Science.gov

Sample records for fhit gene enhances

  1. The FHIT gene product: tumor suppressor and genome ‘caretaker’

    PubMed Central

    Waters, Catherine E.; Saldivar, Joshua C.; Hosseini, Seyed Ali; Huebner, Kay

    2014-01-01

    The FHIT gene at FRA3B is one of the earliest and most frequently altered genes in the majority of human cancers. It was recently discovered that the FHIT gene is not the most fragile locus in epithelial cells, the cell of origin for most Fhit negative cancers, eroding support for past claims that deletions at this locus are simply passenger events that are carried along in expanding cancer clones, due to extreme vulnerability to DNA damage rather than to loss of FHIT function. Indeed, recent reports have reconfirmed FHIT as a tumor suppressor gene with roles in apoptosis and prevention of the epithelial-mesenchymal transition. Other recent works have identified a novel role for the FHIT gene product, Fhit, as a genome ‘caretaker.’ Loss of this caretaker function leads to nucleotide imbalance, spontaneous replication stress, and DNA breaks. Because Fhit loss-induced DNA damage is “checkpoint blind,” cells accumulate further DNA damage during subsequent cell cycles, accruing global genome instability that could facilitate oncogenic mutation acquisition and expedite clonal expansion. Loss of Fhit activity therefore induces a mutator phenotype. Evidence for FHIT as a mutator gene is discussed in light of these recent investigations of Fhit loss and subsequent genome instability. PMID:25283145

  2. Reduced Fhit protein expression and loss of heterozygosity at FHIT gene in tumours from smoking and asbestos-exposed lung cancer patients.

    PubMed

    Pylkkanen, Lea; Wolff, Henrik; Stjernvall, Tuula; Tuominen, Paivi; Sioris, Thanos; Karjalainen, Antti; Anttila, Sisko; Husgafvel-Pursiainen, Kirsti

    2002-02-01

    The FHIT gene, at 3p14.2, has been suggested to form a molecular target to damage induced by human lung carcinogens. We examined aberrant expression of the Fhit protein and allele loss at the FHIT gene in a series of lung cancer cases, mainly of non-small cell carcinoma (NSCLC) histology. We had detailed data on tobacco smoke exposure and occupational asbestos exposure available for the cases. The principal aim of the present study was to investigate whether absent or reduced Fhit expression or FHIT allele loss was associated with exposure to these pulmonary carcinogens. We detected reduced Fhit expression in 62% (33/53) of the cases analysed. Prevalence of allele loss at the FHIT locus was 22% (20/89). Reduced protein expression was common both in the asbestos-exposed (67%) and non-exposed cases (59%); [odds ratio (OR) 1.4, 95% confidence interval (CI) 0.4-4.9]. LOH frequencies differed somewhat between the two groups and were 25% vs. 16%, respectively (OR 1.8; 95% CI 0.5-5.9). Absent or reduced expression was common in smokers, with no significant difference found between current smokers and non-smokers (mainly former smokers) (OR 1.4, 95% CI 0.5-4.5). NSCLCs with squamous cell histology exhibited both aberrant expression (OR 3.1, 95% CI 0.9-10.3) and allele loss (OR 3.3, 95% CI 0.9-12.7) more frequently than adenocarcinoma. Finally, we found that FHIT allele loss was increased in stage II or more advanced disease (OR 2.5, 95% CI 0.9-7.4), and in poorly differentiated tumours (grade 3, OR 2.6, 95% CI 0.8-8.1). In conclusion, our present data support significance of FHIT inactivation in development of lung cancer.

  3. A Fhit-ing role in the DNA damage checkpoint response.

    PubMed

    Ishii, Hideshi; Wang, Ya; Huebner, Kay

    2007-05-02

    The FHIT gene encompasses the most active common fragile site of the human genome and is thus exquisitely sensitive to intragenic alterations by DNA damaging agents, alterations that can lead to FHIT allele loss very early in the preneoplastic phase of cancer development, before or coincident with activation of the DNA damage checkpoint. Fhit protein expression is lost or reduced in many preneoplastic lesions and in >50% of cancers, Fhit knockout mice are highly susceptible to carcinogen induction of tumors and Fhit replacement in these mice by gene therapy induces apoptosis and significantly reduces tumor burden. But learning how Fhit induces apoptosis and suppresses tumors has been a challenge because interacting proteins, effectors of Fhit signals, have not been discovered. Nevertheless, the study of Fhit deficient mouse and human tissue-derived and cancer-derived cells in vitro has led to several important conclusions: repair protein-deficient cancers are more likely to be Fhit-deficient; Fhit-deficient cells show enhanced resistance to UVC, mitomycin C, camptothecin and ionizing radiation-induced cell killing, possibly due to strong activation of the ATR pathway following DNA damage; Fhit-deficient cells show higher efficiency of homologous recombination repair, a double-strand break repair pathway in mammalian cells; Fhit protein indirectly affects S-phase checkpoint and DNA repair. Finally, results of a recent study have suggested that the DNA damage-susceptible FRA3B/FHIT chromosome fragile region, paradoxically, encodes a protein, Fhit, that is necessary for protecting cells from accumulation of DNA damage, through modulation of checkpoint proteins Hus1 and phosphoChk1. Thus, inactivation of Fhit contributes to accumulation of abnormal checkpoint phenotypes in cancer development. It will be very important to determine mechanisms employed by Fhit in modulating checkpoint pathways, and to define consequences of Fhit loss in specific preneoplastic and

  4. A knockdown with smoke model reveals FHIT as a repressor of Heme oxygenase 1.

    PubMed

    Boylston, Jennifer A; Brenner, Charles

    2014-01-01

    Fragile histidine triad (FHIT) gene deletions are among the earliest and most frequent events in carcinogenesis, particularly in carcinogen-exposed tissues. Though FHIT has been established as an authentic tumor suppressor, the mechanism underlying tumor suppression remains opaque. Most experiments designed to clarify FHIT function have analyzed the consequence of re-expressing FHIT in FHIT-negative cells. However, carcinogenesis occurs in cells that transition from FHIT-positive to FHIT-negative. To better understand cancer development, we induced FHIT loss in human bronchial epithelial cells with RNA interference. Because FHIT is a demonstrated target of carcinogens in cigarette smoke, we combined FHIT silencing with cigarette smoke extract (CSE) exposure and measured gene expression consequences by RNA microarray. The data indicate that FHIT loss enhances the expression of a set of oxidative stress response genes after exposure to CSE, including the cytoprotective enzyme heme oxygenase 1 (HMOX1) at the RNA and protein levels. Data are consistent with a mechanism in which Fhit protein is required for accumulation of the transcriptional repressor of HMOX1, Bach1 protein. We posit that by allowing superinduction of oxidative stress response genes, loss of FHIT creates a survival advantage that promotes carcinogenesis.

  5. Simultaneous detection of the tumor suppressor FHIT gene and protein using the multi-functional biochip.

    PubMed

    Askari, Minoo D F; Miller, Gordon H; Vo-Dinh, Tuan

    2002-01-01

    The tumor suppressor gene, fragile histidine triad (FHIT), encompasses the most common human chromosomal fragile site, at 3pl4.2. Detection of FHIT gene is important in cancer diagnostics since its alterations have been associated with several human cancers. A unique multi-functional biochip for simultaneous detection of FHIT DNA and FHIT protein on the same platform was applied. The design of the biochip is based on miniaturization of photodiodes, where functioning of multiple optical sensing elements, amplifiers, discriminators, and logic circuitry are integrated on a single IC board. Performance of biochip is based on biomolecular recognition processes using both DNA and protein bioreceptors, Cy5-labeled probes and laser excitation. Application of biochip for concurrent detection of various immobilized target DNA and protein molecules and multiplex of DNA and protein on the same microarray was accomplished. Linearity of biochip for quantitative measurements was demonstrated. Results demonstrated utility of this multi-functional biochip as a useful detection technology with applications in biological and clinical laboratories.

  6. FHIT — EDRN Public Portal

    Cancer.gov

    FHIT, or fragile histidine triad, is a member of the histidine triad gene family. The FHIT protein is a diadenosine 5',5'''-P1,P3-triphosphate hydrolase involved in purine metabolism. Included in the FHIT gene is the common fragile site FRA3B on chromosome 3, where carcinogen-induced damage can lead to translocations and aberrant transcripts of this gene. Mutated transcripts from this gene have been found in about half of all esophageal, stomach, and colon carcinomas. There are several alternatively spliced transcript variants for this gene.

  7. The hereditary renal cell carcinoma 3;8 translocation fuses FHIT to a patched-related gene, TRC8.

    PubMed

    Gemmill, R M; West, J D; Boldog, F; Tanaka, N; Robinson, L J; Smith, D I; Li, F; Drabkin, H A

    1998-08-04

    The 3;8 chromosomal translocation, t(3;8)(p14.2;q24.1), was described in a family with classical features of hereditary renal cell carcinoma. Previous studies demonstrated that the 3p14.2 breakpoint interrupts the fragile histidine triad gene (FHIT) in its 5' noncoding region. However, evidence that FHIT is causally related to renal or other malignancies is controversial. We now show that the 8q24.1 breakpoint region encodes a 664-aa multiple membrane spanning protein, TRC8, with similarity to the hereditary basal cell carcinoma/segment polarity gene, patched. This similarity involves two regions of patched, the putative sterol-sensing domain and the second extracellular loop that participates in the binding of sonic hedgehog. In the 3;8 translocation, TRC8 is fused to FHIT and is disrupted within the sterol-sensing domain. In contrast, the FHIT coding region is maintained and expressed. In a series of sporadic renal carcinomas, an acquired TRC8 mutation was identified. By analogy to patched, TRC8 might function as a signaling receptor and other pathway members, to be defined, are mutation candidates in malignant diseases involving the kidney and thyroid.

  8. Molecular analysis of the fragile histidine triad (FHIT) tumor suppressor gene in vesical tumors of cattle with chronic enzootic hematuria (CEH).

    PubMed

    Guidi, E; Uboldi, C; Ferretti, L

    2008-01-01

    The FHIT (fragile histidine triad) gene is a tumor suppressor gene known to be inactivated in many tumors including bladder tumors and is spanning FRA3B, a very active common fragile site in the human genome. We have recently isolated the bovine gene, and the aim of this study was to test whether FHIT presents altered expression patterns in vesical tumors of cattle with CEH (chronic enzootic hematuria). CEH is a common syndrome affecting Mediterranean cattle: clastogenic, mutagenic and cancerogenic substances released by the bracken fern (Pteridium spp) grazed by animals induce the formation of neoplastic lesions, among which bladder tumors have a high incidence. We analysed FHIT in 23 bladder tumors of CEH cattle looking at: 1) the methylation status of the CpG island comprising the promoter and part of exon 1; 2) the presence of altered FHIT transcripts; 3) the mRNA expression levels measured with a quantitative real time PCR (QRT-PCR) approach. Our results suggest that unlike in human tumors, FHIT in vesical tumors of CEH cattle is largely unmethylated. Furthermore, the same mRNA isoforms of FHIT were detected in tumors and in healthy tissues, including a novel isoform that was found in this study. Finally, QRT-PCR data did not reveal significantly altered expression profiles of FHIT transcripts. Further studies and larger sets of cases will be useful to confirm this finding, but the data seem to suggest that epigenetic modifications of FHIT and altered expression profiles are not a hallmark of bovine vesical tumors like they are in human tumors.

  9. Novel missense mutation in FHIT gene: interpreting the effect in HPV-mediated cervical cancer in Indian women.

    PubMed

    Neyaz, Md Kausar; Hussain, Showket; Hassan, Md Imtaiyaz; Das, Bhudev C; Husain, Syed Akhtar; Bharadwaj, Mausumi

    2010-02-01

    Human papillomavirus (HPV) is considered to be a major etiological factor but is not sufficient for the development of cervical cancer. Other host factors including altered tumor suppressor gene activities might contribute to the carcinogenic process. Fragile Histidine Triad (FHIT) has been shown to play a pivotal role in carcinogenesis. Therefore, we made an attempt to find out point mutation of FHIT gene in HPV mediated cervical cancer in Indian women. 112 cases of cervical carcinoma tissue biopsies and 38 cervical scrapes samples of normal cytology were employed for this study. Herein, we report a novel mutation identified at nucleotide position 655, at codon 98 from CAT --> CGT with ultimate replacement of amino acid Histidine by Arginine in cervical cancer cases. Molecular modeling was performed to predict the effect of this mutation in disease pathology. We predict that this change, His to Arg substitution in substrate-binding domain may generate catalytically inactive protein with loss of tumor suppressor activity.

  10. Fhit is a physiological target of the protein kinase Src.

    PubMed

    Pekarsky, Yuri; Garrison, Preston N; Palamarchuk, Alexey; Zanesi, Nicola; Aqeilan, Rami I; Huebner, Kay; Barnes, Larry D; Croce, Carlo M

    2004-03-16

    The FHIT gene is a tumor suppressor that is frequently inactivated by genomic alterations at chromosomal region 3p14.2. In the last few years, a considerable amount of data describing inactivation of FHIT in a variety of human malignancies and demonstrating the tumor suppressor potential of Fhit have been reported. Despite the demonstration that FHIT functions as a tumor suppressor, the pathway through which Fhit induces apoptosis and inhibits growth of cancer cells is not known. Our data demonstrate that Fhit is a target of tyrosine phosphorylation by the Src protein kinase. We show that Src phosphorylates Y114 of Fhit in vitro and in vivo, providing insight into a biochemical pathway involved in Fhit signaling.

  11. Homozygous deletion but not mutation of exons 5 and 8 of the fragile histidine triad (FHIT) gene is associated with features of differentiated thyroid carcinoma.

    PubMed

    Yin, De-Tao; Wang, Lin; Sun, Jianrei; Yin, Fengyan; Yan, Qingtao; Shen, Ru-Long; Gao, Jian-Xin; He, Gang

    2010-01-01

    The fragile histidine triad (FHIT) gene encompasses the most common human fragile site, FRA3B at 3p14.2, a region that is involved in homozygous deletions in a variety of human tumors. FHIT is considered to be a tumor suppressor gene that is frequently inactivated in various types of cancer. To study the role of the FHIT gene in thyroid tumorigenesis, we looked for homozygous deletions or mutations of exons 5 and 8 of the FHIT gene in 65 cases of differentiated thyroid carcinoma (DTC) and their matched non-cancerous epithelium (NCE), using exon-specific PCR amplification and PCR single strand conformation polymorphism (PCR-SSCP) techniques. In DTC, the incidence of homozygous deletion of exon 5 was 30.8% (20/65), and it was associated with tumor metastasis to lymph nodes (p <0.05). The incidence of homozygous deletion of exon 8 was 29.2% (19/65), and it was associated with the tumor pathological grade, TNM stage, and lymph node metastasis (p <0.05). There was strong correlation between homozygous deletions of exon 5 and exon 8 (p <0.01). No point mutations were observed in either exon 5 or exon 8. These findings suggest that: (a) exons 5 and 8 of FHIT are key target regions of deletion, (b) homozygous deletions of exon 5 and exon 8 may be good biomarkers for the biological behavior of DTC, and (c) point mutation of these exons may not be involved in the inactivation of the FHIT gene in DTC.

  12. Chromosome 3p loss of heterozygosity and mutation analysis of the FHIT and beta-cat genes in squamous cell carcinoma of the head and neck.

    PubMed Central

    González, M V; Pello, M F; Ablanedo, P; Suárez, C; Alvarez, V; Coto, E

    1998-01-01

    AIMS: To study the loss of heterozygosity at the short arm of chromosome 3 in primary tumours from patients with squamous cell carcinoma of the head and neck; to determine whether the FHIT gene, mapped to 3p14.2 and the CTNNB1 (beta-cat) gene, mapped to 3p21, are deleted or mutated in these tumours. METHODS: DNA was extracted from fresh tumours. Loss of heterozygosity was assessed by microsatellite analysis of the following markers: D3S1283 and D3S1286 (3p24), D3S966 (3p21), and D3S1300 (3P14.2). Homozygous deletion was determined by radioactive multiplex polymerase chain reaction of exons 5 and 6 of the FHIT gene. The presence of mutations in FHIT exon 5 and beta-cat exon 3 was studied by single strand conformation polymorphism. RESULTS: 50% of informative cases (25/50) showed loss of heterozygosity for at least one of the 3p markers. 3p21 was the region with the highest rate of allelic deletion (63%). No point mutation was found in FHIT exon 5 or beta-cat exon 3. No case showed homozygous deletion for the FHIT (exons 5 and 6) or the beta-cat exon 3. CONCLUSIONS: The short arm of chromosome 3 is often deleted in the head and neck squamous cell carcinomas. In the remaining alleles of the FHIT or beta-cat genes, no evidence was found for point mutations or deletions, documented in other common carcinomas. Inactivation could occur by different mechanisms such as methylation, or other genes (not studied here) could be target of allelic losses in squamous cell carcinoma of the head and neck. Images PMID:9797729

  13. Fhit interaction with ferredoxin reductase triggers generation of reactive oxygen species and apoptosis of cancer cells.

    PubMed

    Trapasso, Francesco; Pichiorri, Flavia; Gaspari, Marco; Palumbo, Tiziana; Aqeilan, Rami I; Gaudio, Eugenio; Okumura, Hiroshi; Iuliano, Rodolfo; Di Leva, Giampiero; Fabbri, Muller; Birk, David E; Raso, Cinzia; Green-Church, Kari; Spagnoli, Luigi G; Venuta, Salvatore; Huebner, Kay; Croce, Carlo M

    2008-05-16

    Fhit protein is lost in most cancers, its restoration suppresses tumorigenicity, and virus-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. We have used protein cross-linking and proteomics methods to characterize a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Viral-mediated Fhit restoration increases production of intracellular reactive oxygen species, followed by increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, carrying serious oxidative DNA damage that may contribute to an increased mutation rate. Characterization of Fhit interacting proteins has identified direct effectors of the Fhit-mediated apoptotic pathway that is lost in most cancers through loss of Fhit.

  14. The tumor suppressor protein Fhit. A novel interaction with tubulin.

    PubMed

    Chaudhuri, A R; Khan, I A; Prasad, V; Robinson, A K; Ludueña, R F; Barnes, L D

    1999-08-20

    FHIT (fragile histidine triad) is a candidate human tumor suppressor gene located at chromosome 3p14.2, a location that encompasses the FRA3B chromosomal fragile site. Aberrant transcripts have been detected in a variety of primary tumors, and homozygous deletions in the FHIT locus have been detected in different tumor cell lines. The gene product Fhit in vitro possesses the ability to hydrolyze diadenosine 5',5"'-P(1),P(3)-triphosphate (Ap(3)A). The mechanism of action of Fhit as a tumor suppressor is unknown. Because the tubulin-microtubule system plays an important role in cell division and cell proliferation, we investigated the interaction between wild-type Fhit or mutant Fhit (H96N) and tubulin in vitro. The mutant form of Fhit (H96N) lacks Ap(3)A hydrolase activity but retains tumor suppressor activity. We found that both wild-type and mutated forms of Fhit bind to tubulin strongly and specifically with K(d) values of 1.4 and 2.1 microM, respectively. Neither wild-type nor mutant Fhit cause nucleation or formation of microtubules, but in the presence of microtubule-associated proteins, both wild-type and mutant Fhit promote assembly to a greater extent than do microtubule-associated proteins alone, and the microtubules formed appear normal by electron microscopy. Our results suggest the possibility that Fhit may exert its tumor suppressor activity by interacting with microtubules and also indicate that the interaction between Fhit and tubulin is not related to the Ap(3)A hydrolase activity of Fhit.

