for rice farming in Asian countries. Such deterioration of grain quality is accompanied by the altered expression of starch metabolism-related genes. Here we report the involvement of a starch-hydrolyzing enzyme, α-amylase, in high temperature-triggered grain chalkiness. In developing seeds, high temperature induced the expression of α-amylase genes, namely Amy1A, Amy1C, Amy3A, Amy3D and Amy3E, as well as α-amylase activity, while it decreased an α-amylase-repressing plant hormone, ABA, suggesting starch to be degraded by α-amylase in developing grains under elevated temperature. Furthermore, RNAi-mediated suppression of α-amylase genes in ripening seeds resulted in fewer chalky grains under high-temperature conditions. As the extent of the decrease in chalky grains was highly correlated to decreases in the expression of Amy1A, Amy1C, Amy3A and Amy3B, these genes would be involved in the chalkiness through degradation of starch accumulating in the developing grains. The results show that activation of α-amylase by high temperature is a crucial trigger for grain chalkiness and that its suppression is a potential strategy for ameliorating grain damage from global warming.

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

    PubMed

    Meyer, Michael A

    2014-04-22

    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. PMID:24987507

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

    PubMed Central

    Meyer, Michael A.

    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 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 PMID:24987507

  18. Meta-analysis of clinical data using human meiotic genes identifies a novel cohort of highly restricted cancer-specific marker genes

    PubMed Central

    Feichtinger, Julia; Almutairi, Mikhlid; Almatrafi, Ahmed; Alsiwiehri, Naif; Griffiths, Keith; Stuart, Nicholas; Wakeman, Jane A.; Larcombe, Lee; McFarlane, Ramsay J.

    2012-01-01

    Identifying cancer-specific biomarkers represents an ongoing challenge to the development of novel cancer diagnostic, prognostic and therapeutic strategies. Cancer/testis (CT) genes are an important gene family with expression tightly restricted to the testis in normal individuals but which can also be activated in cancers. Here we develop a pipeline to identify new CT genes. We analysed and validated expression profiles of human meiotic genes in normal and cancerous tissue followed by meta-analyses of clinical data sets from a range of tumour types resulting in the identification of a large cohort of highly specific cancer biomarker genes, including the recombination hot spot activator PRDM9 and the meiotic cohesin genes SMC1beta and RAD21L. These genes not only provide excellent cancer biomarkers for diagnostics and prognostics, but may serve as oncogenes and have excellent drug targeting potential. PMID:22918178

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

    PubMed Central

    Aruga, Jun; Odaka, Yuri S; Kamiya, Akiko; Furuya, Hidetaka

    2007-01-01

    Background Dicyemid mesozoans (Phylum Dicyemida) are simple (8–40-cell) cephalopod endoparasites. They have neither body cavities nor differentiated organs, such as nervous and gastrointestinal systems. Whether dicyemids are intermediate between Protozoa and Metazoa (as represented by their "Mesozoa" classification) or degenerate species of more complex metazoans is controversial. Recent molecular phylogenetic studies suggested that they are simplified bilaterians belonging to the Lophotrochozoa. We cloned two genes developmentally critical in bilaterian animals (Pax6 and Zic), together with housekeeping genes (actin, fructose-bisphosphate aldolase, and ATP synthase beta subunit) from a dicyemid to reveal whether their molecular phylogeny supported the "simplification" hypothesis, and to clarify evolutionary changes in dicyemid gene structure and expression profiles. Results Genomic/cDNA sequence analysis showed that 1) the Pax6 molecular phylogeny and Zic intron positions supported the idea of dicyemids as reduced bilaterians; 2) the aa sequences deduced from the five genes were highly divergent; and 3) Dicyema genes contained very short introns of uniform length. In situ hybridization analyses revealed that Zic genes were expressed in hermaphroditic gonads, and Pax6 was expressed weakly throughout the developmental stages of the 2 types of embryo and in the hermaphroditic gonads. Conclusion The accelerated evolutionary rates and very short and uniform intron may represent a part of Dicyema genomic features. The presence and expression of the two tool-kit genes (Pax6 and Zic) in Dicyema suggests that they can be very versatile genes even required for the highly reduced bilaterian like Dicyema. Dicyemids may be useful models of evolutionary body plan simplification. PMID:17961212

  20. High-resolution chromosome ideogram representation of recognized genes for bipolar disorder.

    PubMed

    Douglas, Lindsay N; McGuire, Austen B; Manzardo, Ann M; Butler, Merlin G

    2016-07-15

    Bipolar disorder (BPD) is genetically heterogeneous with a growing list of BPD associated genes reported in recent years resulting from increased genetic testing using advanced genetic technology, expanded genomic databases, and better awareness of the disorder. We compiled a master list of recognized susceptibility and genes associated with BPD identified from peer-reviewed medical literature sources using PubMed and by searching online databases, such as OMIM. Searched keywords were related to bipolar disorder and genetics. Our compiled list consisted of 290 genes with gene names arranged in alphabetical order in tabular form with source documents and their chromosome location and gene symbols plotted on high-resolution human chromosome ideograms. The identified genes impacted a broad range of biological pathways and processes including cellular signaling pathways particularly cAMP and calcium (e.g., CACNA1C, CAMK2A, CAMK2D, ADCY1, ADCY2); glutamatergic (e.g., GRIK1, GRM3, GRM7), dopaminergic (e.g., DRD2, DRD4, COMT, MAOA) and serotonergic (e.g., HTR1A, HTR2A, HTR3B) neurotransmission; molecular transporters (e.g., SLC39A3, SLC6A3, SLC8A1); and neuronal growth (e.g., BDNF, IGFBP1, NRG1, NRG3). The increasing prevalence of BPD calls for better understanding of the genetic etiology of this disorder and associations between the observed BPD phenotype and genes. Visual representation of genes for bipolar disorder becomes a tool enabling clinical and laboratory geneticists, genetic counselors, and other health care providers and researchers easy access to the location and distribution of currently recognized BPD associated genes. Our study may also help inform diagnosis and advance treatment developments for those affected with this disorder and improve genetic counseling for families. PMID:27063557

  1. High-resolution chromosome ideogram representation of recognized genes for bipolar disorder.

    PubMed

    Douglas, Lindsay N; McGuire, Austen B; Manzardo, Ann M; Butler, Merlin G

    2016-07-15

    Bipolar disorder (BPD) is genetically heterogeneous with a growing list of BPD associated genes reported in recent years resulting from increased genetic testing using advanced genetic technology, expanded genomic databases, and better awareness of the disorder. We compiled a master list of recognized susceptibility and genes associated with BPD identified from peer-reviewed medical literature sources using PubMed and by searching online databases, such as OMIM. Searched keywords were related to bipolar disorder and genetics. Our compiled list consisted of 290 genes with gene names arranged in alphabetical order in tabular form with source documents and their chromosome location and gene symbols plotted on high-resolution human chromosome ideograms. The identified genes impacted a broad range of biological pathways and processes including cellular signaling pathways particularly cAMP and calcium (e.g., CACNA1C, CAMK2A, CAMK2D, ADCY1, ADCY2); glutamatergic (e.g., GRIK1, GRM3, GRM7), dopaminergic (e.g., DRD2, DRD4, COMT, MAOA) and serotonergic (e.g., HTR1A, HTR2A, HTR3B) neurotransmission; molecular transporters (e.g., SLC39A3, SLC6A3, SLC8A1); and neuronal growth (e.g., BDNF, IGFBP1, NRG1, NRG3). The increasing prevalence of BPD calls for better understanding of the genetic etiology of this disorder and associations between the observed BPD phenotype and genes. Visual representation of genes for bipolar disorder becomes a tool enabling clinical and laboratory geneticists, genetic counselors, and other health care providers and researchers easy access to the location and distribution of currently recognized BPD associated genes. Our study may also help inform diagnosis and advance treatment developments for those affected with this disorder and improve genetic counseling for families.

  2. A Comprehensive Analysis of the Combined Effects of High Light and High Temperature Stresses on Gene Expression in Sunflower

    PubMed Central

    Hewezi, Tarek; Léger, Mathieu; Gentzbittel, Laurent

    2008-01-01

    Background and Aims Although high light (HL) and high temperature (HT) stresses have been extensively investigated, a global analysis of their combined effects on the transcriptome of any plant species has not yet been described. Sunflower is an agronomically important oil crop frequently subjected to these stress factors. Because results in model plants may not always translate well to crop plants, responses of sunflower (Helianthus annuus) to HL, HT and a combination of both stresses were analysed by profiling gene expression in leaves and immature seeds. Methods Plants were grown in HL (600 µE m−2 s−1), HT (35 °C) and a combination of HL and HT (HL + HT), and gene expression in leaves and immature seeds was profiled using cDNA microarrays containing more than 8000 putative unigenes. Key Results Using two-way analysis of variance, 105, 55 and 129 cDNA clones were identified showing significant changes in steady-state transcript levels, across the two tissues, in response to HL, HT and HL + HT, respectively. A significant number of these transcripts were found to be specific to each stress. Comparing gene expression profiles between leaves and immature seeds revealed that 89, 113 and 186 cDNA clones can be considered as differentially expressed in response to HL, HT and HL + HT, respectively. More than half of the cDNA clones showing significant differences between embryo and leaf tissues in response to HL + HT were specific to this stress. Significant differences between leaves and seeds shared by all three stress treatments were observed for only eight genes. Conclusions Taken together, these results indicate that vegetative and reproductive tissues employ different transcriptome responses to these stress treatments. Careful examination of the putative functions of these genes revealed novel and specific responses. The potential roles of many of the differentially expressed genes in stress tolerance are mentioned and discussed. PMID:18477560

  3. Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation.

    PubMed

    Beck, Michaël; Rombouts, Charlotte; Moreels, Marjan; Aerts, An; Quintens, Roel; Tabury, Kevin; Michaux, Arlette; Janssen, Ann; Neefs, Mieke; Ernst, Eric; Dieriks, Birger; Lee, Ryonfa; De Vos, Winnok H; Lambert, Charles; Van Oostveldt, Patrick; Baatout, Sarah

    2014-10-01

    Ionizing radiation can elicit harmful effects on the cardiovascular system at high doses. Endothelial cells are critical targets in radiation-induced cardiovascular damage. Astronauts performing a long-term deep space mission are exposed to consistently higher fluences of ionizing radiation that may accumulate to reach high effective doses. In addition, cosmic radiation contains high linear energy transfer (LET) radiation that is known to produce high values of relative biological effectiveness (RBE). The aim of this study was to broaden the understanding of the molecular response to high LET radiation by investigating the changes in gene expression in endothelial cells. For this purpose, a human endothelial cell line (EA.hy926) was irradiated with accelerated nickel ions (Ni) (LET, 183 keV/µm) at doses of 0.5, 2 and 5 Gy. DNA damage was measured 2 and 24 h following irradiation by γ-H2AX foci detection by fluorescence microscopy and gene expression changes were measured by microarrays at 8 and 24 h following irradiation. We found that exposure to accelerated nickel particles induced a persistent DNA damage response up to 24 h after treatment. This was accompanied by a downregulation in the expression of a multitude of genes involved in the regulation of the cell cycle and an upregulation in the expression of genes involved in cell cycle checkpoints. In addition, genes involved in DNA damage response, oxidative stress, apoptosis and cell-cell signaling (cytokines) were found to be upregulated. An in silico analysis of the involved genes suggested that the transcription factors, E2F and nuclear factor (NF)-κB, may be involved in these cellular responses. PMID:25118949

  4. Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes

    PubMed Central

    Ayoub, Nadia A.; Garb, Jessica E.; Tinghitella, Robin M.; Collin, Matthew A.; Hayashi, Cheryl Y.

    2007-01-01

    Spider dragline (major ampullate) silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons), recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs) that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers. PMID:17565367

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

    PubMed Central

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

    2015-01-01

    Multiple myeloma is preceded by an asymptomatic phase, comprising monoclonal gammopathy of uncertain significance and smoldering myeloma. Compared to the former, smoldering myeloma has a higher and non-uniform rate of progression to clinical myeloma, reflecting a subset of patients with higher risk. We evaluated the gene expression profile of smoldering myeloma plasma cells among 105 patients enrolled in a prospective observational trial at our institution, with a view to identifying a high-risk signature. Baseline clinical, bone marrow, cytogenetic and radiologic data were evaluated for their potential to predict time to therapy for symptomatic myeloma. A gene signature derived from four genes, at an optimal binary cut-point of 9.28, identified 14 patients (13%) with a 2-year therapy risk of 85.7%. Conversely, a low four-gene score (<9.28) combined with baseline monoclonal protein <3 g/dL and albumin ≥3.5 g/dL identified 61 patients with low-risk smoldering myeloma with a 5.0% chance of progression at 2 years. The top 40 probe sets showed concordance with indices of chromosome instability. These data demonstrate high discriminatory power of a gene-based assay and suggest a role for dysregulation of mitotic checkpoints in the context of genomic instability as a hallmark of high-risk smoldering myeloma. PMID:26022710

  6. Prolonged Application of High Fluid Shear to Chondrocytes Recapitulates Gene Expression Profiles Associated with Osteoarthritis

    PubMed Central

    Zhu, Fei; Wang, Pu; Lee, Norman H.; Goldring, Mary B.; Konstantopoulos, Konstantinos

    2010-01-01

    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/cm2) 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/progression of OA

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

    PubMed

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

    2014-01-01

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

  8. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors

    PubMed Central

    Colot, Hildur V.; Park, Gyungsoon; Turner, Gloria E.; Ringelberg, Carol; Crew, Christopher M.; Litvinkova, Liubov; Weiss, Richard L.; Borkovich, Katherine A.; Dunlap, Jay C.

    2006-01-01

    The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination. PMID:16801547

  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.

    PubMed

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

    PubMed Central

    Moe, Maren; Meuwissen, Theo; Lien, Sigbjørn; Bendixen, Christian; Wang, Xuefei; Conley, Lene Nagstrup; Berget, Ingunn; Tajet, Håvard; Grindflek, Eli

    2007-01-01

    Background: Boar taint is a major obstacle when using uncastrated male pigs for swine production. One of the main compounds causing this taint is androstenone, a pheromone produced in porcine testis. Here we use microarrays to study the expression of thousands of genes simultaneously in testis of high and low androstenone boars. The study allows identification of genes and pathways associated with elevated androstenone levels, which is essential for recognising potential molecular markers for breeding purposes. Results: Testicular tissue was collected from 60 boars, 30 with extreme high and 30 with extreme low levels of androstenone, from each of the two breeds Duroc and Norwegian Landrace. The samples were hybridised to porcine arrays containing 26,877 cDNA clones, detecting 563 and 160 genes that were differentially expressed (p < 0.01) in Duroc and Norwegian Landrace, respectively. Of these significantly up- and down-regulated clones, 72 were found to be common for the two breeds, suggesting the possibility of both general and breed specific mechanisms in regulation of, or response to androstenone levels in boars. Ten genes were chosen for verification of expression patterns by quantitative real competitive PCR and real-time PCR. As expected, our results point towards steroid hormone metabolism and biosynthesis as important biological processes for the androstenone levels, but other potential pathways were identified as well. Among these were oxidoreductase activity, ferric iron binding, iron ion binding and electron transport activities. Genes belonging to the cytochrome P450 and hydroxysteroid dehydrogenase families were highly up-regulated, in addition to several genes encoding different families of conjugation enzymes. Furthermore, a number of genes encoding transcription factors were found both up- and down-regulated. The high number of clones belonging to ferric iron and iron ion binding suggests an importance of these genes, and the association between

  11. Mammalian ets-1 and ets-2 genes encode highly conserved proteins

    SciTech Connect

    Watson, D.K.; McWilliams, M.J.; Lapis, P.; Lautenberger, J.A.; Schweinfest, C.W.; Papas, T.S. )

    1988-11-01

    Cellular ets sequences homologous to v-ets of the avian leukemia virus E26 are highly conserved. In mammals the ets sequences are dispersed on two separate chromosomal loci, called ets-1 and ets-2. To determine the structure of these two genes and identify the open reading frames that code for the putative proteins, the authors have sequenced human ets-1 cDNAs and ets-2 cDNA clones obtained from both human and mouse. The human ETS1 gene is capable of encoding a protein of 441 amino acids. This protein is >95% identical to the chicken c-ets-1 gene product. Thus, the human ETS1 gene is homologous to the chicken c-ets-1 gene, the protooncogene that the E26 virus transduced. Human and mouse ets-2 cDNA clones are closely related and contain open reading frames capable of encoding proteins of 469 and 468 residues, respectively. Direct comparison of these data with previously published finding indicates that ets is a family of genes whose members share distinct domains.

  12. The Neurospora crassa carotenoid biosynthetic gene (albino 3) reveals highly conserved regions among prenyltransferases.

    PubMed

    Carattoli, A; Romano, N; Ballario, P; Morelli, G; Macino, G

    1991-03-25

    In the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light. Here we report the characterization of the albino-3 (al-3) gene of N. crassa, which encodes the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthetase. This is the first geranylgeranyl-pyrophosphate synthetase gene isolated. Nucleotide sequence comparison of al-3 genomic and cDNA clones revealed that the al-3 gene is not interrupted by introns. Transcription of the al-3 gene has been examined in dark-grown and light-induced mycelia. The analysis revealed that the al-3 gene is not expressed in the dark and that its transcription is induced by blue light (Nelson, M. A., Morelli, G., Carattoli, A., Romano, N., and Macino, G. (1989) Mol. Cell. Biol. 9, 1271-1276). The al-3 gene encodes a polypeptide of 428 amino acids. Comparison of the deduced amino acid sequence of al-3 with the sequences of prenyltransferases of other species, from bacteria to humans, showed three highly conserved homologous regions. These homologous regions may be involved in the formation of the catalytic site of the prenyltransferases.

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

    PubMed

    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

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

    PubMed Central

    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

  15. High levels of variation in Salix lignocellulose genes revealed using poplar genomic resources

    PubMed Central

    2013-01-01

    Background Little is known about the levels of variation in lignin or other wood related genes in Salix, a genus that is being increasingly used for biomass and biofuel production. The lignin biosynthesis pathway is well characterized in a number of species, including the model tree Populus. We aimed to transfer the genomic resources already available in Populus to its sister genus Salix to assess levels of variation within genes involved in wood formation. Results Amplification trials for 27 gene regions were undertaken in 40 Salix taxa. Twelve of these regions were sequenced. Alignment searches of the resulting sequences against reference databases, combined with phylogenetic analyses, showed the close similarity of these Salix sequences to Populus, confirming homology of the primer regions and indicating a high level of conservation within the wood formation genes. However, all sequences were found to vary considerably among Salix species, mainly as SNPs with a smaller number of insertions-deletions. Between 25 and 176 SNPs per kbp per gene region (in predicted exons) were discovered within Salix. Conclusions The variation found is sizeable but not unexpected as it is based on interspecific and not intraspecific comparison; it is comparable to interspecific variation in Populus. The characterisation of genetic variation is a key process in pre-breeding and for the conservation and exploitation of genetic resources in Salix. This study characterises the variation in several lignocellulose gene markers for such purposes. PMID:23924375

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

    ERIC Educational Resources Information Center

    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)

  17. A validated gene expression model of high-risk multiple myeloma is defined by deregulated expression of genes mapping to chromosome 1.

    PubMed

    Shaughnessy, John D; Zhan, Fenghuang; Burington, Bart E; Huang, Yongsheng; Colla, Simona; Hanamura, Ichiro; Stewart, James P; Kordsmeier, Bob; Randolph, Christopher; Williams, David R; Xiao, Yan; Xu, Hongwei; Epstein, Joshua; Anaissie, Elias; Krishna, Somashekar G; Cottler-Fox, Michele; Hollmig, Klaus; Mohiuddin, Abid; Pineda-Roman, Mauricio; Tricot, Guido; van Rhee, Frits; Sawyer, Jeffrey; Alsayed, Yazan; Walker, Ronald; Zangari, Maurizio; Crowley, John; Barlogie, Bart

    2007-03-15

    To molecularly define high-risk disease, we performed microarray analysis on tumor cells from 532 newly diagnosed patients with multiple myeloma (MM) treated on 2 separate protocols. Using log-rank tests of expression quartiles, 70 genes, 30% mapping to chromosome 1 (P < .001), were linked to early disease-related death. Importantly, most up-regulated genes mapped to chromosome 1q, and down-regulated genes mapped to chromosome 1p. The ratio of mean expression levels of up-regulated to down-regulated genes defined a high-risk score present in 13% of patients with shorter durations of complete remission, event-free survival, and overall survival (training set: hazard ratio [HR], 5.16; P < .001; test cohort: HR, 4.75; P < .001). The high-risk score also was an independent predictor of outcome endpoints in multivariate analysis (P < .001) that included the International Staging System and high-risk translocations. In a comparison of paired baseline and relapse samples, the high-risk score frequency rose to 76% at relapse and predicted short postrelapse survival (P < .05). Multivariate discriminant analysis revealed that a 17-gene subset could predict outcome as well as the 70-gene model. Our data suggest that altered transcriptional regulation of genes mapping to chromosome 1 may contribute to disease progression, and that expression profiling can be used to identify high-risk disease and guide therapeutic interventions. PMID:17105813

  18. Clostridium difficile Isolates with High Linezolid MICs Harbor the Multiresistance Gene cfr

    PubMed Central

    Martín, Adoración; Alcalá, Luis; Cercenado, Emilia; Iglesias, Cristina; Reigadas, Elena; Bouza, Emilio

    2014-01-01

    We studied the molecular mechanisms of linezolid resistance in 9 isolates of toxigenic Clostridium difficile with high linezolid MICs. The activity of linezolid was determined against 891 clinical isolates of toxigenic C. difficile. The MIC50 and MIC90 of linezolid were 0.75 μg/ml and 1.5 μg/ml, respectively. Nine strains (1%) showed high linezolid MICs (6 μg/ml to 16 μg/ml) and also were resistant to clindamycin, erythromycin, and chloramphenicol. These strains were selected for molecular studies: sequencing of domain V of the 23 rRNA gene, detection of the cfr methyltransferase gene, and sequencing of the ribosomal protein genes rplC and rplD. Molecular relatedness between strains was assessed using PCR ribotyping and MLVA (multilocus variable-number tandem-repeat analysis) typing. The strains belonged to ribotypes 001 (2/9), 017 (6/9), and 078 (1/9). MLVA showed that strains of ribotype 001 and 017 belonged to the same clonal complex in each ribotype. We did not detect mutations in the 23S rRNA gene. The cfr gene was detected in 7 of 9 strains. Sequencing of cfr amplicons revealed a similarity of 100% to a fragment of transposon Tn6218 of C. difficile, which was annotated as a putative chloramphenicol/florfenicol resistance protein. We were unable to detect mechanisms of resistance to linezolid in the 2 strains belonging to ribotype 001. While the relevance of our results lies in the detection of the cfr gene as a possible mechanism of resistance to linezolid in C. difficile, our findings should be assessed by further investigations to characterize these possible cfr genes and their contribution to linezolid resistance. PMID:25385106

  19. Different gene expressions between cattle and yak provide insights into high-altitude adaptation.

    PubMed

    Wang, K; Yang, Y; Wang, L; Ma, T; Shang, H; Ding, L; Han, J; Qiu, Q

    2016-02-01

    DNA sequence variation has been widely reported as the genetic basis for adaptation, in both humans and other animals, to the hypoxic environment experienced at high altitudes. However, little is known about the patterns of gene expression underlying such hypoxic adaptations. In this study, we examined the differences in the transcriptomes of four organs (heart, kidney, liver and lung) between yak and cattle, a pair of closely related species distributed at high and low altitudes respectively. Of the four organs examined, heart shows the greatest differentiation between the two species in terms of gene expression profiles. Detailed analyses demonstrated that some genes associated with the oxygen supply system and the defense systems that respond to threats of hypoxia are differentially expressed. In addition, genes with significantly differentiated patterns of expression in all organs exhibited an unexpected uniformity of regulation along with an elevated frequency of nonsynonymous substitutions. This co-evolution of protein sequences and gene expression patterns is likely to be correlated with the optimization of the yak metabolic system to resist hypoxia.

  20. Reverse engineering and analysis of genome-wide gene regulatory networks from gene expression profiles using high-performance computing.

    PubMed

    Belcastro, Vincenzo; Gregoretti, Francesco; Siciliano, Velia; Santoro, Michele; D'Angelo, Giovanni; Oliva, Gennaro; di Bernardo, Diego

    2012-01-01

    Regulation of gene expression is a carefully regulated phenomenon in the cell. “Reverse-engineering” algorithms try to reconstruct the regulatory interactions among genes from genome-scale measurements of gene expression profiles (microarrays). Mammalian cells express tens of thousands of genes; hence, hundreds of gene expression profiles are necessary in order to have acceptable statistical evidence of interactions between genes. As the number of profiles to be analyzed increases, so do computational costs and memory requirements. In this work, we designed and developed a parallel computing algorithm to reverse-engineer genome-scale gene regulatory networks from thousands of gene expression profiles. The algorithm is based on computing pairwise Mutual Information between each gene-pair. We successfully tested it to reverse engineer the Mus Musculus (mouse) gene regulatory network in liver from gene expression profiles collected from a public repository. A parallel hierarchical clustering algorithm was implemented to discover “communities” within the gene network. Network communities are enriched for genes involved in the same biological functions. The inferred network was used to identify two mitochondrial proteins.

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

    PubMed Central

    Moe, Maren; Lien, Sigbjørn; Bendixen, Christian; Hedegaard, Jakob; Hornshøj, Henrik; Berget, Ingunn; Meuwissen, Theo HE; Grindflek, Eli

    2008-01-01

    Background Boar taint is the unpleasant odour and flavour of the meat of uncastrated male pigs that is primarily caused by high levels of androstenone and skatole in adipose tissue. Androstenone is a steroid and its levels are mainly genetically determined. Studies on androstenone metabolism have, however, focused on a limited number of genes. Identification of additional genes influencing levels of androstenone may facilitate implementation of marker assisted breeding practices. In this study, microarrays were used to identify differentially expressed genes and pathways related to androstenone metabolism in the liver from boars with extreme levels of androstenone in adipose tissue. Results Liver tissue samples from 58 boars of the two breeds Duroc and Norwegian Landrace, 29 with extreme high and 29 with extreme low levels of androstenone, were selected from more than 2500 individuals. The samples were hybridised to porcine cDNA microarrays and the 1% most significant differentially expressed genes were considered significant. Among the differentially expressed genes were metabolic phase I related genes belonging to the cytochrome P450 family and the flavin-containing monooxygenase FMO1. Additionally, phase II conjugation genes including UDP-glucuronosyltransferases UGT1A5, UGT2A1 and UGT2B15, sulfotransferase STE, N-acetyltransferase NAT12 and glutathione S-transferase were identified. Phase I and phase II metabolic reactions increase the water solubility of steroids and play a key role in their elimination. Differential expression was also found for genes encoding 17beta-hydroxysteroid dehydrogenases (HSD17B2, HSD17B4, HSD17B11 and HSD17B13) and plasma proteins alpha-1-acid glycoprotein (AGP) and orosomucoid (ORM1). 17beta-hydroxysteroid dehydrogenases and plasma proteins regulate the availability of steroids by controlling the amount of active steroids accessible to receptors and available for metabolism. Differences in the expression of FMO1, NAT12, HSD17B2 and

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

    PubMed Central

    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

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

    PubMed

    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.

  4. A single nucleotide polymorphism analysis of the LAMA1 gene in Japanese patients with high myopia

    PubMed Central

    Sasaki, Sayaka; Ota, Masao; Meguro, Akira; Nishizaki, Ritsuko; Okada, Eiichi; Mok, Jeewon; Kimura, Tetusya; Oka, Akira; Katsuyama, Yoshihiko; Ohno, Shigeaki; Inoko, Hidetoshi; Mizuki, Nobuhisa

    2007-01-01

    Although a myopia susceptibility gene has not yet been elucidated, ten candidate regions (MYP1–MYP10) have been associated with myopia by linkage analysis employing large pedigrees. We report herein on the results of our analysis pertaining to polymorphisms of LAMA1 (alpha subunit of laminin), a promising candidate gene for high myopia present in the MYP2 region of Japanese subjects with high myopia. Three hundred and thirty Japanese subjects with high myopia at a level of greater than −9.25 D and ethnically and sex matched 330 normal controls without high myopia was enrolled in this study. The thirteen SNPs located on the LAMA1 gene were analyzed using PCR and SNP-specific fluorogenic probes. Two of the SNPs were monomorphic and none of the 11 SNPs showed statistically significant association with high myopia in the Japanese population. There is no convincing evidence to prove a connection between nucleotide sequence variations in LAMA1 and high myopia. The pairwise linkage disequilibrium (LD) mapping disclosed a strong value (D' > 0.8) and narrow ranged block within these SNPs. PMID:19668483

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

    PubMed

    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

  6. Highly repetitive tRNA(Pro)-tRNA(His) gene cluster from Photobacterium phosphoreum.

    PubMed Central

    Giroux, S; Beaudet, J; Cedergren, R

    1988-01-01

    A DNA fragment comprising the four tRNA gene sequences of the Escherichia coli argT locus hybridized with two Sau3A-generated DNA fragments from the vibrio Photobacterium phosphoreum (ATCC 11040). Detailed sequence analysis of the longer fragment shows the following gene organization: 5'-promoter-tRNA(Pro)-tRNAPro-tRNA(Pro)-tRNA(His)-tRNA(Pro)-tRNA(Pro)- tRNA(His)-tRNA(Pro)-five pseudogenes derived from the upstream tRNAPro interspersed by putative Rho-independent terminators. This sequence demonstrates the presence of highly repetitive, tandem tRNA genes in a bacterial genome. Furthermore, a stretch of 304 nucleotides from this cluster was found virtually unchanged in the other (shorter) fragment which was previously sequenced. The two clusters together contain eight tRNA(Pro) pseudogenes and eight fully intact tRNA(Pro) genes, an unusually high number for a single eubacterial isoacceptor tRNA. These results show that the organization of some tRNA operons is highly variable in eubacteria. Images PMID:3056906

  7. Gene Signature of High White Blood Cell Count in B-Precursor Acute Lymphoblastic Leukemia

    PubMed Central

    Dombkowski, Alan A.; Caldwell, J. Timothy; Chu, Roland; Xavier, Ana C.; Thummel, Ryan; Neely, Melody; Matherly, Larry H.; Ge, Yubin; Taub, Jeffrey W.

    2016-01-01

    In this study we sought to identify genetic factors associated with the presenting white blood cell (WBC) count in B-precursor acute lymphoblastic leukemia (BP-ALL). Using ETV6-RUNX1-positive BP-ALL patient samples, a homogeneous subtype, we identified 16 differentially expressed genes based on the presenting WBC count (< 50,000/cumm vs > 50,000). We further confirmed that IL1R1, BCAR3, KCNH2, PIR, and ZDHHC23 were differentially expressed in a larger cohort of ETV6-RUNX1-negative BP-ALL patient samples. Statistical analysis demonstrated that expression levels of these genes could accurately categorize high and low WBC count subjects using two independent patient sets, representing positive and negative ETV6-RUNX1 cases. Further studies in leukemia cell line models will better delineate the role of these genes in regulating the white blood cell count and potentially identify new therapeutic targets. PMID:27536776

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

    PubMed Central

    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

  9. High initiation rates at the ribosomal gene promoter do not depend upon spacer transcription

    SciTech Connect

    Labhart, P.; Reeder, R.H. )

    1989-05-01

    We report experiments that test the model that in Xenopus laevis, RNA polymerase I is handed over in a conservative fashion from the T3 terminator to the adjacent gene promoter. We have introduced transcription-terminating lesions into the ribosomal DNA repeat by irradiating cultured cells with ultraviolet light. We used isolated nuclei to measure the effect of such lesions on transcription. UV damage sufficient to prevent all elongating RNA polymerase from reaching T3 from upstream had no adverse effect on the density of RNA polymerase at the very 5' end of the gene. We conclude that high rates of transcription initiation at the gene promoter do not depend upon polymerase passing from one repeat to the next or on polymerase initiating at the spacer promoters.

  10. High-Throughput Fluorescent Tagging of Full-Length Arabidopsis Gene Products in Planta1

    PubMed Central

    Tian, Guo-Wei; Mohanty, Amitabh; Chary, S. Narasimha; Li, Shijun; Paap, Brigitte; Drakakaki, Georgia; Kopec, Charles D.; Li, Jianxiong; Ehrhardt, David; Jackson, David; Rhee, Seung Y.; Raikhel, Natasha V.; Citovsky, Vitaly

    2004-01-01

    We developed a high-throughput methodology, termed fluorescent tagging of full-length proteins (FTFLP), to analyze expression patterns and subcellular localization of Arabidopsis gene products in planta. Determination of these parameters is a logical first step in functional characterization of the approximately one-third of all known Arabidopsis genes that encode novel proteins of unknown function. Our FTFLP-based approach offers two significant advantages: first, it produces internally-tagged full-length proteins that are likely to exhibit native intracellular localization, and second, it yields information about the tissue specificity of gene expression by the use of native promoters. To demonstrate how FTFLP may be used for characterization of the Arabidopsis proteome, we tagged a series of known proteins with diverse subcellular targeting patterns as well as several proteins with unknown function and unassigned subcellular localization. PMID:15141064

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

    PubMed

    Wheeler, Marsha M; Robinson, Gene E

    2014-07-17

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

  12. Gene Signature of High White Blood Cell Count in B-Precursor Acute Lymphoblastic Leukemia.

    PubMed

    Edwards, Holly; Rubenstein, Mara; Dombkowski, Alan A; Caldwell, J Timothy; Chu, Roland; Xavier, Ana C; Thummel, Ryan; Neely, Melody; Matherly, Larry H; Ge, Yubin; Taub, Jeffrey W

    2016-01-01

    In this study we sought to identify genetic factors associated with the presenting white blood cell (WBC) count in B-precursor acute lymphoblastic leukemia (BP-ALL). Using ETV6-RUNX1-positive BP-ALL patient samples, a homogeneous subtype, we identified 16 differentially expressed genes based on the presenting WBC count (< 50,000/cumm vs > 50,000). We further confirmed that IL1R1, BCAR3, KCNH2, PIR, and ZDHHC23 were differentially expressed in a larger cohort of ETV6-RUNX1-negative BP-ALL patient samples. Statistical analysis demonstrated that expression levels of these genes could accurately categorize high and low WBC count subjects using two independent patient sets, representing positive and negative ETV6-RUNX1 cases. Further studies in leukemia cell line models will better delineate the role of these genes in regulating the white blood cell count and potentially identify new therapeutic targets.

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

    PubMed

    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

  14. High accuracy mutation detection in leukemia on a selected panel of cancer genes.

    PubMed

    Kalender Atak, Zeynep; De Keersmaecker, Kim; Gianfelici, Valentina; Geerdens, Ellen; Vandepoel, Roel; Pauwels, Daphnie; Porcu, Michaël; Lahortiga, Idoya; Brys, Vanessa; Dirks, Willy G; Quentmeier, Hilmar; Cloos, Jacqueline; Cuppens, Harry; Uyttebroeck, Anne; Vandenberghe, Peter; Cools, Jan; Aerts, Stein

    2012-01-01

    With the advent of whole-genome and whole-exome sequencing, high-quality catalogs of recurrently mutated cancer genes are becoming available for many cancer types. Increasing access to sequencing technology, including bench-top sequencers, provide the opportunity to re-sequence a limited set of cancer genes across a patient cohort with limited processing time. Here, we re-sequenced a set of cancer genes in T-cell acute lymphoblastic leukemia (T-ALL) using Nimblegen sequence capture coupled with Roche/454 technology. First, we investigated how a maximal sensitivity and specificity of mutation detection can be achieved through a benchmark study. We tested nine combinations of different mapping and variant-calling methods, varied the variant calling parameters, and compared the predicted mutations with a large independent validation set obtained by capillary re-sequencing. We found that the combination of two mapping algorithms, namely BWA-SW and SSAHA2, coupled with the variant calling algorithm Atlas-SNP2 yields the highest sensitivity (95%) and the highest specificity (93%). Next, we applied this analysis pipeline to identify mutations in a set of 58 cancer genes, in a panel of 18 T-ALL cell lines and 15 T-ALL patient samples. We confirmed mutations in known T-ALL drivers, including PHF6, NF1, FBXW7, NOTCH1, KRAS, NRAS, PIK3CA, and PTEN. Interestingly, we also found mutations in several cancer genes that had not been linked to T-ALL before, including JAK3. Finally, we re-sequenced a small set of 39 candidate genes and identified recurrent mutations in TET1, SPRY3 and SPRY4. In conclusion, we established an optimized analysis pipeline for Roche/454 data that can be applied to accurately detect gene mutations in cancer, which led to the identification of several new candidate T-ALL driver mutations.

  15. High-resolution gene expression atlases for adult and developing mouse brain and spinal cord.

    PubMed

    Henry, Alex M; Hohmann, John G

    2012-10-01

    Knowledge of the structure, genetics, circuits, and physiological properties of the mammalian brain in both normal and pathological states is ever increasing as research labs worldwide probe the various aspects of brain function. Until recently, however, comprehensive cataloging of gene expression across the central nervous system has been lacking. The Allen Institute for Brain Science, as part of its mission to propel neuroscience research, has completed several large gene-mapping projects in mouse, nonhuman primate, and human brain, producing informative online public resources and tools. Here we present the Allen Mouse Brain Atlas, covering ~20,000 genes throughout the adult mouse brain; the Allen Developing Mouse Brain Atlas, detailing expression of approximately 2,000 important developmental genes across seven embryonic and postnatal stages of brain growth; and the Allen Spinal Cord Atlas, revealing expression for ~20,000 genes in the adult and neonatal mouse spinal cords. Integrated data-mining tools, including reference atlases, informatics analyses, and 3-D viewers, are described. For these massive-scale projects, high-throughput industrial techniques were developed to standardize and reliably repeat experimental goals. To verify consistency and accuracy, a detailed analysis of the 1,000 most viewed genes for the adult mouse brain (according to website page views) was performed by comparing our data with peer-reviewed literature and other databases. We show that our data are highly consistent with independent sources and provide a comprehensive compendium of information and tools used by thousands of researchers each month. All data and tools are freely available via the Allen Brain Atlas portal (www.brain-map.org).

  16. High-resolution gene expression atlases for adult and developing mouse brain and spinal cord.

    PubMed

    Henry, Alex M; Hohmann, John G

    2012-10-01

    Knowledge of the structure, genetics, circuits, and physiological properties of the mammalian brain in both normal and pathological states is ever increasing as research labs worldwide probe the various aspects of brain function. Until recently, however, comprehensive cataloging of gene expression across the central nervous system has been lacking. The Allen Institute for Brain Science, as part of its mission to propel neuroscience research, has completed several large gene-mapping projects in mouse, nonhuman primate, and human brain, producing informative online public resources and tools. Here we present the Allen Mouse Brain Atlas, covering ~20,000 genes throughout the adult mouse brain; the Allen Developing Mouse Brain Atlas, detailing expression of approximately 2,000 important developmental genes across seven embryonic and postnatal stages of brain growth; and the Allen Spinal Cord Atlas, revealing expression for ~20,000 genes in the adult and neonatal mouse spinal cords. Integrated data-mining tools, including reference atlases, informatics analyses, and 3-D viewers, are described. For these massive-scale projects, high-throughput industrial techniques were developed to standardize and reliably repeat experimental goals. To verify consistency and accuracy, a detailed analysis of the 1,000 most viewed genes for the adult mouse brain (according to website page views) was performed by comparing our data with peer-reviewed literature and other databases. We show that our data are highly consistent with independent sources and provide a comprehensive compendium of information and tools used by thousands of researchers each month. All data and tools are freely available via the Allen Brain Atlas portal (www.brain-map.org). PMID:22832508

  17. Reduced Set of Virulence Genes Allows High Accuracy Prediction of Bacterial Pathogenicity in Humans

    PubMed Central

    Iraola, Gregorio; Vazquez, Gustavo; Spangenberg, Lucía; Naya, Hugo

    2012-01-01

    Although there have been great advances in understanding bacterial pathogenesis, there is still a lack of integrative information about what makes a bacterium a human pathogen. The advent of high-throughput sequencing technologies has dramatically increased the amount of completed bacterial genomes, for both known human pathogenic and non-pathogenic strains; this information is now available to investigate genetic features that determine pathogenic phenotypes in bacteria. In this work we determined presence/absence patterns of different virulence-related genes among more than finished bacterial genomes from both human pathogenic and non-pathogenic strains, belonging to different taxonomic groups (i.e: Actinobacteria, Gammaproteobacteria, Firmicutes, etc.). An accuracy of 95% using a cross-fold validation scheme with in-fold feature selection is obtained when classifying human pathogens and non-pathogens. A reduced subset of highly informative genes () is presented and applied to an external validation set. The statistical model was implemented in the BacFier v1.0 software (freely available at ), that displays not only the prediction (pathogen/non-pathogen) and an associated probability for pathogenicity, but also the presence/absence vector for the analyzed genes, so it is possible to decipher the subset of virulence genes responsible for the classification on the analyzed genome. Furthermore, we discuss the biological relevance for bacterial pathogenesis of the core set of genes, corresponding to eight functional categories, all with evident and documented association with the phenotypes of interest. Also, we analyze which functional categories of virulence genes were more distinctive for pathogenicity in each taxonomic group, which seems to be a completely new kind of information and could lead to important evolutionary conclusions. PMID:22916122

  18. Forager bees (Apis mellifera) highly express immune and detoxification genes in tissues associated with nectar processing.

    PubMed

    Vannette, Rachel L; Mohamed, Abbas; Johnson, Brian R

    2015-11-09

    Pollinators, including honey bees, routinely encounter potentially harmful microorganisms and phytochemicals during foraging. However, the mechanisms by which honey bees manage these potential threats are poorly understood. In this study, we examine the expression of antimicrobial, immune and detoxification genes in Apis mellifera and compare between forager and nurse bees using tissue-specific RNA-seq and qPCR. Our analysis revealed extensive tissue-specific expression of antimicrobial, immune signaling, and detoxification genes. Variation in gene expression between worker stages was pronounced in the mandibular and hypopharyngeal gland (HPG), where foragers were enriched in transcripts that encode antimicrobial peptides (AMPs) and immune response. Additionally, forager HPGs and mandibular glands were enriched in transcripts encoding detoxification enzymes, including some associated with xenobiotic metabolism. Using qPCR on an independent dataset, we verified differential expression of three AMP and three P450 genes between foragers and nurses. High expression of AMP genes in nectar-processing tissues suggests that these peptides may contribute to antimicrobial properties of honey or to honey bee defense against environmentally-acquired microorganisms. Together, these results suggest that worker role and tissue-specific expression of AMPs, and immune and detoxification enzymes may contribute to defense against microorganisms and xenobiotic compounds acquired while foraging.

  19. High Rates of Antimicrobial Drug Resistance Gene Acquisition after International Travel, the Netherlands

    PubMed Central

    von Wintersdorff, Christian J.H.; Penders, John; Stobberingh, Ellen E.; Lashof, Astrid M.L. Oude; Hoebe, Christian J.P.A.; Savelkoul, Paul H.M.

    2014-01-01

    We investigated the effect of international travel on the gut resistome of 122 healthy travelers from the Netherlands by using a targeted metagenomic approach. Our results confirm high acquisition rates of the extended-spectrum β-lactamase encoding gene blaCTX-M, documenting a rise in prevalence from 9.0% before travel to 33.6% after travel (p<0.001). The prevalence of quinolone resistance encoding genes qnrB and qnrS increased from 6.6% and 8.2% before travel to 36.9% and 55.7% after travel, respectively (both p<0.001). Travel to Southeast Asia and the Indian subcontinent was associated with the highest acquisition rates of qnrS and both blaCTX-M and qnrS, respectively. Investigation of the associations between the acquisitions of the blaCTX-M and qnr genes showed that acquisition of a blaCTX-M gene was not associated with that of a qnrB (p = 0.305) or qnrS (p = 0.080) gene. These findings support the increasing evidence that travelers contribute to the spread of antimicrobial drug resistance. PMID:24655888

  20. Forager bees (Apis mellifera) highly express immune and detoxification genes in tissues associated with nectar processing

    PubMed Central

    Vannette, Rachel L.; Mohamed, Abbas; Johnson, Brian R.

    2015-01-01

    Pollinators, including honey bees, routinely encounter potentially harmful microorganisms and phytochemicals during foraging. However, the mechanisms by which honey bees manage these potential threats are poorly understood. In this study, we examine the expression of antimicrobial, immune and detoxification genes in Apis mellifera and compare between forager and nurse bees using tissue-specific RNA-seq and qPCR. Our analysis revealed extensive tissue-specific expression of antimicrobial, immune signaling, and detoxification genes. Variation in gene expression between worker stages was pronounced in the mandibular and hypopharyngeal gland (HPG), where foragers were enriched in transcripts that encode antimicrobial peptides (AMPs) and immune response. Additionally, forager HPGs and mandibular glands were enriched in transcripts encoding detoxification enzymes, including some associated with xenobiotic metabolism. Using qPCR on an independent dataset, we verified differential expression of three AMP and three P450 genes between foragers and nurses. High expression of AMP genes in nectar-processing tissues suggests that these peptides may contribute to antimicrobial properties of honey or to honey bee defense against environmentally-acquired microorganisms. Together, these results suggest that worker role and tissue-specific expression of AMPs, and immune and detoxification enzymes may contribute to defense against microorganisms and xenobiotic compounds acquired while foraging. PMID:26549293

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

    PubMed

    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.

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

    PubMed Central

    Bányai, László; Patthy, László

    2016-01-01

    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. PMID:27476717

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

    PubMed

    Bányai, László; Patthy, László

    2016-01-01

    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. PMID:27476717

  4. A stationary-phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved gene family.

    PubMed Central

    Braun, E L; Fuge, E K; Padilla, P A; Werner-Washburne, M

    1996-01-01

    The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms. In unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, growth and proliferation are regulated by nutrient availability. We have described changes in the pattern of protein synthesis during the growth of S. cerevisiae cells to stationary phase (E. K. Fuge, E. L. Braun, and M. Werner-Washburne, J. Bacteriol. 176:5802-5813, 1994) and noted a protein, which we designated Snz1p (p35), that shows increased synthesis after entry into stationary phase. We report here the identification of the SNZ1 gene, which encodes this protein. We detected increased SNZ1 mRNA accumulation almost 2 days after glucose exhaustion, significantly later than that of mRNAs encoded by other postexponential genes. SNZ1-related sequences were detected in phylogenetically diverse organisms by sequence comparisons and low-stringency hybridization. Multiple SNZ1-related sequences were detected in some organisms, including S. cerevisiae. Snz1p was found to be among the most evolutionarily conserved proteins currently identified, indicating that we have identified a novel, highly conserved protein involved in growth arrest in S. cerevisiae. The broad phylogenetic distribution, the regulation of the SNZ1 mRNA and protein in S. cerevisiae, and identification of a Snz protein modified during sporulation in the gram-positive bacterium Bacillus subtilis support the hypothesis that Snz proteins are part of an ancient response that occurs during nutrient limitation and growth arrest. PMID:8955308

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

    USGS Publications Warehouse

    Pearson, T.; Giffard, P.; Beckstrom-Sternberg, S.; Auerbach, R.; Hornstra, H.; Tuanyok, A.; Price, E.P.; Glass, M.B.; Leadem, B.; Beckstrom-Sternberg, J. S.; Allan, G.J.; Foster, J.T.; Wagner, D.M.; Okinaka, R.T.; Sim, S.H.; Pearson, O.; Wu, Z.; Chang, J.; Kaul, R.; Hoffmaster, A.R.; Brettin, T.S.; Robison, R.A.; Mayo, M.; Gee, J.E.; Tan, P.; Currie, B.J.; Keim, P.

    2009-01-01

    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. Conclusion: We describe an

  6. A highly conserved sequence is a novel gene involved in de novo vitamin B6 biosynthesis

    PubMed Central

    Ehrenshaft, Marilyn; Bilski, Piotr; Li, Ming Y.; Chignell, Colin F.; Daub, Margaret E.

    1999-01-01

    The Cercospora nicotianae SOR1 (singlet oxygen resistance) gene was identified previously as a gene involved in resistance of this fungus to singlet-oxygen-generating phototoxins. Although homologues to SOR1 occur in organisms in four kingdoms and encode one of the most highly conserved proteins yet identified, the precise function of this protein has, until now, remained unknown. We show that SOR1 is essential in pyridoxine (vitamin B6) synthesis in C. nicotianae and Aspergillus flavus, although it shows no homology to previously identified pyridoxine synthesis genes identified in Escherichia coli. Sequence database analysis demonstrated that organisms encode either SOR1 or E. coli pyridoxine biosynthesis genes, but not both, suggesting that there are two divergent pathways for de novo pyridoxine biosynthesis in nature. Pathway divergence appears to have occurred during the evolution of the eubacteria. We also present data showing that pyridoxine quenches singlet oxygen at a rate comparable to that of vitamins C and E, two of the most highly efficient biological antioxidants, suggesting a previously unknown role for pyridoxine in active oxygen resistance. PMID:10430950

  7. High-density polymorphisms analysis of 23 candidate genes for association with bone mineral density.

    PubMed

    Giroux, Sylvie; Elfassihi, Latifa; Clément, Valérie; Bussières, Johanne; Bureau, Alexandre; Cole, David E C; Rousseau, François

    2010-11-01

    Osteoporosis is a bone disease characterized by low bone mineral density (BMD), a highly heritable and polygenic trait. Women are more prone than men to develop osteoporosis due to a lower peak bone mass and accelerated bone loss at menopause. Peak bone mass has been convincingly shown to be due to genetic factors with heritability up to 80%. Menopausal bone loss has been shown to have around 38% to 49% heritability depending on the site studied. To have more statistical power to detect small genetic effects we focused on premenopausal women. We studied 23 candidate genes, some involved in calcium and vitamin-D regulation and others because estrogens strongly induced their gene expression in mice where it was correlated with humerus trabecular bone density. High-density polymorphisms were selected to cover the entire gene variability and 231 polymorphisms were genotyped in a first sample of 709 premenopausal women. Positive associations were retested in a second, independent, sample of 673 premenopausal women. Ten polymorphisms remained associated with BMD in the combined samples and one was further associated in a large sample of postmenopausal women (1401 women). This associated polymorphism was located in the gene CSF3R (granulocyte colony stimulating factor receptor) that had never been associated with BMD before. The results reported in this study suggest a role for CSF3R in the determination of bone density in women.

  8. Identification of cis-elements conferring high levels of gene expression in non-green plastids.

    PubMed

    Zhang, Jiang; Ruf, Stephanie; Hasse, Claudia; Childs, Liam; Scharff, Lars B; Bock, Ralph

    2012-10-01

    Although our knowledge about the mechanisms of gene expression in chloroplasts has increased substantially over the past decades, next to nothing is known about the signals and factors that govern expression of the plastid genome in non-green tissues. Here we report the development of a quantitative method suitable for determining the activity of cis-acting elements for gene expression in non-green plastids. The in vivo assay is based on stable transformation of the plastid genome and the discovery that root length upon seedling growth in the presence of the plastid translational inhibitor kanamycin is directly proportional to the expression strength of the resistance gene nptII in transgenic tobacco plastids. By testing various combinations of promoters and translation initiation signals, we have used this experimental system to identify cis-elements that are highly active in non-green plastids. Surprisingly, heterologous expression elements from maize plastids were significantly more efficient in conferring high expression levels in root plastids than homologous expression elements from tobacco. Our work has established a quantitative method for characterization of gene expression in non-green plastid types, and has led to identification of cis-elements for efficient plastid transgene expression in non-green tissues, which are valuable tools for future transplastomic studies in basic and applied research.

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

    PubMed

    Gerber, Sophie; Chadœuf, Joël; Gugerli, Felix; Lascoux, Martin; Buiteveld, Joukje; Cottrell, Joan; Dounavi, Aikaterini; Fineschi, Silvia; Forrest, Laura L; Fogelqvist, Johan; Goicoechea, Pablo G; Jensen, Jan Svejgaard; Salvini, Daniela; Vendramin, Giovanni G; Kremer, Antoine

    2014-01-01

    Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea) distributed across Europe. Adult trees within a given area in each stand were exhaustively sampled (range [239, 754], mean 423), mapped, and acorns were collected ([17,147], 51) from several mother trees ([3], [47], 23). Seedlings ([65,387], 178) were harvested and geo-referenced in six of the eight stands. Genetic information was obtained from screening distinct molecular markers spread across the genome, genotyping each tree, acorn or seedling. All samples were thus genotyped at 5-8 nuclear microsatellite loci. Fathers/parents were assigned to acorns and seedlings using likelihood methods. Mating success of male and female parents, pollen and seed dispersal curves, and also hybridisation rates were estimated in each stand and compared on a continental scale. On average, the percentage of the wind-borne pollen from outside the stand was 60%, with large variation among stands (21-88%). Mean seed immigration into the stand was 40%, a high value for oaks that are generally considered to have limited seed dispersal. However, this estimate varied greatly among stands (20-66%). Gene flow was mostly intraspecific, with large variation, as some trees and stands showed particularly high rates of hybridisation. Our results show that mating success was unevenly distributed among trees. The high levels of gene flow suggest that geographically remote oak stands are unlikely to be genetically isolated, questioning the static definition of gene reserves and seed stands. PMID:24454802

  10. High Rates of Gene Flow by Pollen and Seed in Oak Populations across Europe

    PubMed Central

    Gerber, Sophie; Chadœuf, Joël; Gugerli, Felix; Lascoux, Martin; Buiteveld, Joukje; Cottrell, Joan; Dounavi, Aikaterini; Fineschi, Silvia; Forrest, Laura L.; Fogelqvist, Johan; Goicoechea, Pablo G.; Jensen, Jan Svejgaard; Salvini, Daniela; Vendramin, Giovanni G.; Kremer, Antoine

    2014-01-01

    Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea) distributed across Europe. Adult trees within a given area in each stand were exhaustively sampled (range [239, 754], mean 423), mapped, and acorns were collected ([17,147], 51) from several mother trees ([3], [47], 23). Seedlings ([65,387], 178) were harvested and geo-referenced in six of the eight stands. Genetic information was obtained from screening distinct molecular markers spread across the genome, genotyping each tree, acorn or seedling. All samples were thus genotyped at 5–8 nuclear microsatellite loci. Fathers/parents were assigned to acorns and seedlings using likelihood methods. Mating success of male and female parents, pollen and seed dispersal curves, and also hybridisation rates were estimated in each stand and compared on a continental scale. On average, the percentage of the wind-borne pollen from outside the stand was 60%, with large variation among stands (21–88%). Mean seed immigration into the stand was 40%, a high value for oaks that are generally considered to have limited seed dispersal. However, this estimate varied greatly among stands (20–66%). Gene flow was mostly intraspecific, with large variation, as some trees and stands showed particularly high rates of hybridisation. Our results show that mating success was unevenly distributed among trees. The high levels of gene flow suggest that geographically remote oak stands are unlikely to be genetically isolated, questioning the static definition of gene reserves and seed stands. PMID:24454802

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

    PubMed

    Rohde, Palle Duun; Madsen, Lisbeth Strøm; Neumann Arvidson, Sandra Marie; Loeschcke, Volker; Demontis, Ditte; Kristensen, Torsten Nygaard

    2016-01-01

    Fruit flies are important model organisms for functional testing of candidate genes in multiple disciplines, including the study of human diseases. Here we use a high-throughput locomotor activity assay to test the response on activity behavior of gene disruption in Drosophila melanogaster. The aim was to investigate the impact of disruption of 14 candidate genes for human attention-deficit/hyperactivity disorder (ADHD) on fly behavior. By obtaining a range of correlated measures describing the space of variables for behavioral activity we show, that some mutants display similar phenotypic responses, and furthermore, that the genes disrupted in those mutants had common molecular functions; namely processes related to cGMP activity, cation channels and serotonin receptors. All but one of the candidate genes resulted in aberrant behavioral activity, suggesting involvement of these genes in behavioral activity in fruit flies. Results provide additional support for the investigated genes being risk candidate genes for ADHD in humans.

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

    PubMed

    Rohde, Palle Duun; Madsen, Lisbeth Strøm; Neumann Arvidson, Sandra Marie; Loeschcke, Volker; Demontis, Ditte; Kristensen, Torsten Nygaard

    2016-01-01

    Fruit flies are important model organisms for functional testing of candidate genes in multiple disciplines, including the study of human diseases. Here we use a high-throughput locomotor activity assay to test the response on activity behavior of gene disruption in Drosophila melanogaster. The aim was to investigate the impact of disruption of 14 candidate genes for human attention-deficit/hyperactivity disorder (ADHD) on fly behavior. By obtaining a range of correlated measures describing the space of variables for behavioral activity we show, that some mutants display similar phenotypic responses, and furthermore, that the genes disrupted in those mutants had common molecular functions; namely processes related to cGMP activity, cation channels and serotonin receptors. All but one of the candidate genes resulted in aberrant behavioral activity, suggesting involvement of these genes in behavioral activity in fruit flies. Results provide additional support for the investigated genes being risk candidate genes for ADHD in humans. PMID:26954609

  13. High-throughput genome sequencing of lichenizing fungi to assess gene loss in the ammonium transporter/ammonia permease gene family

    PubMed Central

    2013-01-01

    Background Horizontal gene transfer has shaped the evolution of the ammonium transporter/ammonia permease gene family. Horizontal transfers of ammonium transporter/ammonia permease genes into the fungi include one transfer from archaea to the filamentous ascomycetes associated with the adaptive radiation of the leotiomyceta. The horizontally transferred gene has subsequently been lost in most of the group but has been selectively retained in lichenizing fungi. However, some groups of lichens appear to have secondarily lost the archaeal ammonium transporter. Definitive assessment of gene loss can only be made via whole genome sequencing. Results Ammonium transporter/ammonia permease gene sequences were recovered from the assembled genomes of eight lichenizing fungi in key clades including the Caliciales, the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacterium as a primary photobiont or lichens living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the lichens from the Acarosporomycetidae, Chaetotheriomycetes and Arthoniomycetes. In addition, the four ammonium transporter/ammonia permease genes from Cladonia grayi were shown to be functional by expressing the lichen genes in a strain of Saccharomyces cerevisiae in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source. Conclusions Given sufficient coverage, next-generation sequencing technology can definitively address the loss of a gene in a genome when using environmental DNA isolated from lichen thalli collected from their natural habitats. Lichen-forming fungi have been losing ammonium transporters

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

    PubMed

    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).

  15. Metagenomic analysis of stress genes in microbial mat communities from Antarctica and the High Arctic.

    PubMed

    Varin, Thibault; Lovejoy, Connie; Jungblut, Anne D; Vincent, Warwick F; Corbeil, Jacques

    2012-01-01

    Polar and alpine microbial communities experience a variety of environmental stresses, including perennial cold and freezing; however, knowledge of genomic responses to such conditions is still rudimentary. We analyzed the metagenomes of cyanobacterial mats from Arctic and Antarctic ice shelves, using high-throughput pyrosequencing to test the hypotheses that consortia from these extreme polar habitats were similar in terms of major phyla and subphyla and consequently in their potential responses to environmental stresses. Statistical comparisons of the protein-coding genes showed similarities between the mats from the two poles, with the majority of genes derived from Proteobacteria and Cyanobacteria; however, the relative proportions differed, with cyanobacterial genes more prevalent in the Antarctic mat metagenome. Other differences included a higher representation of Actinobacteria and Alphaproteobacteria in the Arctic metagenomes, which may reflect the greater access to diasporas from both adjacent ice-free lands and the open ocean. Genes coding for functional responses to environmental stress (exopolysaccharides, cold shock proteins, and membrane modifications) were found in all of the metagenomes. However, in keeping with the greater exposure of the Arctic to long-range pollutants, sequences assigned to copper homeostasis genes were statistically (30%) more abundant in the Arctic samples. In contrast, more reads matching the sigma B genes were identified in the Antarctic mat, likely reflecting the more severe osmotic stress during freeze-up of the Antarctic ponds. This study underscores the presence of diverse mechanisms of adaptation to cold and other stresses in polar mats, consistent with the proportional representation of major bacterial groups. PMID:22081564

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

    PubMed

    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.

  17. High capacity nanoporous silicon carrier for systemic delivery of gene silencing therapeutics.

    PubMed

    Shen, Jianliang; Xu, Rong; Mai, Junhua; Kim, Han-Cheon; Guo, Xiaojing; Qin, Guoting; Yang, Yong; Wolfram, Joy; Mu, Chaofeng; Xia, Xiaojun; Gu, Jianhua; Liu, Xuewu; Mao, Zong-Wan; Ferrari, Mauro; Shen, Haifa

    2013-11-26

    Gene silencing agents such as small interfering RNA (siRNA) and microRNA offer the promise to modulate expression of almost every gene for the treatment of human diseases including cancer. However, lack of vehicles for effective systemic delivery to the disease organs has greatly limited their in vivo applications. In this study, we developed a high capacity polycation-functionalized nanoporous silicon (PCPS) platform comprised of nanoporous silicon microparticles functionalized with arginine-polyethyleneimine inside the nanopores for effective delivery of gene silencing agents. Incubation of MDA-MB-231 human breast cancer cells with PCPS loaded with STAT3 siRNA (PCPS/STAT3) or GRP78 siRNA (PCPS/GRP78) resulted in 91 and 83% reduction of STAT3 and GRP78 gene expression in vitro. Treatment of cells with a microRNA-18a mimic in PCPS (PCPS/miR-18) knocked down 90% expression of the microRNA-18a target gene ATM. Systemic delivery of PCPS/STAT3 siRNA in murine model of MDA-MB-231 breast cancer enriched particles in tumor tissues and reduced STAT3 expression in cancer cells, causing significant reduction of cancer stem cells in the residual tumor tissue. At the therapeutic dosage, PCPS/STAT3 siRNA did not trigger acute immune response in FVB mice, including changes in serum cytokines, chemokines, and colony-stimulating factors. In addition, weekly dosing of PCPS/STAT3 siRNA for four weeks did not cause signs of subacute toxicity based on changes in body weight, hematology, blood chemistry, and major organ histology. Collectively, the results suggest that we have developed a safe vehicle for effective delivery of gene silencing agents.

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

    PubMed

    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

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

    PubMed

    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. PMID:25234370

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

    PubMed

    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. PMID:27269511

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

    PubMed

    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.

  2. High gene flow on a continental scale in the polyandrous Kentish plover Charadrius alexandrinus.

    PubMed

    Küpper, Clemens; Edwards, Scott V; Kosztolányi, András; Alrashidi, Monif; Burke, Terry; Herrmann, Philipp; Argüelles-Tico, Araceli; Amat, Juan A; Amezian, Mohamed; Rocha, Afonso; Hötker, Hermann; Ivanov, Anton; Chernicko, Joseph; Székely, Tamás

    2012-12-01

    Gene flow promotes genetic homogeneity of species in time and space. Gene flow can be modulated by sex-biased dispersal that links population genetics to mating systems. We investigated the phylogeography of the widely distributed Kentish plover Charadrius alexandrinus. This small shorebird has a large breeding range spanning from Western Europe to Japan and exhibits an unusually flexible mating system with high female breeding dispersal. We analysed genetic structure and gene flow using a 427-bp fragment of the mitochondrial (mtDNA) control region, 21 autosomal microsatellite markers and a Z microsatellite marker in 397 unrelated individuals from 21 locations. We found no structure or isolation-by-distance over the continental range. However, island populations had low genetic diversity and were moderately differentiated from mainland locations. Genetic differentiation based on autosomal markers was positively correlated with distance between mainland and each island. Comparisons of uniparentally and biparentally inherited markers were consistent with female-biased gene flow. Maternally inherited mtDNA was less structured, whereas the Z-chromosomal marker was more structured than autosomal microsatellites. Adult males were more related than females within genetic clusters. Taken together, our results suggest a prominent role for polyandrous females in maintaining genetic homogeneity across large geographic distances.

  3. Minimum entropy decomposition: Unsupervised oligotyping for sensitive partitioning of high-throughput marker gene sequences

    PubMed Central

    Eren, A Murat; Morrison, Hilary G; Lescault, Pamela J; Reveillaud, Julie; Vineis, Joseph H; Sogin, Mitchell L

    2015-01-01

    Molecular microbial ecology investigations often employ large marker gene datasets, for example, ribosomal RNAs, to represent the occurrence of single-cell genomes in microbial communities. Massively parallel DNA sequencing technologies enable extensive surveys of marker gene libraries that sometimes include nearly identical sequences. Computational approaches that rely on pairwise sequence alignments for similarity assessment and de novo clustering with de facto similarity thresholds to partition high-throughput sequencing datasets constrain fine-scale resolution descriptions of microbial communities. Minimum Entropy Decomposition (MED) provides a computationally efficient means to partition marker gene datasets into ‘MED nodes', which represent homogeneous operational taxonomic units. By employing Shannon entropy, MED uses only the information-rich nucleotide positions across reads and iteratively partitions large datasets while omitting stochastic variation. When applied to analyses of microbiomes from two deep-sea cryptic sponges Hexadella dedritifera and Hexadella cf. dedritifera, MED resolved a key Gammaproteobacteria cluster into multiple MED nodes that are specific to different sponges, and revealed that these closely related sympatric sponge species maintain distinct microbial communities. MED analysis of a previously published human oral microbiome dataset also revealed that taxa separated by less than 1% sequence variation distributed to distinct niches in the oral cavity. The information theory-guided decomposition process behind the MED algorithm enables sensitive discrimination of closely related organisms in marker gene amplicon datasets without relying on extensive computational heuristics and user supervision. PMID:25325381

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

    PubMed Central

    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

  5. High variability and non-neutral evolution of the mammalian avpr1a gene

    PubMed Central

    Fink, Sabine; Excoffier, Laurent; Heckel, Gerald

    2007-01-01

    Background The arginine-vasopressin 1a receptor has been identified as a key determinant for social behaviour in Microtus voles, humans and other mammals. Nevertheless, the genetic bases of complex phenotypic traits like differences in social and mating behaviour among species and individuals remain largely unknown. Contrary to previous studies focusing on differences in the promotor region of the gene, we investigate here the level of functional variation in the coding region (exon 1) of this locus. Results We detected high sequence diversity between higher mammalian taxa as well as between species of the genus Microtus. This includes length variation and radical amino acid changes, as well as the presence of distinct protein variants within individuals. Additionally, negative selection prevails on most parts of the first exon of the arginine-vasopressin receptor 1a (avpr1a) gene but it contains regions with higher rates of change that harbour positively selected sites. Synonymous and non-synonymous substitution rates in the avpr1a gene are not exceptional compared to other genes, but they exceed those found in related hormone receptors with similar functions. Discussion These results stress the importance of considering variation in the coding sequence of avpr1a in regards to associations with life history traits (e.g. social behaviour, mating system, habitat requirements) of voles, other mammals and humans in particular. PMID:17900345

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

    PubMed

    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. Differential gene expression in ovaries of pregnant pigs with high and low prolificacy levels and identification of candidate genes for litter size.