  15. Immunohistochemical characterization of FHIT expression in normal human tissues

    PubMed Central

    Kujan, Omar; Abuderman, Abdulwahab; Al-Shawaf, Ahmad Zahi

    2016-01-01

    Background Fragile histidine triad (FHIT) is a tumor suppressor gene that is commonly inactivated in human tumors. Interestingly, the normal pattern of FHIT expression is largely unknown. Aim This study is aimed to characterize the expression of FHIT protein in normal human tissues. Materials and methods A total of 119 normal human tissue specimens were analyzed for the FHIT expression using immunohistochemistry technique. The inclusion criteria included: normal/inflammatory tissue with no evidence of cellular atypia. Results All studied specimens were stained positively with FHIT and showed either nuclear or cytoplasmic expression. Interestingly, the pattern of FHIT staining was similar among different specimens from each organ. FHIT is located predominantly in the nucleus, although cytoplasmic staining is also present in some cell types. Oral squamous epithelium, breast ductal epithelium, squamous and tubal metaplastic epithelium of the uterine cervix, esophageal squamous epithelium, salivary glands, and bronchial epithelia all strongly expressed the nuclear protein. In connective tissue, FHIT has shown strong cytoplasmic expression in histocytes including macrophages and dendritic cells, fibroblasts, and myofibroblasts. Conclusion Documentation of the pattern of FHIT expression in normal tissues will contribute to our understanding of the normal function of this protein and to interpretation of potentially altered FHIT expression in human tumors. PMID:28250975

  16. Characterization of the role of Fhit in maintenance of genomic integrity following low dose radiation, in vivo and in vitro

    SciTech Connect

    Wang, Ya

    2010-05-14

    The major goal of this study is to determine the effects of the Fhit pathway on low dose (< 0.1 Gy) ionizing radiation (IR)-induced genetic instability. Reduction of Fhit protein expression is observed in most solid tumors particularly in those tumors resulting from exposure to environmental carcinogens. Therefore, characterization of the role of the Fhit-dependent pathway in preventing low dose IR-induced genetic instability will provide useful parameters for evaluating the low dose IR-induced risk of mutagenesis and carcinogenesis. We pursued 3 specific aims to study our hypothesis that the Fhit-dependent pathways maintain genomic integrity through adjusting checkpoint response and repair genes expression following low dose IR. Aim 1: Determine whether Fhit interaction with RPA is necessary for Fhit to affect the cellular response to low dose IR. We combined the approaches of in vitro (GST pull-down and site-directed mutagenesis) and in vivo (observing the co-localization and immunoprecipitation of Fhit and RPA in Fhit knock out mouse cells transfected with mutant Fhit which has lost ability to interact with RPA in vitro). Aim 2: Determine the role of genes whose expression is affected by Fhit in low dose irradiated cells. We analyzed the distinct signature of gene expression in low dose irradiated Fhit-/- cells compared with Fhit+/+ cells by combining microarray, gene transfection and siRNA approaches. Aim 3: Determine the role of Fhit in genetic susceptibility to low dose IR in vivo. We compared the gene mutation frequency and the fragile site stability in the cells isolated from the Fhit+/+ and Fhit-/- mice at 1.5 years following low dose IR. These results determine the role of the Fhit-dependent pathway in maintaining genomic integrity in vitro and in vivo, which provide a basis for choosing surrogate markers in the Fhit-dependent pathway to evaluate low dose IR-induced risk of mutagenesis and carcinogenesis.

  17. Characterization of the role of Fhit in maintenance of genomic integrity following low dose radiation, in vivo and in vitro

    SciTech Connect

    Ya Wang

    2010-05-31

    The major goal of this study is to determine the effects of the Fhit pathway on low dose ({le} 0.1 Gy) ionizing radiation (IR)-induced genetic instability. Reduction of Fhit protein expression is observed in most solid tumors particularly in those tumors resulting from exposure to environmental carcinogens. Therefore, characterization of the role of the Fhit-dependent pathway in preventing low dose IR-induced genetic instability will provide useful parameters for evaluating the low dose IR-induced risk of mutagenesis and carcinogenesis. We pursued 3 specific aims to study our hypothesis that the Fhit-dependent pathways maintain genomic integrity through adjusting checkpoint response and repair genes expression following low dose IR. Aim 1: Determine whether Fhit interaction with RPA is necessary for Fhit to affect the cellular response to low dose IR. We combined the approaches of in vitro (GST pull-down and site-directed mutagenesis) and in vivo (observing the co-localization and immunoprecipitation of Fhit and RPA in Fhit knock out mouse cells transfected with mutant Fhit which has lost ability to interact with RPA in vitro). Aim 2: Determine the role of genes whose expression is affected by Fhit in low dose irradiated cells. We analyzed the distinct signature of gene expression in low dose irradiated Fhit-/- cells compared with Fhit+/+ cells by combining microarray, gene transfection and siRNA approaches. Aim 3: Determine the role of Fhit in genetic susceptibility to low dose IR in vivo. We compared the gene mutation frequency and the fragile site stability in the cells isolated from the Fhit+/+ and Fhit-/- mice at 1.5 years following low dose IR. These results determine the role of the Fhit-dependent pathway in maintaining genomic integrity in vitro and in vivo, which provide a basis for choosing surrogate markers in the Fhit-dependent pathway to evaluate low dose IR-induced risk of mutagenesis and carcinogenesis.

  18. Fhit and CHK1 have opposing effects on homologous recombination repair.

    PubMed

    Hu, Baocheng; Wang, Hongyan; Wang, Xiang; Lu, Hua-Rui; Huang, Cuifen; Powell, Simon N; Huebner, Kay; Wang, Ya

    2005-10-01

    Fragile histidine triad (FHIT) gene deletion or promoter methylation and reduced Fhit protein expression occur in approximately 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified. We previously reported that compared with Fhit+/+ cells, Fhit-/- cells with an overactivated ATR/CHK1 pathway show increased mutation frequency and resistance to DNA damage-induced killing, indicating that Fhit and the CHK1 pathway have opposing roles in cells responding to DNA damage. In this study, we show that cells, with or without Fhit expression, have similar DNA double-strand break induction levels and similar rejoining rates following ionizing radiation, indicating that the effect of Fhit on cell radiosensitivity is independent of nonhomologous end-joining. By combining I-SceI-induced-DNA double-strand break system and small interfering RNA approach, we also show that knocking down Fhit increases the efficiency of homologous recombination repair of cells, but knocking down Chk1 decreases the efficiency of homologous recombination repair, associated with the sensitivity to ionizing radiation-induced killing. Taken together, the results show that the role of Fhit in affecting the sensitivity of cells to ionizing radiation-induced killing is through the CHK1 pathway linked to homologous recombination repair. These results also illustrate the importance of balanced checkpoint activation in genomic stability and suggest a connection between the radioresistance and mutagenesis, carcinogenesis, as well as tumor progression in Fhit-deficient cells or tissue.

  19. Phosphorylation of the human Fhit tumor suppressor on tyrosine 114 in Escherichia coli and unexpected steady state kinetics of the phosphorylated forms.

    PubMed

    Garrison, Preston N; Robinson, Angela K; Pekarsky, Yuri; Croce, Carlo M; Barnes, Larry D

    2005-04-26

    The human tumor suppressor Fhit is a homodimeric histidine triad (HIT) protein of 147 amino acids which has Ap(3)A hydrolase activity. We have recently discovered that Fhit is phosphorylated in vivo and is phosphorylated in vitro by Src kinase [Pekarsky, Y., Garrison, P. N., Palamarchuk, A., Zanesi, N., Aqeilan, R. I., Huebner, K., Barnes, L. D., and Croce, C. M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 3775-3779]. Now we have coexpressed Fhit with the elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit. Unphosphorylated Fhit, Fhit phosphorylated on one subunit, and Fhit phosphorylated on both subunits were purified to apparent homogeneity by column chromatography on anion-exchange and gel filtration resins. MALDI-TOF and HPLC-ESI tandem mass spectrometry of intact Fhit and proteolytic peptides of Fhit demonstrated that Fhit is phosphorylated on Y(114) on either one or both subunits. Monophosphorylated Fhit exhibited monophasic kinetics with K(m) and k(cat) values approximately 2- and approximately 7-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics. One site had K(m) and k(cat) values approximately 2- and approximately 140-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. The second site had a K(m) approximately 60-fold higher and a k(cat) approximately 6-fold lower than the corresponding values for unphosphorylated Fhit. The unexpected kinetic patterns for the phosphorylated forms suggest the system may be enzymologically novel. The decreases in the values of K(m) and k(cat) for the phosphorylated forms in comparison to those of unphosphorylated Fhit favor the formation and lifetime of the Fhit-Ap(3)A complex, which may enhance the tumor suppressor activity of Fhit.

  20. Evaluation of influence of Ap4A analogues on Fhit-positive HEK293T cells; cytotoxicity and ability to induce apoptosis.

    PubMed

    Krakowiak, Agnieszka; Pęcherzewska, Róża; Kaczmarek, Renata; Tomaszewska, Agnieszka; Nawrot, Barbara; Stec, Wojciech J

    2011-08-15

    Fragile histidine triad (Fhit) protein encoded by tumour suppressor FHIT gene is a proapoptotic protein with diadenosine polyphosphate (Ap(n)A, n=2-6) hydrolase activity. It has been hypothesised that formation of Fhit-substrate complex results in an apoptosis initiation signal while subsequent hydrolysis of Ap(n)A terminates this action. A series of Ap(n)A analogues have been identified in vitro as strong Fhit ligands [Varnum, J. M.; Baraniak, J.; Kaczmarek, R.; Stec, W. J.; Brenner, C. BMC Chem. Biol.2001, 1, 3]. We assumed that in Fhit-positive cells these compounds might preferentially bind to Fhit and inhibit its hydrolytic activity what would prolong the lifetime of apoptosis initiation signalling complex. Therefore, several Fhit inhibitors were tested for their cytotoxicity and ability to induce apoptosis in Fhit-positive HEK293T cells. These experiments have shown that Ap(4)A analogue, containing a glycerol residue instead of the central pyrophosphate and two terminal phosphorothioates [A(PS)-CH(2)CH(OH)CH(2)-(PS)A (1)], is the most cytotoxic among test compounds (IC(50)=17.5±4.2 μM) and triggers caspase-dependent cell apoptosis. The Fhit-negative HEK293T cells (in which Fhit was silenced by RNAi) were not sensitive to compound 1. These results indicate that the Ap(4)A analogue 1 induces Fhit-dependent apoptosis and therefore, it can be considered as a drug candidate for anticancer therapy in Fhit-positive cancer cells and in Fhit-negative cancer cells, in which re-expression of Fhit was accomplished by gene therapy.

  1. High throughput tissue microarray analysis of FHIT expression in diffuse large cell B-cell lymphoma from Saudi Arabia.

    PubMed

    Al Kuraya, Khawla; Siraj, Abdul Khalid; Bavi, Prashant; Al-Jomah, Naif; El-Solh, Hassan; Ezzat, Adnan; Al-Dayel, Fouad; Belgaumi, Asim; Al-Kofide, Amani; Sabbah, Rajeh; Sheikh, Salwa; Amr, Samir; Simon, Ronald; Sauter, Guido

    2006-08-01

    Recent studies have suggested a potential prognostic role of alterations of the fragile histidine triad (FHIT) gene in diffuse large B-cell lymphoma. To evaluate possible mechanisms of FHIT inactivation and to further clarify its potential prognostic relevance, we analyzed a set of 114 diffuse large B-cell lymphoma with clinical follow-up information. Tissue microarrays were analyzed by immunohistochemistry for protein expression, and corresponding DNA samples were analyzed for FHIT promotor hypermethlyation. Reduced or absent FHIT expression was found in 75 of 114 diffuse large B-cell lymphoma (66%), but was unrelated to clinical tumor stage or patient prognosis. FHIT promotor hypermethylation was observed in 29 of 93 (23%) interpretable diffuse large B-cell lymphoma. Hypermethylation was not significantly correlated to protein expression loss, which could be explained by competing mechanisms for FHIT inactivation in a substantial fraction of non FHIT hypermethylated diffuse large B-cell lymphoma. Hypermethylation was significantly associated with poor prognosis of diffuse large B-cell lymphoma patients and predominantly seen in nongerminal center diffuse large B-cell lymphoma (27%), but less frequent (13%) in germinal center diffuse large B-cell lymphoma. In summary, these data suggest that promotor hypermethylation is responsible for reduced FHIT expression in a substantial subset of diffuse large B-cell lymphoma, which is primarily composed of nongerminal center subtype with poor patient prognosis.

  2. FHIT suppresses epithelial-mesenchymal transition (EMT) and metastasis in lung cancer through modulation of microRNAs.

    PubMed

    Suh, Sung-Suk; Yoo, Ji Young; Cui, Ri; Kaur, Balveen; Huebner, Kay; Lee, Taek-Kyun; Aqeilan, Rami I; Croce, Carlo M

    2014-10-01

    Metastasis is the principal cause of cancer death and occurs through multiple, complex processes that involve the concerted action of many genes. A number of studies have indicated that the Fragile Histidine Triad (FHIT) gene product, FHIT, functions as a tumor suppressor in a variety of common human cancers. Although there are suggestions of a role for FHIT loss in progression of various cancers, a role for such loss in metastasis has not been defined. Here, via in vivo and in vitro assays, we reveal that the enforced expression of FHIT significantly suppresses metastasis, accompanied by inhibition of the epithelial-mesenchymal transition (EMT), a process involved in metastasis through coordinate modulation of EMT-related genes. Specifically, miR-30c, a FHIT-upregulated microRNA, contributes to FHIT function in suppression of EMT and metastasis by directly targeting metastasis genes Metadherin (MTDH), High-mobility group AT-hook 2 (HMGA2), and the mesenchymal markers, Vimentin (VIM) and Fibronectin (FN1), in human lung cancer. Finally, we demonstrate that the expression pattern of FHIT and miR-30c is inversely correlated with that of MTDH and HMGA2 in normal tissue, non-metastatic and metastatic tumors, serving as a potential biomarker for metastasis in lung cancer.

  3. FHIT Suppresses Epithelial-Mesenchymal Transition (EMT) and Metastasis in Lung Cancer through Modulation of MicroRNAs

    PubMed Central

    Suh, Sung-Suk; Yoo, Ji Young; Cui, Ri; Kaur, Balveen; Huebner, Kay; Lee, Taek-Kyun; Aqeilan, Rami I.; Croce, Carlo M.

    2014-01-01

    Metastasis is the principal cause of cancer death and occurs through multiple, complex processes that involve the concerted action of many genes. A number of studies have indicated that the Fragile Histidine Triad (FHIT) gene product, FHIT, functions as a tumor suppressor in a variety of common human cancers. Although there are suggestions of a role for FHIT loss in progression of various cancers, a role for such loss in metastasis has not been defined. Here, via in vivo and in vitro assays, we reveal that the enforced expression of FHIT significantly suppresses metastasis, accompanied by inhibition of the epithelial-mesenchymal transition (EMT), a process involved in metastasis through coordinate modulation of EMT-related genes. Specifically, miR-30c, a FHIT-upregulated microRNA, contributes to FHIT function in suppression of EMT and metastasis by directly targeting metastasis genes Metadherin (MTDH), High-mobility group AT—hook 2 (HMGA2), and the mesenchymal markers, Vimentin (VIM) and Fibronectin (FN1), in human lung cancer. Finally, we demonstrate that the expression pattern of FHIT and miR-30c is inversely correlated with that of MTDH and HMGA2 in normal tissue, non-metastatic and metastatic tumors, serving as a potential biomarker for metastasis in lung cancer. PMID:25340791

  4. Biological functions of mammalian Nit1, the counterpart of the invertebrate NitFhit Rosetta stone protein, a possible tumor suppressor.

    PubMed

    Semba, Shuho; Han, Shuang-Yin; Qin, Haiyan R; McCorkell, Kelly A; Iliopoulos, Dimitrios; Pekarsky, Yuri; Druck, Teresa; Trapasso, Francesco; Croce, Carlo M; Huebner, Kay

    2006-09-22

    The "Rosetta Stone" hypothesis proposes that the existence of a fusion protein in some organisms predicts that the separate polypeptides function in the same biochemical pathway in other organisms and may physically interact. In Drosophila melanogaster and Caenorhabditis elegans, NitFhit protein is composed of two domains, a fragile histidine triad homolog and a bacterial and plant nitrilase homolog. We assessed the biological effects of mammalian Nit1 expression in comparison with Fhit and observed that: 1) Nit1 expression was observed in most normal tissues and overlapped partially with Fhit expression; 2) Nit1-deficient mouse kidney cells exhibited accelerated proliferation, resistance to DNA damage stress, and increased cyclin D1 expression; 3) cyclin D1 was up-regulated in Nit1 null mammary gland and skin; 4) Nit1 overexpression induced caspase-dependent apoptosis in vitro; and 5) Nit1 allele deficiency led to increased incidence of N-nitrosomethylbenzylamine-induced murine forestomach tumors. Thus, the biological effects of Nit1 expression are similar to Fhit effects. Adenoviruses carrying recombinant NIT1 and FHIT induced apoptosis in Fhit- and Nit1-deficient cells, respectively, suggesting that Nit1-Fhit interaction is not essential for function of either protein. The results suggest that Nit1 and Fhit share tumor suppressor signaling pathways, while localization of the NIT1 gene at a stable, rather than fragile, chromosome site explains the paucity of gene alterations and in frequent loss of expression of the NIT1 gene in human malignancies.

  5. Quantitative assessment of the diagnostic role of FHIT promoter methylation in non-small cell lung cancer

    PubMed Central

    Tan, Yulong; Lu, Zhouyi; Wang, An; Tan, Lixing; Chen, Sidi; Guo, Shicheng; Wang, Jiucun; Chen, Xiaofeng

    2017-01-01

    Aberrant methylation of CpG islands acquired in promoter regions plays an important role in carcinogenesis. Accumulated evidence demonstrates FHIT gene promoter hyper-methylation is involved in non-small cell lung cancer (NSCLC). To test the diagnostic ability of FHIT methylation status on NSCLC, thirteen studies, including 2,119 samples were included in our meta-analysis. Simultaneously, four independent DNA methylation datasets from TCGA and GEO database were analyzed for validation. The pooled odds ratio of FHIT promoter methylation in cancer samples was 3.43 (95% CI: 1.85 to 6.36) compared with that in controls. In subgroup analysis, significant difference of FHIT gene promoter methylation status in NSCLC and controls was found in Asians but not in Caucasian population. In validation stage, 950 Caucasian samples, including 126 paired samples from TCGA, 568 cancer tissues and 256 normal controls from GEO database were analyzed, and all 8 CpG sites near the promoter region of FHIT gene were not significantly differentially methylated. Thus the diagnostic role of FHIT gene in the lung cancer may be relatively limited in the Caucasian population but useful in the Asians. PMID:28036263

  6. Strong Signature of Natural Selection within an FHIT Intron Implicated in Prostate Cancer Risk

    PubMed Central

    Ding, Yan; Larson, Garrett; Rivas, Guillermo; Lundberg, Cathryn; Geller, Louis; Ouyang, Ching; Weitzel, Jeffrey; Archambeau, John; Slater, Jerry; Daly, Mary B.; Benson, Al B.; Kirkwood, John M.; O'Dwyer, Peter J.; Sutphen, Rebecca; Stewart, James A.; Johnson, David; Nordborg, Magnus; Krontiris, Theodore G.

    2008-01-01

    Previously, a candidate gene linkage approach on brother pairs affected with prostate cancer identified a locus of prostate cancer susceptibility at D3S1234 within the fragile histidine triad gene (FHIT), a tumor suppressor that induces apoptosis. Subsequent association tests on 16 SNPs spanning approximately 381 kb surrounding D3S1234 in Americans of European descent revealed significant evidence of association for a single SNP within intron 5 of FHIT. In the current study, re-sequencing and genotyping within a 28.5 kb region surrounding this SNP further delineated the association with prostate cancer risk to a 15 kb region. Multiple SNPs in sequences under evolutionary constraint within intron 5 of FHIT defined several related haplotypes with an increased risk of prostate cancer in European-Americans. Strong associations were detected for a risk haplotype defined by SNPs 138543, 142413, and 152494 in all cases (Pearson's χ2 = 12.34, df 1, P = 0.00045) and for the homozygous risk haplotype defined by SNPs 144716, 142413, and 148444 in cases that shared 2 alleles identical by descent with their affected brothers (Pearson's χ2 = 11.50, df 1, P = 0.00070). In addition to highly conserved sequences encompassing SNPs 148444 and 152413, population studies revealed strong signatures of natural selection for a 1 kb window covering the SNP 144716 in two human populations, the European American (π = 0.0072, Tajima's D = 3.31, 14 SNPs) and the Japanese (π = 0.0049, Fay & Wu's H = 8.05, 14 SNPs), as well as in chimpanzees (Fay & Wu's H = 8.62, 12 SNPs). These results strongly support the involvement of the FHIT intronic region in an increased risk of prostate cancer. PMID:18953408

  7. Characterization of the role of Fhit in suppression of DNA damage

    PubMed Central

    Saldivar, Joshua C.; Bene, Jessica; Hosseini, Seyed Ali; Miuma, Satoshi; Horton, Susan; Heerema, Nyla; Huebner, Kay

    2012-01-01

    The fragile histidine triad protein, Fhit, has a number of reported tumor suppressive functions which include signaling of apoptosis in cancer cells in vitro and in vivo, modulation of the DNA damage response, down-regulation of target oncogene expression, suppression of tumor growth in vivo, and suppression of cancer cell invasion and metastasis. Most of these functions of Fhit have been observed on exogenous re-expression of Fhit in Fhit-negative cancer cells. However, little is known about the tumorigenic changes that occur in normal or precancerous cells following loss of Fhit expression. Recently, we have shown that shortly after loss of Fhit expression, cells exhibit signs of DNA replication stress-induced DNA damage and develop genomic instability. Here, we extend these findings through investigation of different factors that affect Fhit function to prevent DNA damage. We found that Fhit activity is dependent upon a functional HIT domain and the tyrosine-114 residue, previously shown to be required for tumor suppression by Fhit. Furthermore, Fhit function was shown to be independent of exogenous and endogenous sources of oxidative stress. Finally, Fhit function was shown to be dependent upon Chk1 kinase activity, but independent of Atr or Atm kinases. Evidence suggests that Fhit and Chk1 kinase cooperate to prevent replication stress-induced DNA damage. These findings provide important and unexpected insights into the mechanism whereby loss of Fhit expression contributes to cell transformation. PMID:23102829

  8. Fhit loss in lung preneoplasia: relation to DNA damage response checkpoint activation

    PubMed Central

    Cirombella, Roberto; Montrone, Giuseppe; Stoppacciaro, Antonella; Giglio, Simona; Volinia, Stefano; Graziano, Paolo; Huebner, Kay; Vecchione, Andrea

    2009-01-01

    Loss of heterozygosity at the FHIT locus is coincident with activation of DNA damage response checkpoint proteins; thus damage at fragile loci may trigger checkpoint activation. We examined preneoplastic lesions adjacent to non-small cell lung carcinomas for alterations to expression of Fhit and activated checkpoint proteins. Expression scores were analyzed for pair-wise associations and correlations among proteins and type of lesion. Hyperplastic and dysplastic lesions were positive for nuclear H2AX expression; 12/20 dysplastic lesions were negative for Fhit expression. Fhit positive lesions showed expression of most checkpoint proteins examined, while Fhit negative lesions showed absence of expression of Chk1 and phosphoChk1. The results show that loss of expression of Fhit is significantly directly correlated with absence of activated Chk1 in dysplasia, and suggest a connection between loss of Fhit and modulation of checkpoint activity. PMID:19931269

  9. Fhit Nuclear Import Following EGF Stimulation Sustains Proliferation of Breast Cancer Cells.

    PubMed

    Bianchi, Francesca; Sasso, Marianna; Turdo, Federica; Beretta, Giovanni L; Casalini, Patrizia; Ghirelli, Cristina; Sfondrini, Lucia; Ménard, Sylvie; Tagliabue, Elda; Campiglio, Manuela

    2015-11-01

    The tumor-suppressor protein fragile histidine triad (Fhit) exerts its functions in the cytoplasm, although some reports suggest that it may also act in the nucleus. We previously showed that cytosolic Fhit protein levels in cancer cell lines stimulated to proliferate were reduced by proteasomal degradation. Here, we demonstrate that Fhit is physiologically present in the nucleus of breast cancer cell lines and tissues at a low level and that proliferative stimulation increases nuclear levels. Breast cancer cells expressing the FhitY114F mutant, which do not undergo proteasomal degradation, contained mutated Fhit in the nucleus, while cells treated with a proteasome inhibitor accumulated nuclear Fhit during proliferation. Thus, Fhit nuclear shuttling and proteasome degradation phenomena occur independently. When Fhit was coupled to a nuclear localization sequence, the proliferation rate of the transfected cells increased together with levels of proliferation pathway mediators cyclin D1, phospho-MAPK, and phospho-STAT3. Fhit nuclear translocation upon mitogenic stimulation may represent a new regulatory mechanism that allows rapid restoration of Fhit cytoplasmic levels and promotes the proliferation cascade activated by mitogenic stimulation.

  10. Gene and enhancer traps for gene discovery.

    PubMed

    Rojas-Pierce, Marcela; Springer, Patricia S

    2003-01-01

    Gene traps and enhancer traps provide a valuable tool for gene discovery. With this system, genes can be identified based solely on the expression pattern of an inserted reporter gene. The use of a reporter gene, such as beta-glucuoronidase (GUS), provides a very sensitive assay for the identification of tissue- and cell-type specific expression patterns. In this chapter, protocols for examining and documenting GUS reporter gene activity in individual lines are described. Methods for the amplification of sequences flanking transposant insertions and subsequent molecular and genetic characterization of individual insertions are provided.

  11. A Fhit-mimetic peptide suppresses annexin A4-mediated chemoresistance to paclitaxel in lung cancer cells.

    PubMed

    Gaudio, Eugenio; Paduano, Francesco; Ngankeu, Apollinaire; Ortuso, Francesco; Lovat, Francesca; Pinton, Sandra; D'Agostino, Sabrina; Zanesi, Nicola; Aqeilan, Rami I; Campiglia, Pietro; Novellino, Ettore; Alcaro, Stefano; Croce, Carlo M; Trapasso, Francesco

    2016-05-24

    We recently reported that Fhit is in a molecular complex with annexin A4 (ANXA4); following to their binding, Fhit delocalizes ANXA4 from plasma membrane to cytosol in paclitaxel-resistant lung cancer cells, thus restoring their chemosensitivity to the drug. Here, we demonstrate that Fhit physically interacts with A4 through its N-terminus; molecular dynamics simulations were performed on a 3D Fhit model to rationalize its mechanism of action. This approach allowed for the identification of the QHLIKPS heptapeptide (position 7 to 13 of the wild-type Fhit protein) as the smallest Fhit sequence still able to preserve its ability to bind ANXA4. Interestingly, Fhit peptide also recapitulates the property of the native protein in inhibiting Annexin A4 translocation from cytosol to plasma membrane in A549 and Calu-2 lung cancer cells treated with paclitaxel. Finally, the combination of Tat-Fhit peptide and paclitaxel synergistically increases the apoptotic rate of cultured lung cancer cells and blocks in vivo tumor formation.Our findings address to the identification of chemically simplified Fhit derivatives that mimic Fhit tumor suppressor functions; intriguingly, this approach might lead to the generation of novel anticancer drugs to be used in combination with conventional therapies in Fhit-negative tumors to prevent or delay chemoresistance.

  12. A Fhit-mimetic peptide suppresses annexin A4-mediated chemoresistance to paclitaxel in lung cancer cells

    PubMed Central

    Ngankeu, Apollinaire; Ortuso, Francesco; Lovat, Francesca; Pinton, Sandra; D'Agostino, Sabrina; Zanesi, Nicola; Aqeilan, Rami I.; Campiglia, Pietro; Novellino, Ettore; Alcaro, Stefano; Croce, Carlo M.; Trapasso, Francesco

    2016-01-01

    We recently reported that Fhit is in a molecular complex with annexin A4 (ANXA4); following to their binding, Fhit delocalizes ANXA4 from plasma membrane to cytosol in paclitaxel-resistant lung cancer cells, thus restoring their chemosensitivity to the drug. Here, we demonstrate that Fhit physically interacts with A4 through its N-terminus; molecular dynamics simulations were performed on a 3D Fhit model to rationalize its mechanism of action. This approach allowed for the identification of the QHLIKPS heptapeptide (position 7 to 13 of the wild-type Fhit protein) as the smallest Fhit sequence still able to preserve its ability to bind ANXA4. Interestingly, Fhit peptide also recapitulates the property of the native protein in inhibiting Annexin A4 translocation from cytosol to plasma membrane in A549 and Calu-2 lung cancer cells treated with paclitaxel. Finally, the combination of Tat-Fhit peptide and paclitaxel synergistically increases the apoptotic rate of cultured lung cancer cells and blocks in vivo tumor formation. Our findings address to the identification of chemically simplified Fhit derivatives that mimic Fhit tumor suppressor functions; intriguingly, this approach might lead to the generation of novel anticancer drugs to be used in combination with conventional therapies in Fhit-negative tumors to prevent or delay chemoresistance. PMID:27166255

  13. A New Explanation for the Roles of Fhit Protein in the Progress of Breast Cancer

    DTIC Science & Technology

    2005-09-01

    CHK1 protein - -- -- CHK1 •- - Fig. 2. An over-activation of the CHK1 activity- 0 4 W ATR/CHK1 pathway is shown in CDC2A protein - irradiated Fhit...AD Award Number: DAMD17-03-1-650 TITLE: A New Explanation for the Roles of Fhit Protein in the Progress of Breast Cancer PRINCIPAL INVESTIGATOR: Ya...AND SUBTITLE 5a. CONTRACT NUMBER A New Explanation for the Roles of Fhit Protein in the Progress of Breast Cancer 5b. GRANT NUMBER DAMD17-03-1-0650 5c

  14. Effect of FHIT loss and p53 mutation on HPV-infected lung carcinoma development.

    PubMed

    Yu, Yan; Liu, Xiaofei; Yang, Yuxuan; Zhao, Xiaodan; Xue, Jianjun; Zhang, Weixiao; Yang, Aimin

    2015-07-01

    High-risk human papillomavirus (HPV)16/18 infection in the development of lung cancer has previously been identified, and fragile histidine triad (FHIT) loss and p53 mutation are frequently observed in the disease. However, the association between these factors has not been well studied. The present study aimed to further investigate the significance of HPV infection, FHIT loss and p53 mutations in the development of lung cancer and their possible associations. DNA was extracted from paraffin-embedded specimens from 88 cases of squamous cell carcinoma (SCC), 56 of adenocarcinoma (AC), 36 of small cell lung carcinoma (SCLC) and 110 non-cancer control cases of lung neoplasms. The prevalence of HPV infection was determined by polymerase chain reaction analysis, and FHIT loss and p53 mutations were detected by immunohistochemistry. The χ(2), Fisher's exact and Pearson correlation tests were applied for statistical analysis. The results of the present study demonstrated that HPVL1 (the major capsid protein of HPV), HPV16 and HPV18 infection were more prevalent in the lung cancer samples compared with the non-cancer controls (all P<0.001). FHIT loss occurred more frequently in the lung cancer samples (44.44%) compared with the non-cancer controls (7.25%) (P<0.001). FHIT loss in the HPVL1-positive group was significantly increased compared with the HPVL1-negative group in the lung cancer cases and the non-cancer controls (P<0.05). In the lung cancer cases, the p53 mutation rates in the HPVL1- and HPV16/18-positive groups were significantly increased compared with the HPVL1- and HPV16/18-negative groups (P<0.05). In the 180 lung cancer cases, the coexistence rate of FHIT loss and a history of smoking was 38.33% (69/180; Pearson contingency coefficient of r=0.318; P<0.001). FHIT loss and p53 mutation exhibited a synergistic effect on HPV-associated lung cancer (Pearson contingency coefficient r=0.357, P<0.001). The present study demonstrated that FHIT loss may be important

  15. Exome-wide single-base substitutions in tissues and derived cell lines of the constitutive Fhit knockout mouse.

    PubMed

    Paisie, Carolyn A; Schrock, Morgan S; Karras, Jenna R; Zhang, Jie; Miuma, Satoshi; Ouda, Iman M; Waters, Catherine E; Saldivar, Joshua C; Druck, Teresa; Huebner, Kay

    2016-04-01

    Loss of expression of Fhit, a tumor suppressor and genome caretaker, occurs in preneoplastic lesions during development of many human cancers. Furthermore, Fhit-deficient mouse models are exquisitely susceptible to carcinogen induction of cancers of the lung and forestomach. Due to absence of Fhit genome caretaker function, cultured cells and tissues of the constitutive Fhit knockout strain develop chromosome aneuploidy and allele copy number gains and losses and we hypothesized that Fhit-deficient cells would also develop point mutations. On analysis of whole exome sequences of Fhit-deficient tissues and cultured cells, we found 300 to >1000 single-base substitutions associated with Fhit loss in the 2% of the genome included in exomes, relative to the C57Bl6 reference genome. The mutation signature is characterized by increased C>T and T>C mutations, similar to the "age at diagnosis" signature identified in human cancers. The Fhit-deficiency mutation signature also resembles a C>T and T>C mutation signature reported for human papillary kidney cancers and a similar signature recently reported for esophageal and bladder cancers, cancers that are frequently Fhit deficient. The increase in T>C mutations in -/- exomes may be due to dNTP imbalance, particularly in thymidine triphosphate, resulting from decreased expression of thymidine kinase 1 in Fhit-deficient cells. Fhit-deficient kidney cells that survived in vitro dimethylbenz(a)anthracene treatment additionally showed increased T>A mutations, a signature generated by treatment with this carcinogen, suggesting that these T>A transversions may be evidence of carcinogen-induced preneoplastic changes.

  16. Fhit delocalizes annexin a4 from plasma membrane to cytosol and sensitizes lung cancer cells to paclitaxel.

    PubMed

    Gaudio, Eugenio; Paduano, Francesco; Spizzo, Riccardo; Ngankeu, Apollinaire; Zanesi, Nicola; Gaspari, Marco; Ortuso, Francesco; Lovat, Francesca; Rock, Jonathan; Hill, Grace A; Kaou, Mohamed; Cuda, Giovanni; Aqeilan, Rami I; Alcaro, Stefano; Croce, Carlo M; Trapasso, Francesco

    2013-01-01

    Fhit protein is lost or reduced in a large fraction of human tumors, and its restoration triggers apoptosis and suppresses tumor formation or progression in preclinical models. Here, we describe the identification of candidate Fhit-interacting proteins with cytosolic and plasma membrane localization. Among these, Annexin 4 (ANXA4) was validated by co-immunoprecipitation and confocal microscopy as a partner of this novel Fhit protein complex. Here we report that overexpression of Fhit prevents Annexin A4 translocation from cytosol to plasma membrane in A549 lung cancer cells treated with paclitaxel. Moreover, paclitaxel administration in combination with AdFHIT acts synergistically to increase the apoptotic rate of tumor cells both in vitro and in vivo experiments.

  17. Fhit Delocalizes Annexin A4 from Plasma Membrane to Cytosol and Sensitizes Lung Cancer Cells to Paclitaxel

    PubMed Central

    Gaudio, Eugenio; Paduano, Francesco; Spizzo, Riccardo; Ngankeu, Apollinaire; Zanesi, Nicola; Gaspari, Marco; Ortuso, Francesco; Lovat, Francesca; Rock, Jonathan; Hill, Grace A.; Kaou, Mohamed; Cuda, Giovanni; Aqeilan, Rami I.; Alcaro, Stefano; Croce, Carlo M.; Trapasso, Francesco

    2013-01-01

    Fhit protein is lost or reduced in a large fraction of human tumors, and its restoration triggers apoptosis and suppresses tumor formation or progression in preclinical models. Here, we describe the identification of candidate Fhit-interacting proteins with cytosolic and plasma membrane localization. Among these, Annexin 4 (ANXA4) was validated by co-immunoprecipitation and confocal microscopy as a partner of this novel Fhit protein complex. Here we report that overexpression of Fhit prevents Annexin A4 translocation from cytosol to plasma membrane in A549 lung cancer cells treated with paclitaxel. Moreover, paclitaxel administration in combination with AdFHIT acts synergistically to increase the apoptotic rate of tumor cells both in vitro and in vivo experiments. PMID:24223161

  18. Transcriptional enhancer from milk protein genes

    DOEpatents

    Casperson, Gerald F.; Schmidhauser, Christian T.; Bissell, Mina J.

    1999-01-01

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  19. Transcriptional enhancer from milk protein genes

    SciTech Connect

    Casperson, G.F.; Schmidhauser, C.T.; Bissell, M.J.

    1999-12-21

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  20. Gene Editing, Enhancing and Women's Role.

    PubMed

    Simonstein, Frida

    2017-02-02

    A recent article on the front page of The Independent (September 18, 2015) reported that the genetic 'manipulation' of IVF embryos is to start in Britain, using a new revolutionary gene-editing technique, called Crispr/Cas9. About three weeks later (Saturday 10, October 2015), on the front page of the same newspaper, it was reported that the National Health Service (NHS) faces a one billion pound deficit only 3 months into the new year. The hidden connection between these reports is that gene editing could be used to solve issues related to health care allocation. Improving the health of future generations might coincide with public health goals; it might improve the health of individuals and communities, and, if successful, might be seen as a public good. However, enhancing future generations will require In Vitro Fertilisation and Pre-implantation Genetic Diagnosis. Remarkably, the necessary involvement of women in an enhancing scenario has not been discussed by its proponents. The present discourse on moral obligations of future generations, although not referring to women, seems to imply that women might be required, morally, if not legally, to reproduce with IVF. Enhancing future generations will be gendered, unless the artificial womb is developed. These are challenging issues that require a wider perspective, of both women and men. Despite the lack of a unified feminist conclusion in the discussions about the merits and risks of human genome modification, there is an urgent need to clarify the role of women in this scenario.

  1. Enhancing cognition after stress with gene therapy.

    PubMed

    Nicholas, Andrea; Munhoz, Carolina D; Ferguson, Deveroux; Campbell, Laura; Sapolsky, Robert

    2006-11-08

    Hippocampal function is essential for the acquisition, consolidation, and retrieval of spatial memory. High circulating levels of glucocorticoids (GCs), the adrenal steroid hormones secreted during stress, have been shown to impair both acquisition and retrieval and can either impair or enhance consolidation, depending on experimental conditions. In contrast, estrogen can enhance spatial memory performance and can block the deleterious effects of GCs on such performance. We therefore constructed a chimeric gene ("ER/GR") containing the hormone-binding domain of the GC receptor and the DNA binding domain of the estrogen receptor; as a result, ER/GR transduces deleterious GC signals into beneficial estrogenic ones. We show here that acute immobilization stress, before acquisition and retrieval phases, increases latencies for male rats in a hidden platform version of the Morris water maze. This impairment is blocked by hippocampal expression of the ER/GR transgene. ER/GR expression also blocks decreases in platform crossings caused by acute stress, either after acquisition or before retrieval. Three days of stress before acquisition produces an estrogen-like enhancement of performance in ER/GR-treated rats. Moreover, ER/GR blocks the suppressive effects of GCs on expression of brain-derived neurotrophic factor (BDNF), a growth factor central to hippocampal-dependent cognition and plasticity, instead producing an estrogenic increase in BDNF expression. Thus, ER/GR expression enhances spatial memory performance and blocks the impairing effects of GCs on such performance.