    PubMed

    Fernandez-Rodriguez, Amanda; Munoz, Maria; Fernandez, Almudena; Pena, Ramona N; Tomas, Anna; Noguera, Jose L; Ovilo, Cristina; Fernandez, Ana I

    2011-02-01

    Previous results from a genome scan in an F(2) Iberian × Meishan pig intercross showed several chromosome regions associated with litter size traits in this species. In order to identify candidate genes underlying these quantitative trait loci (QTL), we performed an ovary gene expression analysis during the sow's pregnancy. F(2) sows were ranked by their estimated breeding values for prolificacy: six sows with the highest estimated breeding value (EBV) (i.e., high prolificacy) and six sows with the lowest EBV (low prolificacy) were selected. Samples were hybridized using an Affymetrix GeneChip porcine genome array. Statistical analysis with a mixed model approach identified 221 differentially expressed probes, representing 189 genes. These genes were functionally annotated in order to identify genetic pathways overrepresented in this list. Among the functional groups most represented was, in first position, immune system response activation against external stimulus. The second group consisted of integrated genes that regulate maternal homeostasis by complement and coagulation cascades. A third group was involved in lipid and fatty acid enzymes of metabolic processes, which participate in the steroidogenesis pathway. In order to identify powerful candidate genes for prolificacy, the second approach of this study was to merge microarray data with the QTL positional information affecting litter size, previously detected in the same experimental cross. As a result, we have identified 27 differentially expressed genes colocalizing with QTL for litter size traits, which fulfill the biological, positional, and functional criteria. PMID:20926806

  8. GeneChip Expression Profiling Reveals the Alterations of Energy Metabolism Related Genes in Osteocytes under Large Gradient High Magnetic Fields

    PubMed Central

    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

  9. 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.

    PubMed

    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. PMID:26945769

  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.

    PubMed

    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.

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

    PubMed

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

    2014-01-01

    Microbes have great potential for arsenic (As) and antimony (Sb) bioremediation in heavily contaminated soil because they have the ability to biotransform As and Sb to species that have less toxicity or are more easily removed. In this study, we integrated a metagenomic method with physicochemical characterization to elucidate the composition of microbial community and functional genes (related to As and Sb) in a high As (range from 34.11 to 821.23 mg kg-1) and Sb (range from 226.67 to 3923.07 mg kg-1) contaminated mine field. Metagenomic analysis revealed that microbes from 18 phyla were present in the 5 samples of soil contaminated with high As and Sb. Moreover, redundancy analysis (RDA) of the relationship between the 18 phyla and the concentration of As and Sb demonstrated that 5 phyla of microbes, i.e. Actinobacteria, Firmicutes, Nitrospirae, Tenericutes and Gemmatimonadetes were positively correlated with As and Sb concentration. The distribution, diversity and abundance of functional genes (including arsC, arrA, aioA, arsB and ACR3) were much higher for the samples containing higher As and Sb concentrations. Based on correlation analysis, the results showed a positive relationship between arsC-like (R2 = 0.871) and aioA-like (R2 = 0.675) gene abundance and As concentration, and indicated that intracellular As(V) reduction and As(III) oxidation could be the dominant As detoxification mechanism enabling the microbes to survive in the environment. This study provides a direct and reliable reference on the diversity of microbial community and functional genes in an extremely high concentration As- and Sb-contaminated environment. PMID:25299175

  12. Metagenomic Approach Reveals Variation of Microbes with Arsenic and Antimony Metabolism Genes from Highly Contaminated Soil

    PubMed Central

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

    2014-01-01

    Microbes have great potential for arsenic (As) and antimony (Sb) bioremediation in heavily contaminated soil because they have the ability to biotransform As and Sb to species that have less toxicity or are more easily removed. In this study, we integrated a metagenomic method with physicochemical characterization to elucidate the composition of microbial community and functional genes (related to As and Sb) in a high As (range from 34.11 to 821.23 mg kg−1) and Sb (range from 226.67 to 3923.07 mg kg−1) contaminated mine field. Metagenomic analysis revealed that microbes from 18 phyla were present in the 5 samples of soil contaminated with high As and Sb. Moreover, redundancy analysis (RDA) of the relationship between the 18 phyla and the concentration of As and Sb demonstrated that 5 phyla of microbes, i.e. Actinobacteria, Firmicutes, Nitrospirae, Tenericutes and Gemmatimonadetes were positively correlated with As and Sb concentration. The distribution, diversity and abundance of functional genes (including arsC, arrA, aioA, arsB and ACR3) were much higher for the samples containing higher As and Sb concentrations. Based on correlation analysis, the results showed a positive relationship between arsC-like (R2 = 0.871) and aioA-like (R2 = 0.675) gene abundance and As concentration, and indicated that intracellular As(V) reduction and As(III) oxidation could be the dominant As detoxification mechanism enabling the microbes to survive in the environment. This study provides a direct and reliable reference on the diversity of microbial community and functional genes in an extremely high concentration As- and Sb-contaminated environment. PMID:25299175

  13. Comparison of low and high dose ionising radiation using topological analysis of gene coexpression networks

    PubMed Central

    2012-01-01

    Background The growing use of imaging procedures in medicine has raised concerns about exposure to low-dose ionising radiation (LDIR). While the disastrous effects of high dose ionising radiation (HDIR) is well documented, the detrimental effects of LDIR is not well understood and has been a topic of much debate. Since little is known about the effects of LDIR, various kinds of wet-lab and computational analyses are required to advance knowledge in this domain. In this paper we carry out an “upside-down pyramid” form of systems biology analysis of microarray data. We characterised the global genomic response following 10 cGy (low dose) and 100 cGy (high dose) doses of X-ray ionising radiation at four time points by analysing the topology of gene coexpression networks. This study includes a rich experimental design and state-of-the-art computational systems biology methods of analysis to study the differences in the transcriptional response of skin cells exposed to low and high doses of radiation. Results Using this method we found important genes that have been linked to immune response, cell survival and apoptosis. Furthermore, we also were able to identify genes such as BRCA1, ABCA1, TNFRSF1B, MLLT11 that have been associated with various types of cancers. We were also able to detect many genes known to be associated with various medical conditions. Conclusions Our method of applying network topological differences can aid in identifying the differences among similar (eg: radiation effect) yet very different biological conditions (eg: different dose and time) to generate testable hypotheses. This is the first study where a network level analysis was performed across two different radiation doses at various time points, thereby illustrating changes in the cellular response over time. PMID:22594378

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

    SciTech Connect

    Jett, J.

    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.

  15. A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast.

    PubMed Central

    Cullen, C F; May, K M; Hagan, I M; Glover, D M; Ohkura, H

    2000-01-01

    We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases. plo1(+) regulates both spindle formation and septation. We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+). mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes. PMID:10924454

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

    PubMed

    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

  17. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

    PubMed Central

    Horlbeck, Max A; Gilbert, Luke A; Villalta, Jacqueline E; Adamson, Britt; Pak, Ryan A; Chen, Yuwen; Fields, Alexander P; Park, Chong Yon; Corn, Jacob E; Kampmann, Martin; Weissman, Jonathan S

    2016-01-01

    We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites. DOI: http://dx.doi.org/10.7554/eLife.19760.001 PMID:27661255

  18. Reconciling extremely strong barriers with high levels of gene exchange in annual sunflowers.

    PubMed

    Sambatti, Julianno B M; Strasburg, Jared L; Ortiz-Barrientos, Daniel; Baack, Eric J; Rieseberg, Loren H

    2012-05-01

    In several cases, estimates of gene flow between species appear to be higher than we might predict given the strength of interspecific barriers separating these species pairs. However, as far as we are aware, detailed measurements of reproductive isolation have not previously been compared with a coalescent-based assessment of gene flow. Here, we contrast these two measures in two species of sunflower, Helianthus annuus and H. petiolaris. We quantified the total reproductive barrier strength between these species by compounding the contributions of the following prezygotic and postzygotic barriers: ecogeographic isolation, reproductive asynchrony, niche differentiation, pollen competition, hybrid seed formation, hybrid seed germination, hybrid fertility, and extrinsic postzygotic isolation. From this estimate, we calculated the probability that a reproductively successful hybrid is produced: estimates of P(hyb) range from 10(-4) to 10(-6) depending on the direction of the cross and the degree of independence among reproductive barriers. We then compared this probability with population genetic estimates of the per generation migration rate (m). We showed that the relatively high levels of gene flow estimated between these sunflower species (N(e) m= 0.34-0.76) are mainly due to their large effective population sizes (N(e) > 10(6)). The interspecific migration rate (m) is very small (<10(-7)) and an order of magnitude lower than that expected based on our reproductive barrier strength estimates. Thus, even high levels of reproductive isolation (>0.999) may produce genomic mosaics.

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

    PubMed

    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

  20. High glucose driven expression of pro-inflammatory cytokine and chemokine genes in lymphocytes: molecular mechanisms of IL-17 family gene expression.

    PubMed

    Kumar, Prabhakaran; Natarajan, Kartiga; Shanmugam, Narkunaraja

    2014-03-01

    High glucose is an independent risk factor that alters the expression pattern of cytokines/chemokine leading to leukocyte activation in diabetes. Fluctuation of cytokine milieu in lymphocytes may lead to differentiation into a particular subset. Our objectives were to profile high glucose induced inflammatory gene expression in lymphocytes, to examine in vivo relevance in diabetes and to identify the key transcription factors and signaling pathways involved. Cytokine gene arrays and T-helper (Th1/Th2/Th17) cytokine profiler RT(2)-PCR arrays used for cytokine expression profiling followed by validation using Real Time-qPCR and relative RT-PCR in Jurkat T-lymphocytes, peripheral blood lymphocytes (PBLCs) from normal and diabetes subjects. Luciferase reporter plasmid, pharmacological inhibitors and mutant plasmids were used for promoter activation and signaling pathway studies. High glucose induced gene profiling in Jurkat T-lymphocytes showed significantly increased expression of 64 proinflammatory genes including IL-6 and IL-17A and most of these genes were Nuclear Factor (NF)-κB and AP-1 regulated. RT(2)-PCR array results suggested the transcriptional activation of IL-17 and its downstream signaling in Jurkat T-lymphocytes upon high glucose treatment. Candidate genes like Interleukin (IL)-17A, IL-17E IL-17F and IL-6 were up-regulated in both Jurkat T-lymphocytes and PBLCs from normal and diabetes subjects. This high glucose induced cytokine expression was due to promoter activation. Pharmacology inhibitor studies showed the involvement of NF-κB, protein kinase-C, p38 Mitogen activated protein kinase; Janus activated kinase-signal transducer and activator of transcription and extracellular regulated kinase signaling pathways. Further, high glucose treatment increased the adhesion of lymphocytes to human umbilical vein endothelial cells. These results show that IL-17 cytokines are induced by high glucose via key signaling pathways leading to lymphocyte activation

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

    PubMed

    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.

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

    PubMed

    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. PMID:26890482

  3. Strategies for achieving high-level expression of genes in Escherichia coli.

    PubMed Central

    Makrides, S C

    1996-01-01

    Progress in our understanding of several biological processes promises to broaden the usefulness of Escherichia coli as a tool for gene expression. There is an expanding choice of tightly regulated prokaryotic promoters suitable for achieving high-level gene expression. New host strains facilitate the formation of disulfide bonds in the reducing environment of the cytoplasm and offer higher protein yields by minimizing proteolytic degradation. Insights into the process of protein translocation across the bacterial membranes may eventually make it possible to achieve robust secretion of specific proteins into the culture medium. Studies involving molecular chaperones have shown that in specific cases, chaperones can be very effective for improved protein folding, solubility, and membrane transport. Negative results derived from such studies are also instructive in formulating different strategies. The remarkable increase in the availability of fusion partners offers a wide range of tools for improved protein folding, solubility, protection from proteases, yield, and secretion into the culture medium, as well as for detection and purification of recombinant proteins. Codon usage is known to present a potential impediment to high-level gene expression in E. coli. Although we still do not understand all the rules governing this phenomenon, it is apparent that "rare" codons, depending on their frequency and context, can have an adverse effect on protein levels. Usually, this problem can be alleviated by modification of the relevant codons or by coexpression of the cognate tRNA genes. Finally, the elucidation of specific determinants of protein degradation, a plethora of protease-deficient host strains, and methods to stabilize proteins afford new strategies to minimize proteolytic susceptibility of recombinant proteins in E. coli. PMID:8840785

  4. Effects of high fat diet on GPR109A and GPR81 gene expression.

    PubMed

    Wanders, Desiree; Graff, Emily C; Judd, Robert L

    2012-08-24

    GPR109A (PUMA-G, NIACR1, HCA(2)) and GPR81 (HCA(1)) are G protein-coupled receptors located predominantly on adipocytes that mediate anti-lipolytic effects. These cell surface receptors give the adipocyte the ability to "sense" metabolic changes in the environment and respond through lipolytic regulation and release of products including free fatty acids and pro- or anti-inflammatory adipokines. The endogenous ligands for GPR109A and GPR81 are β-hydroxybutyrate and lactate, respectively, both of which are hydroxycarboxylic acids and intermediates of energy metabolism. Circulating β-hydroxybutyrate levels are increased during a 2-3 day fast and prolonged starvation, while lactate levels are elevated during times of intense exercise. Therefore, regulation of expression of these receptors is crucial for the metabolic sensing ability of the adipocyte and ultimately whole body energy homeostasis. We investigated the effects of high fat diet-induced obesity on expression of GPR109A and GPR81. Sixteen male C57BL/6 mice were placed on a control (10% kcal fat; n=8) or a high fat (60% kcal fat; n=8) diet for 11 weeks. Diet-induced obesity significantly reduced GPR109A and GPR81 gene expression in epididymal fat pads. This decrease in GPR109A and GPR81 gene expression was positively correlated with a decrease in adipose tissue PPARγ gene expression. In contrast, acute treatment of both 3T3-L1 adipocytes and RAW 264.7 macrophages with lipopolysaccharide significantly increased GPR109A gene expression, but had no effect on GPR81 expression in 3T3-L1 adipocytes. In conclusion, chronic obesity reduces GPR109A and GPR81 expression in the adipose tissue, while acute in vitro LPS treatment increases expression of GPR109A in adipocytes and macrophages.

  5. High time for a roll call: gene duplication and phylogenetic relationships of TCP-like genes in monocots

    PubMed Central

    Mondragón-Palomino, Mariana; Trontin, Charlotte

    2011-01-01

    Background and Aims The TCP family is an ancient group of plant developmental transcription factors that regulate cell division in vegetative and reproductive structures and are essential in the establishment of flower zygomorphy. In-depth research on eudicot TCPs has documented their evolutionary and developmental role. This has not happened to the same extent in monocots, although zygomorphy has been critical for the diversification of Orchidaceae and Poaceae, the largest families of this group. Investigating the evolution and function of TCP-like genes in a wider group of monocots requires a detailed phylogenetic analysis of all available sequence information and a system that facilitates comparing genetic and functional information. Methods The phylogenetic relationships of TCP-like genes in monocots were investigated by analysing sequences from the genomes of Zea mays, Brachypodium distachyon, Oryza sativa and Sorghum bicolor, as well as EST data from several other monocot species. Key Results All available monocot TCP-like sequences are associated in 20 major groups with an average identity ≥64 % and most correspond to well-supported clades of the phylogeny. Their sequence motifs and relationships of orthology were documented and it was found that 67 % of the TCP-like genes of Sorghum, Oryza, Zea and Brachypodium are in microsyntenic regions. This analysis suggests that two rounds of whole genome duplication drove the expansion of TCP-like genes in these species. Conclusions A system of classification is proposed where putative or recognized monocot TCP-like genes are assigned to a specific clade of PCF-, CIN- or CYC/tb1-like genes. Specific biases in sequence data of this family that must be tackled when studying its molecular evolution and phylogeny are documented. Finally, the significant retention of duplicated TCP genes from Zea mays is considered in the context of balanced gene drive. PMID:21444336

  6. Plasmid metagenome reveals high levels of antibiotic resistance genes and mobile genetic elements in activated sludge.

    PubMed

    Zhang, Tong; Zhang, Xu-Xiang; Ye, Lin

    2011-01-01

    The overuse or misuse of antibiotics has accelerated antibiotic resistance, creating a major challenge for the public health in the world. Sewage treatment plants (STPs) are considered as important reservoirs for antibiotic resistance genes (ARGs) and activated sludge characterized with high microbial density and diversity facilitates ARG horizontal gene transfer (HGT) via mobile genetic elements (MGEs). However, little is known regarding the pool of ARGs and MGEs in sludge microbiome. In this study, the transposon aided capture (TRACA) system was employed to isolate novel plasmids from activated sludge of one STP in Hong Kong, China. We also used Illumina Hiseq 2000 high-throughput sequencing and metagenomics analysis to investigate the plasmid metagenome. Two novel plasmids were acquired from the sludge microbiome by using TRACA system and one novel plasmid was identified through metagenomics analysis. Our results revealed high levels of various ARGs as well as MGEs for HGT, including integrons, transposons and plasmids. The application of the TRACA system to isolate novel plasmids from the environmental metagenome, coupled with subsequent high-throughput sequencing and metagenomic analysis, highlighted the prevalence of ARGs and MGEs in microbial community of STPs.

  7. Fish oil decreases hepatic lipogenic genes in rats fasted and refed on a high fructose diet.

    PubMed

    de Castro, Gabriela S; Cardoso, João Felipe R; Calder, Philip C; Jordão, Alceu A; Vannucchi, Helio

    2015-03-01

    Fasting and then refeeding on a high-carbohydrate diet increases serum and hepatic triacylglycerol (TAG) concentrations compared to standard diets. Fructose is a lipogenic monosaccharide which stimulates de novo fatty acid synthesis. Omega-3 (n-3) fatty acids stimulate hepatic β-oxidation, partitioning fatty acids away from TAG synthesis. This study investigated whether dietary n-3 fatty acids from fish oil (FO) improve the hepatic lipid metabolic response seen in rats fasted and then refed on a high-fructose diet. During the post-prandial (fed) period, rats fed a FO rich diet showed an increase in hepatic peroxisome proliferator-activated receptor α (PPAR-α) gene expression and decreased expression of carbohydrate responsive element binding protein (ChREBP), fatty acid synthase (FAS) and microsomal triglyceride transfer protein (MTTP). Feeding a FO rich diet for 7 days prior to 48 h of fasting resulted in lower hepatic TAG, lower PPAR-α expression and maintenance of hepatic n-3 fatty acid content. Refeeding on a high fructose diet promoted an increase in hepatic and serum TAG and in hepatic PPAR-α, ChREBP and MTTP expression. FO did not prevent the increase in serum and hepatic TAG after fructose refeeding, but did decrease hepatic expression of lipogenic genes and increased the n-3 fatty acid content of the liver. n-3 Fatty acids can modify some components of the hepatic lipid metabolic response to later feeding with a high fructose diet.

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

    PubMed Central

    Li, Wei; Li, Huafei; Li, Jinfeng; Wang, Huajing; Zhao, He; Zhang, Li; Xia, Yu; Ye, Zengwei; Gao, Jie; Dai, Jianxin; Wang, Hao; Guo, Yajun

    2012-01-01

    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-intravenous administration, intra-tumor accumulation of the stealth polyplex was clearly promoted. This study successfully circumvented the traditional dilemma of efficient gene transfection at a high nitrogen-from-polyethylenimine to phosphate-from-DNA ratio that is accompanied with site cytotoxicity and low stability. As such, these simply tailored noncytotoxic nano vesicles show significant potential for use in practical gene therapy. PMID:22977303

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

    NASA Astrophysics Data System (ADS)

    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

  10. Genes that are involved in high hydrostatic pressure treatments in a Listeria monocytogenes Scott A ctsR deletion mutant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Listeria monocytogenes is a food-borne pathogen of significant threat to public health. High Hydrostatic Pressure (HHP) treatment can be used to control L. monocytogenes in food. The CtsR (class three stress gene repressor) protein negatively regulates the expression of class III heat shock genes....

  11. Predicted highly expressed genes in Nocardia farcinica and the implication to its primary metabolism and nocardial virulence

    SciTech Connect

    Wu, Gang; Nie, Lei; Zhang, Weiwen

    2006-02-23

    Nocardia farcinica is a gram positive, filamentous bacterium, and is considered an opportunistic pathogen. In this study, the highly expressed genes in N. farcinica were predicted using the codon adaptation index (CAI) as a numerical estimator of gene expressivity. Using ribosomal protein (RP) genes as references, the top {approx}10% of the genes were predicted to be the predicted highly expressed (PHX) genes in N. farcinica using a CAI cutoff of greater than 0.73. Consistent with early analysis in Streptomyces genomes, most of the PHX genes in N. farcinica were involved in various ''house-keeping'' functions important for cell growth. However, fifteen genes putatively involved in no cardial virulence were predicted as PHX in N. farcinica, which included genes encoding four Mce virulence proteins, cyclopropane fatty acid synthase which is involved in the modification of cell wall important for nocardia virulence, polyketide synthase PKS13 for mycolic acid synthesis and non-ribosomal peptide synthetase involved in biosynthesis of a mycobactin-related siderophore. In addition, multiple genes involved in defense against reactive oxygen species (ROS) produced by the phagocyte were predicted with high expressivity, which included alkylhydroperoxide reductase (ahpC), catalase (katG), superoxide dismutase (sodF), thioredoxin, thioredoxin reductase, glutathione peroxidase, and peptide methionine sulfoxide reductase, suggesting that combating against ROS was essential for survival of N. farcinica in host cells. The study also showed that the distribution of PHX genes in the N. farcinica circular chromosome was uneven, with more PHX genes located in the regions close to replication initiation site. The results provided the first approximates of global gene expression patterns in N. farcinica, which will be useful in guiding experimental design for further investigation.

  12. Comparative Analysis of Mycobacterium tuberculosis pe and ppe Genes Reveals High Sequence Variation and an Apparent Absence of Selective Constraints

    PubMed Central

    McEvoy, Christopher R. E.; Cloete, Ruben; Müller, Borna; Schürch, Anita C.; van Helden, Paul D.; Gagneux, Sebastien; Warren, Robin M.; Gey van Pittius, Nicolaas C.

    2012-01-01

    Mycobacterium tuberculosis complex (MTBC) genomes contain 2 large gene families termed pe and ppe. The function of pe/ppe proteins remains enigmatic but studies suggest that they are secreted or cell surface associated and are involved in bacterial virulence. Previous studies have also shown that some pe/ppe genes are polymorphic, a finding that suggests involvement in antigenic variation. Using comparative sequence analysis of 18 publicly available MTBC whole genome sequences, we have performed alignments of 33 pe (excluding pe_pgrs) and 66 ppe genes in order to detect the frequency and nature of genetic variation. This work has been supplemented by whole gene sequencing of 14 pe/ppe (including 5 pe_pgrs) genes in a cohort of 40 diverse and well defined clinical isolates covering all the main lineages of the M. tuberculosis phylogenetic tree. We show that nsSNP's in pe (excluding pgrs) and ppe genes are 3.0 and 3.3 times higher than in non-pe/ppe genes respectively and that numerous other mutation types are also present at a high frequency. It has previously been shown that non-pe/ppe M. tuberculosis genes display a remarkably low level of purifying selection. Here, we also show that compared to these genes those of the pe/ppe families show a further reduction of selection pressure that suggests neutral evolution. This is inconsistent with the positive selection pressure of “classical” antigenic variation. Finally, by analyzing such a large number of genes we were able to detect large differences in mutation type and frequency between both individual genes and gene sub-families. The high variation rates and absence of selective constraints provides valuable insights into potential pe/ppe function. Since pe/ppe proteins are highly antigenic and have been studied as potential vaccine components these results should also prove informative for aspects of M. tuberculosis vaccine design. PMID:22496726

  13. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression.

    PubMed

    Scorer, C A; Clare, J J; McCombie, W R; Romanos, M A; Sreekrishna, K

    1994-02-01

    Pichia pastoris is a methylotrophic yeast increasingly important in the production of therapeutic proteins. Expression vectors are based on the methanol-inducible AOX1 promoter and are integrated into the host chromosome. In most cases high copy number integration has been shown to be important for high-level expression. Since this occurs at low frequency during transformation, we previously used DNA dot blot screens to identify suitable clones. In this paper we report the use of vectors containing the Tn903 kanr gene conferring G418-resistance. Initial experiments demonstrated that copy number showed a tight correlation with drug-resistance. Using a G418 growth inhibition screen, we readily isolated a series of transformants, containing progressively increasing numbers (1 to 12) of a vector expressing HIV-1 ENV, which we used to examine the relationship between copy number and foreign mRNA levels. Northern blot analysis indicated that ENV mRNA levels from a single-copy clone were nearly as high as AOX1 mRNA, and increased progressively with increasing copy number so as to greatly exceed AOX1 mRNA. We have also developed protocols for the selection, using G418, of high copy number transformants following spheroplast transformation or electroporation. We anticipate that these protocols will simplify the use of Pichia as a biotechnological tool.

  14. Heterogeneous high throughput scientific computing with APM X-Gene and Intel Xeon Phi

    DOE PAGESBeta

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

    2015-01-01

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

  15. Heterogeneous high throughput scientific computing with APM X-Gene and Intel Xeon Phi

    SciTech Connect

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

    2015-01-01

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

  16. A quantitative high-throughput screen identifies potential epigenetic modulators of gene expression.

    PubMed

    Johnson, Ronald L; Huang, Wenwei; Jadhav, Ajit; Austin, Christopher P; Inglese, James; Martinez, Elisabeth D

    2008-04-15

    Epigenetic regulation of gene expression is essential in embryonic development and contributes to cancer pathology. We used a cell-based imaging assay that measures derepression of a silenced green fluorescent protein (GFP) reporter to identify novel classes of compounds involved in epigenetic regulation. This locus derepression (LDR) assay was screened against a 69,137-member chemical library using quantitative high-throughput screening (qHTS), a titration-response method that assays compounds at multiple concentrations. From structure-activity relationships of the 411 actives recovered from the qHTS, 6 distinct chemical series were chosen for further study. A total of 48 qHTS actives and analogs were counterscreened using the parental line of the LDR cells, which lack the GFP reporter. Three series-8-hydroxy quinoline, quinoline-8-thiol, and 1,3,5-thiadiazinane-2-thione-were not fluorescent and reconfirmed activity in the LDR cells. The three active series did not inhibit histone deacetylase activity in nuclear extracts or reactivate the expression of the densely methylated p16 gene in cancer cells. However, one series induced expression of the methylated CDH13 gene and inhibited the viability of several lung cancer lines at submicromolar concentrations. These results suggest that the identified small molecules act on epigenetic or transcriptional components and validate our approach of using a cell-based imaging assay in conjunction with qHTS.

  17. A Quantitative High-Throughput Screen Identifies Potential Epigenetic Modulators of Gene Expression

    PubMed Central

    Johnson, Ronald L.; Huang, Wenwei; Jadhav, Ajit; Austin, Christopher P.; Inglese, James; Martinez, Elisabeth D.

    2008-01-01

    Epigenetic regulation of gene expression is essential in embryonic development and contributes to cancer pathology. We used a cell-based imaging assay that measures derepression of a silenced GFP reporter to identify novel classes of compounds involved in epigenetic regulation. This Locus Derepression (LDR) assay was screened against a 69,137-member chemical library using quantitative high-throughput screening (qHTS), a titration-response method that assays compounds at multiple concentrations. From structure-activity relationships of the 411 actives recovered from the qHTS, six distinct chemical series were chosen for further study. Forty-eight qHTS actives and analogs were counter screened using the parental line of the LDR cells, which lack the GFP reporter. Three series, 8-hydroxy quinoline, quinoline-8-thiol and 1,3,5-thiadiazinane-2-thione, were not fluorescent and re-confirmed activity in the LDR cells. The three active series did not inhibit histone deacetylase activity in nuclear extracts or reactivate the expression of the densely methylated p16 gene in cancer cells. However, one series induced expression of the methylated CDH13 gene and inhibited the viability of several lung cancer lines at submicromolar concentrations. These results suggest that the identified small molecules act on epigenetic or transcriptional components and validate our approach of using a cell-based imaging assay in conjunction with qHTS. PMID:18211814

  18. High-Throughput and Combinatorial Gene Expression on a Chip for Metabolism-Induced Toxicology Screening

    PubMed Central

    Kwon, Seok Joon; Lee, Dong Woo; Shah, Dhiral A.; Ku, Bosung; Jeon, Sang Youl; Solanki, Kusum; Ryan, Jessica D.; Clark, Douglas S.; Dordick, Jonathan S.; Lee, Moo-Yeal

    2014-01-01

    Differential expression of various drug-metabolizing enzymes in the human liver may cause deviations of pharmacokinetic profiles, resulting in inter-individual variability of drug toxicity and/or efficacy. Here we present the “Transfected Enzyme and Metabolism Chip” (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip. The device enables users to manipulate the expression of individual and multiple human metabolizing-enzyme genes (such as CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2E1, and UGT1A4) in THLE-2 cell microarrays. To identify specific enzymes involved in drug detoxification, we created 84 combinations of metabolic-gene expressions in a combinatorial fashion on a single microarray. Thus, the TeamChip platform can provide critical information necessary for evaluating metabolism-induced toxicity in a high-throughput manner. PMID:24799042

  19. High throughput profiling of antibiotic resistance genes in urban park soils with reclaimed water irrigation.

    PubMed

    Wang, Feng-Hua; Qiao, Min; Su, Jian-Qiang; Chen, Zheng; Zhou, Xue; Zhu, Yong-Guan

    2014-08-19

    Reclaimed water irrigation (RWI) in urban environments is becoming popular, due to rapid urbanization and water shortage. The continuous release of residual antibiotics and antibiotic resistance genes (ARGs) from reclaimed water could result in the dissemination of ARGs in the downstream environment. This study provides a comprehensive profile of ARGs in park soils exposed to RWI through a high-throughput quantitative PCR approach. 147 ARGs encoding for resistance to a broad-spectrum of antibiotics were detected among all park soil samples. Aminoglycoside and beta-lactam were the two most dominant types of ARGs, and antibiotic deactivation and efflux pump were the two most dominant mechanisms in these RWI samples. The total enrichment of ARGs varied from 99.3-fold to 8655.3-fold compared to respective controls. Six to 60 ARGs were statistically enriched among these RWI samples. Four transposase genes were detected in RWI samples. TnpA-04 was the most enriched transposase gene with an enrichment was up to 2501.3-fold in Urumqi RWI samples compared with control soil samples. Furthermore, significantly positive correlation was found between ARGs and transposase abundances, indicating that transposase might be involved in the propagation of ARGs. This study demonstrated that RWI resulted in the enrichment of ARGs in urban park soils. PMID:25057898

  20. High throughput profiling of antibiotic resistance genes in urban park soils with reclaimed water irrigation.

    PubMed

    Wang, Feng-Hua; Qiao, Min; Su, Jian-Qiang; Chen, Zheng; Zhou, Xue; Zhu, Yong-Guan

    2014-08-19

    Reclaimed water irrigation (RWI) in urban environments is becoming popular, due to rapid urbanization and water shortage. The continuous release of residual antibiotics and antibiotic resistance genes (ARGs) from reclaimed water could result in the dissemination of ARGs in the downstream environment. This study provides a comprehensive profile of ARGs in park soils exposed to RWI through a high-throughput quantitative PCR approach. 147 ARGs encoding for resistance to a broad-spectrum of antibiotics were detected among all park soil samples. Aminoglycoside and beta-lactam were the two most dominant types of ARGs, and antibiotic deactivation and efflux pump were the two most dominant mechanisms in these RWI samples. The total enrichment of ARGs varied from 99.3-fold to 8655.3-fold compared to respective controls. Six to 60 ARGs were statistically enriched among these RWI samples. Four transposase genes were detected in RWI samples. TnpA-04 was the most enriched transposase gene with an enrichment was up to 2501.3-fold in Urumqi RWI samples compared with control soil samples. Furthermore, significantly positive correlation was found between ARGs and transposase abundances, indicating that transposase might be involved in the propagation of ARGs. This study demonstrated that RWI resulted in the enrichment of ARGs in urban park soils.

  1. High-Affinity PEGylated Polyacridine Peptide Polyplexes Mediate Potent In Vivo Gene Expression

    PubMed Central

    Kizzire, Koby; Khargharia, Sanjib; Rice, Kevin G.