  2. A Weakened Transcriptional Enhancer Yields Variegated Gene Expression

    PubMed Central

    Collins, Cathy; Azmi, Peter; Berru, Maribel; Zhu, Xiaofu; Shulman, Marc J.

    2006-01-01

    Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH) locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life) of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated) forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, ∼1.4 kb 3′ of the promoter, but inactive ∼6 kb 3′, indicating that the activity of the core enhancer was distance-dependent. A longer segment – the core enhancer plus ∼1 kb of 3′ flanking material, including the 3′ matrix attachment region – was active, and the activity of this longer segment was orientation-dependent. Our data suggest that this 3′ flank includes binding sites for at least two activators. PMID:17183661

  3. Cloned Hemoglobin Genes Enhance Growth Of Cells

    NASA Technical Reports Server (NTRS)

    Khosla, Chaitan; Bailey, James E.

    1991-01-01

    Experiments show that portable deoxyribonucleic acid (DNA) sequences incorporated into host cells make them produce hemoglobins - oxygen-binding proteins essential to function of red blood cells. Method useful in several biotechnological applications. One, enhancement of growth of cells at higher densities. Another, production of hemoglobin to enhance supplies of oxygen in cells, for use in chemical reactions requiring oxygen, as additive to serum to increase transport of oxygen, and for binding and separating oxygen from mixtures of gases.

  4. The Gene Ontology: enhancements for 2011.

    PubMed

    2012-01-01

    The Gene Ontology (GO) (http://www.geneontology.org) is a community bioinformatics resource that represents gene product function through the use of structured, controlled vocabularies. The number of GO annotations of gene products has increased due to curation efforts among GO Consortium (GOC) groups, including focused literature-based annotation and ortholog-based functional inference. The GO ontologies continue to expand and improve as a result of targeted ontology development, including the introduction of computable logical definitions and development of new tools for the streamlined addition of terms to the ontology. The GOC continues to support its user community through the use of e-mail lists, social media and web-based resources.

  5. The Gene Ontology: enhancements for 2011

    PubMed Central

    2012-01-01

    The Gene Ontology (GO) (http://www.geneontology.org) is a community bioinformatics resource that represents gene product function through the use of structured, controlled vocabularies. The number of GO annotations of gene products has increased due to curation efforts among GO Consortium (GOC) groups, including focused literature-based annotation and ortholog-based functional inference. The GO ontologies continue to expand and improve as a result of targeted ontology development, including the introduction of computable logical definitions and development of new tools for the streamlined addition of terms to the ontology. The GOC continues to support its user community through the use of e-mail lists, social media and web-based resources. PMID:22102568

  6. Gene Isoform Specificity through Enhancer-Associated Antisense Transcription

    PubMed Central

    Onodera, Courtney S.; Underwood, Jason G.; Katzman, Sol; Jacobs, Frank; Greenberg, David; Salama, Sofie R.; Haussler, David

    2012-01-01

    Enhancers and antisense RNAs play key roles in transcriptional regulation through differing mechanisms. Recent studies have demonstrated that enhancers are often associated with non-coding RNAs (ncRNAs), yet the functional role of these enhancer:ncRNA associations is unclear. Using RNA-Sequencing to interrogate the transcriptomes of undifferentiated mouse embryonic stem cells (mESCs) and their derived neural precursor cells (NPs), we identified two novel enhancer-associated antisense transcripts that appear to control isoform-specific expression of their overlapping protein-coding genes. In each case, an enhancer internal to a protein-coding gene drives an antisense RNA in mESCs but not in NPs. Expression of the antisense RNA is correlated with expression of a shorter isoform of the associated sense gene that is not present when the antisense RNA is not expressed. We demonstrate that expression of the antisense transcripts as well as expression of the short sense isoforms correlates with enhancer activity at these two loci. Further, overexpression and knockdown experiments suggest the antisense transcripts regulate expression of their associated sense genes via cis-acting mechanisms. Interestingly, the protein-coding genes involved in these two examples, Zmynd8 and Brd1, share many functional domains, yet their antisense ncRNAs show no homology to each other and are not present in non-murine mammalian lineages, such as the primate lineage. The lack of homology in the antisense ncRNAs indicates they have evolved independently of each other and suggests that this mode of lineage-specific transcriptional regulation may be more widespread in other cell types and organisms. Our findings present a new view of enhancer action wherein enhancers may direct isoform-specific expression of genes through ncRNA intermediates. PMID:22937057

  7. Surface enhanced Raman gene probe and methods thereof

    DOEpatents

    Vo-Dinh, T.

    1998-09-29

    The subject invention disclosed herein is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

  8. Surface enhanced Raman gene probe and methods thereof

    DOEpatents

    Vo-Dinh, Tuan

    1998-01-01

    The subject invention disclosed herein is a new gene probe biosensor and methods thereof based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays.

  9. Surface enhanced Raman gene probe and methods thereof

    DOEpatents

    Vo-Dinh, T.

    1998-07-21

    The subject invention disclosed is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

  10. Surface enhanced Raman gene probe and methods thereof

    DOEpatents

    Vo-Dinh, T.

    1998-02-24

    The subject invention disclosed is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

  11. Enhancer Complexes Located Downstream of Both Human Immunoglobulin Cα Genes

    PubMed Central

    Mills, Frederick C.; Harindranath, Nagaradona; Mitchell, Mary; Max, Edward E.

    1997-01-01

    To investigate regulation of human immunoglobulin heavy chain expression, we have cloned DNA downstream from the two human Cα genes, corresponding to the position in the mouse IgH cluster of a locus control region (LCR) that includes an enhancer which regulates isotype switching. Within 25 kb downstream of both the human immunoglobulin Cα1 and Cα2 genes we identified several segments of DNA which display B lymphoid–specific DNase I hypersensitivity as well as enhancer activity in transient transfections. The corresponding sequences downstream from each of the two human Cα genes are nearly identical to each other. These enhancers are also homologous to three regions which lie in similar positions downstream from the murine Cα gene and form the murine LCR. The strongest enhancers in both mouse and human have been designated HS12. Within a 135-bp core homology region, the human HS12 enhancers are ∼90% identical to the murine homolog and include several motifs previously demonstrated to be important for function of the murine enhancer; additional segments of high sequence conservation suggest the possibility of previously unrecognized functional motifs. On the other hand, certain functional elements in the murine enhancer, including a B cell–specific activator protein site, do not appear to be conserved in human HS12. The human homologs of the murine enhancers designated HS3 and HS4 show lower overall sequence conservation, but for at least two of the functional motifs in the murine HS4 (a κB site and an octamer motif  ) the human HS4 homologs are exactly conserved. An additional hypersensitivity site between human HS3 and HS12 in each human locus displays no enhancer activity on its own, but includes a region of high sequence conservation with mouse, suggesting the possibility of another novel functional element. PMID:9294139

  12. Mechanically enhanced microcapsules for cellular gene therapy.

    PubMed

    Shen, F; Mazumder, M A J; Burke, N A D; Stöver, H D H; Potter, M A

    2009-07-01

    Microcapsules bearing a covalently cross-linked coating have been developed for cellular gene therapy as an improvement on alginate-poly(L-lysine)-alginate (APA) microcapsules that only have ionic cross-linking. In this study, two mutually reactive polyelectrolytes, a polycation (designated C70), poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride-co-2-aminoethyl methacrylate hydrochloride) and a polyanion (designated A70), poly(sodium methacrylate-co-2-(methacryloyloxy)ethyl acetoacetate), were used during the microcapsule fabrication. Ca-alginate beads were sequentially laminated with C70, A70, poly(L-lysine) (PLL), and alginate. The A70 reacts with both C70 and PLL to form a approximately 30 microm thick covalently cross-linked interpenetrating polymer network on the surface of the capsules. Confocal images confirmed the location of the C70/A70/PLL network and the stability of the network after 4 weeks implantation in mice. The mechanical and chemical resistance of the capsules was tested with a "stress test" where microcapsules were gently shaken in 0.003% EDTA for 15 min. APA capsules disappeared during this treatment, whereas the modified capsules, even those that had been retrieved from mice after 4-weeks implantation, remained intact. Analysis of solutions passing through model flat membranes showed that the molecular weight cut-off of alginate-C70-A70-PLL-alginate is similar to that of alginate-PLL-alginate. Recombinant cells encapsulated in APA and modified capsules were able to secrete luciferase into culture media. The modified capsules were found to capture some components of regular culture media used during preparation, causing an immune reaction in implanted mice, but use of UltraCulture serum-free medium was found to prevent this immune reaction. In vivo biocompatibility of the new capsules was similar to the APA capsules, with no sign of clinical toxicity on complete blood counts and liver function tests. The increased stability of the

  13. An autoregulatory enhancer element of the Drosophila homeotic gene Deformed.

    PubMed

    Bergson, C; McGinnis, W

    1990-12-01

    The stable determination of different anterior-posterior regions of the Drosophila embryo is controlled by the persistent expression of homeotic selector genes. One mechanism that has been proposed to explain the persistent expression of the homeotic gene Deformed is an autoactivation circuit that would be used once Deformed expression had been established by earlier acting patterning genes. Here we show that a large cis-regulatory element mapping approximately 5 kb upstream of the Deformed transcription start has the properties predicted for a Deformed autoregulatory enhancer. This element provides late, spatially localized expression in the epidermal cells of the maxillary and mandibular segments which is wholly dependent upon endogenous Deformed function. In addition, the autoregulatory enhancer can be activated ectopically in embryos and in imaginal disc cells by ectopic expression of Deformed protein. Deletion analysis of the autoregulatory element indicates that it contains compartment specific sub-elements similar to those of other homeotic loci.

  14. The insulation of genes from external enhancers and silencing chromatin

    PubMed Central

    Burgess-Beusse, Bonnie; Farrell, Catherine; Gaszner, Miklos; Litt, Michael; Mutskov, Vesco; Recillas-Targa, Felix; Simpson, Melanie; West, Adam; Felsenfeld, Gary

    2002-01-01

    Insulators are DNA sequence elements that can serve in some cases as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin. Some insulators also can act as blocking elements to protect against the activating influence of distal enhancers associated with other genes. Although most of the insulators identified so far derive from Drosophila, they also are found in vertebrates. An insulator at the 5′ end of the chicken β-globin locus marks a boundary between an open chromatin domain and a region of constitutively condensed chromatin. Detailed analysis of this element shows that it possesses both enhancer blocking activity and the ability to screen reporter genes against position effects. Enhancer blocking is associated with binding of the protein CTCF; sites that bind CTCF are found at other critical points in the genome. Protection against position effects involves other properties that appear to be associated with control of histone acetylation and methylation. Insulators thus are complex elements that can help to preserve the independent function of genes embedded in a genome in which they are surrounded by regulatory signals they must ignore. PMID:12154228

  15. Enhancer Runaway and the Evolution of Diploid Gene Expression

    PubMed Central

    Fyon, Frédéric; Cailleau, Aurélie; Lenormand, Thomas

    2015-01-01

    Evidence is mounting that the evolution of gene expression plays a major role in adaptation and speciation. Understanding the evolution of gene regulatory regions is indeed an essential step in linking genotypes and phenotypes and in understanding the molecular mechanisms underlying evolutionary change. The common view is that expression traits (protein folding, expression timing, tissue localization and concentration) are under natural selection at the individual level. Here, we use a theoretical approach to show that, in addition, in diploid organisms, enhancer strength (i.e., the ability of enhancers to activate transcription) may increase in a runaway process due to competition for expression between homologous enhancer alleles. These alleles may be viewed as self-promoting genetic elements, as they spread without conferring a benefit at the individual level. They gain a selective advantage by getting associated to better genetic backgrounds: deleterious mutations are more efficiently purged when linked to stronger enhancers. This process, which has been entirely overlooked so far, may help understand the observed overrepresentation of cis-acting regulatory changes in between-species phenotypic differences, and sheds a new light on investigating the contribution of gene expression evolution to adaptation. PMID:26561855

  16. Macrophage mediated PCI enhanced gene-directed enzyme prodrug therapy

    NASA Astrophysics Data System (ADS)

    Christie, Catherine E.; Zamora, Genesis; Kwon, Young J.; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

    2015-03-01

    Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. Prodrug activating gene therapy (suicide gene therapy) employing the transduction of the E. coli cytosine deaminase (CD) gene into tumor cells, is a promising method. Expression of this gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-FC, to the toxic metabolite, 5-fluorouracil (5-FU). 5-FC may be particularly suitable for brain tumors, because it can readily cross the bloodbrain barrier (BBB). In addition the bystander effect, where activated drug is exported from the transfected cancer cells into the tumor microenvironment, plays an important role by inhibiting growth of adjacent tumor cells. Tumor-associated macrophages (TAMs) are frequently found in and around glioblastomas. Monocytes or macrophages (Ma) loaded with drugs, nanoparticles or photosensitizers could therefore be used to target tumors by local synthesis of chemo attractive factors. The basic concept is to combine PCI, to enhance the ex vivo transfection of a suicide gene into Ma, employing specially designed core/shell NP as gene carrier.

  17. Epigenetic engineering of ribosomal RNA genes enhances protein production.

    PubMed

    Santoro, Raffaella; Lienemann, Philipp; Fussenegger, Martin

    2009-08-14

    Selection of mammalian high-producer cell lines remains a major challenge for the biopharmaceutical manufacturing industry. Ribosomal RNA (rRNA) genes encode the major component of the ribosome but many rRNA gene copies are not transcribed due to epigenetic silencing by the nucleolar remodelling complex (NoRC) [6], which may limit the cell's full production capacity. Here we show that the knockdown of TIP5, a subunit of NoRC, decreases the number of silent rRNA genes, upregulates rRNA transcription, enhances ribosome synthesis and increases production of recombinant proteins. However, general enhancement of rRNA transcription rate did not stimulate protein synthesis. Our data demonstrates that the number of transcriptionally competent rRNA genes limits efficient ribosome synthesis. Epigenetic engineering of ribosomal RNA genes offers new possibilities for improving biopharmaceutical manufacturing and provides novel insights into the complex regulatory network which governs the translation machinery in normal cellular processes as well as in pathological conditions like cancer.

  18. Deciphering the combinatorial architecture of a Drosophila homeotic gene enhancer

    PubMed Central

    Drewell, Robert A.; Nevarez, Michael J.; Kurata, Jessica S.; Winkler, Lauren N.; Li, Lily; Dresch, Jacqueline M.

    2013-01-01

    Summary In Drosophila, the 330 kb bithorax complex regulates cellular differentiation along the anterio-posterior axis during development in the thorax and abdomen and is comprised of three homeotic genes: Ultrabithorax, abdominal-A, and Abdominal-B. The expression of each of these genes is in turn controlled through interactions between transcription factors and a number of cis-regulatory modules in the neighboring intergenic regions. In this study, we examine how the sequence architecture of transcription factor binding sites mediates the functional activity of one of these cis-regulatory modules. Using computational, mathematical modeling and experimental molecular genetic approaches we investigate the IAB7b enhancer, which regulates Abdominal-B expression specifically in the presumptive seventh and ninth abdominal segments of the early embryo. A cross-species comparison of the IAB7b enhancer reveals an evolutionarily conserved signature motif containing two FUSHI-TARAZU activator transcription factor binding sites. We find that the transcriptional repressors KNIRPS, KRUPPEL and GIANT are able to restrict reporter gene expression to the posterior abdominal segments, using different molecular mechanisms including short-range repression and competitive binding. Additionally, we show the functional importance of the spacing between the two FUSHI-TARAZU binding sites and discuss the potential importance of cooperativity for transcriptional activation. Our results demonstrate that the transcriptional output of the IAB7b cis-regulatory module relies on a complex set of combinatorial inputs mediated by specific transcription factor binding and that the sequence architecture at this enhancer is critical to maintain robust regulatory function. PMID:24514265

  19. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    SciTech Connect

    Salem, Tamer Z.; Zhang, Fengrui; Thiem, Suzanne M.

    2013-01-20

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  20. Hyaluronic acid enhances gene delivery into the cochlea.

    PubMed

    Shibata, Seiji B; Cortez, Sarah R; Wiler, James A; Swiderski, Donald L; Raphael, Yehoash

    2012-03-01

    Cochlear gene therapy can be a new avenue for the treatment of severe hearing loss by inducing regeneration or phenotypic rescue. One necessary step to establish this therapy is the development of a safe and feasible inoculation surgery, ideally without drilling the bony cochlear wall. The round window membrane (RWM) is accessible in the middle-ear space, but viral vectors placed on this membrane do not readily cross the membrane to the cochlear tissues. In an attempt to enhance permeability of the RWM, we applied hyaluronic acid (HA), a nontoxic and biodegradable reagent, onto the RWM of guinea pigs, prior to delivering an adenovirus carrying enhanced green fluorescent protein (eGFP) reporter gene (Ad-eGFP) at the same site. We examined distribution of eGFP in the cochlea 1 week after treatment, comparing delivery of the vector via the RWM, with or without HA, to delivery by a cochleostomy into the perilymph. We found that cochlear tissue treated with HA-assisted delivery of Ad-eGFP demonstrated wider expression of transgenes in cochlear cells than did tissue treated by cochleostomy injection. HA-assisted vector delivery facilitated expression in cells lining the scala media, which are less accessible and not transduced after perilymphatic injection. We assessed auditory function by measuring auditory brainstem responses and determined that thresholds were significantly better in the ears treated with HA-assisted Ad-eGFP placement on the RWM as compared with cochleostomy. Together, these data demonstrate that HA-assisted delivery of viral vectors provides an atraumatic and clinically feasible method to introduce transgenes into cochlear cells, thereby enhancing both research methods and future clinical application.

  1. Demystifying the secret mission of enhancers: linking distal regulatory elements to target genes

    PubMed Central

    Yao, Lijing; Berman, Benjamin P.; Farnham, Peggy J.