    2012-01-01

    PEGylated polyacridine peptides bind to plasmid DNA with high affinity to form unique polyplexes that possess a long circulatory half-life and are hydrodynamically (HD)-stimulated to produce efficient gene expression in the liver of mice. We previously demonstrated that (Acr-Lys)6-Cys-PEG5kDa stabilizes a 1 μg pGL3 dose for up to 1 hr in the circulation, resulting in HD-stimulated (saline only) gene expression in the liver, equivalent in magnitude to direct-HD dosing of 1 μg of pGL3 (Fernandez C.A. et al. Gene Therapy 2011). In the present study we report that increasing the spacing of Acr with either 4 or 5 Lys residues, dramatically increases the stability of PEGylated polyacridine peptide polyplexes in the circulation allowing maximal HD-stimulated expression for up to 5 hrs post-DNA administration. Co-administration of a decoy dose of 9 μg of non-expressing DNA polyplex with 1 μg of pGL3 polyplex further extended the HD-stimulated expression to 9 hrs. This structure-activity relationship study defines the PEGylated polyacridine peptide requirements for maintaining fully transfection competent plasmid DNA in the circulation for 5 hrs and provides an understanding as to why polyplexes or lipoplexes prepared with PEI, chitosan or Lipofectamine are inactive within 5 min following i.v. dosing. PMID:22786534

  2. Highly efficient method for gene delivery into mouse dorsal root ganglia neurons.

    PubMed

    Yu, Lingli; Reynaud, Florie; Falk, Julien; Spencer, Ambre; Ding, Yin-Di; Baumlé, Véronique; Lu, Ruisheng; Castellani, Valérie; Yuan, Chonggang; 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 mouse Day 13.5-16. 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 (6 × 10(4)) 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. PMID:25698920

  3. High-density SNP genotyping array for hexaploid wheat and its secondary and tertiary gene pool.

    PubMed

    Winfield, Mark O; Allen, Alexandra M; Burridge, Amanda J; Barker, Gary L A; Benbow, Harriet R; Wilkinson, Paul A; Coghill, Jane; Waterfall, Christy; Davassi, Alessandro; Scopes, Geoff; Pirani, Ali; Webster, Teresa; Brew, Fiona; Bloor, Claire; King, Julie; West, Claire; Griffiths, Simon; King, Ian; Bentley, Alison R; Edwards, Keith J

    2016-05-01

    In wheat, a lack of genetic diversity between breeding lines has been recognized as a significant block to future yield increases. Species belonging to bread wheat's secondary and tertiary gene pools harbour a much greater level of genetic variability, and are an important source of genes to broaden its genetic base. Introgression of novel genes from progenitors and related species has been widely employed to improve the agronomic characteristics of hexaploid wheat, but this approach has been hampered by a lack of markers that can be used to track introduced chromosome segments. Here, we describe the identification of a large number of single nucleotide polymorphisms that can be used to genotype hexaploid wheat and to identify and track introgressions from a variety of sources. We have validated these markers using an ultra-high-density Axiom(®) genotyping array to characterize a range of diploid, tetraploid and hexaploid wheat accessions and wheat relatives. To facilitate the use of these, both the markers and the associated sequence and genotype information have been made available through an interactive web site. PMID:26466852

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

    PubMed

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

    2003-02-01

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

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

    PubMed

    Lu, Cairui; Zou, Changsong; Song, Guoli

    2015-08-01

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

  6. High-density SNP genotyping array for hexaploid wheat and its secondary and tertiary gene pool.

    PubMed

    Winfield, Mark O; Allen, Alexandra M; Burridge, Amanda J; Barker, Gary L A; Benbow, Harriet R; Wilkinson, Paul A; Coghill, Jane; Waterfall, Christy; Davassi, Alessandro; Scopes, Geoff; Pirani, Ali; Webster, Teresa; Brew, Fiona; Bloor, Claire; King, Julie; West, Claire; Griffiths, Simon; King, Ian; Bentley, Alison R; Edwards, Keith J

    2016-05-01

    In wheat, a lack of genetic diversity between breeding lines has been recognized as a significant block to future yield increases. Species belonging to bread wheat's secondary and tertiary gene pools harbour a much greater level of genetic variability, and are an important source of genes to broaden its genetic base. Introgression of novel genes from progenitors and related species has been widely employed to improve the agronomic characteristics of hexaploid wheat, but this approach has been hampered by a lack of markers that can be used to track introduced chromosome segments. Here, we describe the identification of a large number of single nucleotide polymorphisms that can be used to genotype hexaploid wheat and to identify and track introgressions from a variety of sources. We have validated these markers using an ultra-high-density Axiom(®) genotyping array to characterize a range of diploid, tetraploid and hexaploid wheat accessions and wheat relatives. To facilitate the use of these, both the markers and the associated sequence and genotype information have been made available through an interactive web site.

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

    PubMed

    Stefanetti, Valentina; Marenzoni, Maria Luisa; Passamonti, Fabrizio; Cappelli, Katia; Garcia-Etxebarria, Koldo; Coletti, Mauro; Capomaccio, Stefano

    2016-01-01

    Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses that have co-evolved with vertebrate genomes for millions of years. Previous studies have identified the envelope (env) protein genes of retroviral origin preferentially expressed in the placenta which suggests a role in placentation based on their membrane fusogenic capacity and therefore they have been named syncytins. Until now, all the characterized syncytins have been associated with three invasive placentation types: the endotheliochorial (Carnivora), the synepitheliochorial (Ruminantia), and the hemochorial placentation (human, mouse) where they play a role in the syncytiotrophoblast formation. The purpose of the present study was to evaluate whether EqERV env RNA is expressed in horse tissues as well and investigate if the horse, possessing an epitheliochorial placenta, has "captured" a common retroviral env gene with syncytin-like properties in placental tissues. Interestingly, although in the equine placenta there is no syncytiotrophoblast layer at the maternal-fetal interface, our results showed that EqERV env RNA is highly expressed at that level, as expected for a candidate syncytin-like gene but with reduced abundance in the other somatic tissues (nearly 30-fold lower) thus suggesting a possible role in the placental tissue. Although the horse is one of the few domestic animals with a sequenced genome, few studies have been conducted about the EqERV and their expression in placental tissue has never been investigated. PMID:27176223

  8. Aspergillus glaucus Aquaglyceroporin Gene glpF Confers High Osmosis Tolerance in Heterologous Organisms.

    PubMed

    Liu, Xiao-Dan; Wei, Yi; Zhou, Xiao-Yang; Pei, Xue; Zhang, Shi-Hong

    2015-10-01

    Aquaglyceroporins (GlpFs) that transport glycerol along with water and other uncharged solutes are involved in osmoregulation in myriad species. Fungal species form a large group of eukaryotic organisms, and their GlpFs may be diverse, exhibiting various activities. However, few filamentous fungal GlpFs have been biologically investigated. Here, a glpF gene from the halophilic fungus Aspergillus glaucus (AgglpF) was verified to be a channel of water or glycerol in Xenopus laevis oocytes and was further functionally analyzed in three heterologous systems. In Saccharomyces cerevisiae, cells overexpressing AgglpF possessed significant tolerance of drought, salt, and certain metal ions. AgglpF was then characterized in the filamentous fungus of Neurospora crassa. Based on the N. crassa aquaporin gene (NcAQP) disruption mutant (the Δaqp mutant), a series of complementary strains carrying NcAQP and AgglpF and three asparagine-proline-alanine-gene (NPA)-deleted AgglpF fragments were created. As revealed by salt resistance analysis, the AgglpF complementary strain possessed the highest salt resistance among the tested strains. In addition, the intracellular glycerol content in the AgglpF complementary strain was markedly higher than that in the other strains. The AgGlpF-green fluorescent protein (GFP) fusion protein was subcellularly localized in the plasma membrane of onion epidermal cells, suggesting that AgglpF functions in plants. Indeed, when AgglpF was expressed in Arabidopsis thaliana, transgenic lines survived under conditions of high osmotic stress and under conditions of drought stress in particular. Overall, our results revealed that AgGlpF as a water/glycerol transporter is required for survival of both fungi and plants under conditions of high osmotic stress and may have value in applications in genetic engineering for generating high salt and drought resistance.

  9. Aspergillus glaucus Aquaglyceroporin Gene glpF Confers High Osmosis Tolerance in Heterologous Organisms.

    PubMed

    Liu, Xiao-Dan; Wei, Yi; Zhou, Xiao-Yang; Pei, Xue; Zhang, Shi-Hong

    2015-10-01

    Aquaglyceroporins (GlpFs) that transport glycerol along with water and other uncharged solutes are involved in osmoregulation in myriad species. Fungal species form a large group of eukaryotic organisms, and their GlpFs may be diverse, exhibiting various activities. However, few filamentous fungal GlpFs have been biologically investigated. Here, a glpF gene from the halophilic fungus Aspergillus glaucus (AgglpF) was verified to be a channel of water or glycerol in Xenopus laevis oocytes and was further functionally analyzed in three heterologous systems. In Saccharomyces cerevisiae, cells overexpressing AgglpF possessed significant tolerance of drought, salt, and certain metal ions. AgglpF was then characterized in the filamentous fungus of Neurospora crassa. Based on the N. crassa aquaporin gene (NcAQP) disruption mutant (the Δaqp mutant), a series of complementary strains carrying NcAQP and AgglpF and three asparagine-proline-alanine-gene (NPA)-deleted AgglpF fragments were created. As revealed by salt resistance analysis, the AgglpF complementary strain possessed the highest salt resistance among the tested strains. In addition, the intracellular glycerol content in the AgglpF complementary strain was markedly higher than that in the other strains. The AgGlpF-green fluorescent protein (GFP) fusion protein was subcellularly localized in the plasma membrane of onion epidermal cells, suggesting that AgglpF functions in plants. Indeed, when AgglpF was expressed in Arabidopsis thaliana, transgenic lines survived under conditions of high osmotic stress and under conditions of drought stress in particular. Overall, our results revealed that AgGlpF as a water/glycerol transporter is required for survival of both fungi and plants under conditions of high osmotic stress and may have value in applications in genetic engineering for generating high salt and drought resistance. PMID:26209670

  10. Aspergillus glaucus Aquaglyceroporin Gene glpF Confers High Osmosis Tolerance in Heterologous Organisms

    PubMed Central

    Liu, Xiao-Dan; Wei, Yi; Zhou, Xiao-Yang; Pei, Xue

    2015-01-01

    Aquaglyceroporins (GlpFs) that transport glycerol along with water and other uncharged solutes are involved in osmoregulation in myriad species. Fungal species form a large group of eukaryotic organisms, and their GlpFs may be diverse, exhibiting various activities. However, few filamentous fungal GlpFs have been biologically investigated. Here, a glpF gene from the halophilic fungus Aspergillus glaucus (AgglpF) was verified to be a channel of water or glycerol in Xenopus laevis oocytes and was further functionally analyzed in three heterologous systems. In Saccharomyces cerevisiae, cells overexpressing AgglpF possessed significant tolerance of drought, salt, and certain metal ions. AgglpF was then characterized in the filamentous fungus of Neurospora crassa. Based on the N. crassa aquaporin gene (NcAQP) disruption mutant (the Δaqp mutant), a series of complementary strains carrying NcAQP and AgglpF and three asparagine-proline-alanine-gene (NPA)-deleted AgglpF fragments were created. As revealed by salt resistance analysis, the AgglpF complementary strain possessed the highest salt resistance among the tested strains. In addition, the intracellular glycerol content in the AgglpF complementary strain was markedly higher than that in the other strains. The AgGlpF-green fluorescent protein (GFP) fusion protein was subcellularly localized in the plasma membrane of onion epidermal cells, suggesting that AgglpF functions in plants. Indeed, when AgglpF was expressed in Arabidopsis thaliana, transgenic lines survived under conditions of high osmotic stress and under conditions of drought stress in particular. Overall, our results revealed that AgGlpF as a water/glycerol transporter is required for survival of both fungi and plants under conditions of high osmotic stress and may have value in applications in genetic engineering for generating high salt and drought resistance. PMID:26209670

  11. Gene expression profiling defines a high-risk entity of multiple myeloma.

    PubMed

    Zhan, Feng-Huang; Barlogie, Bart; John D, Shaughnessy

    2007-04-01

    Multiple myeloma (MM) is the second most common hematological malignancy and remains incurable. The marked variation in survival of patients with symptomatic myeloma ranging from few months to more than 15 years can be explained by differences in tumor mass, proliferative activity and, more recently, by cytogenetic and molecular genetic characteristics of the myeloma clone. Oligonucleotide microarray-based gene expression analysis was applied to CD138-enriched plasma cells from newly diagnosed patients with symptomatic or progressive multiple myeloma treated with melphalan-based high-dose therapy. Here we discuss recent progress made in the development of molecular-based diagnostics and prognostics for MM from Myeloma Institute for Research and Therapy of University Arkansas for Medical Sciences, where we treat more patients with myeloma than anywhere else in the world. Seven distinct entities of myeloma were elucidated by genomic profiling. Expression extremes of 70 genes from a high-risk signature profile,30% of which were derived from chromosome 1, were strongly linked to disease-related survival. CKS1B located on chromosome 1q21, responsible for promoting cell cycle progression by inducing the degradation of p27Kip1, represented a strong candidate gene related to rapid patient death and was studied in detail. The data suggest that CKS1B influences myeloma cell growth and survival through SKP2j and P27(Kip1) -dependent and independent mechanisms and that therapeutic strategies aimed at abolishing CKS1B function may hold promise for the treatment of high-risk disease for which effective therapies are currently lacking.

  12. Effects of High Fat Feeding on Adipose Tissue Gene Expression in Diabetic Goto-Kakizaki Rats

    PubMed Central

    Xue, Bai; Nie, Jing; Wang, Xi; DuBois, Debra C; Jusko, William J; Almon, Richard R

    2015-01-01

    Development and progression of type 2 diabetes is a complex interaction between genetics and environmental influences. High dietary fat is one environmental factor that is conducive to the development of insulin-resistant diabetes. In the present report, we compare the responses of lean poly-genic, diabetic Goto-Kakizaki (GK) rats to those of control Wistar-Kyoto (WKY) rats fed a high fat diet from weaning to 20 weeks of age. This comparison included a wide array of physiological measurements along with gene expression profiling of abdominal adipose tissue using Affymetrix gene array chips. Animals of both strains fed a high fat diet or a normal diet were sacrificed at 4, 8, 12, 16, and 20 weeks for this comparison. The microarray analysis revealed that the two strains developed different adaptations to increased dietary fat. WKY rats decrease fatty acid synthesis and lipogenic processes whereas GK rats increase lipid elimination. However, on both diets the major differences between the two strains remained essentially the same. Specifically relative to the WKY strain, the GK strain showed lipoatrophy, chronic inflammation, and insulin resistance. PMID:26309393

  13. Finding differentially expressed genes in high dimensional data: Rank based test statistic via a distance measure.

    PubMed

    Mathur, Sunil; Sadana, Ajit

    2015-12-01

    We present a rank-based test statistic for the identification of differentially expressed genes using a distance measure. The proposed test statistic is highly robust against extreme values and does not assume the distribution of parent population. Simulation studies show that the proposed test is more powerful than some of the commonly used methods, such as paired t-test, Wilcoxon signed rank test, and significance analysis of microarray (SAM) under certain non-normal distributions. The asymptotic distribution of the test statistic, and the p-value function are discussed. The application of proposed method is shown using a real-life data set.

  14. A visible, targeted high-efficiency gene delivery and transfection strategy

    PubMed Central

    2011-01-01

    Background To enhance myocardial angiogenic gene expression, a novel gene delivery strategy was tested. Direct intramyocardial injection of an angiogenic gene with microbubbles and insonation were applied in a dog animal model. Dogs received one of the four different treatments in conjunction with either the enhanced green fluorescence protein (EGFP) gene or the hepatocyte growth factor (HGF) gene: gene with microbubbles (MB) and ultrasound (US); gene with US; gene with MB; or the gene alone. Results Distribution of MB and the gene in the myocardium was visualized during the experiment. Compared with the EGFP gene group, an average 14.7-fold enhancement in gene expression was achieved in the EGFP+MB/US group (P < 0.01). Compared with the HGF gene group, an average 10.7-fold enhancement in gene expression was achieved in the HGF+MB/US group (P < 0.01). In addition, capillary density increased from 20.8 ± 3.4/mm2 in the HGF gene group to 146.7 ± 31.4/mm2 in HGF+MB/US group (P < 0.01). Conclusions Thus, direct intramyocardial injection of an angiogenic gene in conjunction with microbubbles plus insonation synergistically enhances angiogenesis. This method offers an observable gene delivery procedure with enhanced expression efficiency of the delivered gene. PMID:21600027

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

    PubMed

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

    2013-03-01

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

  16. Selection of reference genes for qRT-PCR in high fat diet-induced hepatic steatosis mice model.

    PubMed

    Xu, Lingyan; Ma, Xinran; Cui, Bin; Li, Xiaoying; Ning, Guang; Wang, Shu

    2011-07-01

    With the epidemic proportions of obesity worldwide and the concurrent prevalence of hepatic steatosis, there is an urgent need for better understanding the intrinsic mechanism of hepatic steatosis, especially the changes of gene expression underlying the development of hepatic steatosis and its associated abnormal liver function. Quantitative real-time PCR (qRT-PCR) is a sensitive and highly reproducible technique of gene expression analysis. However, for accurate and reliable gene expression results, it is vital to have an internal control gene expressed at constant levels under all the experimental conditions being analyzed for. In this study, the authors validated candidate reference genes suitable for qRT-PCR profiling experiments using livers from control mice and high fat diet-induced obese mice. Cross-validation of expression stability of ten selected reference genes using three popular algorithms, GeNorm, NormFinder, and BestKeeper found HPRT1 and GAPDH as most stable reference genes. Thus, HPRT1 and GAPDH are recommended as stable reference genes most suitable for gene expression studies in the development of hepatic steatosis.

  17. Highly specific expression of luciferase gene in lungs of naive nude mice directed by prostate-specific antigen promoter

    SciTech Connect

    Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng . E-mail: Dongfeng_pan@yahoo.com

    2005-09-09

    PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10{sup 9} PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.

  18. Targeted and highly efficient gene transfer into CD4+ cells by a recombinant human immunodeficiency virus retroviral vector.

    PubMed Central

    Shimada, T; Fujii, H; Mitsuya, H; Nienhuis, A W

    1991-01-01

    We have established a recombinant HIV gene transfer system based on transient expression of the HIV packaging functions and a recombinant vector genome in monkey kidney Cos cells. The recombinant HIV retroviral vector introduced the neoR gene into CD4+ cells with high efficiency, comparable to that achieved with the highest titer amphotropic murine recombinant retrovirus. Vector preparations were devoid of replication competent, infectious HIV. Gene transfer was dependent on CD4 expression, as shown by expression of the CD4 gene in HeLa cells, and could be inhibited by soluble CD4. This specific and efficient gene transfer system may be useful for development of gene therapy for which T cells are the desired targets. Images PMID:1885765

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

    PubMed

    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. PMID:26736050

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

    PubMed

    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.

  1. Toward an ultra-high resolution community climate system model for the BlueGene platform

    NASA Astrophysics Data System (ADS)

    Dennis, John M.; Jacob, Robert; Vertenstein, Mariana; Craig, Tony; Loy, Raymond

    2007-07-01

    Global climate models need to simulate several small, regional-scale processes which affect the global circulation in order to accurately simulate the climate. This is particularly important in the ocean where small scale features such as oceanic eddies are currently represented with adhoc parameterizations. There is also a need for higher resolution to provide climate predictions at small, regional scales. New high-performance computing platforms such as the IBM BlueGene can provide the necessary computational power to perform ultra-high resolution climate model integrations. We have begun to investigate the scaling of the individual components of the Community Climate System Model to prepare it for integrations on BlueGene and similar platforms. Our investigations show that it is possible to successfully utilize Script O(32K) processors. We describe the scalability of five models: the Parallel Ocean Program (POP), the Community Ice CodE (CICE), the Community Land Model (CLM), and the new CCSM sequential coupler (CPL7) which are components of the next generation Community Climate System Model (CCSM); as well as the High-Order Method Modeling Environment (HOMME) which is a dynamical core currently being evaluated within the Community Atmospheric Model. For our studies we concentrate on 1/10° resolution for CICE, POP, and CLM models and 1/4° resolution for HOMME. The ability to simulate high resolutions on the massively parallel petascale systems that will dominate high-performance computing for the foreseeable future is essential to the advancement of climate science.

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

    PubMed Central

    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. PMID:25429295

  3. Expression of nitric oxide synthase (NOS) genes in channel catfish is highly regulated and time dependent after bacterial challenges.

    PubMed

    Yao, Jun; Li, Chao; Zhang, Jiaren; Liu, Shikai; Feng, Jianbin; Wang, Ruijia; Li, Yun; Jiang, Chen; Song, Lin; Chen, Ailu; Liu, Zhanjiang

    2014-07-01

    Nitric oxide is well known for its roles in immune responses. As such, its synthesizing enzymes have been extensively studied from various species including some teleost fish species. However, the NOS genes have not been characterized in channel catfish (Ictalurus punctatus). In this study, we identified and characterized three NOS genes including one NOS1 and two NOS2 genes in channel catfish. Comparing with the NOS genes from other fish species, the catfish NOS genes are highly conserved in their structural features. Phylogenetic and syntenic analyses allowed determination of NOS1 and NOS2 genes of channel catfish and their orthology relationships. Syntenic analysis, as well as the phylogenetic analysis, indicated that the two NOS2 genes of catfish were lineage-specific duplication. The NOS genes were broadly expressed in most tested tissues, with NOS1 being expressed at the highest levels in the brain, NOS2b1 highly expressed in the skin and gill, and NOS2b2 lowly expressed in most of the tested tissues. The most striking findings of this study was that the expression of the NOS genes are highly regulated after bacterial infection, with time-dependent expression patterns that parallel the migration of macrophages. After Edwardsiella ictaluri challenge, dramatically different responses among the three NOS genes were observed. NOS1 was only significantly in the skin early after infection, while NOS2b1 was rapidly upregulated in gill, but more up-regulated in trunk kidney with the progression of the disease, suggesting such differences in gene expression may be reflective of the migration of macrophages among various tissues of the infected fish. In contrast to NOS1 and NOS2b1, NOS2b2 was normally expressed at very low levels, but it is induced in the brain and liver while significantly down-regulated in most other tissues.

  4. High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization

    PubMed Central

    Yoshikawa, Toshiyuki; Piao, Yulan; Zhong, Jinhui; Matoba, Ryo; Carter, Mark G.; Wang, Yuxia; Goldberg, Ilya; Ko, Minoru S.H.

    2006-01-01

    Mammalian preimplantation embryos provide an excellent opportunity to study temporal and spatial gene expression in whole mount in situ hybridization (WISH). However, large-scale studies are made difficult by the size of the embryos (∼60 μm diameter) and their fragility. We have developed a chamber system that allows parallel processing of embryos without the aid of a microscope. We first selected 91 candidate genes that were transcription factors highly expressed in blastocysts, and more highly expressed in embryonic (ES) than in trophoblast (TS) stem cells. We then used the WISH to identify 48 genes expressed predominantly in the ICM and to follow several of these genes in all seven preimplantation stages. The ICM-predominant expressions of these genes suggest their involvement in the pluripotency of embryonic cells. This system provides a useful tool to a systematic genome-scale analysis of preimplantation embryos. PMID:16325481

  5. Identifier (ID) elements are not preferentially located to brain-specific genes: high ID element representation in other tissue-specific- and housekeeping genes of the rat.

    PubMed

    Goldman, Andrés; Capoano, Carlos A; González-López, Evangelina; Geisinger, Adriana

    2014-01-01

    BC1 is a short non-coding RNA from rodents, which is transcribed by RNA pol III. Its RNA is highly abundant in the brain, where it exerts a post-transcriptional regulatory role in dendrites. Upon transcription, retroposition and insertion, BC1 gives rise to a subclass of short interspersed repetitive sequences (SINEs) named identifier (ID) elements. IDs can become integrated inside non-coding regions of RNA pol II transcription units, and - although challenged by a couple of reports - their preferential location to brain-specific genes has been long proposed. Furthermore, an additional, cis-regulatory role in the control of brain-specific pol II-directed transcripts has been suggested for these sequences. In this work we used Northern blot and in silico analyses to examine IDs' location among pol II transcription units in different tissues, and in housekeeping genes. ID sequences appeared distributed in a similar fashion within tissue-specific hnRNA populations of the brain, testis and liver, and within housekeeping primary transcripts as well. Moreover, when the lengths of the unprocessed transcripts were considered, ID representation was higher in housekeeping ones. On the other hand, ID elements appeared similarly distributed among the different gene regions, with the obvious exclusion of those sequences where strict constraints for proper gene expression exist. Altogether, the widespread distribution of ID elements in all the analyzed genes - including housekeeping - and in all gene regions, suggests a random location, raising questions about the specific cis-regulatory role of those sequences.

  6. Effects of curcumin on global gene expression profiles in the highly invasive human breast carcinoma cell line MDA-MB 231: A gene network-based microarray analysis.

    PubMed

    Cine, Naci; Limtrakul, Pornngarm; Sunnetci, Deniz; Nagy, Balint; Savli, Hakan

    2013-01-01

    Curcumin, or diferuloylmethane, is a major chemical component of turmeric (Curcuma longa Linn.) that has been consumed as a dietary spice through the ages. This yellow-colored polyphenol has a notably wide range of beneficial properties, including anti-inflammatory, antioxidant, antitumoral, anti-invasive and anti-metastatic activity. In the present study, microarray gene expression analysis was applied to identify the curcumin-regulated genes in a highly invasive human breast carcinoma cell line (MDA-MB 231). Cells were cultured with curcumin (20 μM) for 24 h; total RNA was isolated and hybridized to Whole Human Genome Microarray slides. Gene set enrichment analyses on our whole genome expression data revealed downregulation of the EGF pathway elements following curcumin treatment. Furthermore, gene network analysis identified a significantly relevant network among the differentially expressed genes, centered on the EGR1 and FOS genes. The members of these pathways and networks play an essential role in the regulation of cancer cell growth and development; the majority exhibited decreased expression levels following treatment with curcumin. These observations suggest that curcumin is an excellent candidate for the prevention and treatment of breast cancer. PMID:23251236

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

    NASA Astrophysics Data System (ADS)

    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.

  8. High-resolution mapping of the gene for cystinosis, using combined biochemical and linkage analysis

    SciTech Connect

    Jean, G.; Fuchshuber, A.; Gribouval, O.

    1996-03-01

    Infantile nephropathic cystinosis is an autosomal recessive disorder characterized biochemically by an abnormally high intracellular content of free cystine in different organs and tissues due to a transport defect of cystine through the lysosomal membrane. Affected children present with the Fanconi syndrome and usually develop progressive renal failure within the 1st decade of life. Measurement of free cystine in purified polymorphonuclear leukocytes provides an accurate method for diagnosis and detection of heterozygous carriers previously determined by their leukocyte cystine content in the linkage analysis. This approach allowed us to obtain highly significant results, confirming the localization of the cystinosis gene locus recently mapped to the short arm of chromosome 17 by the Cystinosis Collaborative Research Group. Crucial recombination events allowed us to refine the interval of the cystinosis gene to a genetic distance of 1 cM. No evidence of genetic heterogeneity was found. Our results demonstrate that the use of the previously determined phenotypes of heterozygous carriers in linkage analysis provides a reliable method for the investigation of simplex families in autosomal recessive traits. 25 refs., 4 figs., 1 tab.

  9. 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

    PubMed Central

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

    2011-01-01

    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. PMID:21705439

  10. Improve carbon metabolic flux in Saccharomyces cerevisiae at high temperature by overexpressed TSL1 gene.

    PubMed

    Ge, Xiang-Yang; Xu, Yan; Chen, Xiang

    2013-04-01

    This study describes a novel strategy to improve the glycolysis flux of Saccharomyces cerevisiae at high temperature. The TSL1 gene-encoding regulatory subunit of the trehalose synthase complex was overexpressed in S. cerevisiae Z-06, which increased levels of trehalose synthase activity in extracts, enhanced stress tolerance and glucose consuming rate of the yeast cells. As a consequence, the final ethanol concentration of 185.5 g/L was obtained at 38 °C for 36 h (with productivity up to 5.2 g/L/h) in 7-L fermentor, and the ethanol productivity was 92.7 % higher than that of the parent strain. The results presented here provide a novel way to enhance the carbon metabolic flux at high temperature, which will be available for the purposes of producing other primary metabolites of commercial interest using S. cerevisiae as a host.

  11. Fabricating Genetically Engineered High-Power Lithium-Ion Batteries Using Multiple Virus Genes

    NASA Astrophysics Data System (ADS)

    Lee, Yun Jung; Yi, Hyunjung; Kim, Woo-Jae; Kang, Kisuk; Yun, Dong Soo; Strano, Michael S.; Ceder, Gerbrand; Belcher, Angela M.

    2009-05-01

    Development of materials that deliver more energy at high rates is important for high-power applications, including portable electronic devices and hybrid electric vehicles. For lithium-ion (Li+) batteries, reducing material dimensions can boost Li+ ion and electron transfer in nanostructured electrodes. By manipulating two genes, we equipped viruses with peptide groups having affinity for single-walled carbon nanotubes (SWNTs) on one end and peptides capable of nucleating amorphous iron phosphate(a-FePO4) fused to the viral major coat protein. The virus clone with the greatest affinity toward SWNTs enabled power performance of a-FePO4 comparable to that of crystalline lithium iron phosphate (c-LiFePO4) and showed excellent capacity retention upon cycling at 1C. This environmentally benign low-temperature biological scaffold could facilitate fabrication of electrodes from materials previously excluded because of extremely low electronic conductivity.

  12. Identification of Transcription Factor Genes and Their Correlation with the High Diversity of Stramenopiles

    PubMed Central

    Buitrago-Flórez, Francisco Javier; Restrepo, Silvia; Riaño-Pachón, Diego Mauricio

    2014-01-01

    The biological diversity among Stramenopiles is striking; they range from large multicellular seaweeds to tiny unicellular species, they embrace many ecologically important autothrophic (e.g., diatoms, brown algae), and heterotrophic (e.g., oomycetes) groups. Transcription factors (TFs) and other transcription regulators (TRs) regulate spatial and temporal gene expression. A plethora of transcriptional regulatory proteins have been identified and classified into families on the basis of sequence similarity. The purpose of this work is to identify the TF and TR complement in diverse species belonging to Stramenopiles in order to understand how these regulators may contribute to their observed diversity. We identified and classified 63 TF and TR families in 11 species of Stramenopiles. In some species we found gene families with high relative importance. Taking into account the 63 TF and TR families identified, 28 TF and TR families were established to be positively correlated with specific traits like number of predicted proteins, number of flagella and number of cell types during the life cycle. Additionally, we found gains and losses in TF and TR families specific to some species and clades, as well as, two families with high abundance specific to the autotrophic species and three families with high abundance specific to the heterotropic species. For the first time, there is a systematic search of TF and TR families in Stramenopiles. The attempts to uncover relationships between these families and the complexity of this group may be of great impact, considering that there are several important pathogens of plants and animals, as well as, important species involved in carbon cycling. Specific TF and TR families identified in this work appear to be correlated with particular traits in the Stramenopiles group and may be correlated with the high complexity and diversity in Stramenopiles. PMID:25375671

  13. High-Stearic and High-Oleic Cottonseed Oils Produced by Hairpin RNA-Mediated Post-Transcriptional Gene Silencing1

    PubMed Central

    Liu, Qing; Singh, Surinder P.; Green, Allan G.

    2002-01-01

    We have genetically modified the fatty acid composition of cottonseed oil using the recently developed technique of hairpin RNA-mediated gene silencing to down-regulate the seed expression of two key fatty acid desaturase genes, ghSAD-1-encoding stearoyl-acyl-carrier protein Δ9-desaturase and ghFAD2-1-encoding oleoyl-phosphatidylcholine ω6-desaturase. Hairpin RNA-encoding gene constructs (HP) targeted against either ghSAD-1 or ghFAD2-1 were transformed into cotton (Gossypium hirsutum cv Coker 315). The resulting down-regulation of the ghSAD-1 gene substantially increased stearic acid from the normal levels of 2% to 3% up to as high as 40%, and silencing of the ghFAD2-1 gene resulted in greatly elevated oleic acid content, up to 77% compared with about 15% in seeds of untransformed plants. In addition, palmitic acid was significantly lowered in both high-stearic and high-oleic lines. Similar fatty acid composition phenotypes were also achieved by transformation with conventional antisense constructs targeted against the same genes, but at much lower frequencies than were achieved with the HP constructs. By intercrossing the high-stearic and high-oleic genotypes, it was possible to simultaneously down-regulate both ghSAD-1 and ghFAD2-1 to the same degree as observed in the individually silenced parental lines, demonstrating for the first time, to our knowledge, that duplex RNA-induced posttranslational gene silencing in independent genes can be stacked without any diminution in the degree of silencing. The silencing of ghSAD-1 and/or ghFAD2-1 to various degrees enables the development of cottonseed oils having novel combinations of palmitic, stearic, oleic, and linoleic contents that can be used in margarines and deep frying without hydrogenation and also potentially in high-value confectionery applications. PMID:12177486

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

    PubMed Central

    2010-01-01

    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. PMID:20929578

  15. Targeted gene enrichment and high-throughput sequencing for environmental biomonitoring: a case study using freshwater macroinvertebrates.

    PubMed

    Dowle, Eddy J; Pochon, Xavier; C Banks, Jonathan; Shearer, Karen; Wood, Susanna A

    2016-09-01

    Recent studies have advocated biomonitoring using DNA techniques. In this study, two high-throughput sequencing (HTS)-based methods were evaluated: amplicon metabarcoding of the cytochrome C oxidase subunit I (COI) mitochondrial gene and gene enrichment using MYbaits (targeting nine different genes including COI). The gene-enrichment method does not require PCR amplification and thus avoids biases associated with universal primers. Macroinvertebrate samples were collected from 12 New Zealand rivers. Macroinvertebrates were morphologically identified and enumerated, and their biomass determined. DNA was extracted from all macroinvertebrate samples and HTS undertaken using the illumina miseq platform. Macroinvertebrate communities were characterized from sequence data using either six genes (three of the original nine were not used) or just the COI gene in isolation. The gene-enrichment method (all genes) detected the highest number of taxa and obtained the strongest Spearman rank correlations between the number of sequence reads, abundance and biomass in 67% of the samples. Median detection rates across rare (<1% of the total abundance or biomass), moderately abundant (1-5%) and highly abundant (>5%) taxa were highest using the gene-enrichment method (all genes). Our data indicated primer biases occurred during amplicon metabarcoding with greater than 80% of sequence reads originating from one taxon in several samples. The accuracy and sensitivity of both HTS methods would be improved with more comprehensive reference sequence databases. The data from this study illustrate the challenges of using PCR amplification-based methods for biomonitoring and highlight the potential benefits of using approaches, such as gene enrichment, which circumvent the need for an initial PCR step. PMID:26583904

  16. Targeted gene enrichment and high-throughput sequencing for environmental biomonitoring: a case study using freshwater macroinvertebrates.

    PubMed

    Dowle, Eddy J; Pochon, Xavier; C Banks, Jonathan; Shearer, Karen; Wood, Susanna A

    2016-09-01

    Recent studies have advocated biomonitoring using DNA techniques. In this study, two high-throughput sequencing (HTS)-based methods were evaluated: amplicon metabarcoding of the cytochrome C oxidase subunit I (COI) mitochondrial gene and gene enrichment using MYbaits (targeting nine different genes including COI). The gene-enrichment method does not require PCR amplification and thus avoids biases associated with universal primers. Macroinvertebrate samples were collected from 12 New Zealand rivers. Macroinvertebrates were morphologically identified and enumerated, and their biomass determined. DNA was extracted from all macroinvertebrate samples and HTS undertaken using the illumina miseq platform. Macroinvertebrate communities were characterized from sequence data using either six genes (three of the original nine were not used) or just the COI gene in isolation. The gene-enrichment method (all genes) detected the highest number of taxa and obtained the strongest Spearman rank correlations between the number of sequence reads, abundance and biomass in 67% of the samples. Median detection rates across rare (<1% of the total abundance or biomass), moderately abundant (1-5%) and highly abundant (>5%) taxa were highest using the gene-enrichment method (all genes). Our data indicated primer biases occurred during amplicon metabarcoding with greater than 80% of sequence reads originating from one taxon in several samples. The accuracy and sensitivity of both HTS methods would be improved with more comprehensive reference sequence databases. The data from this study illustrate the challenges of using PCR amplification-based methods for biomonitoring and highlight the potential benefits of using approaches, such as gene enrichment, which circumvent the need for an initial PCR step.

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

    PubMed Central

    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

  18. Lipoic acid modified low molecular weight polyethylenimine mediates nontoxic and highly potent in vitro gene transfection.

    PubMed

    Zheng, Meng; Zhong, Yinan; Meng, Fenghua; Peng, Rui; Zhong, Zhiyuan

    2011-12-01

    The clinical success of gene therapy intimately relies on the development of safe and efficient gene carrier systems. We found here that 1.8 kDa polyethylenimine (PEI) following hydrophobic modification with lipoic acid (LA) mediated nontoxic and highly potent in vitro gene transfection in both HeLa and 293T cells. 1.8 kDa PEI-LA conjugates were prepared with controlled degree of substitution (DS) by coupling LA to PEI using carbodiimide chemistry. Gel electrophoresis measurements showed that the DNA binding ability of 1.8 kDa PEI was impaired by lipoylation, in which an N/P ratio of 2/1 and 4-6/1 was required for 1.8 kDa PEI and 1.8 kDa PEI-LA conjugates, respectively, to completely inhibit DNA migration. Interestingly, dynamic light scattering measurements (DLS) revealed that PEI-LA conjugates condensed DNA into much smaller sizes (183-84 nm) than unmodified 1.8 kDa PEI (444-139 nm) at N/P ratios ranging from 20/1 to 60/1. These polyplexes revealed similar surface charges of ca. +22 to +30 mV. 1.8 kDa PEI-LA(2) polyplexes formed at an N/P ratio of 10/1 were stable against exchange with 12-fold excess of negatively charged dextran sodium sulfate (DSS) relative to DNA phosphate groups while 1.8 kDa PEI controls dissociated at 6-fold excess of DSS, indicating that lipoylation of 1.8 kDa PEI resulted in stronger binding with DNA. Importantly, DNA was released from 1.8 kDa PEI-LA(2) polyplexes upon addition of 10 mM dithiothreitol (DTT). Reduction-triggered unpacking of 1.8 kDa PEI-LA(2) polyplexes was also confirmed by DLS. MTT assays demonstrated that all PEI-LA conjugates and polyplexes were essentially nontoxic to HeLa and 293T cells up to a tested concentration of 50 μg/mL and an N/P ratio of 80/1, respectively. The in vitro gene transfection studies in HeLa and 293T cells showed that lipoylation of 1.8 kDa PEI markedly boosted its transfection activity. For example, 1.8 kDa PEI-LA(2) polyplexes displayed 400-fold and 500-fold higher levels of gene expression

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

    PubMed

    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

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

    PubMed

    Szafron, Lukasz Michal; Balcerak, Anna; Grzybowska, Ewa Anna; Pienkowska-Grela, Barbara; Felisiak-Golabek, Anna; Podgorska, Agnieszka; Kulesza, Magdalena; Nowak, Natalia; Pomorski, Pawel; Wysocki, Juliusz; Rubel, Tymon; Dansonka-Mieszkowska, Agnieszka; Konopka, Bozena; Lukasik, Martyna; Kupryjanczyk, Jolanta

    2015-01-01

    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.

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

    PubMed Central

    Szafron, Lukasz Michal; Balcerak, Anna; Grzybowska, Ewa Anna; Pienkowska-Grela, Barbara; Felisiak-Golabek, Anna; Podgorska, Agnieszka; Kulesza, Magdalena; Nowak, Natalia; Pomorski, Pawel; Wysocki, Juliusz; Rubel, Tymon; Dansonka-Mieszkowska, Agnieszka; Konopka, Bozena; Lukasik, Martyna; Kupryjanczyk, Jolanta

    2015-01-01

    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. PMID:25978564

  2. Highly restricted gene flow and deep evolutionary lineages in the giant clam Tridacna maxima

    NASA Astrophysics Data System (ADS)

    Nuryanto, A.; Kochzius, M.

    2009-09-01

    The tropical Indo-West Pacific is the biogeographic region with the highest diversity of marine shallow water species, with its centre in the Indo-Malay Archipelago. However, due to its high endemism, the Red Sea is also considered as an important centre of evolution. Currently, not much is known about exchange among the Red Sea, Indian Ocean and West Pacific, as well as connectivity within the Indo-Malay Archipelago, even though such information is important to illuminate ecological and evolutionary processes that shape marine biodiversity in these regions. In addition, the inference of connectivity among populations is important for conservation. This study aims to test the hypothesis that the Indo-Malay Archipelago and the Red Sea are important centres of evolution by studying the genetic population structure of the giant clam Tridacna maxima. This study is based on a 484-bp fragment of the cytochrome c oxidase I gene from 211 individuals collected at 14 localities in the Indo-West Pacific to infer lineage diversification and gene flow as a measure for connectivity. The analysis showed a significant genetic differentiation among sample sites in the Indo-West Pacific (Φst = 0.74, P < 0.001) and across the Indo-Malay Archipelago (Φst = 0.72, P < 0.001), indicating restricted gene flow. Hierarchical AMOVA revealed the highest fixation index (Φct = 0.8, P < 0.001) when sample sites were assigned to the following regions: (1) Red Sea, (2) Indian Ocean and Java Sea, (3) Indonesian throughflow and seas in the East of Sulawesi, and (4) Western Pacific. Geological history as well as oceanography are important factors that shape the genetic structure of T. maxima in the Indo-Malay Archipelago and Red Sea. The observed deep evolutionary lineages might include cryptic species and this result supports the notion that the Indo-Malay Archipelago and the Red Sea are important centres of evolution.

  3. High Producing Tumor Necrosis Factor Alpha Gene Alleles in Protection against Severe Manifestations of Dengue

    PubMed Central

    Sam, Sing-Sin; Teoh, Boon-Teong; Chinna, Karuthan; AbuBakar, Sazaly

    2015-01-01

    Dengue virus (DENV) infection usually presents with mild self-limiting dengue fever (DF). Few however, would present with the more severe form of the disease, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In the present study, the association between IL-12B, IL-10 and TNF-α gene polymorphisms and dengue severity was investigated. Methods: A case-control study was performed on a total of 120 unrelated controls, 86 DF patients and 196 DHF/DSS patients. The polymorphisms in IL-12B, IL-10 and TNF-α genes were genotyped using PCR-RFLP and PCR-sequencing methods. Results: A protective association of TNF-α -308A allele and -308GA genotype against DHF/DSS was observed, while TNF-α -238A allele and -238GA genotype were associated with DHF/DSS. A combination of TNF-α -308GA+AA genotype and IL-10 non-GCC haplotypes, IL-12B pro homozygotes (pro1/pro1, pro2/pro2) and IL-12B 3'UTR AC were significantly correlated with protective effects against DHF/DSS. An association between the cytokine gene polymorphisms and protection against the clinical features of severe dengue including thrombocytopenia and increased liver enzymes was observed in this study. Conclusion: The overall findings of the study support the correlation of high-producer TNF-α genotypes combined with low-producer IL-10 haplotypes and IL-12B genotypes in reduced risk of DHF/DSS. PMID:25589894

  4. The implication of life style on codon usage patterns and predicted highly expressed genes for three Frankia genomes.

    PubMed

    Sen, Arnab; Sur, Saubashya; Bothra, Asim K; Benson, David R; Normand, Philippe; Tisa, Louis S

    2008-05-01

    Frankia are nitrogen-fixing actinomycetes that form a symbiotic association with over 200 species of woody dicotyledonous plants. Recently, three Frankia genomes were completely sequenced. In this study, the synonymous codon usage patterns of three Frankia genomes (strains CcI3, ACN14a, and EAN1pec) were determined and compared to each other and to other actinobacteria. As expected for a high G+C organism, codon usage by Frankia was highly biased, but differences were observed among the three strains. Using the codon adaptation index (CAI) as a numerical estimator of gene expression level, highly expressed genes in Frankia were predicted with ribosomal protein genes as a reference. The analysis of the predicted highly expressed genes showed that Frankia strain CcI3 had a different profile from the other two strains. Strain CcI3 had fewer predicted highly expressed genes in several COG categories including lipid transport and metabolism, secondary metabolites biosynthesis, inorganic ion transport and metabolism, and general function prediction only than Frankia strains EAN1pec and ACN14a. Interestingly, Frankia EAN1pec had more predicted highly expressed genes in transcription and signal transduction mechanisms than the other two strains. These differences were not just a reflection in total gene numbers, but also based on percentage of genes within a category. These results support the hypothesis that strain CcI3 is becoming a symbiotic specialist and the other two facultative symbiotic strains are maintaining their capacity to exist as free-living soil dwellers.

  5. Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements

    PubMed Central

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

    2009-01-01

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

  6. SINA: Accurate high-throughput multiple sequence alignment of ribosomal RNA genes

    PubMed Central

    Pruesse, Elmar; Peplies, Jörg; Glöckner, Frank Oliver

    2012-01-01

    Motivation: In the analysis of homologous sequences, computation of multiple sequence alignments (MSAs) has become a bottleneck. This is especially troublesome for marker genes like the ribosomal RNA (rRNA) where already millions of sequences are publicly available and individual studies can easily produce hundreds of thousands of new sequences. Methods have been developed to cope with such numbers, but further improvements are needed to meet accuracy requirements. Results: In this study, we present the SILVA Incremental Aligner (SINA) used to align the rRNA gene databases provided by the SILVA ribosomal RNA project. SINA uses a combination of k-mer searching and partial order alignment (POA) to maintain very high alignment accuracy while satisfying high throughput performance demands. SINA was evaluated in comparison with the commonly used high throughput MSA programs PyNAST and mothur. The three BRAliBase III benchmark MSAs could be reproduced with 99.3, 97.6 and 96.1 accuracy. A larger benchmark MSA comprising 38 772 sequences could be reproduced with 98.9 and 99.3% accuracy using reference MSAs comprising 1000 and 5000 sequences. SINA was able to achieve higher accuracy than PyNAST and mothur in all performed benchmarks. Availability: Alignment of up to 500 sequences using the latest SILVA SSU/LSU Ref datasets as reference MSA is offered at http://www.arb-silva.de/aligner. This page also links to Linux binaries, user manual and tutorial. SINA is made available under a personal use license. Contact: epruesse@mpi-bremen.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22556368

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

    PubMed

    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.

  8. Diagnosis of canine leptospirosis by a highly sensitive FRET-PCR targeting the lig genes.

    PubMed

    Xu, Chuanling; Loftis, Amanda; Ahluwalia, Sudhir K; Gao, Dongya; Verma, Ashutosh; Wang, Chengming; Kaltenboeck, Bernhard

    2014-01-01

    Canine leptospirosis is underdiagnosed due to its wide spectrum of clinical presentations and the lack of a rapid and sensitive test for the accurate diagnosis of acute and chronic infections. In this study, we developed a highly sensitive and specific fluorescence resonance energy transfer (FRET)-PCR to detect common pathogenic leptospires in dogs, including Leptospira interrogans serovars Autumnalis, Canicola, Copenhageni (Icterohaemorrhagiae serogroup) and Pomona, and Leptospira kirschneri serovar Grippotyphosa. This PCR targets the lig genes, exclusively found in the pathogenic Leptospira species but not in saprophytic species (L. biflexa). A robust, high-stringency step-down real-time platform was coupled to the highly specific detection of leptospiral DNA by fluorescently labeled FRET probes. This enabled the detection of a single copy of the lig gene in a PCR containing DNA from up to 50 µL canine blood or 400 µL urine. Sensitivity determination by use of limiting serial dilutions of extracted leptospiral DNA indicated that the lig FRET-PCR we established was almost 100-fold more sensitive than the widely accepted lipL32 SYBR assay and 10-fold more sensitive than a 16S rRNA TaqMan assay. Application of this method to 207 dogs with potential leptospiral infection enabled us to diagnose three cases of canine leptospirosis characterized by low amounts of leptospiral DNA in body fluids. Detection of canine leptospirosis with the lig FRET-PCR was more sensitive with the lig FRET-PCR than with the 16S rRNA TaqMan PCR, which detected only 2 of the 3 cases, and the lipL32 SYBR PCR, which detected none of the 3 dogs with leptospirosis. PMID:24586833

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

    PubMed

    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.

  10. Suppression of Ripening-Associated Gene Expression in Tomato Fruits Subjected to a High CO2 Concentration.

    PubMed Central

    Rothan, C.; Duret, S.; Chevalier, C.; Raymond, P.

    1997-01-01

    High concentrations of CO2 block or delay the ripening of fruits. In this study we investigated the effects of high CO2 on ripening and on the expression of stress- and ripening-inducible genes in cherry tomato (Lycopersicon esculentum Mill.) fruit. Mature-green tomato fruits were submitted to a high CO2 concentration (20%) for 3 d and then transferred to air. These conditions effectively inhibited ripening-associated color changes and ethylene production, and reduced the protein content. No clear-cut effect was observed on the expression of two proteolysis-related genes, encoding polyubiquitin and ubiquitin-conjugating enzyme E2, respectively. Exposure of fruit to high CO2 also resulted in the strong induction of two genes encoding stress-related proteins: a ripening-regulated heat-shock protein and glutamate decarboxylase. Induction of these two genes indicated that high CO2 had a stress effect, most likely through cytosolic acidification. In addition, high CO2 blocked the accumulation of mRNAs for genes involved in the main ripening-related changes: ethylene synthesis (1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase), color (phytoene synthase), firmness (polygalacturonase), and sugar accumulation (acid invertase). The expression of ripening-specific genes was affected by CO2 regardless of whether their induction was ethylene- or development-dependent. It is proposed that the inhibition of tomato fruit ripening by high CO2 is due, in part, to the suppression of the expression of ripening-associated genes, which is probably related to the stress effect exerted by high CO2. PMID:12223703

  11. A Novel Tryptophanyl-tRNA Synthetase Gene Confers High-Level Resistance to Indolmycin▿ †

    PubMed Central

    Vecchione, James J.; Sello, Jason K.

    2009-01-01

    Indolmycin, a potential antibacterial drug, competitively inhibits bacterial tryptophanyl-tRNA synthetases. An effort to identify indolmycin resistance genes led to the discovery of a gene encoding an indolmycin-resistant isoform of tryptophanyl-tRNA synthetase. Overexpression of this gene in an indolmycin-sensitive strain increased the indolmycin MIC 60-fold. Its transcription and distribution in various bacterial genera were assessed. The level of resistance conferred by this gene was compared to that of a known indolmycin resistance gene and to those of genes with resistance-conferring point mutations. PMID:19546369

  12. Molecular evolutionary analysis of the high-affinity K+ transporter gene family in angiosperms.

    PubMed

    Yang, P; Hua, C; Zhou, F; Zhang, B-J; Cai, X-N; Chen, Q-Z; Wang, R-L

    2016-07-15

    The high-affinity K(+) transporter (HKT) family comprises a group of multifunctional cation transporters widely distributed in organisms ranging from Bacteria to Eukarya. In angiosperms, the HKT family consists primarily of nine types, whose evolutionary relationships are not fully understood. The available sequences from 31 plant species were used to perform a comprehensive evolutionary analysis, including an examination of selection pressure and estimating phylogenetic tree and gene duplication events. Our results show that a gene duplication in the HKT1;5/HKT1;4 cluster might have led to the divergence of the HKT1;5 and HKT1;4 subfamilies. Additionally, maximum likelihood analysis revealed that the HKT family has undergone a strong purifying selection. An analysis of the amino acids provided strong statistical evidence for a functional divergence between subfamilies 1 and 2. Our study was the first to provide evidence of this functional divergence between these two subfamilies. Analysis of co-evolution in HKT identified 25 co-evolved groups. These findings expanded our understanding of the evolutionary mechanisms driving functional diversification of HKT proteins.

  13. The human archain gene, ARCN1, has highly conserved homologs in rice and drosophila

    SciTech Connect

    Radice, P.; Jones, C.; Perry, H.

    1995-03-01

    A novel human gene, ARCN1, has been identified in chromosome band 11q23.3. It maps approximately 50 kb telomeric to MLL, a gene that is disrupted in a number of leukemia-associated translocation chromosomes. cDNA clones representing ARCN1 hybridize to 4-kb mRNA species present in all tissues tested. Sequencing of cDNAs suggests that at least two forms of mRNA with alternative 5 {prime} ends are present within the cell. The mRNA with the longest open reading frame gives rise to a protein of 57 kDa. Although the sequence reported is novel, remarkable similarity is observed with two predicted protein sequences from partial DNA sequences generated by rice (Oryza sativa) and fruit fly (Drosophila melanogaster) genome projects. The degree of sequence conservation is comparable to that observed for highly conserved structural proteins, such as heat shock protein HSP70, and is greater than that of {gamma}-gubulin and heat shock protein HSP60. A more distant relationship to the group of clathrin-associated proteins suggests a possible role in vesicle structure or trafficking. In view of its ancient pedigree and a potential involvement in cellular architecture, the authors propose that the ARCN1 protein be named archain. 20 refs., 5 figs.

  14. Genetic analysis of axial length genes in high grade myopia from Indian population☆

    PubMed Central

    Sharmila, Ferdinamarie; Abinayapriya; Ramprabhu, Karthikeyan; Kumaramanickavel, Govindasamy; R.R.Sudhir; Sripriya, Sarangapani

    2014-01-01

    Purpose To study the putative association of Membrane frizzled related protein (MFRP) and Visual system homeobox protein (VSX2) gene variants with axial length (AL) in myopia. Method A total of 189 samples with (N = 98) and without (N = 91) myopia were genotyped for the MRFP and VSX2 variations in ABI Prism 3100 AVANT genetic analyzer. Genotype/haplotype analysis was performed using PLINK, Haploview and THESIAS softwares. Results Fifteen variations were observed in the MFRP gene of which, rs36015759 (c.492C > T, T164T) in exon 5 was distributed at a high frequency in the controls and significantly associated with a low risk for myopia (P = 4.10 ∗ e− 07 OR < 1.0). An increased frequency for the coding haplotype block [CGTCGG] harboring rs36015759 was observed in controls (31%) than cases (8%) that also correlated with a decreased mean AL (− 1.35085; P = 0.000444) by THESIAS analysis. The ‘T’ allele of rs36015759 was predicted to abolish the binding site for splicing enhancer (SRp40) by FASTSNP analysis. Conclusion Myopia is a complex disorder influenced by genetic and environmental factors. Our work shows evidence of association of a specific MFRP haplotype which was more prevalent in controls with decreased AL. However, replication and functional studies are warranted to confirm these findings. PMID:25606400

  15. High transcript levels of heat-shock genes are associated with shorter lifespan of Caenorhabditis elegans.

    PubMed

    Manière, X; Krisko, A; Pellay, F X; Di Meglio, J-M; Hersen, P; Matic, I

    2014-12-01

    Individual lifespans of isogenic organisms, such as Caenorhabditis elegans nematodes, fruit flies, and mice, vary greatly even under identical environmental conditions. To study the molecular mechanisms responsible for such variability, we used an assay based on the measurement of post-reproductive nematode movements stimulated by a moderate electric field. This assay allows for the separation of individual nematodes based on their speed. We show that this phenotype could be used as a biomarker for aging because it is a better predictor of lifespan than chronological age. Fast nematodes have longer lifespans, fewer protein carbonyls, higher heat-shock resistance, and higher transcript levels of the daf-16 and hsf-1 genes, which code for the stress response transcription factors, than slow nematodes. High transcript levels of the genes coding for heat-shock proteins observed in slow nematodes correlate with lower heat-shock resistance, more protein carbonyls, and shorter lifespan. Taken together, our data suggests that shorter lifespan results from early-life damage accumulation that causes subsequent faster age-related deterioration.

  16. Molecular evolutionary analysis of the high-affinity K+ transporter gene family in angiosperms.

    PubMed

    Yang, P; Hua, C; Zhou, F; Zhang, B-J; Cai, X-N; Chen, Q-Z; Wang, R-L

    2016-01-01

    The high-affinity K(+) transporter (HKT) family comprises a group of multifunctional cation transporters widely distributed in organisms ranging from Bacteria to Eukarya. In angiosperms, the HKT family consists primarily of nine types, whose evolutionary relationships are not fully understood. The available sequences from 31 plant species were used to perform a comprehensive evolutionary analysis, including an examination of selection pressure and estimating phylogenetic tree and gene duplication events. Our results show that a gene duplication in the HKT1;5/HKT1;4 cluster might have led to the divergence of the HKT1;5 and HKT1;4 subfamilies. Additionally, maximum likelihood analysis revealed that the HKT family has undergone a strong purifying selection. An analysis of the amino acids provided strong statistical evidence for a functional divergence between subfamilies 1 and 2. Our study was the first to provide evidence of this functional divergence between these two subfamilies. Analysis of co-evolution in HKT identified 25 co-evolved groups. These findings expanded our understanding of the evolutionary mechanisms driving functional diversification of HKT proteins. PMID:27525850

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

    PubMed

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

    2003-09-15

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

  18. Association between PKA gene polymorphism and NTDs in high risk Chinese population in Shanxi

    PubMed Central

    Wu, Jian; Lu, Xiaolin; Wang, Zhen; Shangguan, Shaofang; Chang, Shaoyan; Li, Rui; Wu, Lihua; Bao, Yihua; Niu, Bo; Wang, Li; Zhang, Ting

    2013-01-01

    Objective: This study aimed to investigate the single nucleotide polymorphisms (SNPs) of PKA and neural tube defects (NTDs) in Chinese population. Method: A total of 183 NTDs cases and 200 healthy controls were used in this study. 7 selected single nucleotide polymorphism (SNP) sites in the PKA gene were analyzed with MassArray high-throughput DNA analyzer with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. A series of statistical methods were carried out to investigate the correlation between the SNPs and the patient susceptibility to NTDs. Results: Statistical analysis showed a significant correlation between the SNP sites rs12132032 in PRKACB and NTDs. The AA genotype, A-allele and dominant AA in rs12132032 significantly increased the incidence of NTDs especially anencephaly (OR=3.87, 95% CI: 1.80-8.34 with genotype; OR=2.08, 95% CI: 1.43-3.04 with allele; OR=3.10, 95% CI: 1.53-6.26 with dominant). The T-allele of rs594631 in PRKACB was correlative with NTDs in male but not in female. Conclusions: The gene polymorphism loci rs12132032 in PRKACB maybe a potential risk factor for anencephaly in Chinese population from Shanxi, while gender susceptibility may influence the correlation. PMID:24294386

  19. High-molecular-weight polyethyleneimine conjuncted pluronic for gene transfer agents.

    PubMed

    Liang, Wenqing; Gong, Haiyang; Yin, Dongfeng; Lu, Shiyong; Fu, Qiang

    2011-01-01

    In order to enhance the gene delivery efficiency and decrease cytotoxicity of polyplexes, copolymers consisting of branched polyethyleneimine (PEI) 25 kDa grafted with Pluronic (F127, F68, P105) were successfully synthesized using a simple two-step procedure. The copolymers were tested for cytotoxicity and DNA condensation and complexation properties. Their polyplexes with plasmid DNA were characterized in terms of DNA size and surface charge and transfection efficiency. The complex sizes were below 300 nm, which implicated their potential for intracellular delivery. The Pluronic-g-PEI exhibited better condensation and complexation properties than PEI 25 kDa. The cytotoxicity of PEI was strongly reduced after copolymerization. The Pluronic-g-PEI showed lower cytotoxicity in three different cell lines (Hela, MCF-7, and HepG2) than PEI 25 kDa. pGL3-lus was used as a reporter gene, and the transfection efficiency was in vitro measured in HeLa cells. Compared with unmodified PEI 25 kDa Pluronic-g-PEI showed much higher transfection efficiency. These results demonstrate that polyplexes prepared using a combined strategy of surface crosslinking and grafted with Pluronic seem to provide promising properties as stable, high transfection efficiency vectors.

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

    SciTech Connect

    Wright, J.; Teraoka, S.; Concannon, P.

    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.

  1. Highly Efficient Gene Suppression by Chemically Modified 27 Nucleotide Double-Stranded RNAs

    NASA Astrophysics Data System (ADS)

    Kubo, Takanori; Zhelev, Zhivko; Bakalova, Rumiana; Ohba, Hideki

    2008-02-01

    RNA interference (RNAi) technology, described by Fire and Mello in 1998, is a powerful tool for the suppression of gene expression in mammalian cells. RNAi technology has several advantages over other chemical and genetic drugs. However, several problems in RNAi technology, such as cellular delivery, nuclease stability, and side effects, should be solved before applying it in the clinic. In this study, we focused on the development of novel chemically modified 27 nucleotide (nt) double-stranded RNAs (dsRNAs) with improved biological properties. Our chemically modified 27 nt dsRNAs exhibited an enhanced RNAi activity and a markedly increased stability in cell culture medium (containing 10% serum) in comparison with widely used 21 nt siRNAs and recently reported nonmodified 27 nt dsRNAs. The chemically modified 27 nt dsRNAs also exhibited a strong high long-term gene silencing effect after the 7 d treatment of viable cells. The chemically modified 27 nt dsRNAs in specific positions could be processed to 21 nt siRNAs by a recombinant Dicer enzyme. We suggested that the chemically modified 27 nt dsRNAs could be used for therapeutic applications (as genetic drugs) and bioanalyses.

  2. Genome-scale gene function prediction using multiple sources of high-throughput data in yeast Saccharomyces cerevisiae.

    PubMed

    Joshi, Trupti; Chen, Yu; Becker, Jeffrey M; Alexandrov, Nickolai; Xu, Dong

    2004-01-01

    Characterizing gene function is one of the major challenging tasks in the post-genomic era. To address this challenge, we have developed GeneFAS (Gene Function Annotation System), a new integrated probabilistic method for cellular function prediction by combining information from protein-protein interactions, protein complexes, microarray gene expression profiles, and annotations of known proteins through an integrative statistical model. Our approach is based on a novel assessment for the relationship between (1) the interaction/correlation of two proteins' high-throughput data and (2) their functional relationship in terms of their Gene Ontology (GO) hierarchy. We have developed a Web server for the predictions. We have applied our method to yeast Saccharomyces cerevisiae and predicted functions for 1548 out of 2472 unannotated proteins.

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

    SciTech Connect

    Patrick, N.; Miyakawa, F.; Hunt, J.A.

    1994-09-01

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

  4. Long QT Interval in Turner Syndrome – A High Prevalence of LQTS Gene Mutations

    PubMed Central

    Trolle, Christian; Mortensen, Kristian H.; Pedersen, Lisbeth N.; Berglund, Agnethe; Jensen, Henrik K.; Andersen, Niels H.; Gravholt, Claus H.

    2013-01-01

    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 (p<0.001) and did not change over time (416.9±22.6 vs. 415.6±25.5 ms; p = 0.4). 45,X karyotype was associated with increased hQTc prolongation compared to other Turner syndrome karyotypes (418.2±24.8 vs. 407.6±25.5 ms; p = 0.055). In women with Turner syndrome and a bQTc >432 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. PMID:23936059

  5. The SKP1-Like Gene Family of Arabidopsis Exhibits a High Degree of Differential Gene Expression and Gene Product Interaction during Development

    PubMed Central

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

    2012-01-01

    The Arabidopsis thaliana genome encodes several families of polypeptides that are known or predicted to participate in the formation of the SCF-class of E3-ubiquitin ligase complexes. One such gene family encodes the Skp1-like class of polypeptide subunits, where 21 genes have been identified and are known to be expressed in Arabidopsis. Phylogenetic analysis based on deduced polypeptide sequence organizes the family of ASK proteins into 7 clades. The complexity of the ASK gene family, together with the close structural similarity among its members raises the prospect of significant functional redundancy among select paralogs. We have assessed the potential for functional redundancy within the ASK gene family by analyzing an expanded set of criteria that define redundancy with higher resolution. The criteria used include quantitative expression of locus-specific transcripts using qRT-PCR, assessment of the sub-cellular localization of individual ASK:YFP auto-fluorescent fusion proteins expressed in vivo as well as the in planta assessment of individual ASK-F-Box protein interactions using bimolecular fluorescent complementation techniques in combination with confocal imagery in live cells. The results indicate significant functional divergence of steady state transcript abundance and protein-protein interaction specificity involving ASK proteins in a pattern that is poorly predicted by sequence-based phylogeny. The information emerging from this and related studies will prove important for defining the functional intersection of expression, localization and gene product interaction that better predicts the formation of discrete SCF complexes, as a prelude to investigating their molecular mode of action. PMID:23226441

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

    PubMed

    Mok, Wendy W K; Li, Yingfu

    2013-04-15

    Molecular cloning technologies that have emerged in recent years are more efficient and simpler to use than traditional strategies, but many have the disadvantages of requiring multiple steps and expensive proprietary enzymes. We have engineered cloning vectors containing variants of IbsC, a 19-residue toxin from Escherichia coli K-12. These toxic peptides offer selectivity to minimise the background, labour, and cost associated with conventional molecular cloning. As demonstrated with the cloning of reporter genes, this "detox cloning" system consistently produced over 95 % positive clones. Purification steps between digestion and ligation are not necessary, and the total time between digestion and plating of transformants can be as little as three hours. Thus, these IbsC-based cloning vectors are as reliable and amenable to high-throughput cloning as commercially available systems, and have the advantage of being more time-efficient and cost-effective. PMID:23512843

  7. Phenotype MicroArrays for High-Throughput Phenotypic Testing and Assay of Gene Function

    PubMed Central

    Bochner, Barry R.; Gadzinski, Peter; Panomitros, Eugenia

    2001-01-01

    The bacterium Escherichia coli is used as a model cellular system to test and validate a new technology called Phenotype MicroArrays (PMs). PM technology is a high-throughput technology for simultaneous testing of a large number of cellular phenotypes. It consists of preconfigured well arrays in which each well tests a different cellular phenotype and an automated instrument that continuously monitors and records the response of the cells in all wells of the arrays. For example, nearly 700 phenotypes of E. coli can be assayed by merely pipetting a cell suspension into seven microplate arrays. PMs can be used to directly assay the effects of genetic changes on cells, especially gene knock-outs. Here, we provide data on phenotypic analysis of six strains and show that we can detect expected phenotypes as well as, in some cases, unexpected phenotypes. PMID:11435407

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

    PubMed

    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

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

    PubMed

    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.

  10. High-throughput transcriptomic analysis nominates proteasomal genes as age-specific biomarkers and therapeutic targets in prostate cancer

    PubMed Central

    Zhao, S G; Jackson, W C; Kothari, V; Schipper, M J; Erho, N; Evans, J R; Speers, C; Hamstra, D A; Niknafs, Y S; Nguyen, P L; Schaeffer, E M; Ross, A E; Den, R B; Klein, E A; Jenkins, R B; Davicioni, E; Feng, F Y

    2015-01-01

    Background: Although prostate cancer (PCa) is hypothesized to differ in nature between younger versus older patients, the underlying molecular distinctions are poorly understood. We hypothesized that high-throughput transcriptomic analysis would elucidate biological differences in PCas arising in younger versus older men, and would nominate potential age-specific biomarkers and therapeutic targets. Methods: The high-density Affymetrix GeneChip platform, encompassing >1 million genomic loci, was utilized to assess gene expression in 1090 radical prostatectomy samples from patients with long-term follow-up. We identified genes associated with metastatic progression by 10 years post-treatment in younger (age<65) versus older (age⩾65) patients, and ranked these genes by their prognostic value. We performed Gene Set Enrichment Analysis (GSEA) to nominate biological concepts that demonstrated age-specific effects, and validated a target by treating with a clinically available drug in three PCa cell lines derived from younger men. Results: Over 80% of the top 1000 prognostic genes in younger and older men were specific to that age group. GSEA nominated the proteasome pathway as the most differentially prognostic in younger versus older patients. High expression of proteasomal genes conferred worse prognosis in younger but not older men on univariate and multivariate analysis. Bortezomib, a Food and Drug Administration approved proteasome inhibitor, decreased proliferation in three PCa cell lines derived from younger patients. Conclusions: Our data show significant global differences in prognostic genes between older versus younger men. We nominate proteasomeal gene expression as an age-specific biomarker and potential therapeutic target specifically in younger men. Limitations of our study include clinical differences between cohorts, and increased comorbidities and lower survival in older patients. These intriguing findings suggest that current models of PCa biology do

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

    PubMed Central

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

    2015-01-01

    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

  12. Cross-species hybridisation of human and bovine orthologous genes on high density cDNA microarrays

    PubMed Central

    Adjaye, James; Herwig, Ralf; Herrmann, Doris; Wruck, Wasco; BenKahla, Alia; Brink, Thore C; Nowak, Monika; Carnwath, Joseph W; Hultschig, Claus; Niemann, Heiner; Lehrach, Hans

    2004-01-01

    Background Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. The objective of this study was to investigate the feasibility and reproducibility of cross-species analysis employing a human cDNA microarray as probe. Results As a proof of principle, total RNA derived from human and bovine fetal brains was used as a source of labelled targets for hybridisation onto a human cDNA microarray composed of 349 characterised genes. Each gene was spotted 20 times representing 6,980 data points thus enabling highly reproducible spot quantification. Employing high stringency hybridisation and washing conditions, followed by data analysis, revealed slight differences in the expression levels and reproducibility of the signals between the two species. We also assigned each of the genes into three expression level categories- i.e. high, medium and low. The correlation co-efficient of cross hybridisation between the orthologous genes was 0.94. Verification of the array data by semi-quantitative RT-PCR using common primer sequences enabled co-amplification of both human and bovine transcripts. Finally, we were able to assign gene names to previously uncharacterised bovine ESTs. Conclusions Results of our study demonstrate the harnessing and utilisation power of comparative genomics and prove the feasibility of using human microarrays to facilitate the identification of co-expressed orthologous genes in common tissues derived from different

  13. Does global gene expression analysis in type 2 diabetes provide an opportunity to identify highly promising drug targets?