    2015-01-01

    Abstract Enhancers are short regulatory sequences bound by sequence-specific transcription factors and play a major role in the spatiotemporal specificity of gene expression patterns in development and disease. While it is now possible to identify enhancer regions genomewide in both cultured cells and primary tissues using epigenomic approaches, it has been more challenging to develop methods to understand the function of individual enhancers because enhancers are located far from the gene(s) that they regulate. However, it is essential to identify target genes of enhancers not only so that we can understand the role of enhancers in disease but also because this information will assist in the development of future therapeutic options. After reviewing models of enhancer function, we discuss recent methods for identifying target genes of enhancers. First, we describe chromatin structure-based approaches for directly mapping interactions between enhancers and promoters. Second, we describe the use of correlation-based approaches to link enhancer state with the activity of nearby promoters and/or gene expression. Third, we describe how to test the function of specific enhancers experimentally by perturbing enhancer–target relationships using high-throughput reporter assays and genome editing. Finally, we conclude by discussing as yet unanswered questions concerning how enhancers function, how target genes can be identified, and how to distinguish direct from indirect changes in gene expression mediated by individual enhancers. PMID:26446758

  2. A Photo-Degradable Gene Delivery System for Enhanced Nuclear Gene Transcription

    PubMed Central

    Hoyoung, Lee; Yeji, Kim; Patrick G., Schweickert; Stephen F., Konieczny; You-Yeon, Won

    2013-01-01

    There currently exists a significant gap in our understanding of how the detailed chemical characteristics of polycation gene carriers influence their delivery performances in overcoming an important cellular-level transport barrier, i.e., intranuclear gene transcription. In this study, a UV-degradable gene carrier material (ENE4-1) was synthesized by crosslinking low molecular weight branched polyethylenimine (bPEI-2k) molecules using UV-cleavable o-nitrobenzyl urethane (NBU) as the linker molecule. NBU degrades upon exposure to mild UV irradiation. Therefore, this UV-degradable carrier allows us to control the chemical characteristics of the polymer/DNA complex (polyplex) particles at desired locations within the intracellular environment. By using this photolytic DNA carrier, we found that the exact timing of the UV degradation significantly influences the gene transfection efficiencies of ENE4-1/DNA(pGL2) polyplexes in HeLa cells. Interestingly, even if the polyplexes were UV-degraded at different intracellular locations/times, their nuclear entry efficiency was not influenced by the location/timing of UV degradation. The UV treatment did not influence the size or binding strength of the polyplexes. However, we confirmed that the degradation of the carrier molecules impacts the chemical characteristics of the polyplexes (it produces carbamic acid and nitrosobenzyl aldehyde groups on ENE4-1). We believe that these anionic acid groups enhance the interaction of the polyplexes with nuclear transcription proteins and thus the final gene expression levels; this effect was found to occur, even though UV irradiation itself has a general effect of reducing transfection efficiencies. Excess (uncomplexed) ENE4-1 polymers appear to not play any role in the UV-enhanced gene transcription phenomenon. PMID:24172855

  3. RFLP mapping of the sugary enhancer1 gene in maize.

    PubMed

    Tadmor, Y; Azanza, F; Han, T; Rocheford, T R; Juvik, J A

    1995-08-01

    RFLP marker data from an F2∶3 population derived from a cross between a sugary1 (su1) and a sugary enhancer1 (su1, sel) inbred were used to construct a genetic linkage map of maize. This map includes 93 segregating marker loci distributed throughout the maize genome, providing a saturated linkage map that is suitable for linkage analysis with quantitative trait loci (QTL). This population, which has been immortalized in the form of sibbed F2∶3 families, was derived from each of the 214 F2 plants and along with probe data are available to the scientific community. QTL analysis for kernel sucrose (the primary form of sugar) concentration at 20 days after pollination (DAP) uncovered the segregation of seven major QTL influencing sucrose concentration; a locus linked to umc36a described the greatest proportion of the variation (24.7%). Since maltose concentration has previously been reported to be associated with the se1 phenotype, an analysis of probe associations with maltose concentration at 40 DAP was also conducted. The highly significant association of umc36a with maltose and sucrose concentrations provided evidence that this probe is linked to se1. Phenotypic evaluation for the se1 genotype in each F2∶3 family enabled us to map the gene 12.1 cM distal to umc36a. In contrast to previous work where se1 was reported to be located on chromosome four, our data strongly suggest that the sugary enhancer1 locus maps on the the distal portion of the long arm of chromosome 2 in the maize genome.

  4. Dual-therapeutic reporter genes fusion for enhanced cancer gene therapy and imaging.

    PubMed

    Sekar, T V; Foygel, K; Willmann, J K; Paulmurugan, R

    2013-05-01

    Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus-thymidine kinase (HSV1-TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK-NTR gene- along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK-GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK-NTR-prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals.

  5. Functional Enhancers As Master Regulators of Tissue-Specific Gene Regulation and Cancer Development

    PubMed Central

    Ko, Je Yeong; Oh, Sumin; Yoo, Kyung Hyun

    2017-01-01

    Tissue-specific transcription is critical for normal development, and abnormalities causing undesirable gene expression may lead to diseases such as cancer. Such highly organized transcription is controlled by enhancers with specific DNA sequences recognized by transcription factors. Enhancers are associated with chromatin modifications that are distinct epigenetic features in a tissue-specific manner. Recently, super-enhancers comprising enhancer clusters co-occupied by lineage-specific factors have been identified in diverse cell types such as adipocytes, hair follicle stem cells, and mammary epithelial cells. In addition, noncoding RNAs, named eRNAs, are synthesized at super-enhancer regions before their target genes are transcribed. Many functional studies revealed that super-enhancers and eRNAs are essential for the regulation of tissue-specific gene expression. In this review, we summarize recent findings concerning enhancer function in tissue-specific gene regulation and cancer development. PMID:28359147

  6. GOseek: a gene ontology search engine using enhanced keywords.

    PubMed

    Taha, Kamal

    2013-01-01

    We propose in this paper a biological search engine called GOseek, which overcomes the limitation of current gene similarity tools. Given a set of genes, GOseek returns the most significant genes that are semantically related to the given genes. These returned genes are usually annotated to one of the Lowest Common Ancestors (LCA) of the Gene Ontology (GO) terms annotating the given genes. Most genes have several annotation GO terms. Therefore, there may be more than one LCA for the GO terms annotating the given genes. The LCA annotating the genes that are most semantically related to the given gene is the one that receives the most aggregate semantic contribution from the GO terms annotating the given genes. To identify this LCA, GOseek quantifies the contribution of the GO terms annotating the given genes to the semantics of their LCAs. That is, it encodes the semantic contribution into a numeric format. GOseek uses microarray experiment data to rank result genes based on their significance. We evaluated GOseek experimentally and compared it with a comparable gene prediction tool. Results showed marked improvement over the tool.

  7. Enhancer-core-promoter specificity separates developmental and housekeeping gene regulation.

    PubMed

    Zabidi, Muhammad A; Arnold, Cosmas D; Schernhuber, Katharina; Pagani, Michaela; Rath, Martina; Frank, Olga; Stark, Alexander

    2015-02-26

    Gene transcription in animals involves the assembly of RNA polymerase II at core promoters and its cell-type-specific activation by enhancers that can be located more distally. However, how ubiquitous expression of housekeeping genes is achieved has been less clear. In particular, it is unknown whether ubiquitously active enhancers exist and how developmental and housekeeping gene regulation is separated. An attractive hypothesis is that different core promoters might exhibit an intrinsic specificity to certain enhancers. This is conceivable, as various core promoter sequence elements are differentially distributed between genes of different functions, including elements that are predominantly found at either developmentally regulated or at housekeeping genes. Here we show that thousands of enhancers in Drosophila melanogaster S2 and ovarian somatic cells (OSCs) exhibit a marked specificity to one of two core promoters--one derived from a ubiquitously expressed ribosomal protein gene and another from a developmentally regulated transcription factor--and confirm the existence of these two classes for five additional core promoters from genes with diverse functions. Housekeeping enhancers are active across the two cell types, while developmental enhancers exhibit strong cell-type specificity. Both enhancer classes differ in their genomic distribution, the functions of neighbouring genes, and the core promoter elements of these neighbouring genes. In addition, we identify two transcription factors--Dref and Trl--that bind and activate housekeeping versus developmental enhancers, respectively. Our results provide evidence for a sequence-encoded enhancer-core-promoter specificity that separates developmental and housekeeping gene regulatory programs for thousands of enhancers and their target genes across the entire genome.

  8. Enhanced transfection of brain tumor suppressor genes by photochemical internalization

    NASA Astrophysics Data System (ADS)

    Chou, Chih H.; Sun, Chung-Ho; Zhou, Yi-Hong; Madsen, Steen J.; Hirschberg, Henry

    2011-03-01

    One of many limitations for cancer gene therapy is the inability of the therapeutic gene to transfect a sufficient number of tumor cells. Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. The utility of PCI for the delivery of a tumor suppressor gene (PAX-6) was investigated in monolayers and spheroids consisting of F98 rat glioma cells.

  9. Standardized Plant Disease Evaluations will Enhance Resistance Gene Discovery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gene discovery and marker development using DNA based tools require plant populations with well-documented phenotypes. Related crops such as apples and pears may share a number of genes, for example resistance to common diseases, and data mining in one crop may reveal genes for the other. However, u...

  10. Intragenic Locus in Human PIWIL2 Gene Shares Promoter and Enhancer Functions

    PubMed Central

    Zinovyeva, Marina V.; Nikolaev, Lev G.; Azhikina, Tatyana L.

    2016-01-01

    Recently, more evidence supporting common nature of promoters and enhancers has been accumulated. In this work, we present data on chromatin modifications and non-polyadenylated transcription characteristic for enhancers as well as results of in vitro luciferase reporter assays suggesting that PIWIL2 alternative promoter in exon 7 also functions as an enhancer for gene PHYHIP located 60Kb upstream. This finding of an intragenic enhancer serving as a promoter for a shorter protein isoform implies broader impact on understanding enhancer-promoter networks in regulation of gene expression. PMID:27248499

  11. MBD3 Localizes at Promoters, Gene Bodies and Enhancers of Active Genes

    PubMed Central

    Shimbo, Takashi; Du, Ying; Grimm, Sara A.; Dhasarathy, Archana; Mav, Deepak; Shah, Ruchir R.; Shi, Huidong; Wade, Paul A.

    2013-01-01

    The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. Recent reports describing localization of NuRD provide new insights that question previous models on NuRD action, but are not in complete agreement. Here, we provide location analysis of endogenous MBD3, a component of NuRD complex, in two human breast cancer cell lines (MCF-7 and MDA-MB-231) using two independent genomic techniques: DNA adenine methyltransferase identification (DamID) and ChIP-seq. We observed concordance of the resulting genomic localization, suggesting that these studies are converging on a robust map for NuRD in the cancer cell genome. MBD3 preferentially associated with CpG rich promoters marked by H3K4me3 and showed cell-type specific localization across gene bodies, peaking around the transcription start site. A subset of sites bound by MBD3 was enriched in H3K27ac and was in physical proximity to promoters in three-dimensional space, suggesting function as enhancers. MBD3 enrichment was also noted at promoters modified by H3K27me3. Functional analysis of chromatin indicated that MBD3 regulates nucleosome occupancy near promoters and in gene bodies. These data suggest that MBD3, and by extension the NuRD complex, may have multiple roles in fine tuning expression for both active and silent genes, representing an important step in defining regulatory mechanisms by which NuRD complex controls chromatin structure and modification status. PMID:24385926

  12. Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants

    PubMed Central

    Parker, Stephen C. J.; Stitzel, Michael L.; Taylor, D. Leland; Orozco, Jose Miguel; Erdos, Michael R.; Akiyama, Jennifer A.; van Bueren, Kelly Lammerts; Chines, Peter S.; Narisu, Narisu; Black, Brian L.; Visel, Axel; Pennacchio, Len A.; Collins, Francis S.; Becker, Jesse; Benjamin, Betty; Blakesley, Robert; Bouffard, Gerry; Brooks, Shelise; Coleman, Holly; Dekhtyar, Mila; Gregory, Michael; Guan, Xiaobin; Gupta, Jyoti; Han, Joel; Hargrove, April; Johnson, Taccara; Legaspi, Richelle; Lovett, Sean; Maduro, Quino; Masiello, Cathy; Maskeri, Baishali; McDowell, Jenny; Montemayor, Casandra; Mullikin, James; Park, Morgan; Riebow, Nancy; Schandler, Karen; Schmidt, Brian; Sison, Christina; Stantripop, Mal; Thomas, James; Thomas, Pam; Vemulapalli, Meg; Young, Alice

    2013-01-01

    Chromatin-based functional genomic analyses and genomewide association studies (GWASs) together implicate enhancers as critical elements influencing gene expression and risk for common diseases. Here, we performed systematic chromatin and transcriptome profiling in human pancreatic islets. Integrated analysis of islet data with those from nine cell types identified specific and significant enrichment of type 2 diabetes and related quantitative trait GWAS variants in islet enhancers. Our integrated chromatin maps reveal that most enhancers are short (median = 0.8 kb). Each cell type also contains a substantial number of more extended (≥3 kb) enhancers. Interestingly, these stretch enhancers are often tissue-specific and overlap locus control regions, suggesting that they are important chromatin regulatory beacons. Indeed, we show that (i) tissue specificity of enhancers and nearby gene expression increase with enhancer length; (ii) neighborhoods containing stretch enhancers are enriched for important cell type–specific genes; and (iii) GWAS variants associated with traits relevant to a particular cell type are more enriched in stretch enhancers compared with short enhancers. Reporter constructs containing stretch enhancer sequences exhibited tissue-specific activity in cell culture experiments and in transgenic mice. These results suggest that stretch enhancers are critical chromatin elements for coordinating cell type–specific regulatory programs and that sequence variation in stretch enhancers affects risk of major common human diseases. PMID:24127591

  13. Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants.

    PubMed

    Parker, Stephen C J; Stitzel, Michael L; Taylor, D Leland; Orozco, Jose Miguel; Erdos, Michael R; Akiyama, Jennifer A; van Bueren, Kelly Lammerts; Chines, Peter S; Narisu, Narisu; Black, Brian L; Visel, Axel; Pennacchio, Len A; Collins, Francis S

    2013-10-29

    Chromatin-based functional genomic analyses and genomewide association studies (GWASs) together implicate enhancers as critical elements influencing gene expression and risk for common diseases. Here, we performed systematic chromatin and transcriptome profiling in human pancreatic islets. Integrated analysis of islet data with those from nine cell types identified specific and significant enrichment of type 2 diabetes and related quantitative trait GWAS variants in islet enhancers. Our integrated chromatin maps reveal that most enhancers are short (median = 0.8 kb). Each cell type also contains a substantial number of more extended (≥ 3 kb) enhancers. Interestingly, these stretch enhancers are often tissue-specific and overlap locus control regions, suggesting that they are important chromatin regulatory beacons. Indeed, we show that (i) tissue specificity of enhancers and nearby gene expression increase with enhancer length; (ii) neighborhoods containing stretch enhancers are enriched for important cell type-specific genes; and (iii) GWAS variants associated with traits relevant to a particular cell type are more enriched in stretch enhancers compared with short enhancers. Reporter constructs containing stretch enhancer sequences exhibited tissue-specific activity in cell culture experiments and in transgenic mice. These results suggest that stretch enhancers are critical chromatin elements for coordinating cell type-specific regulatory programs and that sequence variation in stretch enhancers affects risk of major common human diseases.

  14. [PC-1 enhances c-myc gene expression in prostate cancer cells].

    PubMed

    Yu, Lan; Shi, Qing-Guo; Qian, Xiao-Long; Li, Shan-Hu; Wang, Hong-Tao; Wang, Le-Le; Zhou, Jian-Guang

    2010-04-01

    PC-1(Prostate and colon gene 1) gene belongs to TPD52 (Tumor Protein D52) gene family. The expression of PC-1 is found to promote androgen-independent progression. This study was conducted to assess the mechnism of promotion of androgen-independent progression in PC-1 gene. The c-myc gene expression was tested by RT-PCR and Western blotting analyses in the LNCaP-pc-1 and LNCaP-zero cell line. After separation of cytoplasm and nulear proteins of the LNCaP-pc-1 and LNCaP-zero cell line, the beta-catenin protein was detected by Western blotting. C4-2 cell line was used to examine the effects of 10058-F4 on the PC-1 gene expression. The results of RT-PCR and Western blotting indicated that PC-1 enhanced c-myc gene expression in prostate cancer cells, PC-1 was also found to enhance beta-catenin expression in nuclear. Furthermore, a small-molecule c-Myc inhibitor, 10058-F4 represses PC-1 gene expression in C4-2 cell line. Our findings suggest that PC-1 enhances c-myc gene expression in prostate cancer cells through the Wnt/beta-catenin pathway. Meanwhile, c-myc plays a feed-forward role in enhancing PC-1 driven c-myc gene expression, and promotes prostate an-drogen-independent progression.

  15. Growth enhancement and gene expression of Arabidopsis thaliana irradiated with active oxygen species

    NASA Astrophysics Data System (ADS)

    Watanabe, Satoshi; Ono, Reoto; Hayashi, Nobuya; Shiratani, Masaharu; Tashiro, Kosuke; Kuhara, Satoru; Inoue, Asami; Yasuda, Kaori; Hagiwara, Hiroko

    2016-07-01

    The characteristics of plant growth enhancement effect and the mechanism of the enhancement induced by plasma irradiation are investigated using various active species in plasma. Active oxygen species in oxygen plasma are effective for growth enhancement of plants. DNA microarray analysis of Arabidopsis thaliana indicates that the genes coding proteins that counter oxidative stresses by eliminating active oxygen species are expressed at significantly high levels. The size of plant cells increases owing to oxygen plasma irradiation. The increases in gene expression levels and cell size suggest that the increase in the expression level of the expansin protein is essential for plant growth enhancement phenomena.

  16. Genome organization and long-range regulation of gene expression by enhancers

    PubMed Central

    Smallwood, Andrea; Ren, Bing

    2014-01-01

    It is now well accepted that cell-type specific gene regulation is under the purview of enhancers. Great strides have been made recently to characterize and identify enhancers both genetically and epigenetically for multiple cell types and species, but efforts have just begun to link enhancers to their target promoters. Mapping these interactions and understanding how the 3D landscape of the genome constrains such interactions is fundamental to our understanding of mammalian gene regulation. Here, we review recent progress in mapping long-range regulatory interactions in mammalian genomes, focusing on transcriptional enhancers and chromatin organization principles. PMID:23465541

  17. SAFETY AND EFFICIENCY OF MODULATING PARACELLULAR PERMEABILITY TO ENHANCE AIRWAY EPITHELIAL GENE TRANSFER IN VIVO

    EPA Science Inventory


    ABSTRACT

    We evaluated the safety of agents that enhance gene transfer by modulating paracellular permeability. Lactate dehydrogenase (LDH) and cytokine release were measured in polarized primary human airway epithelial (HAE) cells after luminal application of vehicle, ...

  18. Genes related to xylose fermentation and methods of using same for enhanced biofuel production

    DOEpatents

    Wohlbach, Dana J.; Gasch, Audrey P.

    2014-08-05

    The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

  19. Genes related to xylose fermentation and methods of using same for enhanced biofuel production

    SciTech Connect

    Wohlbach, Dana J.; Gasch, Audrey P.

    2015-09-29

    The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

  20. Genes related to xylose fermentation and methods of using same for enhanced biofuel production

    DOEpatents

    Wohlbach, Dana J.; Gasch, Audrey P.

    2016-11-29

    The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

  1. Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish.

    PubMed

    Wang, Xiaodan; Kültz, Dietmar

    2017-03-28

    Fish respond to salinity stress by transcriptional induction of many genes, but the mechanism of their osmotic regulation is unknown. We developed a reporter assay using cells derived from the brain of the tilapia Oreochromis mossambicus (OmB cells) to identify osmolality/salinity-responsive enhancers (OSREs) in the genes of Omossambicus Genomic DNA comprising the regulatory regions of two strongly salinity-induced genes, inositol monophosphatase 1 (IMPA1.1) and myo-inositol phosphate synthase (MIPS), was isolated and analyzed with dual luciferase enhancer trap reporter assays. We identified five sequences (two in IMPA1.1 and three in MIPS) that share a common consensus element (DDKGGAAWWDWWYDNRB), which we named "OSRE1." Additional OSREs that were less effective in conferring salinity-induced trans-activation and do not match the OSRE1 consensus also were identified in both MIPS and IMPA1.1 Although OSRE1 shares homology with the mammalian osmotic-response element/tonicity-responsive enhancer (ORE/TonE) enhancer, the latter is insufficient to confer osmotic induction in fish. Like other enhancers, OSRE1 trans-activates genes independent of orientation. We conclude that OSRE1 is a cis-regulatory element (CRE) that enhances the hyperosmotic induction of osmoregulated genes in fish. Our study also shows that tailored reporter assays developed for OmB cells facilitate the identification of CREs in fish genomes. Knowledge of the OSRE1 motif allows affinity-purification of the corresponding transcription factor and computational approaches for enhancer screening of fish genomes. Moreover, our study enables targeted inactivation of OSRE1 enhancers, a method superior to gene knockout for functional characterization because it confines impairment of gene function to a specific context (salinity stress) and eliminates pitfalls of constitutive gene knockouts (embryonic lethality, developmental compensation).