    PubMed

    Buechler, C; Schäffler, A

    2007-12-01

    The recent technological advances in high-throughput gene expression analysis allow the simultaneous investigation of thousands of genes. These technologies represent promising tools for the identification of new drug targets and considerable progress has been achieved in cancer research where microarray data provide a basis to design new drugs and to predict adverse reactions and the efficacy of chemotherapy. The metabolic syndrome represents a cluster of disorders including high blood pressure, insulin resistance/type 2 diabetes mellitus, visceral obesity and dyslipidaemia with fatty liver disease being a common associated complication. High-throughput gene expression analyses using GeneChips, microarrays and serial analysis of gene expression (SAGE) have been applied to study global gene expression in insulin resistance/type 2 diabetes mellitus. Type 2 diabetes mellitus is a multifactorial and polygenic disease by which several organs are affected. Therefore, the identification of both, disease causing and therapeutically relevant target genes is an ambitious challenge. In the present review we focus on genomic approaches that used biopsies from human skeletal muscle, liver and adipose tissue, the main organs affected by insulin resistance. Members of the PPARgamma coactivator-1 (PGC-1) family of transcriptional coactivators are decreased in skeletal muscle in insulin resistance accounting for the reduced expression of genes involved in mitochondrial oxidative phosphorylation. Hepatic steatosis is also linked to alterations in mitochondrial phosphorylation and oxidative metabolism. An up regulation of pro-inflammatory genes can be detected in early stages of fatty liver disease without histological signs of inflammation. Impaired adipogenesis, intra-adipose accumulation of macrophages and a sustained release of inflammatory and acute phase proteins are characteristic features of adipose tissue in obesity and may aggravate systemic insulin resistance.

  14. Microfluidic droplet-based PCR instrumentation for high-throughput gene expression profiling and biomarker discovery.

    PubMed

    Hayes, Christopher J; Dalton, Tara M

    2015-06-01

    PCR is a common and often indispensable technique used in medical and biological research labs for a variety of applications. Real-time quantitative PCR (RT-qPCR) has become a definitive technique for quantitating differences in gene expression levels between samples. Yet, in spite of this importance, reliable methods to quantitate nucleic acid amounts in a higher throughput remain elusive. In the following paper, a unique design to quantify gene expression levels at the nanoscale in a continuous flow system is presented. Fully automated, high-throughput, low volume amplification of deoxynucleotides (DNA) in a droplet based microfluidic system is described. Unlike some conventional qPCR instrumentation that use integrated fluidic circuits or plate arrays, the instrument performs qPCR in a continuous, micro-droplet flowing process with droplet generation, distinctive reagent mixing, thermal cycling and optical detection platforms all combined on one complete instrument. Detailed experimental profiling of reactions of less than 300 nl total volume is achieved using the platform demonstrating the dynamic range to be 4 order logs and consistent instrument sensitivity. Furthermore, reduced pipetting steps by as much as 90% and a unique degree of hands-free automation makes the analytical possibilities for this instrumentation far reaching. In conclusion, a discussion of the first demonstrations of this approach to perform novel, continuous high-throughput biological screens is presented. The results generated from the instrument, when compared with commercial instrumentation, demonstrate the instrument reliability and robustness to carry out further studies of clinical significance with added throughput and economic benefits. PMID:27077035

  15. Microfluidic droplet-based PCR instrumentation for high-throughput gene expression profiling and biomarker discovery

    PubMed Central

    Hayes, Christopher J.; Dalton, Tara M.

    2015-01-01

    PCR is a common and often indispensable technique used in medical and biological research labs for a variety of applications. Real-time quantitative PCR (RT-qPCR) has become a definitive technique for quantitating differences in gene expression levels between samples. Yet, in spite of this importance, reliable methods to quantitate nucleic acid amounts in a higher throughput remain elusive. In the following paper, a unique design to quantify gene expression levels at the nanoscale in a continuous flow system is presented. Fully automated, high-throughput, low volume amplification of deoxynucleotides (DNA) in a droplet based microfluidic system is described. Unlike some conventional qPCR instrumentation that use integrated fluidic circuits or plate arrays, the instrument performs qPCR in a continuous, micro-droplet flowing process with droplet generation, distinctive reagent mixing, thermal cycling and optical detection platforms all combined on one complete instrument. Detailed experimental profiling of reactions of less than 300 nl total volume is achieved using the platform demonstrating the dynamic range to be 4 order logs and consistent instrument sensitivity. Furthermore, reduced pipetting steps by as much as 90% and a unique degree of hands-free automation makes the analytical possibilities for this instrumentation far reaching. In conclusion, a discussion of the first demonstrations of this approach to perform novel, continuous high-throughput biological screens is presented. The results generated from the instrument, when compared with commercial instrumentation, demonstrate the instrument reliability and robustness to carry out further studies of clinical significance with added throughput and economic benefits. PMID:27077035

  16. High-Frequency Variation of Purine Biosynthesis Genes Is a Mechanism of Success in Campylobacter jejuni

    PubMed Central

    Cameron, Andrew; Huynh, Steven; Scott, Nichollas E.; Frirdich, Emilisa; Apel, Dmitry; Foster, Leonard J.; Parker, Craig T.

    2015-01-01

    ABSTRACT Phenotypic variation is prevalent in the zoonotic pathogen Campylobacter jejuni, the leading agent of enterocolitis in the developed world. Heterogeneity enhances the survival and adaptive malleability of bacterial populations because variable phenotypes may allow some cells to be protected against future stress. Exposure to hyperosmotic stress previously revealed prevalent differences in growth between C. jejuni strain 81-176 colonies due to resistant or sensitive phenotypes, and these isolated colonies continued to produce progeny with differential phenotypes. In this study, whole-genome sequencing of isolated colonies identified allelic variants of two purine biosynthesis genes, purF and apt, encoding phosphoribosyltransferases that utilize a shared substrate. Genetic analyses determined that purF was essential for fitness, while apt was critical. Traditional and high-depth amplicon-sequencing analyses confirmed extensive intrapopulation genetic variation of purF and apt that resulted in viable strains bearing alleles with in-frame insertion duplications, deletions, or missense polymorphisms. Different purF and apt alleles were associated with various stress survival capabilities under several niche-relevant conditions and contributed to differential intracellular survival in an epithelial cell infection model. Amplicon sequencing revealed that intracellular survival selected for stress-fit purF and apt alleles, as did exposure to oxygen and hyperosmotic stress. Putative protein recognition direct repeat sequences were identified in purF and apt, and a DNA-protein affinity screen captured a predicted exonuclease that promoted the global spontaneous mutation rate. This work illustrates the adaptive properties of high-frequency genetic variation in two housekeeping genes, which influences C. jejuni survival under stress and promotes its success as a pathogen. PMID:26419875

  17. Comparative Mitogenomics of the Genus Odontobutis (Perciformes: Gobioidei: Odontobutidae) Revealed Conserved Gene Rearrangement and High Sequence Variations

    PubMed Central

    Ma, Zhihong; Yang, Xuefen; Bercsenyi, Miklos; Wu, Junjie; Yu, Yongyao; Wei, Kaijian; Fan, Qixue; Yang, Ruibin

    2015-01-01

    To understand the molecular evolution of mitochondrial genomes (mitogenomes) in the genus Odontobutis, the mitogenome of Odontobutis yaluensis was sequenced and compared with those of another four Odontobutis species. Our results displayed similar mitogenome features among species in genome organization, base composition, codon usage, and gene rearrangement. The identical gene rearrangement of trnS-trnL-trnH tRNA cluster observed in mitogenomes of these five closely related freshwater sleepers suggests that this unique gene order is conserved within Odontobutis. Additionally, the present gene order and the positions of associated intergenic spacers of these Odontobutis mitogenomes indicate that this unusual gene rearrangement results from tandem duplication and random loss of large-scale gene regions. Moreover, these mitogenomes exhibit a high level of sequence variation, mainly due to the differences of corresponding intergenic sequences in gene rearrangement regions and the heterogeneity of tandem repeats in the control regions. Phylogenetic analyses support Odontobutis species with shared gene rearrangement forming a monophyletic group, and the interspecific phylogenetic relationships are associated with structural differences among their mitogenomes. The present study contributes to understanding the evolutionary patterns of Odontobutidae species. PMID:26492246

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

    PubMed

    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.

  19. Validation of a Prognostic Multi-gene Signature in High-risk Neuroblastoma using the High Throughput Digital NanoString nCounter™ System

    PubMed Central

    Stricker, Thomas P.; La Madrid, Andres Morales; 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.

    2014-01-01

    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. PMID:24560446

  20. Novel Genes Associated with Colorectal Cancer Are Revealed by High Resolution Cytogenetic Analysis in a Patient Specific Manner

    PubMed Central

    Al Qarni, Saeed; Al Rodayyan, Maha; Muhammed Mustafa, Sabeena; Deeb, Ahmad; Al Sheikh, Ebthehal; Afzal Khan, Mohammed; Johani, Mishal; Yousef, Zeyad

    2013-01-01

    Genomic abnormalities leading to colorectal cancer (CRC) include somatic events causing copy number aberrations (CNAs) as well as copy neutral manifestations such as loss of heterozygosity (LOH) and uniparental disomy (UPD). We studied the causal effect of these events by analyzing high resolution cytogenetic microarray data of 15 tumor-normal paired samples. We detected 144 genes affected by CNAs. A subset of 91 genes are known to be CRC related yet high GISTIC scores indicate 24 genes on chromosomes 7, 8, 18 and 20 to be strongly relevant. Combining GISTIC ranking with functional analyses and degree of loss/gain we identify three genes in regions of significant loss (ATP8B1, NARS, and ATP5A1) and eight in regions of gain (CTCFL, SPO11, ZNF217, PLEKHA8, HOXA3, GPNMB, IGF2BP3 and PCAT1) as novel in their association with CRC. Pathway and target prediction analysis of CNA affected genes and microRNAs, respectively indicates TGF-β signaling pathway to be involved in causing CRC. Finally, LOH and UPD collectively affected nine cancer related genes. Transcription factor binding sites on regions of >35% copy number loss/gain influenced 16 CRC genes. Our analysis shows patient specific CRC manifestations at the genomic level and that these different events affect individual CRC patients differently. PMID:24204606

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

    PubMed

    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.

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

    SciTech Connect

    Lindor, N.M.; Sobell, J.L.; Thibodeau, S.N.

    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.

  3. Ancora: a web resource for exploring highly conserved noncoding elements and their association with developmental regulatory genes

    PubMed Central

    Engström, Pär G; Fredman, David; Lenhard, Boris

    2008-01-01

    Metazoan genomes contain arrays of highly conserved noncoding elements (HCNEs) that span developmental regulatory genes and define regulatory domains. We describe Ancora , a web resource that provides data and tools for exploring genomic organization of HCNEs for multiple genomes. Ancora includes a genome browser that shows HCNE locations and features novel HCNE density plots as a powerful tool to discover developmental regulatory genes and distinguish their regulatory elements and domains. PMID:18279518

  4. Analysis of mammalian gene batteries reveals both stable ancestral cores and highly dynamic regulatory sequences

    PubMed Central

    Ettwiller, Laurence; Budd, Aidan; Spitz, François; Wittbrodt, Joachim

    2008-01-01

    Background Changes in gene regulation are suspected to comprise one of the driving forces for evolution. To address the extent of cis-regulatory changes and how they impact on gene regulatory networks across eukaryotes, we systematically analyzed the evolutionary dynamics of target gene batteries controlled by 16 different transcription factors. Results We found that gene batteries show variable conservation within vertebrates, with slow and fast evolving modules. Hence, while a key gene battery associated with the cell cycle is conserved throughout metazoans, the POU5F1 (Oct4) and SOX2 batteries in embryonic stem cells show strong conservation within mammals, with the striking exception of rodents. Within the genes composing a given gene battery, we could identify a conserved core that likely reflects the ancestral function of the corresponding transcription factor. Interestingly, we show that the association between a transcription factor and its target genes is conserved even when we exclude conserved sequence similarities of their promoter regions from our analysis. This supports the idea that turnover, either of the transcription factor binding site or its direct neighboring sequence, is a pervasive feature of proximal regulatory sequences. Conclusions Our study reveals the dynamics of evolutionary changes within metazoan gene networks, including both the composition of gene batteries and the architecture of target gene promoters. This variation provides the playground required for evolutionary innovation around conserved ancestral core functions. PMID:19087242

  5. Identification and analysis of a highly conserved chemotaxis gene cluster in Shewanella species.

    SciTech Connect

    Li, J.; Romine, Margaret F.; Ward, M.

    2007-08-01

    A conserved cluster of chemotaxis genes was identified from the genome sequences of fifteen Shewanella species. An in-frame deletion of the cheA-3 gene, which is located in this cluster, was created in S. oneidensis MR-1 and the gene shown to be essential for chemotactic responses to anaerobic electron acceptors. The CheA-3 protein showed strong similarity to Vibrio cholerae CheA-2 and P. aeruginosa CheA-1, two proteins that are also essential for chemotaxis. The genes encoding these proteins were shown to be located in chemotaxis gene clusters closely related to the cheA-3-containing cluster in Shewanella species. The results of this study suggest that a combination of gene neighborhood and homology analyses may be used to predict which cheA genes are essential for chemotaxis in groups of closely related microorganisms.

  6. Isolation of high salinity stress tolerant genes from Pisum sativum by random overexpression in Escherichia coli and their functional validation

    PubMed Central

    Joshi, Amita; Dang, Hung Quang; Vaid, Neha

    2009-01-01

    Salinity stress is one of the major factors which reduce crop plants growth and productivity resulting in significant economic losses worldwide. Therefore, it would be fruitful to isolate and functionally identify new salinity stress-induced genes for understanding the mechanism and developing salinity stress tolerant plants. Based on functional gene screening assay, we have isolated few salinity tolerant genes out of one million Escherichia coli (SOLR) transformants containing pea cDNAs. Sequence analysis of three of these genes revealed homology to Ribosomal-L30E (RPL30E), Chlorophyll-a/b-binding protein (Chla/bBP) and FIDDLEHEAD (FDH). The salinity tolerance of these genes in bacteria was further confirmed by using another strain of E. coli (DH5α) transformants. The homology based computational modeling of these proteins suggested the high degree of conservation with the conserved domains of their homologous partners. The reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression of these cDNAs (except the FDH) was upregulated in pea plants in response to NaCl stress. We observed that there was no significant effect of Li+ ion on the expression level of these genes, while an increase in response to K+ ion was observed. Overall, this study provides an evidence for a novel function of these genes in high salinity stress tolerance. The PsFDH showed constitutive expression in planta suggesting that it can be used as constitutively expressed marker gene for salinity stress tolerance in plants. This study brings new direction in identifying novel function of unidentified genes in abiotic stress tolerance without previous knowledge of the genome sequence. PMID:19816097

  7. Pathways-Driven Sparse Regression Identifies Pathways and Genes Associated with High-Density Lipoprotein Cholesterol in Two Asian Cohorts

    PubMed Central

    Silver, Matt; Chen, Peng; Li, Ruoying; Cheng, Ching-Yu; Wong, Tien-Yin; Tai, E-Shyong; Teo, Yik-Ying; Montana, Giovanni

    2013-01-01

    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 signalling and immune

  8. A systematic exploration of high-temperature stress-responsive genes in potato using large-scale yeast functional screening.

    PubMed

    Gangadhar, Baniekal Hiremath; Yu, Jae Woong; Sajeesh, Kappachery; Park, Se Won

    2014-04-01

    Potato (S. tuberosum) is a highly heat-sensitive crop; a slight rise from optimal temperature can lead to drastic decline in tuber yield. Despite several advancements made in breeding for thermo-tolerant potato, molecular mechanisms governing thermo-tolerance is poorly understood. The first step towards understanding the thermo-tolerance mechanism is to identify the key genes involved in it. Here we used a yeast-based functional screening method to identify, characterize and classify potato genes with potentials to impart heat tolerance. We constructed two cDNA expression libraries from heat-stressed potato plants (35 °C) after 2 and 48 h of treatment. 95 potential candidate genes were identified based on enhanced ability of yeast cells over-expressing heterologous potato cDNA sequences to tolerate heat stress. Cross-resistance analysis of these heat-tolerant yeast clones to other abiotic stresses indicated that 20 genes were responsive to drought, 14 to salt and 11 to heat/drought/salt stresses. Comparison of 95 genes with reported whole potato transcriptome data showed that majority of them have varying expression patterns under heat, drought and salt stresses. The expression pattern was validated by analyzing the expression of 22 randomly selected genes under various stresses using qPCR. Gene ontology (GO) enrichment analysis of these 95 genes indicated that most of them are involved in various cellular metabolism, signal transduction, response to stress and protein folding, suggesting possible role of these genes in heat tolerance of potato. Genes identified from this study can be potential candidates for engineering heat tolerance as well as broad-spectrum abiotic stress tolerance of potato. PMID:24357347

  9. Identification of colorectal cancer-restricted microRNAs and their target genes based on high-throughput sequencing data

    PubMed Central

    Chang, Jing; Huang, Liya; Cao, Qing; Liu, Fang

    2016-01-01

    To identify potential key microRNAs (miRNAs) and their target genes for colorectal cancer (CRC). High-throughput sequencing data of miRNA expression and gene expression (ID: GSE46622) were downloaded from Gene Expression Omnibus, including matched colon tumor, normal colon epithelium, and liver metastasis tissues from eight CRC patients. Paired t-test and NOISeq separately were utilized to identify differentially expressed miRNAs (DE-miRNAs) and genes. Then, target genes with differential expression and opposite expression trends were identified for DE-miRNAs. Combined with tumor suppressor gene, tumor-associated gene, and TRANSFAC databases, CRC-restricted miRNAs were screened out based on miRNA-target pairs. Compared with normal tissues, there were 56 up- and 37 downregulated miRNAs in metastasis tissues, as well as eight up- and 30 downregulated miRNAs in tumor tissues. miRNA-1 was downregulated in tumor and metastasis tissues, while its target oncogenes TWIST1 and GATA4 were upregulated. Besides, miRNA-let-7f-1-3p was downregulated in tumor tissues, which also targeted TWIST1. In addition, miRNA-133b and miRNA-4458 were downregulated in tumor tissues, while their common target gene DUSP9 was upregulated. Conversely, miRNA-450-b-3p was upregulated in metastasis tissues, while its target tumor suppressor gene CEACAM7 showed downregulation. The identified CRC-restricted miRNAs might be implicated in cancer progression via their target genes, suggesting their potential usage in CRC treatment. PMID:27069368

  10. Identification of colorectal cancer-restricted microRNAs and their target genes based on high-throughput sequencing data.

    PubMed

    Chang, Jing; Huang, Liya; Cao, Qing; Liu, Fang

    2016-01-01

    To identify potential key microRNAs (miRNAs) and their target genes for colorectal cancer (CRC). High-throughput sequencing data of miRNA expression and gene expression (ID: GSE46622) were downloaded from Gene Expression Omnibus, including matched colon tumor, normal colon epithelium, and liver metastasis tissues from eight CRC patients. Paired t-test and NOISeq separately were utilized to identify differentially expressed miRNAs (DE-miRNAs) and genes. Then, target genes with differential expression and opposite expression trends were identified for DE-miRNAs. Combined with tumor suppressor gene, tumor-associated gene, and TRANSFAC databases, CRC-restricted miRNAs were screened out based on miRNA-target pairs. Compared with normal tissues, there were 56 up- and 37 downregulated miRNAs in metastasis tissues, as well as eight up- and 30 downregulated miRNAs in tumor tissues. miRNA-1 was downregulated in tumor and metastasis tissues, while its target oncogenes TWIST1 and GATA4 were upregulated. Besides, miRNA-let-7f-1-3p was downregulated in tumor tissues, which also targeted TWIST1. In addition, miRNA-133b and miRNA-4458 were downregulated in tumor tissues, while their common target gene DUSP9 was upregulated. Conversely, miRNA-450-b-3p was upregulated in metastasis tissues, while its target tumor suppressor gene CEACAM7 showed downregulation. The identified CRC-restricted miRNAs might be implicated in cancer progression via their target genes, suggesting their potential usage in CRC treatment. PMID:27069368

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

    PubMed

    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 < 0.01) was observed between the sequence read numbers and the relative quantitative values for the expressed classical 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.

  12. The transition from linear to highly branched poly(β-amino ester)s: Branching matters for gene delivery.

    PubMed

    Zhou, Dezhong; Cutlar, Lara; Gao, Yongsheng; Wang, Wei; O'Keeffe-Ahern, Jonathan; McMahon, Sean; Duarte, Blanca; Larcher, Fernando; Rodriguez, Brian J; Greiser, Udo; Wang, Wenxin

    2016-06-01

    Nonviral gene therapy holds great promise but has not delivered treatments for clinical application to date. Lack of safe and efficient gene delivery vectors is the major hurdle. Among nonviral gene delivery vectors, poly(β-amino ester)s are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. However, to date, all research has been focused on vectors with a linear structure. A well-accepted view is that dendritic or branched polymers have greater potential as gene delivery vectors because of their three-dimensional structure and multiple terminal groups. Nevertheless, to date, the synthesis of dendritic or branched polymers has been proven to be a well-known challenge. We report the design and synthesis of highly branched poly(β-amino ester)s (HPAEs) via a one-pot "A2 + B3 + C2"-type Michael addition approach and evaluate their potential as gene delivery vectors. We find that the branched structure can significantly enhance the transfection efficiency of poly(β-amino ester)s: Up to an 8521-fold enhancement in transfection efficiency was observed across 12 cell types ranging from cell lines, primary cells, to stem cells, over their corresponding linear poly(β-amino ester)s (LPAEs) and the commercial transfection reagents polyethyleneimine, SuperFect, and Lipofectamine 2000. Moreover, we further demonstrate that HPAEs can correct genetic defects in vivo using a recessive dystrophic epidermolysis bullosa graft mouse model. Our findings prove that the A2 + B3 + C2 approach is highly generalizable and flexible for the design and synthesis of HPAEs, which cannot be achieved by the conventional polymerization approach; HPAEs are more efficient vectors in gene transfection than the corresponding LPAEs. This provides valuable insight into the development and applications of nonviral gene delivery and demonstrates great prospect for their

  13. Multiplexed chemiluminescent assays in ArrayPlates for high-throughput measurement of gene expression

    NASA Astrophysics Data System (ADS)

    Martel, Ralph R.; Rounseville, Matthew P.; Botros, Ihab W.; Seligmann, Bruce E.

    2002-06-01

    Multiplexed Molecular Profiling (MMP) assays for drug discovery are performed in ArrayPlates. ArrayPlates are 96- well microtiter plates that contain a 16-element array at the bottom of each well. Each element within an array measures one analyte in a sample. A CCD imager records the quantitative chemiluminescent readout of all 1,536 elements in a 96-well plate simultaneously. Since array elements are reagent modifiable by the end-user, ArrayPlates can be adapted to a broad range of nucleic acid- and protein-based assays. Such multiplexed assays are rapidly established, flexible, robust, automation-friendly and cost-effective. Nucleic acid assays in ArrayPlates can detect DNA and RNA, including SNPs and ESTs. A multiplexed mRNA assay to measure the expression of 16 genes is described. The assay combines a homogeneous nuclease protection assay with subsequent probe immobilization to the array by means of a sandwich hybridization followed with chemiluminescent detection. This assay was used to examine cells grown and treated in microplates and avoided cloning, transfection, RNA insolation, reverse transcription, amplification and fluorochrome labeling. Standard deviations for the measurement of 16 genes ranged from 3 percent to 13 percent in samples of 30,000 cells. Such ArrayPlates transcription assays are useful in drug discovery and development for target validation, screening, lead optimization, metabolism and toxicity profiling. Chemiluminescent detection provides ArrayPlates assays with high signal-to-noise readout and simplifies imager requirements. Imaging a 2D surface that contains arrays simplifies lens requirements relative to imaging columns of liquid in microtiter plate wells. The Omix imager for ArrayPlates is described.

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

    PubMed

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

    2012-07-01

    Seabirds are highly vagile and can disperse up to thousands of kilometers, making it difficult to identify the factors that promote isolation between populations. The endemic Hawaiian petrel (Pterodroma sandwichensis) is one such species. Today it is endangered, and known to breed only on the islands of Hawaii, Maui, Lanai and Kauai. Historical records indicate that a large population formerly bred on Molokai as well, but this population has recently been extirpated. Given the great dispersal potential of these petrels, it remains unclear if populations are genetically distinct and which factors may contribute to isolation between them. We sampled petrels from across their range, including individuals from the presumably extirpated Molokai population. We sequenced 524 bp of mitochondrial DNA, 741 bp from three nuclear introns, and genotyped 18 microsatellite loci in order to examine the patterns of divergence in this species and to investigate the potential underlying mechanisms. Both mitochondrial and nuclear data sets indicated significant genetic differentiation among all modern populations, but no differentiation was found between historic samples from Molokai and modern birds from Lanai. Population-specific nonbreeding distribution and strong natal philopatry may reduce gene flow between populations. However, the lack of population structure between extirpated Molokai birds and modern birds on Lanai indicates that there was substantial gene flow between these populations and that petrels may be able to overcome barriers to dispersal prior to complete extirpation. Hawaiian petrel populations could be considered distinct management units, however, the dwindling population on Hawaii may require translocation to prevent extirpation in the near future. PMID:22434012

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

    PubMed

    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 < 0.05). However, these bone microstructural parameters were increased by 53%, 34% and 40%, respectively, in IL6(-/-) mice than those in WT mice on the HFD (P < 0.05). IL6(-/-) osteoblasts displayed higher 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.

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

    PubMed

    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 < 0.05). However, these bone microstructural parameters were increased by 53%, 34% and 40%, respectively, in IL6(-/-) mice than those in WT mice on the HFD (P < 0.05). IL6(-/-) osteoblasts displayed higher 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

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

    USGS Publications Warehouse

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

    2012-01-01

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

  18. Local adaptation despite high gene flow in the waterfall-climbing Hawaiian goby, Sicyopterus stimpsoni.

    PubMed

    Moody, K N; Hunter, S N; Childress, M J; Blob, R W; Schoenfuss, H L; Blum, M J; Ptacek, M B

    2015-02-01

    Environmental heterogeneity can promote the emergence of locally adapted phenotypes among subpopulations of a species, whereas gene flow can result in phenotypic and genotypic homogenization. For organisms like amphidromous fishes that change habitats during their life history, the balance between selection and migration can shift through ontogeny, making the likelihood of local adaptation difficult to predict. In Hawaiian waterfall-climbing gobies, it has been hypothesized that larval mixing during oceanic dispersal counters local adaptation to contrasting topographic features of streams, like slope gradient, that can select for predator avoidance or climbing ability in juvenile recruits. To test this hypothesis, we used morphological traits and neutral genetic markers to compare phenotypic and genotypic distributions in recruiting juveniles and adult subpopulations of the waterfall-climbing amphidromous goby, Sicyopterus stimpsoni, from the islands of Hawai'i and Kaua'i. We found that body shape is significantly different between adult subpopulations from streams with contrasting slopes and that trait divergence in recruiting juveniles tracked stream topography more so than morphological measures of adult subpopulation differentiation. Although no evidence of population genetic differentiation was observed among adult subpopulations, we observed low but significant levels of spatially and temporally variable genetic differentiation among juvenile cohorts, which correlated with morphological divergence. Such a pattern of genetic differentiation is consistent with chaotic genetic patchiness arising from variable sources of recruits to different streams. Thus, at least in S. stimpsoni, the combination of variation in settlement cohorts in space and time coupled with strong postsettlement selection on juveniles as they migrate upstream to adult habitats provides the opportunity for morphological adaptation to local stream environments despite high gene flow. PMID

  19. Local adaptation despite high gene flow in the waterfall-climbing Hawaiian goby, Sicyopterus stimpsoni.

    PubMed

    Moody, K N; Hunter, S N; Childress, M J; Blob, R W; Schoenfuss, H L; Blum, M J; Ptacek, M B

    2015-02-01

    Environmental heterogeneity can promote the emergence of locally adapted phenotypes among subpopulations of a species, whereas gene flow can result in phenotypic and genotypic homogenization. For organisms like amphidromous fishes that change habitats during their life history, the balance between selection and migration can shift through ontogeny, making the likelihood of local adaptation difficult to predict. In Hawaiian waterfall-climbing gobies, it has been hypothesized that larval mixing during oceanic dispersal counters local adaptation to contrasting topographic features of streams, like slope gradient, that can select for predator avoidance or climbing ability in juvenile recruits. To test this hypothesis, we used morphological traits and neutral genetic markers to compare phenotypic and genotypic distributions in recruiting juveniles and adult subpopulations of the waterfall-climbing amphidromous goby, Sicyopterus stimpsoni, from the islands of Hawai'i and Kaua'i. We found that body shape is significantly different between adult subpopulations from streams with contrasting slopes and that trait divergence in recruiting juveniles tracked stream topography more so than morphological measures of adult subpopulation differentiation. Although no evidence of population genetic differentiation was observed among adult subpopulations, we observed low but significant levels of spatially and temporally variable genetic differentiation among juvenile cohorts, which correlated with morphological divergence. Such a pattern of genetic differentiation is consistent with chaotic genetic patchiness arising from variable sources of recruits to different streams. Thus, at least in S. stimpsoni, the combination of variation in settlement cohorts in space and time coupled with strong postsettlement selection on juveniles as they migrate upstream to adult habitats provides the opportunity for morphological adaptation to local stream environments despite high gene flow.

  20. An innovative plant genomics and gene annotation program for high school, community college, and university faculty.

    PubMed

    Hacisalihoglu, Gokhan; Hilgert, Uwe; Nash, E Bruce; Micklos, David A

    2008-01-01

    Today's biology educators face the challenge of training their students in modern molecular biology techniques including genomics and bioinformatics. The Dolan DNA Learning Center (DNALC) of Cold Spring Harbor Laboratory has developed and disseminated a bench- and computer-based plant genomics curriculum for biology faculty. In 2007, a five-day "Plant Genomics and Gene Annotation" workshop was held at Florida A&M University in Tallahassee, FL, to enhance participants' knowledge and understanding of plant molecular genetics and assist them in developing and honing their laboratory and computer skills. Florida A&M University is a historically black university with over 95% African-American student enrollment. Sixteen participants, including high school (56%) and community college faculty (25%), attended the workshop. Participants carried out in vitro and in silico experiments with maize, Arabidopsis, soybean, and food products to determine the genotype of the samples. Benefits of the workshop included increased awareness of plant biology research for high school and college level students. Participants completed pre- and postworkshop evaluations for the measurement of effectiveness. Participants demonstrated an overall improvement in their postworkshop evaluation scores. This article provides a detailed description of workshop activities, as well as assessment and long-term support for broad classroom implementation.

  1. Prognostically relevant gene signatures of high-grade serous ovarian carcinoma

    PubMed Central

    Verhaak, Roel G.W.; Tamayo, Pablo; Yang, Ji-Yeon; Hubbard, Diana; Zhang, Hailei; Creighton, Chad J.; Fereday, Sian; Lawrence, Michael; Carter, Scott L.; Mermel, Craig H.; Kostic, Aleksandar D.; Etemadmoghadam, Dariush; Saksena, Gordon; Cibulskis, Kristian; Duraisamy, Sekhar; Levanon, Keren; Sougnez, Carrie; Tsherniak, Aviad; Gomez, Sebastian; Onofrio, Robert; Gabriel, Stacey; Chin, Lynda; Zhang, Nianxiang; Spellman, Paul T.; Zhang, Yiqun; Akbani, Rehan; Hoadley, Katherine A.; Kahn, Ari; Köbel, Martin; Huntsman, David; Soslow, Robert A.; Defazio, Anna; Birrer, Michael J.; Gray, Joe W.; Weinstein, John N.; Bowtell, David D.; Drapkin, Ronny; Mesirov, Jill P.; Getz, Gad; Levine, Douglas A.; Meyerson, Matthew

    2012-01-01

    Because of the high risk of recurrence in high-grade serous ovarian carcinoma (HGS-OvCa), the development of outcome predictors could be valuable for patient stratification. Using the catalog of The Cancer Genome Atlas (TCGA), we developed subtype and survival gene expression signatures, which, when combined, provide a prognostic model of HGS-OvCa classification, named “Classification of Ovarian Cancer” (CLOVAR). We validated CLOVAR on an independent dataset consisting of 879 HGS-OvCa expression profiles. The worst outcome group, accounting for 23% of all cases, was associated with a median survival of 23 months and a platinum resistance rate of 63%, versus a median survival of 46 months and platinum resistance rate of 23% in other cases. Associating the outcome prediction model with BRCA1/BRCA2 mutation status, residual disease after surgery, and disease stage further optimized outcome classification. Ovarian cancer is a disease in urgent need of more effective therapies. The spectrum of outcomes observed here and their association with CLOVAR signatures suggests variations in underlying tumor biology. Prospective validation of the CLOVAR model in the context of additional prognostic variables may provide a rationale for optimal combination of patient and treatment regimens. PMID:23257362

  2. Express Primer Tool for high-throughput gene cloning and expression

    2002-12-01

    A tool to assist in the design of primers for DNA amplification. The Express Primer web-based tool generates primer sequences specifically for the generation of expression clones for both lab scale and high-throughput projects. The application is designed not only to allow the user complete flexibility to specify primer design parameters but also to minimize the amount of manual intervention needed to generate a large number of primers for simultaneous amplification of multiple target genes.more » The Express Primer Tool enables the user to specify various experimental parameters (e.g. optimal Tm, Tm range, maximum Tm difference) for single or multiple candidate sequence(s) in FASTA format input as a flat text (ASCII) file. The application generates condidate primers, selects optimal primer pairs, and writes the forward and reverse primers pairs to an Excel file that is suitable for electronic submission to a synthesis facility. The program parameters emphasize high-throughput but allow for target atrition at various stages of the project.« less

  3. Novel, highly expressed late nodulin gene (LjNOD16) from Lotus japonicus

    SciTech Connect

    Kapranov, P.; Bruijn, F.J. de; Szczyglowski, K.