  2. Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish

    PubMed Central

    Wang, Xiaodan; Kültz, Dietmar

    2017-01-01

    Fish respond to salinity stress by transcriptional induction of many genes, but the mechanism of their osmotic regulation is unknown. We developed a reporter assay using cells derived from the brain of the tilapia Oreochromis mossambicus (OmB cells) to identify osmolality/salinity-responsive enhancers (OSREs) in the genes of O. mossambicus. Genomic DNA comprising the regulatory regions of two strongly salinity-induced genes, inositol monophosphatase 1 (IMPA1.1) and myo-inositol phosphate synthase (MIPS), was isolated and analyzed with dual luciferase enhancer trap reporter assays. We identified five sequences (two in IMPA1.1 and three in MIPS) that share a common consensus element (DDKGGAAWWDWWYDNRB), which we named “OSRE1.” Additional OSREs that were less effective in conferring salinity-induced trans-activation and do not match the OSRE1 consensus also were identified in both MIPS and IMPA1.1. Although OSRE1 shares homology with the mammalian osmotic-response element/tonicity-responsive enhancer (ORE/TonE) enhancer, the latter is insufficient to confer osmotic induction in fish. Like other enhancers, OSRE1 trans-activates genes independent of orientation. We conclude that OSRE1 is a cis-regulatory element (CRE) that enhances the hyperosmotic induction of osmoregulated genes in fish. Our study also shows that tailored reporter assays developed for OmB cells facilitate the identification of CREs in fish genomes. Knowledge of the OSRE1 motif allows affinity-purification of the corresponding transcription factor and computational approaches for enhancer screening of fish genomes. Moreover, our study enables targeted inactivation of OSRE1 enhancers, a method superior to gene knockout for functional characterization because it confines impairment of gene function to a specific context (salinity stress) and eliminates pitfalls of constitutive gene knockouts (embryonic lethality, developmental compensation). PMID:28289196

  3. The Igκ Gene Enhancers, E3′ and Ed, Are Essential for Triggering Transcription

    PubMed Central

    Zhou, Xiaorong; Xiang, Yougui; Garrard, William T.

    2010-01-01

    The mouse Igκ gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3′ enhancer (E3′), and a further downstream enhancer (Ed). Previous studies on B lymphocytes derived from mutant ES cells have shown that deletion of either Ei or E3′ significantly reduces Igκ gene rearrangement, whereas the combined deletion of both Ei and E3′ eliminates such recombination. Furthermore, deletion of either E3′ or Ed significantly reduces rearranged Igκ gene transcription. To determine whether the combined presence of both E3′ and Ed are essential for Igκ gene expression, we generated homozygous double knockout mice (DKO) with targeted deletions in both elements. Significantly, homozygous DKO mice were unable to generate κ+ B cells both in bone marrow and the periphery, and exhibited surface expression almost exclusively of Igλ chains, in spite of the fact that they possessed potentially functional rearranged Igκ genes. Compared with their single-enhancer-deleted counterparts, Igκ loci in homozygous DKO mice exhibited dramatically reduced germline and rearranged gene transcription, lower levels of gene rearrangement and histone H3 acetylation, and markedly increased DNA methylation. This contributed to a partial developmental block at the pre-B cell stage of development. We conclude that the two downstream enhancers are essential in Igκ gene expression and that Ei in homozygous DKO mice is incapable of triggering Igκ gene transcription. Furthermore, these results reveal unexpected compensatory roles for Ed in E3′ knockout mice in triggering germline transcription, and Vκ gene rearrangements to both Jκ and RS elements. PMID:21076060

  4. Identification of nonviable genes affecting touch sensitivity in Caenorhabditis elegans using neuronally enhanced feeding RNA interference.

    PubMed

    Chen, Xiaoyin; Cuadros, Margarete Diaz; Chalfie, Martin

    2015-01-09

    Caenorhabditis elegans senses gentle touch along the body via six touch receptor neurons. Although genetic screens and microarray analyses have identified several genes needed for touch sensitivity, these methods miss pleiotropic genes that are essential for the viability, movement, or fertility of the animals. We used neuronally enhanced feeding RNA interference to screen genes that cause lethality or paralysis when mutated, and we identified 61 such genes affecting touch sensitivity, including five positive controls. We confirmed 18 genes by using available alleles, and further studied one of them, tag-170, now renamed txdc-9. txdc-9 preferentially affects anterior touch response but is needed for tubulin acetylation and microtubule formation in both the anterior and posterior touch receptor neurons. Our results indicate that neuronally enhanced feeding RNA interference screens complement traditional mutageneses by identifying additional nonviable genes needed for specific neuronal functions.

  5. Identification of a retinoic acid-responsive neural enhancer in the Ciona intestinalis Hox1 gene.

    PubMed

    Kanda, Miyuki; Ikeda, Taku; Fujiwara, Shigeki

    2013-02-01

    The Hox1 gene in the urochordate ascidian Ciona intestinalis (Ci-Hox1) is expressed in the nerve cord and epidermis. We identified a nerve cord enhancer in the second intron of Ci-Hox1, and demonstrated that retinoic acid (RA) plays a major role in activating this enhancer. The enhancer contained a putative retinoic acid-response element (RARE). Mutation of the RARE in the Ci-Hox1 nerve cord enhancer only partially abolished the enhancer activity. Genes encoding RA synthase and the RA receptor were knocked down using specific antisense morpholino oligos (MOs), and injection of embryos with these MOs resulted in the complete disappearance of epidermal expression of Ci-Hox1 and reduction of neural expression. However, nerve cord expression was not completely repressed. These results suggest that the nerve cord enhancer is activated by two partially redundant pathways; one RA-dependent and one RA-independent.

  6. Standardized plant disease evaluations will enhance resistance gene discovery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gene discovery and marker development using DNA-based tools require plant populations with well documented phenotypes. If dissimilar phenotype evaluation methods or data scoring techniques are employed with different crops, or at different labs for the same crops, then data mining for genetic marker...

  7. Identification of Genes That Enhance the Nutritional Value of Rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Consumers in developed countries such as the U.S. are willing to pay premium prices for food products having enhanced nutritional value. Grocery stores contain a myriad of calcium fortified foods ranging from orange juice to margarine to frozen waffles. Cereal aisles and bottled water aisles are lin...

  8. Finding genes that enhance the nutritional value of rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofortification refers to natural enhancement of the nutritional value of grain or food products. This can be accomplished through traditional breeding and selection for plants that accumulate more nutrients in their edible portions. Since biofortification does not require genetic engineering or sy...

  9. Heterogeneity of neural progenitor cells revealed by enhancers in the nestin gene

    PubMed Central

    Yaworsky, Paul J.; Kappen, Claudia

    2014-01-01

    Using transgenic embryos, we have identified two distinct CNS progenitor cell-specific enhancers, each requiring the cooperation of at least two independent regulatory sites, within the second intron of the rat nestin gene. One enhancer is active throughout the developing CNS while the other is specifically active in the ventral midbrain. These experiments demonstrate that neural progenitor cells in the midbrain constitute a unique subpopulation based upon their ability to activate the midbrain regulatory elements. Our finding of differential enhancer activity from a gene encoding a structural protein reveals a previously unrecognized diversity in neural progenitor cell populations. PMID:9917366

  10. Embryonic Stem Cell (ES)-Specific Enhancers Specify the Expression Potential of ES Genes in Cancer

    PubMed Central

    Levy, Revital; Meron, Nurit; Toperoff, Gidon; Edrei, Yifat; Bergman, Yehudit; Hellman, Asaf

    2016-01-01

    Cancers often display gene expression profiles resembling those of undifferentiated cells. The mechanisms controlling these expression programs have yet to be identified. Exploring transcriptional enhancers throughout hematopoietic cell development and derived cancers, we uncovered a novel class of regulatory epigenetic mutations. These epimutations are particularly enriched in a group of enhancers, designated ES-specific enhancers (ESSEs) of the hematopoietic cell lineage. We found that hematopoietic ESSEs are prone to DNA methylation changes, indicative of their chromatin activity states. Strikingly, ESSE methylation is associated with gene transcriptional activity in cancer. Methylated ESSEs are hypermethylated in cancer relative to normal somatic cells and co-localized with silenced genes, whereas unmethylated ESSEs tend to be hypomethylated in cancer and associated with reactivated genes. Constitutive or hematopoietic stem cell-specific enhancers do not show these trends, suggesting selective reactivation of ESSEs in cancer. Further analyses of a hypomethylated ESSE downstream to the VEGFA gene revealed a novel regulatory circuit affecting VEGFA transcript levels across cancers and patients. We suggest that the discovered enhancer sites provide a framework for reactivation of ES genes in cancer. PMID:26886256

  11. Genes and signaling pathways involved in memory enhancement in mutant mice

    PubMed Central

    2014-01-01

    Mutant mice have been used successfully as a tool for investigating the mechanisms of memory at multiple levels, from genes to behavior. In most cases, manipulating a gene expressed in the brain impairs cognitive functions such as memory and their underlying cellular mechanisms, including synaptic plasticity. However, a remarkable number of mutations have been shown to enhance memory in mice. Understanding how to improve a system provides valuable insights into how the system works under normal conditions, because this involves understanding what the crucial components are. Therefore, more can be learned about the basic mechanisms of memory by studying mutant mice with enhanced memory. This review will summarize the genes and signaling pathways that are altered in the mutants with enhanced memory, as well as their roles in synaptic plasticity. Finally, I will discuss how knowledge of memory-enhancing mechanisms could be used to develop treatments for cognitive disorders associated with impaired plasticity. PMID:24894914

  12. Genes and signaling pathways involved in memory enhancement in mutant mice.

    PubMed

    Lee, Yong-Seok

    2014-06-04

    Mutant mice have been used successfully as a tool for investigating the mechanisms of memory at multiple levels, from genes to behavior. In most cases, manipulating a gene expressed in the brain impairs cognitive functions such as memory and their underlying cellular mechanisms, including synaptic plasticity. However, a remarkable number of mutations have been shown to enhance memory in mice. Understanding how to improve a system provides valuable insights into how the system works under normal conditions, because this involves understanding what the crucial components are. Therefore, more can be learned about the basic mechanisms of memory by studying mutant mice with enhanced memory. This review will summarize the genes and signaling pathways that are altered in the mutants with enhanced memory, as well as their roles in synaptic plasticity. Finally, I will discuss how knowledge of memory-enhancing mechanisms could be used to develop treatments for cognitive disorders associated with impaired plasticity.

  13. Enhancers and chromatin structures: regulatory hubs in gene expression and diseases.

    PubMed

    Hu, Zhenhua; Tee, Wee Wei

    2017-03-28

    Gene expression requires successful communication between enhancer and promoter regions, whose activities are regulated by a variety of factors and associated with distinct chromatin structures; in addition, functionally related genes and their regulatory repertoire tend to be arranged in the same sub-chromosomal regulatory domains. In this review, we discuss the importance of enhancers, especially clusters of enhancers (such as super-enhancers), as key regulatory hubs to integrate environmental cues and encode spatiotemporal instructions for genome expression, which are critical for a variety of biological processes governing mammalian development. Furthermore, we emphasize that the enhancer-promoter interaction landscape provides a critical context to understand the etiologies and mechanisms behind numerous complex human diseases, and provides new avenues for effective transcriptional-based interventions.

  14. Novel Cationic Lipids with Enhanced Gene Delivery and Antimicrobial Activity

    PubMed Central

    Fein, David E.; Bucki, Robert; Byfield, Fitzroy; Leszczynska, Katarzyna; Janmey, Paul A.

    2010-01-01

    Cationic lipids facilitate plasmid delivery, and some cationic sterol-based compounds have antimicrobial activity because of their amphiphilic character. These dual functions are relevant in the context of local ongoing infection during intrapulmonary gene transfer for cystic fibrosis. The transfection activities of two cationic lipids, dexamethasone spermine (DS) and disubstituted spermine (D2S), were tested as individual components and mixtures in bovine aortic endothelial cells and A549 cells. The results showed a 3- to 7-fold improvement in transgene expression for mixtures of DS with 20 to 40 mol% D2S. D2S and coformulations with DS, dioleoyl phosphatidylethanolamine, and DNA exhibited potent bactericidal activity against Escherichia coli MG1655, Bacillus subtilis, and Pseudomonas aeruginosa PAO1, which was maintained in bronchoalveolar lavage fluid. Complete bacterial killing was demonstrated at ∼5 μM, including gene delivery formulations, with 2 orders of magnitude higher tolerance before eukaryotic membrane disruption (erythrocyte hemolysis). D2S also exhibited lipopolysaccharide (LPS) scavenging activity resulting in significant inhibition of LPS-mediated activation of human neutrophils with 85 and 65% lower interleukin-8 released at 12 and 24 h, respectively. Mixtures of DS and D2S can improve transfection activity over common lipofection reagents, and D2S has strong antimicrobial action suited for the suppression of bacterial-mediated inflammation. PMID:20573781

  15. Structural requirements for the functional activity of a U1 snRNA gene enhancer.

    PubMed Central

    Cheung, C H; Fan, Q N; Stumph, W E

    1993-01-01

    The transcriptional enhancer of a chicken U1 small nuclear RNA (snRNA) gene contains a GC-box, an octamer motif, and an SPH motif that are recognized by the transcription factors Sp1, Oct-1, and SBF respectively. Previous work indicated that the octamer and the SPH motifs were both required for U1 gene enhancer activity in frog oocytes when the U1 gene was coinjected with a competing snRNA gene template. Here we show that neither two copies of the octamer motif, nor two copies of the SPH motif, can effectively substitute for the natural combination of octamer and SPH. Furthermore, neither the octamer nor the SPH motif (in the absence of the other) functioned efficiently in combination with a GC-box. Alteration of the spacing between the octamer and SPH motifs also reduced U1 template activity. Several potential cis-acting elements other than the SPH motif, with one possible exception among those tested, were unable to cooperate with the octamer motif to effectively enhance U1 gene expression. These results indicate that rather stringent structural requirements exist with respect to the essential cis-acting motifs present in the U1 enhancer, possibly reflecting the unique properties of the transcription complexes assembled on snRNA gene promoters. Images PMID:8441636

  16. Activation of Ftz-F1-Responsive Genes through Ftz/Ftz-F1 Dependent Enhancers

    PubMed Central

    Field, Amanda; Xiang, Jie; Anderson, W. Ray; Graham, Patricia; Pick, Leslie

    2016-01-01

    The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either ftz-f1 or ftz mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of ftz itself and engrailed by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success. To discern rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and ftz-f1 mutant embryos. Ftz-F1-responsive genes most highly regulated included engrailed and nine additional genes expressed in patterns dependent on both ftz and ftz-f1. Candidate enhancers for these genes were identified by combining BDTNP Ftz ChIP-chip data with a computational search for Ftz-F1 binding sites. Of eight enhancer reporter genes tested in transgenic embryos, six generated expression patterns similar to the corresponding endogenous gene and expression was lost in ftz mutants. These studies identified a new set of Ftz-F1 targets, all of which are co-regulated by Ftz. Comparative analysis of enhancers containing Ftz/Ftz-F1 binding sites that were or were not bona fide targets in vivo suggested that GAF negatively regulates enhancers that contain Ftz/Ftz-F1 binding sites but are not actually utilized. These targets include other regulatory factors as well as genes involved directly in morphogenesis, providing insight into how pair-rule genes establish the body pattern. PMID:27723822

  17. DNA Hypomethylation in Intragenic and Intergenic Enhancer Chromatin of Muscle-Specific Genes Usually Correlates with their Expression.

    PubMed

    Ehrlich, Kenneth C; Paterson, Heather L; Lacey, Michelle; Ehrlich, Melanie

    2016-12-01

    Tissue-specific enhancers are critical for gene regulation. In this study, we help elucidate the contribution of muscle-associated differential DNA methylation to the enhancer activity of highly muscle-specific genes. By bioinformatic analysis of 44 muscle-associated genes, we show that preferential gene expression in skeletal muscle (SkM) correlates with SkM-specific intragenic and intergenic enhancer chromatin and overlapping foci of DNA hypomethylation. Some genes, e.g., CASQ1 and FBXO32, displayed broad regions of both SkM- and heart-specific enhancer chromatin but exhibited focal SkM-specific DNA hypomethylation. Half of the genes had SkM-specific super-enhancers. In contrast to simple enhancer/gene-expression correlations, a super-enhancer was associated with the myogenic MYOD1 gene in both SkM and myoblasts even though SkM has < 1 percent as much MYOD1 expression. Local chromatin differences in this super-enhancer probably contribute to the SkM/myoblast differential expression. Transfection assays confirmed the tissue-specificity of the 0.3-kb core enhancer within MYOD1's super-enhancer and demonstrated its repression by methylation of its three CG dinucleotides. Our study suggests that DNA hypomethylation increases enhancer tissue-specificity and that SkM super-enhancers sometimes are poised for physiologically important, rapid up-regulation.

  18. DNA Hypomethylation in Intragenic and Intergenic Enhancer Chromatin of Muscle-Specific Genes Usually Correlates with their Expression

    PubMed Central

    Ehrlich, Kenneth C.; Paterson, Heather L.; Lacey, Michelle; Ehrlich, Melanie

    2016-01-01

    Tissue-specific enhancers are critical for gene regulation. In this study, we help elucidate the contribution of muscle-associated differential DNA methylation to the enhancer activity of highly muscle-specific genes. By bioinformatic analysis of 44 muscle-associated genes, we show that preferential gene expression in skeletal muscle (SkM) correlates with SkM-specific intragenic and intergenic enhancer chromatin and overlapping foci of DNA hypomethylation. Some genes, e.g., CASQ1 and FBXO32, displayed broad regions of both SkM- and heart-specific enhancer chromatin but exhibited focal SkM-specific DNA hypomethylation. Half of the genes had SkM-specific super-enhancers. In contrast to simple enhancer/gene-expression correlations, a super-enhancer was associated with the myogenic MYOD1 gene in both SkM and myoblasts even though SkM has < 1 percent as much MYOD1 expression. Local chromatin differences in this super-enhancer probably contribute to the SkM/myoblast differential expression. Transfection assays confirmed the tissue-specificity of the 0.3-kb core enhancer within MYOD1’s super-enhancer and demonstrated its repression by methylation of its three CG dinucleotides. Our study suggests that DNA hypomethylation increases enhancer tissue-specificity and that SkM super-enhancers sometimes are poised for physiologically important, rapid up-regulation. PMID:28018137

  19. WWOX, large common fragile site genes, and cancer

    PubMed Central

    Gao, Ge

    2015-01-01

    WWOX is a gene that spans an extremely large chromosomal region. It is derived from within chromosomal band 16q23.2 which is a region with frequent deletions and other alterations in a variety of different cancers. This chromosomal band also contains the FRA16D common fragile site (CFS). CFSs are chromosomal regions found in all individuals which are highly unstable. WWOX has also been demonstrated to function as a tumor suppressor that is involved in the development of many cancers. Two other highly unstable CFSs, FRA3B (3p14.2) and FRA6E (6q26), also span extremely large genes, FHIT and PARK2, respectively, and these two genes are also found to be important tumor suppressors. There are a number of interesting similarities between these three large CFS genes. In spite of the fact that they are derived from some of the most unstable chromosomal regions in the genome, they are found to be highly evolutionarily conserved and the chromosomal region spanning the mouse homologs of both WWOX and FHIT are also CFSs in mice. Many of the other CFSs also span extremely large genes and many of these are very attractive tumor suppressor candidates. WWOX is therefore a member of a very interesting family of very large CFS genes. PMID:25595185

  20. Enhanced osteoblast proliferation and collagen gene expression by estradiol

    SciTech Connect

    Ernest, M.; Schmid, Ch.; Froesch, E.R. )

    1988-04-01

    Estrogens play a crucial role in the development of postmenopausal osteoporosis. However, the mechanism by which estrogens exert their effects on bone is unknown. To examine possible direct effects of 17{beta}-estradiol on bone-forming cells, the authors used pure rat osteoblast-like cells in vitro as a model. Osteoblast-like cells prepared from calvaria of newborn rats were cultured serum-free in methylcellulose-containing medium for 21 days. Osteoblast-like cells proliferate selectively into clonally derived cell clusters of spherical morphorlogy. 17{beta}-Estradiol at concentrations of 0.1 nM and 1 nM enhanced osteoblast-like cell proliferation by 41% and 68% above vehicle-treated controls. The biologically inactive stereoisomer 17{alpha}-estradiol (same concentrations) had no effect. Moreover, the antiestrogen tamoxifen abolished the stimulation of osteoblast-like cell proliferation by 17{beta}-estradiol. After 21 days of culture, RNA was prepared and analyzed in a dot-hybridization assay for the abundance of pro{alpha}1(I) collagen mRNA. Steady-state mRNA levels were increased in cultures treated with 17{beta}-estradiol in a dose-dependent manner with maximal stimulation at 1 nM and 10 nM. At the same concentrations, the percentage of synthesized protein (labeled by ({sup 3}H)proline pulse) that was digestible by collagenase was increased, indicating that 17{beta}-estradiol acts as pretranslational levels to enhance synthesis of bone collagen. These data show that the osteoblast is a direct target for 17{beta}-estradiol.

  1. Gene Therapy of Breast Cancer: Studies of Selective Promoter/Enhancer-Modified Vectors to Deliver Suicide Genes

    DTIC Science & Technology

    1997-09-01

    gene therapy strategies for breast cancer by translation of studies derived from the DF3/MUCl gene. We have completed Tasks 1 and 2 as outlined in the Statement of Work using the DF3 promoter to selectively drive transgenes in breast cancer cells. The DF3 promoter has been used in an adenoviral vector to selectively detect and eliminate breast cancer cells that contaminate hematopoietic stem cell preparations used in autologous bone marrow transplantation. More recent work has involved modification of the DF3 promoter by adding a Tet-enhancer system to increase expression

  2. Transcriptional enhancers in protein-coding exons of vertebrate developmental genes.

    PubMed

    Ritter, Deborah I; Dong, Zhiqiang; Guo, Su; Chuang, Jeffrey H

    2012-01-01

    Many conserved noncoding sequences function as transcriptional enhancers that regulate gene expression. Here, we report that protein-coding DNA also frequently contains enhancers functioning at the transcriptional level. We tested the enhancer activity of 31 protein-coding exons, which we chose based on strong sequence conservation between zebrafish and human, and occurrence in developmental genes, using a Tol2 transposable GFP reporter assay in zebrafish. For each exon we measured GFP expression in hundreds of embryos in 10 anatomies via a novel system that implements the voice-recognition capabilities of a cellular phone. We find that 24/31 (77%) exons drive GFP expression compared to a minimal promoter control, and 14/24 are anatomy-specific (expression in four anatomies or less). GFP expression driven by these coding enhancers frequently overlaps the anatomies where the host gene is expressed (60%), suggesting self-regulation. Highly conserved coding sequences and highly conserved noncoding sequences do not significantly differ in enhancer activity (coding: 24/31 vs. noncoding: 105/147) or tissue-specificity (coding: 14/24 vs. noncoding: 50/105). Furthermore, coding and noncoding enhancers display similar levels of the enhancer-related histone modification H3K4me1 (coding: 9/24 vs noncoding: 34/81). Meanwhile, coding enhancers are over three times as likely to contain an H3K4me1 mark as other exons of the host gene. Our work suggests that developmental transcriptional enhancers do not discriminate between coding and noncoding DNA and reveals widespread dual functions in protein-coding DNA.

  3. Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells

    PubMed Central

    Soucie, Erinn L.; Weng, Ziming; Geirsdóttir, Laufey; Molawi, Kaaweh; Maurizio, Julien; Fenouil, Romain; Mossadegh-Keller, Noushine; Gimenez, Gregory; VanHille, Laurent; Beniazza, Meryam; Favret, Jeremy; Berruyer, Carole; Perrin, Pierre; Hacohen, Nir; Andrau, J.-C.; Ferrier, Pierre; Dubreuil, Patrice; Sidow, Arend; Sieweke, Michael H.

    2016-01-01

    Differentiated macrophages can self-renew in tissues and expand long-term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network controlling self-renewal. Single cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells. PMID:26797145

  4. Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

    SciTech Connect

    Perez, C.F.

    1984-08-01

    The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT/sup +/ colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references.

  5. Gene pyramiding enhances durable blast disease resistance in rice.

    PubMed

    Fukuoka, Shuichi; Saka, Norikuni; Mizukami, Yuko; Koga, Hironori; Yamanouchi, Utako; Yoshioka, Yosuke; Hayashi, Nagao; Ebana, Kaworu; Mizobuchi, Ritsuko; Yano, Masahiro

    2015-01-14

    Effective control of blast, a devastating fungal disease of rice, would increase and stabilize worldwide food production. Resistance mediated by quantitative trait loci (QTLs), which usually have smaller individual effects than R-genes but confer broad-spectrum or non-race-specific resistance, is a promising alternative to less durable race-specific resistance for crop improvement, yet evidence that validates the impact of QTL combinations (pyramids) on the durability of plant disease resistance has been lacking. Here, we developed near-isogenic experimental lines representing all possible combinations of four QTL alleles from a durably resistant cultivar. These lines enabled us to evaluate the QTLs singly and in combination in a homogeneous genetic background. We present evidence that pyramiding QTL alleles, each controlling a different response to M. oryzae, confers strong, non-race-specific, environmentally stable resistance to blast disease. Our results suggest that this robust defence system provides durable resistance, thus avoiding an evolutionary "arms race" between a crop and its pathogen.

  6. Gene Therapy of Breast Cancer: Studies of Selective Promoter/Enhancer-Modified Vectors to Deliver Suicide Genes

    DTIC Science & Technology

    1998-09-01

    antigen - expressing tu- 36. Wickham. TJ.. P. Mathias. D.A. Cheresh. and G.R. Nemerow. 1993. In- mor cells in bone marrow aspirates by polymerase chain ...transduction of Escherichia Coi lacZ. Proc." 1992. Detection of tumor cells in bone marrow of patients with primary breast Nail. Acad. Sci. USA. 85:2603-2607...tissue specific /selective promoter or enhancer to direct the expression of a therapeutic gene in the desired target

  7. Functional analysis of an eye specific enhancer of the eyeless gene in Drosophila

    PubMed Central

    Hauck, Bernd; Gehring, Walter J.; Walldorf, Uwe

    1999-01-01

    The development of the Drosophila compound eye requires the function of a set of evolutionarily conserved genes. Among these, the Drosophila Pax-6 gene eyeless (ey) plays a major role. ey has been considered a master control gene of eye development in the animal kingdom because targeted expression of ey and vertebrate as well as invertebrate homologs lead to the formation of ectopic eyes in Drosophila. We demonstrate that an intron of the ey gene contains an enhancer that regulates the eye specific expression of the gene in the eye disc primordia of embryos and in the eye imaginal discs of third instar larvae. Moreover, a 212-bp enhancer element is necessary and sufficient for the enhancer function. It is partially conserved in Drosophila hydei and contains putative Pax-6 Paired domain binding sites. We show that several binding sites are required for the eye specific expression, and, therefore, we propose a Pax-6-like molecule to be a positive transactivator for the eye specific ey expression. This transactivator recently has been identified as twin of eyeless, the second Pax-6 gene in Drosophila. PMID:9892673

  8. Identification of Gene Transcription Start Sites and Enhancers Responding to Pulmonary Carbon Nanotube Exposure in Vivo.

    PubMed

    Bornholdt, Jette; Saber, Anne Thoustrup; Lilje, Berit; Boyd, Mette; Jørgensen, Mette; Chen, Yun; Vitezic, Morana; Jacobsen, Nicklas Raun; Poulsen, Sarah Søs; Berthing, Trine; Bressendorff, Simon; Vitting-Seerup, Kristoffer; Andersson, Robin; Hougaard, Karin Sørig; Yauk, Carole L; Halappanavar, Sabina; Wallin, Håkan; Vogel, Ulla; Sandelin, Albin

    2017-03-31

    Increased use of nanomaterials in industry, medicine, and consumer products has raised concerns over their toxicity. To ensure safe use of nanomaterials, understanding their biological effects at the molecular level is crucial. In particular, the regulatory mechanisms responsible for the cascade of genes activated by nanomaterial exposure are not well-characterized. To this end, we profiled the genome-wide usage of gene transcription start sites and linked active enhancer regions in lungs of C57BL/6 mice 24 h after intratracheal instillation of a single dose of the multiwalled carbon nanotube (MWCNT) Mitsui-7. Our results revealed a massive gene regulatory response, where expression of key inflammatory genes (e.g., Csf3, Il24, and Fgf23) was increased >100-fold 24 h after Mitsui-7 exposure. Many of the Mitsui-7-responsive transcription start sites were alternative transcription start sites for known genes, and the number of alternative transcription start sites used in a given gene was correlated with overall Mitsui-7 response. Strikingly, genes that were up-regulated after Mitsui-7 exposure only through their main annotated transcription start site were linked to inflammatory and defense responses, while genes up-regulated only through alternative transcription start sites were functionally heterogeneous and not inflammation-associated. Furthermore, we identified almost 12 000 active enhancers, many of which were Mitsui-7-responsive, and we identified similarly responding putative target genes. Overall, our study provides the location and activity of Mitsui-7-induced enhancers and transcription start sites, providing a useful resource for targeted experiments elucidating the biological effects of nanomaterials and the identification of biomarkers for early detection of MWCNT-induced inflammation.

  9. Distinct parasegmental and imaginal enhancers and the establishment of the expression pattern of the Ubx gene

    SciTech Connect

    Pirrotta, V.; Chan, Chi Shing; McCabe, D.; Qian, Su

    1995-12-01

    The expression domain of the Ubx gene in Drosophila embryos is bounded by the product of the hb gene, acting as a repressor. We show that all Ubx fragments that bind Hb protein in vitro contain parasegmental enhancers active in the embryo in specific parasegmental patterns. We have found three new embryonic enhancer elements in the upstream region, in addition to the two previously identified. Each produces a pattern initially bounded at PS6 by Hb but sooner or later breaks down this boundary and begins to express in the anterior region. These enhancers do not respond to the long-term maintenance mediated by the Polycomb group of genes. They also cease functioning after germ band extension. Expression in imaginal tissues is due to a set of entirely separate and independent imaginal disc enhancers. These do not contain Hb binding sites and by themselves have no anterior/posterior positional information, although some distinguish between ventral and dorsal discs. A third kind of element, the Polycomb Response Element (PRE), has no enhancer activity but causes long-term maintenance of the expression domain of other enhancers present in the vicinity. The interaction of these elements results in the correct expression of Ubx in imaginal tissues. 40 refs., 7 figs.

  10. The mouse Enhancer trap locus 1 (Etl-1): a novel mammalian gene related to Drosophila and yeast transcriptional regulator genes.

    PubMed

    Soininen, R; Schoor, M; Henseling, U; Tepe, C; Kisters-Woike, B; Rossant, J; Gossler, A

    1992-11-01

    A novel mouse gene, Enhancer trap locus 1 (Etl-1), was identified in close proximity to a lacZ enhancer trap integration in the mouse genome showing a specific beta-galactosidase staining pattern during development. In situ analysis revealed a widespread but not ubiquitous expression of Etl-1 throughout development with particularly high levels in the central nervous system and epithelial cells. The amino acid sequence of the Etl-1 protein deduced from the cDNA shows strong similarity, over a stretch of 500 amino acids, to the Drosophila brahma protein involved in the regulation of homeotic genes and to the yeast transcriptional activator protein SNF2/SWI2 as well as to the RAD54 protein and the recently described helicase-related yeast proteins STH1 and MOT1. Etl-1 is the first mammalian member of this group of proteins that are implicated in gene regulation and/or influencing chromatin structure. The homology to the regulatory proteins SNF2/SWI2 and brahma and the expression pattern during embryogenesis suggest that Etl-1 protein might be involved in gene regulating pathways during mouse development.

  11. Applying horizontal gene transfer phenomena to enhance non-viral gene therapy

    PubMed Central

    Elmer, Jacob J.; Christensen, Matthew D.; Rege, Kaushal

    2014-01-01

    Horizontal gene transfer (HGT) is widespread amongst prokaryotes, but eukaryotes tend to be far less promiscuous with their genetic information. However, several examples of HGT from pathogens into eukaryotic cells have been discovered and mimicked to improve non-viral gene delivery techniques. For example, several viral proteins and DNA sequences have been used to significantly increase cytoplasmic and nuclear gene delivery. Plant genetic engineering is routinely performed with the pathogenic bacterium Agrobacterium tumefaciens and similar pathogens (e.g. Bartonella henselae) may also be able to transform human cells. Intracellular parasites like Trypanosoma cruzi may also provide new insights into overcoming cellular barriers to gene delivery. Finally, intercellular nucleic acid transfer between host cells will also be briefly discussed. This article will review the unique characteristics of several different viruses and microbes and discuss how their traits have been successfully applied to improve non-viral gene delivery techniques. Consequently, pathogenic traits that originally caused diseases may eventually be used to treat many genetic diseases. PMID:23994344

  12. Nacystelyn enhances adenoviral vector-mediated gene delivery to mouse airways.

    PubMed

    Kushwah, R; Oliver, J R; Cao, H; Hu, J

    2007-08-01

    Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors.

  13. Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin.

    PubMed

    Hu, Li-Zhong; Wan, Ning; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-03-24

    Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

  14. Hippocampal gene expression patterns underlying the enhancement of memory by running in aged mice

    PubMed Central

    Stranahan, Alexis M.; Lee, Kim; Becker, Kevin G.; Zhang, Yonqing; Maudsley, Stuart; Martin, Bronwen; Cutler, Roy G.; Mattson, Mark P.

    2009-01-01

    Physical activity preserves cognition in the aging brain, but the mechanisms remain obscure. In order to identify candidate genes and pathways responsible for the preservation of cognitive function by exercise, we trained mice that had been exposed to lifelong running or sedentary lifestyle for 16 months in the hippocampus-dependent water maze. After water maze training, we analyzed the expression of 24,000 genes in the hippocampus using Illumina bead microarray. Runners show greater activation of genes associated with synaptic plasticity and mitochondrial function, and also exhibit significant downregulation of genes associated with oxidative stress and lipid metabolism. Running also modified the effects of learning on the expression of genes involved in cell excitability, energy metabolism, and insulin, MAP kinase and Wnt signaling. These results suggest that the enhancement of cognitive function by lifelong exercise is associated with an altered transcriptional profile following learning. PMID:19070401

  15. Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin

    NASA Astrophysics Data System (ADS)

    Hu, Li-Zhong; Wan, Ning; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-03-01

    Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

  16. Thixotropic solutions enhance viral-mediated gene transfer to airway epithelia.

    PubMed

    Seiler, Michael P; Luner, Paul; Moninger, Thomas O; Karp, Philip H; Keshavjee, Shaf; Zabner, Joseph

    2002-08-01

    Adenovirus-mediated gene transfer to airway epithelia is inefficient in part because its receptor is absent on the apical surface of the airways. Targeting adenovirus to other receptors, increasing the viral concentration, and even prolonging the incubation time with adenovirus vectors can partially overcome the lack of receptors and facilitate gene transfer. Unfortunately, mucociliary clearance would prevent prolonged incubation time in vivo. Thixotropic solutions (TS) are gels that upon a vigorous shearing force reversibly become liquid. We hypothesized that formulating recombinant adenoviruses in TS would decrease virus clearance and thus enhance gene transfer to the airway epithelia. We found that clearance of virus-sized fluorescent beads by human airway epithelia in vitro and by monkey trachea in vivo were markedly decreased when the beads were formulated in TS compared with phosphate-buffered saline (PBS). Adenovirus formulated in TS significantly increased adenovirus-mediated gene transfer of a reporter gene in human airway epithelia in vitro and in murine airway epithelia in vivo. Furthermore, an adenovirus encoding the cystic fibrosis transmembrane regulator (CFTR) gene (AdCFTR) formulated in TS was more efficient in correcting the chloride transport defect in cystic fibrosis airway epithelia than AdCFTR formulated in PBS. These data indicate a novel strategy to augment the efficiency of gene transfer to the airways that may be applicable to a number of different gene transfer vectors and could be of value in gene transfer to cystic fibrosis (CF) airway epithelia in vivo.

  17. Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides

    PubMed Central

    Liu, Yarong; Kim, Young Joo; Ji, Man; Fang, Jinxu; Siriwon, Natnaree; Zhang, Li I; Wang, Pin

    2014-01-01

    Adeno-associated virus type 2 (AAV2) is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs). We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy. PMID:26015948

  18. Enhanced seed oil content by overexpressing genes related to triacylglyceride synthesis.

    PubMed

    Liu, Fang; Xia, Yuping; Wu, Lei; Fu, Donghui; Hayward, Alice; Luo, Junling; Yan, Xiaohong; Xiong, Xiaojuan; Fu, Ping; Wu, Gang; Lu, Changming

    2015-02-25

    Oilseed rape (Brassica napus) is one of the most important oilseed crops globally. To meet increasing demand for oil-based products, the ability to enhance desirable oil content in the seed is required. This study assessed the capability of five genes in the triacylglyceride (TAG) synthesis pathway to enhance oil content. The genes BnGPDH, BnGPAT, BnDGAT, ScGPDH and ScLPAAT were overexpressed separately in a tobacco (Nicotiana benthamiana) model system, and simultaneously by pyramiding in B. napus, under the control of a seed specific Napin promoter. ScLPAAT transgenic plants showed a significant increase of 6.84% to 8.55% in oil content in tobacco seeds, while a ~4% increase was noted for BnGPDH and BnGPAT transgenic seeds. Seed-specific overexpression of all four genes in B. napus resulted in as high a 12.57% to 14.46% increased in seed oil content when compared to WT, equaling close to the sum of the single-gene overexpression increases in tobacco. Taken together, our study demonstrates that BnGPDH, BnGPAT and ScLPAAT may effectively increase seed oil content, and that simultaneous overexpression of these in transgenic B. napus may further enhance the desirable oil content relative to single-gene overexpressors.

  19. Overexpression of Actinidia deliciosa pyruvate decarboxylase 1 gene enhances waterlogging stress in transgenic Arabidopsis thaliana.