    1997-04-01

    We have isolated a Lotus japonicus cDNA corresponding to a highly abundant, late nodule-specific RNA species that encodes a polypeptide with a predicted molecular mass of 15.6 kD. The protein and its corresponding gene were designated NIj16 and LjNOD16, respectively. LjNOD16 was found to be expressed only in the infected cells of L. japonicus nodules. Related DNA sequences could be identified in the genomes of both Glycine max and Medicago sativa. In the latter, a homologous mRNA species was detected in the nodules. Unlike LiNOD16, its alfalfa homologs appear to represent low-abundance mRNA species. However, the proteins corresponding to the LjNOD16 and its alfalfa homolog could be detected at similar levels in nodules but not in roots of both legume species. The predicted amino acid sequence analysis of nodulin NIj16 revealed the presence of a long {alpha}-helical region and a positively charged C terminus. The former domain has a very high propensity to form a coiled-coil type structure, indicating that nodulin NIj16 may interact with an as-yet-unidentified protein target(s) in the nodule-infected cells. Homology searches revealed no significant similarities to any known sequences in the databases, with the exception of two related, anonymous Arabidopsis expressed sequence tags.

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

    PubMed Central

    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.

    PubMed

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

    PubMed

    Sadelain, M; Wang, C H; Antoniou, M; Grosveld, F; Mulligan, R C

    1995-07-18

    Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of

  7. Identification of differentially expressed genes related to metabolic syndrome induced with high-fat diet in E3 rats

    PubMed Central

    Lan, Xi; Li, Dongmin; Zhong, Bo; Ren, Juan; Wang, Xuan; Sun, Qingzhu; Li, Yue; Liu, Lee; Liu, Li

    2015-01-01

    Understanding the genes differentially expressing in aberrant organs of metabolic syndrome (MetS) facilitates the uncovering of molecular mechanisms and the identification of novel therapeutic targets for the disease. This study aimed to identify differentially expressed genes related to MetS in livers of E3 rats with high-fat-diet-induced metabolic syndrome (HFD-MetS). E3 rats were fed with high-fat diet for 24 weeks to induce MetS. Then, suppression subtractive hybridization (SSH) technology was used to identify the genes differentially expressed between HFD-MetS and control E3 rat livers. Twenty positive recombinant clones were chosen randomly from forward subtractive library and sent to sequence. BLAST analysis in GenBank database was used to determine the property of each cDNA fragment. In total, 11 annotated genes, 3 ESTs, and 2 novel gene fragments were identified by SSH technology. The expression of four genes (Alb, Pip4k2a, Scd1, and Tf) known to be associated with MetS and other five genes (Eif1, Rnase4, Rps12, Rup2, and Tmsb4) unknown to be relevant to MetS was significantly up-regulated in the livers of HFD-MetS E3 rats compared with control rats using real-time quantitative PCR (RT-qPCR). By analyzing the correlations between the expression of these nine genes and serum concentrations of TG, Tch, HDL-C, and LDL-C, we found that there were significant positive correlations between TG and the expression of five genes (Alb, Eif1, Pip4k2a, Rps12, and Tmsb4x), Tch and three genes (Rnase4, Scd1, and Tmsb4x), and LDL-C and two genes (Rnase4 and Scd1), as well there were significant negative correlations between HDL-C and the expression of three genes (Rup2, Scd1, and Tf). This study provides important clues for unraveling the molecular mechanisms of MetS. PMID:25294893

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

    PubMed Central

    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-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 fourfold 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. PMID:26528311

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

    PubMed

    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.

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

    PubMed

    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-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 fourfold 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.

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

    PubMed

    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.

  12. Localization of a highly conserved human potassium channel gene (NGK2-KV4-KCNC1) to chromosome 11p15

    SciTech Connect

    Ried, T.; Ward, D.C. ); Rudy, B.; Miera, V.S. de; Lau, D.; Sen, K. )

    1993-02-01

    Several genes (the Shaker or Sh gene family) encoding components of voltage-gated K[sub +] channels have been identified in various species. Based on sequence similarities Sh genes are classified into four groups or subfamilies. Mammalian genes of each one of these subfamilies also show high levels of sequence similarity to one of four related Drosophila genes: Shaker, Shab, Shaw, and Shal. Here we report the isolation of human cDNAs for a Shaw-related product (NGK2,KV2.1a) previously identified in rat and mice. A comparison of the nucleotide and deduced amino acid sequence of NGK2 in rodents and humans shows that this product is highly conserved in mammals; the human NGK2 protein shows over 99% amino acid sequence identity to its rodent homologue. The gene (NGK2-KV4; KCNC1) encoding NGK2 was mapped to human chromosome 11p15 by fluorescence in situ hybridization with the human NGK2 cDNAs. 65 refs., 2 figs., 1 tab.

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

    PubMed

    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-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 fourfold 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. PMID:26528311

  14. Early diffusion of gene expression profiling in breast cancer patients associated with areas of high income inequality.

    PubMed

    Ponce, Ninez A; Ko, Michelle; Liang, Su-Ying; Armstrong, Joanne; Toscano, Michele; Chanfreau-Coffinier, Catherine; Haas, Jennifer S

    2015-04-01

    With the Affordable Care Act reducing coverage disparities, social factors could prominently determine where and for whom innovations first diffuse in health care markets. Gene expression profiling is a potentially cost-effective innovation that guides chemotherapy decisions in early-stage breast cancer, but adoption has been uneven across the United States. Using a sample of commercially insured women, we evaluated whether income inequality in metropolitan areas was associated with receipt of gene expression profiling during its initial diffusion in 2006-07. In areas with high income inequality, gene expression profiling receipt was higher than elsewhere, but it was associated with a 10.6-percentage-point gap between high- and low-income women. In areas with low rates of income inequality, gene expression profiling receipt was lower, with no significant differences by income. Even among insured women, income inequality may indirectly shape diffusion of gene expression profiling, with benefits accruing to the highest-income patients in the most unequal places. Policies reducing gene expression profiling disparities should address low-inequality areas and, in unequal places, practice settings serving low-income patients.

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

    PubMed

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

    2014-11-20

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

  16. Exploring the transcription factor activity in high-throughput gene expression data using RLQ analysis

    PubMed Central

    2013-01-01

    Background Interpretation of gene expression microarray data in the light of external information on both columns and rows (experimental variables and gene annotations) facilitates the extraction of pertinent information hidden in these complex data. Biologists classically interpret genes of interest after retrieving functional information from a subset of genes of interest. Transcription factors play an important role in orchestrating the regulation of gene expression. Their activity can be deduced by examining the presence of putative transcription factors binding sites in the gene promoter regions. Results In this paper we present the multivariate statistical method RLQ which aims to analyze microarray data where additional information is available on both genes and samples. As an illustrative example, we applied RLQ methodology to analyze transcription factor activity associated with the time-course effect of steroids on the growth of primary human lung fibroblasts. RLQ could successfully predict transcription factor activity, and could integrate various other sources of external information in the main frame of the analysis. The approach was validated by means of alternative statistical methods and biological validation. Conclusions RLQ provides an efficient way of extracting and visualizing structures present in a gene expression dataset by directly modeling the link between experimental variables and gene annotations. PMID:23742070

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

    PubMed

    Sheng, Ping; Li, Yushan; Marshall, Sean D G; Zhang, Hongyu

    2015-01-01

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

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

    PubMed

    Sheng, Ping; Li, Yushan; Marshall, Sean D G; Zhang, Hongyu

    2015-01-01

    In this study, we used a culture-independent method based on library construction and sequencing to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the hindgut of Holotrichia parallela larvae. The results indicate that there is a large, diverse set of bacterial genes encoding lignocellulose hydrolysis enzymes in the hindgut of H. parallela. The total of 101 distinct gene fragments (similarity <95%) of glycosyl hydrolase families including GH2 (24 genes), GH8 (27 genes), GH10 (19 genes), GH11 (14 genes) and GH36 (17 genes) families was retrieved, and certain sequences of GH2 (10.61%), GH8 (3.33%), and GH11 (18.42%) families had <60% identities with known sequences in GenBank, indicating their novelty. Based on phylogenetic analysis, sequences from hemicellulase families were related to enzymes from Bacteroidetes and Firmicutes. Fragments from cellulase family were most associated with the phylum of Proteobacteria. Furthermore, a full-length endo-xylanase gene was obtained, and the enzyme exhibited activity over a broad range of pH levels. Our results indicate that there are large number of cellulolytic and xylanolytic bacteria in the hindgut of H. parallela larvae, and these symbiotic bacteria play an important role in the degradation of roots and other organic matter for the host insect. PMID:26197317

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

    PubMed Central

    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 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. PMID:22152034

  20. Differential expression of hepatic genes involved in cholesterol homeostasis in high- and low-responding strains of laboratory opossums.

    PubMed

    Chan, Jeannie; Donalson, Lisa M; Kushwaha, Rampratap S; Ferdinandusse, Sacha; VandeBerg, Jane F; VandeBerg, John L

    2008-05-01

    Plasma very low-density lipoprotein and low-density lipoprotein (VLDL+LDL) cholesterol levels of 2 partially inbred strains of opossums (Monodelphis domestica) differ markedly when they are fed a high-cholesterol and low-fat (HCLF) diet. High-responding opossums exhibit a dramatic increase (>10-fold) in VLDL+LDL cholesterol, whereas low-responding opossums exhibit a minimal increase (<2-fold) in VLDL+LDL cholesterol. The genes responsible for the accumulation of high levels of plasma VLDL+LDL cholesterol in high-responding opossums have not yet been identified. In this study, we analyzed the expression of genes encoding for (1) 4 bile acid synthesis enzymes (CYP7A1, CYP27A1, CYP8B1, and CYP7B1); (2) 3 cholesterol synthesis enzymes (HMGCR, HMGCS1, and SQLE); (3) the LDL receptor (LDLR); (4) 2 sterol transporters (ABCG5 and ABCG8); and (5) 2 bile acid transporters (ABCB11 and SLC10A1) to determine how the expression of these genes was affected by dietary cholesterol in the 2 strains of opossums. We found differences between high and low responders in the expression of cholesterol synthesis genes on the basal diet, as well as differences in the expression of the CYP27A1, ABCG5, ABCG8, and SLC10A1 genes on the HCLF diet. CYP27A1 messenger RNA levels were lower in the livers of high responders compared with low responders, whereas CYP27A1 messenger RNA levels in extrahepatic tissues were similar in high and low responders on the HCLF diet. Low levels of CYP27A1, ABCG5, and ABCG8 expression in the liver may contribute to hypercholesterolemia in high-responding opossums.

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

    PubMed

    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

  2. lambda altSF: a phage variant that acquired the ability to substitute specific sets of genes at high frequency.

    PubMed Central

    Friedman, D; Tomich, P; Parsons, C; Olson, E; Deans, R; Flamm, E

    1981-01-01

    We report the isolation of lambda altSF, a variant of Escherichia coli phage lambda that substitutes sets of genes at high frequency. Two forms of the variant phage have been studied: lambda altSF lambda, which exhibits the immunity (repressor recognition) of phage lambda, and lambda altSF22, which exhibits the immunity of Salmonella phage P22. Lysates made from single plaques of lambda altSF lambda contain 10-30% phage of the P22 form. Similarly, lysates from single plaques of lambda altSF22 contain as much as 1% phage of the lambda form. Heteroduplex analyses reveal the following features of the lambda altSF chromosomes: (i) each form has the immunity genes appropriate to its immune phenotype, (ii) the substituted segments include genes involved in regulation and replication, and (iii) the alt phages have unusual additions and substitutions of DNA not normally found associated with either immunity region. In the case of lambda altSF lambda, there is a small insertion in the region of the cI gene. Because revertants that lose this inserted DNA concomitantly lose the ability to substitute, we conclude that the insertion plays a role in the substitution process. In the case of change from lambda altSF lambda to lambda altSF22, the substituting P22 genes are derived from the E. coli host. We have identified a set of Salmonella phage P22 genes in a standard nonlysogenic strain of E. coli K-12 that is apparently carried in a silent form. The reason for this lack of expression is not obvious, because this P22 material includes structural genes and associated promoters and is potentially active. When this set of genes substitutes for the analogous set of genetic material on the genome of lambda altSF lambda, the P22 genes are expressed in a normal manner. Images PMID:6454136

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

    PubMed

    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.

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

    PubMed

    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

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

    PubMed

    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.

  6. Codon usage in highly expressed genes of Haemophillus influenzae and Mycobacterium tuberculosis: translational selection versus mutational bias.

    PubMed

    Pan, A; Dutta, C; Das, J

    1998-07-30

    Biases in the codon usage and base compositions at three codon sites in different genes of A+T-rich Gram-negative bacterium Haemophillus influenzae and G+C-rich Gram-positive bacterium Mycobacterium tuberculosis have been examined to address the following questions: (1) whether the synonymous codon usage in organisms having highly skewed base compositions is totally dictated by the mutational bias as reported previously (Sharp, P.M., Devine, K.M., 1989. Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do 'prefer' optimal codons. Nucleic Acids Res. 17, 5029-5039), or is also controlled by translational selection; (2) whether preference of G in the first codon positions by highly expressed genes, as reported in Escherichia coli (Gutierrez, G., Marquez, L., Marin, A., 1996. Preference for guanosine at first codon position in highly expressed Escherichia coli genes. A relationship with translational efficiency. Nucleic Acids Res. 24, 2525-2527), is true in other bacteria; and (3) whether the usage of bases in three codon positions is species-specific. Result presented here show that even in organisms with high mutational bias, translational selection plays an important role in dictating the synonymous codon usage, though the set of optimal codons is chosen in accordance with the mutational pressure. The frequencies of G-starting codons are positively correlated to the level of expression of genes, as estimated by their Codon Adaptation Index (CAI) values, in M. tuberculosis as well as in H. influenzae in spite of having an A+T-rich genome. The present study on the codon preferences of two organisms with oppositely skewed base compositions thus suggests that the preference of G-starting codons by highly expressed genes might be a general feature of bacteria, irrespective of their overall G+C contents. The ranges of variations in the frequencies of individual bases at the first and second codon positions of genes of both H

  7. Genome-Wide Identification of New Reference Genes for qRT-PCR Normalization under High Temperature Stress in Rice Endosperm.

    PubMed

    Xu, Heng; Bao, Jian-Dong; Dai, Ji-Song; Li, Yongqing; Zhu, Ying

    2015-01-01

    qRT-PCR is one of the most popular approaches to analyze specific gene expression level, and stably expressed reference genes are essential to obtain reliable results. However, many reference genes are only stable under certain circumstances and different reference genes might be required in different experiments. High temperature is a common stress that affects rice endosperm development and it has become a hot topic recently. Although study about reference genes at different developmental stages in rice has been reported, these genes may not be suitable to study high temperature mediated responses especially in endosperm. In our quest for proper reference genes to quantify gene expression in rice endosperm under high temperature, we studied 6 candidate genes selected from the transcriptome data and 11 housekeeping genes. All genes were analyzed with qRT-PCR and the expression stability was assessed with software geNorm and NormFinder. Fb15 and eIF-4a were identified as the two most stable genes in endosperm at different developmental stages, while high temperature treatment has a least effect on expression of Fb15 and UBQ5 in rice endosperm. Our results provide some good candidate reference genes for qRT-PCR normalization in rice endosperm under different temperatures.

  8. Genome-Wide Identification of New Reference Genes for qRT-PCR Normalization under High Temperature Stress in Rice Endosperm.

    PubMed

    Xu, Heng; Bao, Jian-Dong; Dai, Ji-Song; Li, Yongqing; Zhu, Ying

    2015-01-01

    qRT-PCR is one of the most popular approaches to analyze specific gene expression level, and stably expressed reference genes are essential to obtain reliable results. However, many reference genes are only stable under certain circumstances and different reference genes might be required in different experiments. High temperature is a common stress that affects rice endosperm development and it has become a hot topic recently. Although study about reference genes at different developmental stages in rice has been reported, these genes may not be suitable to study high temperature mediated responses especially in endosperm. In our quest for proper reference genes to quantify gene expression in rice endosperm under high temperature, we studied 6 candidate genes selected from the transcriptome data and 11 housekeeping genes. All genes were analyzed with qRT-PCR and the expression stability was assessed with software geNorm and NormFinder. Fb15 and eIF-4a were identified as the two most stable genes in endosperm at different developmental stages, while high temperature treatment has a least effect on expression of Fb15 and UBQ5 in rice endosperm. Our results provide some good candidate reference genes for qRT-PCR normalization in rice endosperm under different temperatures. PMID:26555942

  9. High throughput genome-specific and gene-specific molecular markers for erucic acid genes in Brassica napus (L.) for marker-assisted selection in plant breeding.

    PubMed

    Rahman, Mukhlesur; Sun, Zudong; McVetty, Peter B E; Li, Genyi

    2008-10-01

    A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1 from a B. rapa BAC library and a BAC clone anchoring Bn-FAE1.2 from a B. oleracea BAC library were used in this research. After sequencing the gene flanking regions, it was found that the dissimilarity of the flanking sequences of these two FAE1 homologs facilitated the design of genome-specific primers that could amplify the corresponding genome in allotetraploid B. napus. The two-base deletion in the C genome gene was detected as a sequence-characterized amplified region (SCAR) marker. To increase the throughput, one genome-specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. Eventually, a super pool of 80 samples was detected simultaneously. This dramatically reduces the cost of marker detection. The single base change in the Bn-FAE1.1 gene was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5' primer end to increase SNP detection throughput through sample pooling. Furthermore, the Bn-FAE1.1 and Bn-FAE1.2 were integrated into the N8 and N13 linkage groups of our previously reported high-density sequence-related amplified polymorphism (SRAP) map, respectively. There were 124 SRAP markers in a N8 bin in which the Bn-FAE1.1 gene-specific SCAR marker was located and 46 SRAP markers in a N13 bin into which the Bn-FAE1.2 SNP marker was integrated. These three kinds of high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs.

  10. High-Density Microarray-Mediated Gene Expression Profiling of Escherichia coli

    PubMed Central

    Wei, Yan; Lee, Jian-Ming; Richmond, Craig; Blattner, Frederick R.; Rafalski, J. Antoni; LaRossa, Robert A.

    2001-01-01

    A nearly complete collection of 4,290 Escherichia coli open reading frames was amplified and arrayed in high density on glass slides. To exploit this reagent, conditions for RNA isolation from E. coli cells, cDNA production with attendant fluorescent dye incorporation, DNA-DNA hybridization, and hybrid quantitation have been established. A brief isopropyl-β-d-thiogalactopyranoside (IPTG) treatment elevated lacZ, lacY, and lacA transcript content about 30-fold; in contrast, most other transcript titers remained unchanged. Distinct RNA expression patterns between E. coli cultures in the exponential and transitional phases of growth were catalogued, as were differences associated with culturing in minimal and rich media. The relative abundance of each transcript was estimated by using hybridization of a genomic DNA-derived, fluorescently labeled probe as a correction factor. This inventory provided a quantitative view of the steady-state level of each mRNA species. Genes the expression of which was detected by this method were enumerated, and results were compared with the current understanding of E. coli physiology. PMID:11133948

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

    PubMed

    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.

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

    PubMed Central

    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

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

    PubMed

    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

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

    PubMed

    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

  15. Metabolic bacterial genes and the construction of high-level composite lineages of life.

    PubMed

    Méheust, Raphaël; Lopez, Philippe; Bapteste, Eric

    2015-03-01

    Understanding how major organismal lineages originated is fundamental for understanding processes by which life evolved. Major evolutionary transitions, like eukaryogenesis, merging genetic material from distantly related organisms, are rare events, hence difficult ones to explain causally. If most archaeal lineages emerged after massive acquisitions of bacterial genes, a rule however arises: metabolic bacterial genes contributed to all major evolutionary transitions.

  16. Developing Pedagogical Tools to Improve Teaching Multiple Models of the Gene in High School

    ERIC Educational Resources Information Center

    Auckaraaree, Nantaya

    2013-01-01

    Multiple models of the gene are used to explore genetic phenomena in scientific practices and in the classroom. In genetics curricula, the classical and molecular models are presented in disconnected domains. Research demonstrates that, without explicit connections, students have difficulty developing an understanding of the gene that spans…

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

    PubMed Central

    Sadelain, M; Wang, C H; Antoniou, M; Grosveld, F; Mulligan, R C

    1995-01-01

    Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of

  18. Two Brassica napus genes encoding NAC transcription factors are involved in response to high-salinity stress.

    PubMed

    Zhong, Hui; Guo, Qian-Qian; Chen, Liang; Ren, Feng; Wang, Qing-Qing; Zheng, Yong; Li, Xue-Bao

    2012-11-01

    The NAC protein family is one of the novel classes of plant-specific transcription factors. In this study, two genes (BnNAC2 and BnNAC5) encoding the putative NAC transcription factors were identified in Brassica napus. Sequence analysis revealed that the deduced BnNAC proteins contain conserved N-terminal region (NAC domain) and highly divergent C-terminal domain. Yeast transactivation analysis showed that BnNAC2 could activate reporter gene expression, suggesting that BnNAC2 functions as a transcriptional activator. Quantitative RT-PCR analysis revealed that BnNAC2 was preferentially expressed in flowers, whereas BnNAC5 mRNAs accumulated at the highest level in stems. Further experimental results indicated that the two genes are high-salinity-, drought- and abscisic acid (ABA)-induced. Overexpression of BnNAC2 and BnNAC5 genes in yeast (Schizosaccharomyces pombe) remarkably inhibited the growth rate of the host cells, and enhanced the cells sensitive to high-salinity and osmotic stresses. Complementation test indicated that BnNAC5 could recover the defects such as salt-hypersensitivity and accelerated-leaf senescence of vni2 T-DNA insertion mutant. Several stress-responsive genes including COR15A and RD29A were enhanced in the complemented plants. These results suggest that BnNAC5 may perform the similar function of VNI2 in response to high-salinity stress and regulation of leaf aging. Key message BnNAC2 and BnNAC5 are salt-, drought- and ABA-induced genes. Overexpression of BnNAC5 in Arabidopsis vni2 mutant recovered the mutant defects (salt-hypersensitivity and accelerated-leaf senescence) to the phenotype of wild type. PMID:22801866

  19. A novel mammalian protein kinase gene (mak) is highly expressed in testicular germ cells at and after meiosis.

    PubMed Central

    Matsushime, H; Jinno, A; Takagi, N; Shibuya, M

    1990-01-01

    We isolated a novel gene designated mak (male germ cell-associated kinase) by using weak cross-hybridization with a tyrosine kinase gene (v-ros). Sequence analysis of the cDNA corresponding to the 2.6-kilobase transcript revealed that the predicted product of rat mak consisted of 622 amino acids and contained protein kinase consensus motifs in its amino-terminal region. Comparison of the deduced amino acid sequence of mak in the kinase domain with those of other protein kinase genes demonstrated that mak was approximately 40% identical to the cdc2-CDC28 gene family in Schizosaccharomyces pombe, Saccharomyces cerevisiae, and humans but less identical to most other protein kinase gene products. Expression of mak was highly tissue specific, and its transcripts were detected almost exclusively in testicular cells entering and after meiosis but hardly detectable in ovarian cells including oocytes, after the dictyotene stage. These results suggest that the mak gene plays an important role in spermatogenesis. Images PMID:2183027

  20. Structural Characterization and Evolutionary Relationship of High-Molecular-Weight Glutenin Subunit Genes in Roegneria nakaii and Roegneria alashanica

    PubMed Central

    Zhang, Lujun; Li, Zhixin; Fan, Renchun; Wei, Bo; Zhang, Xiangqi

    2016-01-01

    The Roegneria of Triticeae is a large genus including about 130 allopolyploid species. Little is known about its high-molecular-weight glutenin subunits (HMW-GSs). Here, we reported six novel HMW-GS genes from R. nakaii and R. alashanica. Sequencing indicated that Rny1, Rny3, and Ray1 possessed intact open reading frames (ORFs), whereas Rny2, Rny4, and Ray2 harbored in-frame stop codons. All of the six genes possessed a similar primary structure to known HMW-GS, while showing some unique characteristics. Their coding regions were significantly shorter than Glu-1 genes in wheat. The amino acid sequences revealed that all of the six genes were intermediate towards the y-type. The phylogenetic analysis showed that the HMW-GSs from species with St, StY, or StH genome(s) clustered in an independent clade, varying from the typical x- and y-type clusters. Thus, the Glu-1 locus in R. nakaii and R. alashanica is a very primitive glutenin locus across evolution. The six genes were phylogenetically split into two groups clustered to different clades, respectively, each of the two clades included the HMW-GSs from species with St (diploid and tetraploid species), StY, and StH genomes. Hence, it is concluded that the six Roegneria HMW-GS genes are from two St genomes undergoing slight differentiation. PMID:27447615

  1. Lipoic acid increases the expression of genes involved in bone formation in mice fed a high-fat diet.

    PubMed

    Xiao, Ying; Cui, Jue; Shi, Yonghui; Le, Guowei

    2011-04-01

    Antioxidant lipoic acid (LA) has been reported to have a potential prophylactic effect on bone loss induced by high-fat diet (HFD). The aim of this work was to examine the hypothesis that LA decreases bone resorption-related gene expression and increases bone formation-related gene expression in HFD-fed mice, preventing a shift in the bone metabolism balance toward resorption. Male C57BL/6 mice were fed a normal diet, HFD, or HFD plus 0.1% LA for 12 weeks. The bone metabolism-related genes differentially expressed between mice fed HFD and those fed HFD supplemented with LA were identified through complementary DNA microarray. The supplemental LA significantly increased bone mineral density and bone antioxidant capacity in mice fed HFD (P < .05). Compared with the HFD-fed mice, LA induced the decreased expression of genes associated with bone resorption, such as Mmp9 (1.9-fold) and Ctsk (2.3-fold), and increased those genes associated with bone formation, such as Col1a1 (1.3-fold) and Alp1 (1.5-fold). Furthermore, LA upregulated many genes involved in the Igf signaling pathway, such as Igf-1 (increased 1.7-fold), and downregulated genes involved in the p53 apoptotic pathway, such as p53 (decreased 2.3-fold), thus attenuating the HFD-induced inhibition of bone formation. Lipoic acid induced upregulation of Il12a (2.1-fold) and downregulation of Tgfbr1 (4.3-fold) and Il17a (11.3-fold), which may reduce bone resorption. In summary, LA supplementation during HFD could affect bone density, altering gene expression.

  2. Hepatic Gene Expression Related to Lower Plasma Cholesterol in Hamsters Fed High Fat Diets Supplemented with Blueberry Pomace and Extract

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We analyzed plasma lipid profiles, and genes related to cholesterol and bile acid metabolism, and inflammation in livers as well as adipose tissue from Syrian Golden hamsters fed high-fat diets supplemented with blueberry (BB) pomace byproducts including 8% dried whole blueberry peels (BBPWHL), 2% d...

  3. Increased nitric oxide availability attenuates high fat diet metabolic alterations and gene expression associated with insulin resistance

    PubMed Central

    2011-01-01

    Background High fat diet impairs nitric oxide (NO) bioavailability, and induces insulin resistance. The link between NO availability and the metabolic adaptation to a high fat diet is not well characterized. The purpose of this study was to investigate the effect of high fat diet on metabolism in mice with decreased (eNOS-/-) and increased (DDAH overexpressed) NO bioavailability. Methods eNOS-/- (n = 16), DDAH (n = 24), and WT (n = 19) mice were fed a high fat diet (HFD) for 13 weeks. Body weight, biochemical parameters, adipokines and insulin were monitored. The matrigel in vivo model with CD31 immunostaining was used to assess angiogenesis. Gene expression in adipose tissues was analyzed by microarray and Real Time PCR. Comparisons of the mean values were made using the unpaired Student t test and p < 0.05 were considered statistically significant. Results eNOS-/- mice gained less weight than control WT and DDAH mice. In DDAH mice, a greater increase in serum adiponectin and a lesser increment in glucose level was observed. Fasting insulin and cholesterol levels remained unchanged. The angiogenic response was increased in DDAH mice. In adipose tissue of DDAH mice, genes characteristic of differentiated adipocytes were down-regulated, whereas in eNOS-/- mice, genes associated with adipogenesis, fatty acid and triglyceride synthesis were upregulated. Conclusions Our results indicate that increased NO availability attenuates some HFD induced alterations in metabolism and gene expression associated with insulin resistance. PMID:21781316

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

    PubMed Central

    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

  5. High-Affinity Glucose Transport in Aspergillus nidulans Is Mediated by the Products of Two Related but Differentially Expressed Genes

    PubMed Central

    Ventura, Luisa; González, Ramón; Ramón, Daniel; MacCabe, Andrew P.

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  6. High-affinity glucose transport in Aspergillus nidulans is mediated by the products of two related but differentially expressed genes.

    PubMed

    Forment, Josep V; Flipphi, Michel; Ventura, Luisa; González, Ramón; Ramón, Daniel; Maccabe, Andrew P

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  7. Global Gene Expression Responses to Low- or High-Dose Radiation in a Human Three-Dimensional Tissue Model

    PubMed Central

    Mezentsev, Alexandre; Amundson, Sally A.

    2011-01-01

    Accumulating data suggest that the biological responses to high and low doses of radiation are qualitatively different, necessitating the direct study of low-dose responses to better understand potential risks. Most such studies have used two-dimensional culture systems, which may not fully represent responses in three-dimensional tissues. To gain insight into low-dose responses in tissue, we have profiled global gene expression in EPI-200, a three-dimensional tissue model that imitates the structure and function of human epidermis, at 4, 16 and 24 h after exposure to high (2.5 Gy) and low (0.1 Gy) doses of low-LET protons. The most significant gene ontology groups among genes altered in expression were consistent with effects observed at the tissue level, where the low dose was associated with recovery and tissue repair, while the high dose resulted in loss of structural integrity and terminal differentiation. Network analysis of the significantly responding genes suggested that TP53 dominated the response to 2.5 Gy, while HNF4A, a novel transcription factor not previously associated with radiation response, was most prominent in the low-dose response. HNF4A protein levels and phosphorylation were found to increase in tissues and cells after low- but not high-dose irradiation. PMID:21486161

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

    PubMed

    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. PMID:19578720

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

    PubMed

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

    2015-03-01

    We report the use of arginine-glycine-aspartic (Arg-Gly-Asp, RGD) peptide-modified dendrimer-entrapped gold nanoparticles (Au DENPs) for highly efficient and specific gene delivery to stem cells. In this study, generation 5 poly(amidoamine) dendrimers modified with RGD via a poly(ethylene glycol) (PEG) spacer and with PEG monomethyl ether were used as templates to entrap gold nanoparticles (AuNPs). The native and the RGD-modified PEGylated dendrimers and the respective well characterized Au DENPs were used as vectors to transfect human mesenchymal stem cells (hMSCs) with plasmid DNA (pDNA) carrying both the enhanced green fluorescent protein and the luciferase (pEGFPLuc) reporter genes, as well as pDNA encoding the human bone morphogenetic protein-2 (hBMP-2) gene. We show that all vectors are capable of transfecting the hMSCs with both pDNAs. Gene transfection using pEGFPLuc was demonstrated by quantitative Luc activity assay and qualitative evaluation by fluorescence microscopy. For the transfection with hBMP-2, the gene delivery efficiency was evaluated by monitoring the hBMP-2 concentration and the level of osteogenic differentiation of the hMSCs via alkaline phosphatase activity, osteocalcin secretion, calcium deposition, and von Kossa staining assays. Our results reveal that the stem cell gene delivery efficiency is largely dependent on the composition and the surface functionality of the dendrimer-based vectors. The coexistence of RGD and AuNPs rendered the designed dendrimeric vector with specific stem cell binding ability likely via binding of integrin receptor on the cell surface and improved three-dimensional conformation of dendrimers, which is beneficial for highly efficient and specific stem cell gene delivery applications. PMID:25658033

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

    PubMed

    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

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

    PubMed

    Nakajima, Keisuke; Yaoita, Yoshio

    2015-01-16

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

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

    PubMed

    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.

  13. A gene stacking approach leads to engineered plants with highly increased galactan levels in Arabidopsis

    DOE PAGESBeta

    Gondolf, Vibe M.; Stoppel, Rhea; Ebert, Berit; Rautengarten, Carsten; Liwanag, April J.M.; Loqué, Dominique; Scheller, Henrik V.

    2014-12-10

    Background: Engineering of plants with a composition of lignocellulosic biomass that is more suitable for downstream processing is of high interest for next-generation biofuel production. Lignocellulosic biomass contains a high proportion of pentose residues, which are more difficult to convert into fuels than hexoses. Therefore, increasing the hexose/pentose ratio in biomass is one approach for biomass improvement. A genetic engineering approach was used to investigate whether the amount of pectic galactan can be specifically increased in cell walls of Arabidopsis fiber cells, which in turn could provide a potential source of readily fermentable galactose. Results: First it was tested ifmore » overexpression of various plant UDP-glucose 4-epimerases (UGEs) could increase the availability of UDP-galactose and thereby increase the biosynthesis of galactan. Constitutive and tissue-specific expression of a poplar UGE and three Arabidopsis UGEs in Arabidopsis plants could not significantly increase the amount of cell wall bound galactose. We then investigated co-overexpression of AtUGE2 together with the β-1,4-galactan synthase GalS1. Co-overexpression of AtUGE2 and GalS1 led to over 80% increase in cell wall galactose levels in Arabidopsis stems, providing evidence that these proteins work synergistically. Furthermore, AtUGE2 and GalS1 overexpression in combination with overexpression of the NST1 master regulator for secondary cell wall biosynthesis resulted in increased thickness of fiber cell walls in addition to the high cell wall galactose levels. Immunofluorescence microscopy confirmed that the increased galactose was present as β-1,4-galactan in secondary cell walls. Conclusions: This approach clearly indicates that simultaneous overexpression of AtUGE2 and GalS1 increases the cell wall galactose to much higher levels than can be achieved by overexpressing either one of these proteins alone. Moreover, the increased galactan content in fiber cells while

  14. A gene stacking approach leads to engineered plants with highly increased galactan levels in Arabidopsis

    SciTech Connect

    Gondolf, Vibe M.; Stoppel, Rhea; Ebert, Berit; Rautengarten, Carsten; Liwanag, April J.M.; Loqué, Dominique; Scheller, Henrik V.

    2014-12-10

    Background: Engineering of plants with a composition of lignocellulosic biomass that is more suitable for downstream processing is of high interest for next-generation biofuel production. Lignocellulosic biomass contains a high proportion of pentose residues, which are more difficult to convert into fuels than hexoses. Therefore, increasing the hexose/pentose ratio in biomass is one approach for biomass improvement. A genetic engineering approach was used to investigate whether the amount of pectic galactan can be specifically increased in cell walls of Arabidopsis fiber cells, which in turn could provide a potential source of readily fermentable galactose. Results: First it was tested if overexpression of various plant UDP-glucose 4-epimerases (UGEs) could increase the availability of UDP-galactose and thereby increase the biosynthesis of galactan. Constitutive and tissue-specific expression of a poplar UGE and three Arabidopsis UGEs in Arabidopsis plants could not significantly increase the amount of cell wall bound galactose. We then investigated co-overexpression of AtUGE2 together with the β-1,4-galactan synthase GalS1. Co-overexpression of AtUGE2 and GalS1 led to over 80% increase in cell wall galactose levels in Arabidopsis stems, providing evidence that these proteins work synergistically. Furthermore, AtUGE2 and GalS1 overexpression in combination with overexpression of the NST1 master regulator for secondary cell wall biosynthesis resulted in increased thickness of fiber cell walls in addition to the high cell wall galactose levels. Immunofluorescence microscopy confirmed that the increased galactose was present as β-1,4-galactan in secondary cell walls. Conclusions: This approach clearly indicates that simultaneous overexpression of AtUGE2 and GalS1 increases the cell wall galactose to much higher levels than can be achieved by overexpressing either one of these proteins alone. Moreover, the increased galactan content in

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

    PubMed

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

    2013-12-01

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

  16. PCR Primers to Study the Diversity of Expressed Fungal Genes Encoding Lignocellulolytic Enzymes in Soils Using High-Throughput Sequencing

    PubMed Central

    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

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

    PubMed

    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

  18. Assessment of the Internal Genes of Influenza A (H7N9) Virus Contributing to High Pathogenicity in Mice

    PubMed Central

    Bi, Yuhai; Xie, Qing; Zhang, Shuang; Li, Yun; Xiao, Haixia; Jin, Tao; Zheng, Weinan; Li, Jing; Jia, Xiaojuan; Sun, Lei; Liu, Jinhua; Qin, Chuan

    2014-01-01

    ABSTRACT The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants was generated by reverse genetics, with each virus containing a single internal gene of the typical A/Anhui/1/2013 (H7N9) (AH-H7N9) virus in the genetic background of the A/chicken/Shandong/lx1023/2007 (H9N2) virus. The replication ability, polymerase activity, and pathogenicity of these viruses were then evaluated in vitro and in vivo. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed that the PB2 K627 residue is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/PB2). Notably, the H7N9-derived PB2 gene displayed greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface hemagglutinin (HA) and neuraminidase (NA) proteins, contributing to the high infectivity of the H7N9 virus in humans. IMPORTANCE To date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been

  19. Taxonomic and gene-centric metagenomics of the fecal microbiome of low and high feed conversion ratio (FCR) broilers.

    PubMed

    Singh, K M; Shah, T M; Reddy, Bhaskar; Deshpande, S; Rank, D N; Joshi, C G

    2014-02-01

    Individual weight gain in broiler growers appears to vary, which may in part be due to variation in their gut microbiota. In this paper we analyse the fecal microbiota of low and high feed conversion ratio (FCR) broilers. After shotgun sequencing of the fecal microbiome, we used the SEED database to identify the microbial diversity and metabolic potential in low and high FCR birds. The domain-level breakdown of our samples was bacteria (>95 %), eukaryotes (>2 %), archaea (>0.2 %), and viruses (>0.2 %). At the phylum level, Proteobacteria (78.83 % in low and 52.04 % in high FCR), Firmicutes (11.97 % in low and 27.53 % in high FCR) and Bacteroidetes (7.10 % in low FCR and 17.53 % in high FCR) predominated in the fecal microbial community. Poultry fecal metagenomes revealed the sequences related to 33 genera in both low and high FCR with significantly different proportion. Functional analysis revealed that genes for the metabolism of carbohydrates, amino acids and derivatives and protein metabolism were most abundant in SEED subsystem in both samples. Genes associated with stress, virulence, cell wall and cell capsule were also abundant. Indeed, genes associated with sulphur assimilation, flagellum and flagellar motility were over represented in low FCR birds. This information could help in developing strategies to improve feed efficiency and feed formulation for broiler chickens.

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

    PubMed

    Li, Rongbo; Zhuang, Yuan; Han, Min; Xu, Tian; Wu, Xiaohui

    2013-05-01

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

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

    PubMed

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

    2015-07-30

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

  2. Simulation of High-Resolution Magnetic Resonance Images on the IBM Blue Gene/L Supercomputer Using SIMRI

    DOE PAGESBeta

    Baum, K. G.; Menezes, G.; Helguera, M.

    2011-01-01

    Medical imaging system simulators are tools that provide a means to evaluate system architecture and create artificial image sets that are appropriate for specific applications. We have modified SIMRI, a Bloch equation-based magnetic resonance image simulator, in order to successfully generate high-resolution 3D MR images of the Montreal brain phantom using Blue Gene/L systems. Results show that redistribution of the workload allows an anatomically accurate 256 3 voxel spin-echo simulation in less than 5 hours when executed on an 8192-node partition of a Blue Gene/L system.

  3. Gene expression profiles underlying alternative caste phenotypes in a highly eusocial bee, Melipona quadrifasciata.

    PubMed

    Judice, C C; Carazzole, M F; Festa, F; Sogayar, M C; Hartfelder, K; Pereira, G A G

    2006-02-01

    To evaluate caste-biased gene expression in Melipona quadrifasciata, a stingless bee, we generated 1278 ESTs using Representational Difference Analysis. Most annotated sequences were similar to honey bee genes of unknown function. Only few queen-biased sequences had their putative function assigned by sequence comparison, contrasting with the worker-biased ESTs. The expression of six annotated genes connected to caste specificity was validated by real time PCR. Interestingly, queens that were developmentally induced by treatment with a juvenile hormone analogue displayed an expression profile clearly different from natural queens for this set of genes. In summary, this study represents an important first step in applying a comparative genomic approach to queen/worker polyphenism in the bee.

  4. Gene therapies: the challenge of super-high-cost treatments and how to pay for them.

    PubMed

    Carr, David R; Bradshaw, Steven E

    2016-06-01

    Gene therapies have the potential to cure rare conditions that often have no current efficacious treatments with a one-time treatment episode, relieving substantial unmet need and having profound positive impact on patients' lives. However, with the first gene therapy now licensed and priced at around US$1 million per patient, cost and uncertain funding mechanisms present a potential barrier to patient access. In this article, we discuss the unique challenges presented by gene therapies, particularly concerning the uncertainty inherent in their clinical evidence package at launch and their affordability within strained healthcare budgets. We present several payment models that would allow for sustainable reimbursement of these innovative technologies and make recommendations pertinent both to those developing gene therapies and to those paying for them. PMID:27185544

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

    PubMed

    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

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

    PubMed Central

    Si, Hongzong; Liu, Shihai; Li, Xianchao; Gao, Caihong; Cui, Lianhua; Li, Chuan; Yang, Xue; Yao, Xiaojun

    2015-01-01

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

  7. High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates.

    PubMed

    Nosaka, K

    1990-02-01

    The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

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

    PubMed

    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.

  9. High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay.

    PubMed

    Dölken, Lars; Ruzsics, Zsolt; Rädle, Bernd; Friedel, Caroline C; Zimmer, Ralf; Mages, Jörg; Hoffmann, Reinhard; Dickinson, Paul; Forster, Thorsten; Ghazal, Peter; Koszinowski, Ulrich H

    2008-09-01

    RNA levels in a cell are determined by the relative rates of RNA synthesis and decay. State-of-the-art transcriptional analyses only employ total cellular RNA. Therefore, changes in RNA levels cannot be attributed to RNA synthesis or decay, and temporal resolution is poor. Recently, it was reported that newly transcribed RNA can be biosynthetically labeled for 1-2 h using thiolated nucleosides, purified from total cellular RNA and subjected to microarray analysis. However, in order to study signaling events at molecular level, analysis of changes occurring within minutes is required. We developed an improved approach to separate total cellular RNA into newly transcribed and preexisting RNA following 10-15 min of metabolic labeling. Employing new computational tools for array normalization and half-life determination we simultaneously study short-term RNA synthesis and decay as well as their impact on cellular transcript levels. As an example we studied the response of fibroblasts to type I and II interferons (IFN). Analysis of RNA transcribed within 15-30 min at different times during the first three hours of interferon-receptor activation resulted in a >10-fold increase in microarray sensitivity and provided a comprehensive profile of the kinetics of IFN-mediated changes in gene expression. We identify a previously undisclosed highly connected network of short-lived transcripts selectively down-regulated by IFNgamma in between 30 and 60 min after IFN treatment showing strong associations with cell cycle and apoptosis, indicating novel mechanisms by which IFNgamma affects these pathways.

  10. High Frequency of Inherited Variants in the MEFV Gene in Acute Lymphocytic Leukemia.

    PubMed

    Sayan, Ozkan; Kilicaslan, Emrah; Celik, Serkan; Tangi, Fatih; Erikci, Alev A; Ipcioglu, Osman; Sanisoglu, Yavuz S; Nalbant, Selim; Oktenli, Cagatay

    2011-09-01

    In the present study, we aimed to determine the frequency of inherited variants in the MEFV (Mediterranean FeVer), the gene responsible for familial Mediterranean fever (FMF), gene in patients with acute lymphocytic leukemia (ALL). The eight MEFV gene variants (M694I, M694V, M680I (G/C-A), V726A, R761H, E148Q and P369S) were detected in 36 patients with ALL and 65 healthy controls; none had own and/or family history compatible with FMF. We identified 11 heterozygous inherited variants in the MEFV gene in both ALL patients and controls. The mean overall frequency of inherited variants in the MEFV gene rate was higher in ALL patients than healthy controls (P = 0.040). It is interesting to note that M680I/0 is predominant variant in patients with ALL. In addition, E148Q variant frequency was also significantly higher in the patient group than the controls (P = 0.012). In conclusion, overall frequency of inherited variants in the MEFV gene was found to be higher in patients with ALL. Based on the present data, it is difficult to reach a definitive conclusion regarding the possibility that inherited variants in the MEFV gene could represent a causative role in ALL. However, the data of our study may provide some new insights in understanding of individual genetic differences in susceptibility to these neoplasms. Further investigations are needed to determine the actual role of inherited variants in the MEFV gene in pathogenesis of ALL.

  11. High-resolution analysis of chromosomal breakpoints and genomic instability identifies PTPRD as a candidate tumor suppressor gene in neuroblastoma.

    PubMed

    Stallings, Raymond L; Nair, Prakash; Maris, John M; Catchpoole, Daniel; McDermott, Michael; O'Meara, Anne; Breatnach, Fin

    2006-04-01

    Although neuroblastoma is characterized by numerous recurrent, large-scale chromosomal imbalances, the genes targeted by such imbalances have remained elusive. We have applied whole-genome oligonucleotide array comparative genomic hybridization (median probe spacing 6 kb) to 56 neuroblastoma tumors and cell lines to identify genes involved with disease pathogenesis. This set of tumors was selected for having either 11q loss or MYCN amplification, abnormalities that define the two most common genetic subtypes of metastatic neuroblastoma. Our analyses have permitted us to map large-scale chromosomal imbalances and high-level amplifications at exon-level resolution and to identify novel microdeletions and duplications. Chromosomal breakpoints (n = 467) generating imbalances >2 Mb were mapped to intervals ranging between 6 and 50 kb in size, providing substantial information on each abnormality. For example, breakpoints leading to large-scale hemizygous loss of chromosome 11q were highly clustered and preferentially associated with segmental duplications. High-level amplifications of MYCN were extremely complex, often resulting in a series of discontinuous regions of amplification. Imbalances (n = 540) <2 Mb long were also detected. Although the majority (78%) of these imbalances mapped to segmentally duplicated regions and primarily reflect constitutional copy number polymorphisms, many subtle imbalances were detected that are likely somatically acquired alterations and include genes involved with tumorigenesis, apoptosis, or neural cell differentiation. The most frequent microdeletion involved the PTPRD locus, indicating a possible tumor suppressor function for this gene.

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

    PubMed

    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.

  13. A cosmid contig and high resolution restriction map of the 2 megabase region containing the Huntington's disease gene.

    PubMed

    Baxendale, S; MacDonald, M E; Mott, R; Francis, F; Lin, C; Kirby, S F; James, M; Zehetner, G; Hummerich, H; Valdes, J

    1993-06-01

    The quest for the mutation responsible for Huntington's disease (HD) has required an exceptionally detailed analysis of a large part of 4p16.3 by molecular genetic techniques, making this stretch of 2.2 megabases one of the best characterized regions of the human genome. Here we describe the construction of a cosmid and P1 clone contig spanning the region containing the HD gene, and the establishment of a detailed, high resolution restriction map. This ordered clone library has allowed the identification of several genes from the region, and has played a vital role in the recent identification of the Huntington's disease gene. The restriction map provides the framework for the detailed analysis of a region extremely rich in coding sequences. This study also exemplifies many of the strategies to be used in the analysis of larger regions of the human genome. PMID:8348156

  14. Human and rat mast cell high-affinity immunoglobulin E receptors: Characterization of putative. alpha. -chain gene products

    SciTech Connect

    Shimizu, Akira; Benfey, P.N.; Leder, P. ); Tepler, I. Brigham and Women's Hospital, Boston, MA ); Berenstein, E.H.; Siraganian, R.P. )

    1988-03-01

    The authors have cloned and determined the entire nucleotide sequence of cDNAs corresponding to the putative {alpha} subunits of the human and rat mast cell high-affinity IgE receptors. Both human and rat cDNAs encode an NH{sub 2}-terminal signal peptide, two immunoglobulin-like extracellular domains (encoded by discrete exons), a hydrophobic transmembrane region, and a positively charged cytoplasmic tail. The human and rat {alpha} subunits share an overall homology with one another and the immunoglobulin gene family, suggesting that they arose from a common ancestral gene and continue to share structural homology with their ligands. In addition, the rat gene is transcribed into at least three distinct forms, each of which yields a somewhat different coding sequence.

  15. A cosmid contig and high resolution restriction map of the 2 megabase region containing the Huntington's disease gene.

    PubMed

    Baxendale, S; MacDonald, M E; Mott, R; Francis, F; Lin, C; Kirby, S F; James, M; Zehetner, G; Hummerich, H; Valdes, J

    1993-06-01

    The quest for the mutation responsible for Huntington's disease (HD) has required an exceptionally detailed analysis of a large part of 4p16.3 by molecular genetic techniques, making this stretch of 2.2 megabases one of the best characterized regions of the human genome. Here we describe the construction of a cosmid and P1 clone contig spanning the region containing the HD gene, and the establishment of a detailed, high resolution restriction map. This ordered clone library has allowed the identification of several genes from the region, and has played a vital role in the recent identification of the Huntington's disease gene. The restriction map provides the framework for the detailed analysis of a region extremely rich in coding sequences. This study also exemplifies many of the strategies to be used in the analysis of larger regions of the human genome.

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

    PubMed

    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

  17. SEPALLATA1/2-suppressed mature apples have low ethylene, high auxin and reduced transcription of ripening-related genes

    PubMed Central

    Schaffer, Robert J.; Ireland, Hilary S.; Ross, John J.; Ling, Toby J.; David, Karine M.

    2012-01-01

    Background and aims Fruit ripening is an important developmental trait in fleshy fruits, making the fruit palatable for seed dispersers. In some fruit species, there is a strong association between auxin concentrations and fruit ripening. We investigated the relationship between auxin concentrations and the onset of ethylene-related ripening in Malus × domestica (apples) at both the hormone and transcriptome levels. Methodology Transgenic apples suppressed for the SEPALLATA1/2 (SEP1/2) class of gene (MADS8/9) that showed severely reduced ripening were compared with untransformed control apples. In each apple type, free indole-3-acetic acid (IAA) concentrations were measured during early ripening. The changes observed in auxin were assessed in light of global changes in gene expression. Principal results It was found that mature MADS8/9-suppressed apples had a higher concentration of free IAA. This was associated with increased expression of the auxin biosynthetic genes in the indole-3-acetamide pathway. Additionally, in the MADS8/9-suppressed apples, there was less expression of the GH3 auxin-conjugating enzymes. A number of genes involved in the auxin-regulated transcription (AUX/IAA and ARF classes of genes) were also observed to change in expression, suggesting a mechanism for signal transduction at the start of ripening. Conclusions The delay in ripening observed in MADS8/9-suppressed apples may be partly due to high auxin concentrations. We propose that, to achieve low auxin associated with fruit maturation, the auxin homeostasis is controlled in a two-pronged manner: (i) by the reduction in biosynthesis and (ii) by an increase in auxin conjugation. This is associated with the change in expression of auxin-signalling genes and the up-regulation of ripening-related genes. PMID:23346344

  18. Mining genes involved in the stratification of Paris Polyphylla seeds using high-throughput embryo Transcriptome sequencing

    PubMed Central

    2013-01-01

    Background Paris polyphylla var. yunnanensis is an important medicinal plant. Seed dormancy is one of the main factors restricting artificial cultivation. The molecular mechanisms of seed dormancy remain unclear, and little genomic or transcriptome data are available for this plant. Results In this study, massive parallel pyrosequencing on the Roche 454-GS FLX Titanium platform was used to generate a substantial sequence dataset for the P. polyphylla embryo. 369,496 high quality reads were obtained, ranging from 50 to 1146 bp, with a mean of 219 bp. These reads were assembled into 47,768 unigenes, which included 16,069 contigs and 31,699 singletons. Using BLASTX searches of public databases, 15,757 (32.3%) unique transcripts were identified. Gene Ontology and Cluster of Orthologous Groups of proteins annotations revealed that these transcripts were broadly representative of the P. polyphylla embryo transcriptome. The Kyoto Encyclopedia of Genes and Genomes assigned 5961 of the unique sequences to specific metabolic pathways. Relative expression levels analysis showed that eleven phytohormone-related genes and five other genes have different expression patterns in the embryo and endosperm in the seed stratification process. Conclusions Gene annotation and quantitative RT-PCR expression analysis identified 464 transcripts that may be involved in phytohormone catabolism and biosynthesis, hormone signal, seed dormancy, seed maturation, cell wall growth and circadian rhythms. In particular, the relative expression analysis of sixteen genes (CYP707A, NCED, GA20ox2, GA20ox3, ABI2, PP2C, ARP3, ARP7, IAAH, IAAS, BRRK, DRM, ELF1, ELF2, SFR6, and SUS) in embryo and endosperm and at two temperatures indicated that these related genes may be candidates for clarifying the molecular basis of seed dormancy in P. polyphlla var. yunnanensis. PMID:23718911

  19. Rapid High Resolution Genotyping of Francisella tularensis by Whole Genome Sequence Comparison of Annotated Genes (“MLST+”)

    PubMed Central

    Mellmann, Alexander; Höppner, Sebastian; Splettstoesser, Wolf D.; Harmsen, Dag

    2015-01-01

    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. PMID:25856198

  20. The transition from linear to highly branched poly(β-amino ester)s: Branching matters for gene delivery

    PubMed Central

    Zhou, Dezhong; Cutlar, Lara; Gao, Yongsheng; Wang, Wei; O’Keeffe-Ahern, Jonathan; McMahon, Sean; Duarte, Blanca; Larcher, Fernando; Rodriguez, Brian J.; Greiser, Udo; Wang, Wenxin

    2016-01-01

    Nonviral gene therapy holds great promise but has not delivered treatments for clinical application to date. Lack of safe and efficient gene delivery vectors is the major hurdle. Among nonviral gene delivery vectors, poly(β-amino ester)s are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. However, to date, all research has been focused on vectors with a linear structure. A well-accepted view is that dendritic or branched polymers have greater potential as gene delivery vectors because of their three-dimensional structure and multiple terminal groups. Nevertheless, to date, the synthesis of dendritic or branched polymers has been proven to be a well-known challenge. We report the design and synthesis of highly branched poly(β-amino ester)s (HPAEs) via a one-pot “A2 + B3 + C2”–type Michael addition approach and evaluate their potential as gene delivery vectors. We find that the branched structure can significantly enhance the transfection efficiency of poly(β-amino ester)s: Up to an 8521-fold enhancement in transfection efficiency was observed across 12 cell types ranging from cell lines, primary cells, to stem cells, over their corresponding linear poly(β-amino ester)s (LPAEs) and the commercial transfection reagents polyethyleneimine, SuperFect, and Lipofectamine 2000. Moreover, we further demonstrate that HPAEs can correct genetic defects in vivo using a recessive dystrophic epidermolysis bullosa graft mouse model. Our findings prove that the A2 + B3 + C2 approach is highly generalizable and flexible for the design and synthesis of HPAEs, which cannot be achieved by the conventional polymerization approach; HPAEs are more efficient vectors in gene transfection than the corresponding LPAEs. This provides valuable insight into the development and applications of nonviral gene delivery and demonstrates great prospect for their

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

    PubMed Central

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

    2015-01-01

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

  2. The high-affinity phosphate-binding protein PstS is accumulated under high fructose concentrations and mutation of the corresponding gene affects differentiation in Streptomyces lividans.

    PubMed

    Díaz, Margarita; Esteban, Ana; Fernández-Abalos, José Manuel; Santamaría, Ramón I

    2005-08-01

    The secreted protein pattern of Streptomyces lividans depends on the carbon source present in the culture media. One protein that shows the most dramatic change is the high-affinity phosphate-binding protein PstS, which is strongly accumulated in the supernatant of liquid cultures containing high concentrations (>3 %) of certain sugars, such as fructose, galactose and mannose. The promoter region of this gene and that of its Streptomyces coelicolor homologue were used to drive the expression of a xylanase in S. lividans that was accumulated in the culture supernatant when grown in the presence of fructose. PstS accumulation was dramatically increased in a S. lividans polyphosphate kinase null mutant (Deltappk) and was impaired in a deletion mutant lacking phoP, the transcriptional regulator gene of the two-component phoR-phoP system that controls the Pho regulon. Deletion of the pstS genes in S. lividans and S. coelicolor impaired phosphate transport and accelerated differentiation and sporulation on solid media. Complementation with a single copy in a S. lividans pstS null mutant returned phosphate transport and sporulation to levels similar to those of the wild-type strain. The present work demonstrates that carbon and phosphate metabolism are linked in the regulation of genes and that this can trigger the genetic switch towards morphogenesis.

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

    PubMed

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

    2013-11-01

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

  4. Identification of highly-disrupted tRNA genes in nuclear genome of the red alga, Cyanidioschyzon merolae 10D

    PubMed Central

    Soma, Akiko; Sugahara, Junichi; Onodera, Akinori; Yachie, Nozomu; Kanai, Akio; Watanabe, Satoru; Yoshikawa, Hirofumi; Ohnuma, Mio; Kuroiwa, Haruko; Kuroiwa, Tsuneyoshi; Sekine, Yasuhiko

    2013-01-01

    The limited locations of tRNA introns are crucial for eukaryal tRNA-splicing endonuclease recognition. However, our analysis of the nuclear genome of an early-diverged red alga, Cyanidioschyzon merolae, demonstrated the first evidence of nuclear-encoded tRNA genes that contain ectopic and/or multiple introns. Some genes exhibited both intronic and permuted structures in which the 3′-half of the tRNA coding sequence lies upstream of the 5′-half, and an intron is inserted into either half. These highly disrupted tRNA genes, which account for 63% of all nuclear tRNA genes, are expressed via the orderly and sequential processing of bulge-helix-bulge (BHB) motifs at intron-exon junctions and termini of permuted tRNA precursors, probably by a C. merolae tRNA-splicing endonuclease with an unidentified subunit architecture. The results revealed a considerable diversity in eukaryal tRNA intron properties and endonuclease architectures, which will help to elucidate the acquisition mechanism of the BHB-mediated disrupted tRNA genes. PMID:23900518

  5. Characterization of DNA fragment from Chlamydia psittaci avian strain which shows high homology with hypB gene of Chlamydia.

    PubMed

    Sato, C; Katumata, A; Takashima, I; Hashimoto, N

    1991-12-01

    A study was performed to characterize DNA fragment No. 17 of C. psittaci strain P-1041 which encoded 42 KD beta-galactosidase fusion protein with type-specific antigenicity. Sequence determination identified a partial open reading frame that spanned about 1,200b. p. nucleotides. Screening the literatures for the nucleotide and deduced amino acid sequences revealed extensive similarity between the DNA fragment of P-1041 and two chlamydial hypB genes. This DNA showed 91.5% homology with C. psittaci GPIC hypB gene in nucleotide sequence and 96.4% homology in deduced amino acid sequence. The hypB gene of C. trachomatis serovar A and the P-1041 DNA fragment showed 81.2% and 91.3% homology in nucleotide and amino acid sequences, respectively. Dot enzyme-linked immunosorbent assay, for the products of deleted DNA fragments defined the coding region for type-specific antigenic polypeptide. In addition, the P-1041 DNA fragment carried a sequence highly homologous (greater than 49%) with other bacterial and plant genes called chaperonin which responds to various stress in cells. From these results, the P-1041 DNA fragment was found to be a part of hypB gene and to encode the region critical for type-specific antigenicity.

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

    PubMed

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

    2016-01-01

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

  7. Isolated trisomy 13 defines a homogeneous AML subgroup with high frequency of mutations in spliceosome genes and poor prognosis.

    PubMed

    Herold, Tobias; Metzeler, Klaus H; Vosberg, Sebastian; Hartmann, Luise; Röllig, Christoph; Stölzel, Friedrich; Schneider, Stephanie; Hubmann, Max; Zellmeier, Evelyn; Ksienzyk, Bianka; Jurinovic, Vindi; Pasalic, Zlatana; Kakadia, Purvi M; Dufour, Annika; Graf, Alexander; Krebs, Stefan; Blum, Helmut; Sauerland, Maria Cristina; Büchner, Thomas; Berdel, Wolfgang E; Woermann, Bernhard J; Bornhäuser, Martin; Ehninger, Gerhard; Mansmann, Ulrich; Hiddemann, Wolfgang; Bohlander, Stefan K; Spiekermann, Karsten; Greif, Philipp A

    2014-08-21

    In acute myeloid leukemia (AML), isolated trisomy 13 (AML+13) is a rare chromosomal abnormality whose prognostic relevance is poorly characterized. We analyzed the clinical course of 34 AML+13 patients enrolled in the German AMLCG-1999 and SAL trials and performed exome sequencing, targeted candidate gene sequencing and gene expression profiling. Relapse-free (RFS) and overall survival (OS) of AML+13 patients were inferior compared to other ELN Intermediate-II patients (n=855) (median RFS, 7.8 vs 14.1 months, P = .006; median OS 9.3 vs. 14.8 months, P = .004). Besides the known high frequency of RUNX1 mutations (75%), we identified mutations in spliceosome components in 88%, including SRSF2 codon 95 mutations in 81%. Recurring mutations were detected in ASXL1 (44%) and BCOR (25%). Two patients carried mutations in CEBPZ, suggesting that CEBPZ is a novel recurrently mutated gene in AML. Gene expression analysis revealed a homogeneous expression profile including upregulation of FOXO1 and FLT3 and downregulation of SPRY2. This is the most comprehensive clinical and biological characterization of AML+13 to date, and reveals a striking clustering of lesions in a few genes, defining AML+13 as a genetically homogeneous subgroup with alterations in a few critical cellular pathways. Clinicaltrials.gov identifiers: AMLCG-1999: NCT00266136; AML96: NCT00180115; AML2003: NCT00180102; and AML60+: NCT00893373. PMID:24923295

  8. Identification and High-Density Mapping of Gene-Rich Regions in Chromosome Group 1 of Wheat

    PubMed Central

    Gill, K. S.; Gill, B. S.; Endo, T. R.; Taylor, T.

    1996-01-01

    We studied the distribution of genes and recombination in wheat (Triticum aestivum) group 1 chromosomes by comparing high-density physical and genetic maps. Physical maps of chromosomes 1A, 1B, and 1D were generated by mapping 50 DNA markers on 56 single-break deletion lines. A consensus physical map was compared with the 1D genetic map of Triticum tauschii (68 markers) and a Triticeae group 1 consensus map (288 markers) to generate a cytogenetic ladder map (CLM). Most group 1 markers (86%) were present in five clusters that encompassed only 10% of the group 1 chromosome. This distribution may reflect that of genes because more than half of the probes were cDNA clones and 30% were PstI genomic. All 14 agronomically important genes in group 1 chromosomes were present in these clusters. Most recombination occurred in gene-cluster regions. Markers fell at an average distance of 244 kb in these regions. The CLM involving the Triticeae consensus genetic map revealed that the above distribution of genes and recombination is the same in other Triticeae species. Because of a significant number of common markers, our CLM can be used for comparative mapping and to estimate physical distances among markers in many Poaceae species including rice and maize. PMID:8978071

  9. Complete mitochondrial genomes of Trisidos kiyoni and Potiarca pilula: Varied mitochondrial genome size and highly rearranged gene order in Arcidae

    PubMed Central

    Sun, Shao’e; Li, Qi; Kong, Lingfeng; Yu, Hong

    2016-01-01

    We present the complete mitochondrial genomes (mitogenomes) of Trisidos kiyoni and Potiarca pilula, both important species from the family Arcidae (Arcoida: Arcacea). Typical bivalve mtDNA features were described, such as the relatively conserved gene number (36 and 37), a high A + T content (62.73% and 61.16%), the preference for A + T-rich codons, and the evidence of non-optimal codon usage. The mitogenomes of Arcidae species are exceptional for their extraordinarily large and variable sizes and substantial gene rearrangements. The mitogenome of T. kiyoni (19,614 bp) and P. pilula (28,470 bp) are the two smallest Arcidae mitogenomes. The compact mitogenomes are weakly associated with gene number and primarily reflect shrinkage of the non-coding regions. The varied size in Arcidae mitogenomes reflect a dynamic history of expansion. A significant positive correlation is observed between mitogenome size and the combined length of cox1-3, the lengths of Cytb, and the combined length of rRNAs (rrnS and rrnL) (P < 0.001). Both protein coding genes (PCGs) and tRNA rearrangements is observed in P. pilula and T. kiyoni mitogenomes. This analysis imply that the complicated gene rearrangement in mitochondrial genome could be considered as one of key characters in inferring higher-level phylogenetic relationship of Arcidae. PMID:27653979

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  11. High level expression in Saccharomyces cerevisiae of an artificial gene encoding a repeated tripeptide aspartyl-phenylyalanyl-lysine.

    PubMed

    Choi, S Y; Lee, S Y; Bock, R M

    1993-08-01

    A chemically synthesized gene, which encodes a 64 or 128 times-repeated tripeptide, aspartyl-phenylalanyl-lysine, has been cloned onto the yeast expression vector pAM82 containing the PHO5 promoter. The artificial gene (LAP gene) contains the untranslated leader sequence of the E. coli lipoprotein gene (lpp) with its transcription terminator sequence. When yeast AH22 cells transformed by recombinant plasmid containing repeated tripeptide gene were derepressed in low phosphate medium, the artificial polypeptides were synthesized to the amounts of about 30% of the total cell protein. SDS-polyacrylamide gel electrophoresis and immunoblot analysis indicated that the artificial polypeptides synthesized in yeast have molecular weights ranging from about 30,000 and 60,000 and have immunoreactivity with the artificial polypeptides expressed in E. coli. The artificial popypeptides in whole cell extract were insoluble and seem to be synthesized as insoluble aggregates. Electron microscopy showed the presence of inclusion bodies in the cell. These polypeptides can be hydrolyzed to tripeptides with trypsin or chymotrypsin. These properties along with the high expression and easy separation may make the artificial polypeptides a potential raw material for the production of an artificial sweetener, Aspartame.

  12. Pathway-based factor analysis of gene expression data produces highly heritable phenotypes that associate with age.

    PubMed

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

    2015-03-09

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    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

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

    PubMed

    Godoy, Patricio; Widera, Agata; Schmidt-Heck, Wolfgang; Campos, Gisela; Meyer, Christoph; Cadenas, Cristina; Reif, Raymond; Stöber, Regina; Hammad, Seddik; Pütter, Larissa; Gianmoena, Kathrin; Marchan, Rosemarie; Ghallab, Ahmed; Edlund, Karolina; Nüssler, Andreas; Thasler, Wo