    PubMed

    Zhang, Ji-Yu; Huang, Sheng-Nan; Wang, Gang; Xuan, Ji-Ping; Guo, Zhong-Ren

    2016-09-01

    Ethanolic fermentation is classically associated with waterlogging tolerance when plant cells switch from respiration to anaerobic fermentation. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we cloned a full-length PDC cDNA sequence from kiwifruit, named AdPDC1. We determined the expression of the AdPDC1 gene in kiwifruit under different environmental stresses using qRT-PCR, and the results showed that the increase of AdPDC1 expression during waterlogging stress was much higher than that during salt, cold, heat and drought stresses. Overexpression of kiwifruit AdPDC1 in transgenic Arabidopsis enhanced the resistance to waterlogging stress but could not enhance resistance to cold stress at five weeks old seedlings. Overexpression of kiwifruit AdPDC1 in transgenic Arabidopsis could not enhance resistance to NaCl and mannitol stresses at the stage of seed germination and in early seedlings. These results suggested that the kiwifruit AdPDC1 gene is required during waterlogging but might not be required during other environmental stresses. Expression of the AdPDC1 gene was down-regulated by abscisic acid (ABA) in kiwifruit, and overexpression of the AdPDC1 gene in Arabidopsis inhibited seed germination and root length under ABA treatment, indicating that ABA might negatively regulate the AdPDC1 gene under waterlogging stress.

  20. Metazoan Nuclear Pores Provide a Scaffold for Poised Genes and Mediate Induced Enhancer-Promoter Contacts.

    PubMed

    Pascual-Garcia, Pau; Debo, Brian; Aleman, Jennifer R; Talamas, Jessica A; Lan, Yemin; Nguyen, Nha H; Won, Kyoung J; Capelson, Maya

    2017-04-06

    Nuclear pore complex components (Nups) have been implicated in transcriptional regulation, yet what regulatory steps are controlled by metazoan Nups remains unclear. We identified the presence of multiple Nups at promoters, enhancers, and insulators in the Drosophila genome. In line with this binding, we uncovered a functional role for Nup98 in mediating enhancer-promoter looping at ecdysone-inducible genes. These genes were found to be stably associated with nuclear pores before and after activation. Although changing levels of Nup98 disrupted enhancer-promoter contacts, it did not affect ongoing transcription but instead compromised subsequent transcriptional activation or transcriptional memory. In support of the enhancer-looping role, we found Nup98 to gain and retain physical interactions with architectural proteins upon stimulation with ecdysone. Together, our data identify Nups as a class of architectural proteins for enhancers and supports a model in which animal genomes use the nuclear pore as an organizing scaffold for inducible poised genes.

  1. Coupled enhancer and coding sequence evolution of a homeobox gene shaped leaf diversity

    PubMed Central

    Vuolo, Francesco; Mentink, Remco A.; Hajheidari, Mohsen; Bailey, C. Donovan; Filatov, Dmitry A.; Tsiantis, Miltos

    2016-01-01

    Here we investigate mechanisms underlying the diversification of biological forms using crucifer leaf shape as an example. We show that evolution of an enhancer element in the homeobox gene REDUCED COMPLEXITY (RCO) altered leaf shape by changing gene expression from the distal leaf blade to its base. A single amino acid substitution evolved together with this regulatory change, which reduced RCO protein stability, preventing pleiotropic effects caused by its altered gene expression. We detected hallmarks of positive selection in these evolved regulatory and coding sequence variants and showed that modulating RCO activity can improve plant physiological performance. Therefore, interplay between enhancer and coding sequence evolution created a potentially adaptive path for morphological evolution. PMID:27852629

  2. Heterologous Overexpression of Poplar SnRK2 Genes Enhanced Salt Stress Tolerance in Arabidopsis thaliana

    PubMed Central

    Song, Xueqing; Yu, Xiang; Hori, Chiaki; Demura, Taku; Ohtani, Misato; Zhuge, Qiang

    2016-01-01

    Subfamily 2 of SNF1-related protein kinase (SnRK2) plays important roles in plant abiotic stress responses as a global positive regulator of abscisic acid signaling. In the genome of the model tree Populus trichocarpa, 12 SnRK2 genes have been identified, and some are upregulated by abiotic stresses. In this study, we heterologously overexpressed the PtSnRK2 genes in Arabidopsis thaliana and found that overexpression of PtSnRK2.5 and PtSnRK2.7 genes enhanced stress tolerance. In the PtSnRK2.5 and PtSnRK2.7 overexpressors, chlorophyll content, and root elongation were maintained under salt stress conditions, leading to higher survival rates under salt stress compared with those in the wild type. Transcriptomic analysis revealed that PtSnRK2.7 overexpression affected stress-related metabolic genes, including lipid metabolism and flavonoid metabolism, even under normal growth conditions. However, the stress response genes reported to be upregulated in Arabidopsis SRK2C/SnRK2.6 and wheat SnRK2.8 overexpressors were not changed by PtSnRK2.7 overexpression. Furthermore, PtSnRK2.7 overexpression widely and largely influenced the transcriptome in response to salt stress; genes related to transport activity, including anion transport-related genes, were characteristically upregulated, and a variety of metabolic genes were specifically downregulated. We also found that the salt stress response genes were greatly upregulated in the PtSnRK2.7 overexpressor. Taken together, poplar subclass 2 PtSnRK2 genes can modulate salt stress tolerance in Arabidopsis, through the activation of cellular signaling pathways in a different manner from that by herbal subclass 2 SnRK2 genes. PMID:27242819

  3. Mediator Kinase Inhibition Further Activates Super-Enhancer Associated Genes in AML

    PubMed Central

    Nitulescu, Ioana I.; Tangpeerachaikul, Anupong; Poss, Zachary C.; Da Silva, Diogo H.; Caruso, Brittany T.; Arefolov, Alexander; Fadeyi, Olugbeminiyi; Christie, Amanda L.; Du, Karrie; Banka, Deepti; Schneider, Elisabeth V.; Jestel, Anja; Zou, Ge; Si, Chong; Ebmeier, Christopher C.; Bronson, Roderick T.; Krivtsov, Andrei V.; Myers, Andrew G.; Kohl, Nancy E.; Kung, Andrew L.; Armstrong, Scott A.; Lemieux, Madeleine E.; Taatjes, Dylan J.; Shair, Matthew D.

    2015-01-01

    Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors (TFs), and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling TFs and oncogenes 1, 2. BRD4 and CDK7 are positive regulators of SE-mediated transcription3,4,5. In contrast, negative regulators of SE-associated genes have not been well described. Here we report that Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We determined that the natural product cortistatin A (CA) selectively inhibited Mediator kinases, had antileukaemic activity in vitro and in vivo, and disproportionately induced upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the TFs CEBPA, IRF8, IRF1 and ETV6 6, 7, 8. The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has antileukaemic activity. Individually increasing or decreasing expression of these TFs suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types and can be pharmacologically targeted as a therapeutic approach to AML. PMID:26416749

  4. Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression.

    PubMed

    Macková, Hana; Hronková, Marie; Dobrá, Jana; Turečková, Veronika; Novák, Ondřej; Lubovská, Zuzana; Motyka, Václav; Haisel, Daniel; Hájek, Tomáš; Prášil, Ilja Tom; Gaudinová, Alena; Štorchová, Helena; Ge, Eva; Werner, Tomáš; Schmülling, Thomas; Vanková, Radomíra

    2013-07-01

    Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.

  5. Specific contribution of the erythropoietin gene 3' enhancer to hepatic erythropoiesis after late embryonic stages.

    PubMed

    Suzuki, Norio; Obara, Naoshi; Pan, Xiaoqing; Watanabe, Miho; Jishage, Kou-Ichi; Minegishi, Naoko; Yamamoto, Masayuki

    2011-09-01

    Erythropoietin (Epo) is secreted from the liver and kidney, where Epo production is strictly regulated at the transcriptional level in a hypoxia- and/or anemia-inducible manner. Here, we examined the in vivo function of the enhancer located 3' to the Epo gene (EpoE-3'). Reporter transgenic-mouse analyses revealed that the EpoE-3' enhancer is necessary and sufficient for the liver-specific and hypoxia-responsive expression of the gene after embryonic day 14.5 (E14.5). However, the enhancer is dispensable for Epo gene expression in the kidney and early-stage embryonic liver. Genetic removal of EpoE-3' from the endogenous Epo gene resulted in mice with severe anemia at late embryonic and neonatal stages due to defects in hepatic erythropoiesis, but early hepatic and splenic erythropoiesis was not affected. The mutant mice recover from the anemia in the juvenile period when major Epo production switches from the liver to the kidney. These results demonstrate that EpoE-3' is necessary for late hepatic erythropoiesis by specifically supporting paracrine production of Epo in the liver. In contrast, Epo production in the kidney utilizes distinct regulatory machinery and supports erythropoiesis in the bone marrow and spleen in adult animals.

  6. Overexpression of a Harpin-encoding gene hrf1 in rice enhances drought tolerance

    PubMed Central

    Zhang, Lei; Xiao, Shanshan; Feng, Wei; Wu, Zhidan; Gao, Xuewen; Liu, Fengquan; Shao, Min

    2011-01-01

    Harpin proteins are well known as eliciters that induce multiple responses in plants, such as systemic acquired resistance, hypersensitive response, enhancement of growth, resistance to the green peach aphid, and tolerance to drought. Overexpression of Harpin-encoding genes enhances plant resistance to diseases in tobacco, rice, rape, and cotton; however, it is not yet known whether the expression of Harpin-encoding genes in vivo improves plant tolerance to abiotic stresses. The results of this study showed that overexpression of a Harpin-encoding gene hrf1 in rice increased drought tolerance through abscisic acid (ABA) signalling. hrf1- overexpression induces an increase in ABA content and promotes stomatal closure in rice. The hrf1 transgenic rice lines exhibited a significant increase in water retention ability, levels of free proline and soluble sugars, tolerance to oxidative stress, reactive oxygen species-scavenging ability, and expression levels of four stress-related genes, OsLEA3-1, OsP5CS, Mn-SOD, and NM_001074345, under drought stress. The study confirmed that hrf1 conferred enhanced tolerance to drought stress on transgenic crops. These results suggest that Harpins may offer new opportunities for generating drought resistance in other crops. PMID:21527628

  7. Comparative genomic analysis reveals a distant liver enhancer upstream of the COUP-TFII gene

    SciTech Connect

    Baroukh, Nadine; Ahituv, Nadav; Chang, Jessie; Shoukry, Malak; Afzal, Veena; Rubin, Edward M.; Pennacchio, Len A.

    2004-08-20

    COUP-TFII is a central nuclear hormone receptor that tightly regulates the expression of numerous target lipid metabolism genes in vertebrates. However, it remains unclear how COUP-TFII itself is transcriptionally controlled since studies with its promoter and upstream region fail to recapitulate the genes liver expression. In an attempt to identify liver enhancers in the vicinity of COUP-TFII, we employed a comparative genomic approach. Initial comparisons between humans and mice of the 3,470kb gene poor region surrounding COUP-TFII revealed 2,023 conserved non-coding elements. To prioritize a subset of these elements for functional studies, we performed further genomic comparisons with the orthologous pufferfish (Fugu rubripes) locus and uncovered two anciently conserved non-coding sequences (CNS) upstream of COUP-TFII (CNS-62kb and CNS-66kb). Testing these two elements using reporter constructs in liver (HepG2) cells revealed that CNS-66kb, but not CNS-62kb, yielded robust in vitro enhancer activity. In addition, an in vivo reporter assay using naked DNA transfer with CNS-66kb linked to luciferase displayed strong reproducible liver expression in adult mice, further supporting its role as a liver enhancer. Together, these studies further support the utility of comparative genomics to uncover gene regulatory sequences based on evolutionary conservation and provide the substrates to better understand the regulation and expression of COUP-TFII.

  8. Enhanced salt tolerance of alfalfa (Medicago sativa) by rstB gene transformation.

    PubMed

    Zhang, Wan-Jun; Wang, Tao

    2015-05-01

    Generating salt tolerance forage plant is essential for use of the land affected by high salinity. A salt tolerance gene rstB was used as a selectable marker gene in Agrobacterium-mediated transformation of tobacco under a selective regime of 170mM NaCl. The transgenic plants showed clear improvement in salt tolerance. To improve salt tolerance of alfalfa (Medicago sativa L.), rstB gene was introduced into alfalfa genome by Agrobacterium-mediated transformation. No abnormal phenotype was observed among the transgenic plants when compared with wild type (wt) plants. Significant enhancement of resistance to salt-shock treatment was noted on the rstB transgenic (T0) plants. Transgenic second-generation (T1) seeds showed improved germination rate and seedling growth under salt-stress condition. Hindered Na(+) accumulation, but enhanced Ca(2+) accumulation was observed on the rstB T1 plants when subjected to salt-stresses. Enhanced calcium accumulation in transgenic plants was also verified by cytohistochemical localization of calcium. Under salt-stress of 50mM NaCl, about 15% of the transgenic plants finished their life-cycle but the wt plants had no flower formation. The results demonstrated that the expression of rstB gene improved salt tolerance in transgenic alfalfa.

  9. Focused ultrasound enhanced molecular imaging and gene therapy for multifusion reporter gene in glioma-bearing rat model.

    PubMed

    Yang, Feng-Yi; Chang, Wen-Yuan; Lin, Wei-Ting; Hwang, Jeng-Jong; Chien, Yi-Chun; Wang, Hsin-Ell; Tsai, Min-Lan

    2015-11-03

    The ability to monitor the responses of and inhibit the growth of brain tumors during gene therapy has been severely limited due to the blood-brain barrier (BBB). A previous study has demonstrated the feasibility of noninvasive in vivo imaging with 123I-2'-fluoro-2'-deoxy-5-iodo-1-β-D-arabinofuranosyluracil (123I-FIAU) for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) cancer gene expression in an experimental animal model. Here, we tested the enhancement of SPECT with 123I-FIAU and ganciclovir (GCV) treatment in brain tumors after BBB disruption induced by focused ultrasound (FUS) in the presence of microbubbles. We established an orthotopic F98 glioma-bearing rat model with trifusion reporter genes. The results of this study showed that the rat model of HSV1-tk-expressing glioma cells could be successfully detected by SPECT imaging after FUS-induced BBB disruption on day 10 after implantation. Compared to the control group, animals receiving the GCV with or without sonication exhibited a significant antitumor activity (P < 0.05) of glioma cells on day 16 after implantation. Moreover, combining sonication with GCV significantly inhibited tumor growth compared with GCV alone. This study demonstrated that FUS may be used to deliver a wide variety of theranostic agents to the brain for molecular imaging and gene therapy in brain diseases.

  10. Multiparameter evaluation of in vivo gene delivery using ultrasound-guided, microbubble-enhanced sonoporation.

    PubMed

    Shapiro, Galina; Wong, Andrew W; Bez, Maxim; Yang, Fang; Tam, Sarah; Even, Lisa; Sheyn, Dmitriy; Ben-David, Shiran; Tawackoli, Wafa; Pelled, Gadi; Ferrara, Katherine W; Gazit, Dan

    2016-02-10

    More than 1800 gene therapy clinical trials worldwide have targeted a wide range of conditions including cancer, cardiovascular diseases, and monogenic diseases. Biological (i.e. viral), chemical, and physical approaches have been developed to deliver nucleic acids into cells. Although viral vectors offer the greatest efficiency, they also raise major safety concerns including carcinogenesis and immunogenicity. The goal of microbubble-mediated sonoporation is to enhance the uptake of drugs and nucleic acids. Insonation of microbubbles is thought to facilitate two mechanisms for enhanced uptake: first, deflection of the cell membrane inducing endocytotic uptake, and second, microbubble jetting inducing the formation of pores in the cell membrane. We hypothesized that ultrasound could be used to guide local microbubble-enhanced sonoporation of plasmid DNA. With the aim of optimizing delivery efficiency, we used nonlinear ultrasound and bioluminescence imaging to optimize the acoustic pressure, microbubble concentration, treatment duration, DNA dosage, and number of treatments required for in vivo Luciferase gene expression in a mouse thigh muscle model. We found that mice injected with 50μg luciferase plasmid DNA and 5×10(5) microbubbles followed by ultrasound treatment at 1.4MHz, 200kPa, 100-cycle pulse length, and 540 Hz pulse repetition frequency (PRF) for 2min exhibited superior transgene expression compared to all other treatment groups. The bioluminescent signal measured for these mice on Day 4 post-treatment was 100-fold higher (p<0.0001, n=5 or 6) than the signals for controls treated with DNA injection alone, DNA and microbubble injection, or DNA injection and ultrasound treatment. Our results indicate that these conditions result in efficient gene delivery and prolonged gene expression (up to 21days) with no evidence of tissue damage or off-target delivery. We believe that these promising results bear great promise for the development of microbubble-enhanced

  11. B29 Gene Silencing in Pituitary Cells is Regulated by Its 3′ Enhancer

    PubMed Central

    Malone, Cindy S.; Kuraishy, Ali I.; Fike, Francesca M.; Loya, Ruchika G.; Mikkili, Minil R.; Teitell, Michael A.; Wall, Randolph

    2007-01-01

    Summary B cell-specific B29 (Igβ, CD79b) genes in rat, mouse, and human are situated between the 5′ growth hormone (GH) locus control region (LCR) and the 3′ GH gene cluster. The entire GH genomic region is DNase1 hypersensitive in GH-expressing pituitary cells, which predicts an “open” chromatin configuration, and yet B29 is not expressed. The B29 promoter and enhancers exhibit histone deacetylation in pituitary cells, but histone deacetylase inhibition failed to activate B29 expression. The B29 promoter and a 3′ enhancer showed local dense DNA methylation in both pituitary and non-lymphoid cells consistent with gene silencing. However, DNA methyltransferase inhibition did not activate B29 expression either. B29 promoter constructs were minimally activated in transfected pituitary cells. Co-transfection of the B cell-specific octamer transcriptional co-activator Bob1 with the B29 promoter construct resulted in high level promoter activity in pituitary cells comparable to B29 promoter activity in transfected B cells. Unexpectedly, inclusion of the B29 3′ enhancer in B29 promoter constructs strongly inhibited B29 transcriptional activity even when pituitary cells were co-transfected with Bob1. Both Oct-1 and Pit-1 bind the B29 3′ enhancer in in vitro EMSA and in in vivo chromatin immunoprecipitation analyses. These data indicate that the GH locus-embedded, tissue-specific B29 gene is silenced in GH-expressing pituitary cells by epigenetic mechanisms, the lack of a B cell-specific transcription factor, and likely by the B29 3′ enhancer acting as a powerful silencer in a context and tissue-specific manner. PMID:16920149

  12. Enhancing gene regulatory network inference through data integration with markov random fields.

    PubMed

    Banf, Michael; Rhee, Seung Y

    2017-02-01

    A gene regulatory network links transcription factors to their target genes and represents a map of transcriptional regulation. Much progress has been made in deciphering gene regulatory networks computationally. However, gene regulatory network inference for most eukaryotic organisms remain challenging. To improve the accuracy of gene regulatory network inference and facilitate candidate selection for experimentation, we developed an algorithm called GRACE (Gene Regulatory network inference ACcuracy Enhancement). GRACE exploits biological a priori and heterogeneous data integration to generate high- confidence network predictions for eukaryotic organisms using Markov Random Fields in a semi-supervised fashion. GRACE uses a novel optimization scheme to integrate regulatory evidence and biological relevance. It is particularly suited for model learning with sparse regulatory gold standard data. We show GRACE's potential to produce high confidence regulatory networks compared to state of the art approaches using Drosophila melanogaster and Arabidopsis thaliana data. In an A. thaliana developmental gene regulatory network, GRACE recovers cell cycle related regulatory mechanisms and further hypothesizes several novel regulatory links, including a putative control mechanism of vascular structure formation due to modifications in cell proliferation.

  13. Enhancing gene regulatory network inference through data integration with markov random fields

    PubMed Central

    Banf, Michael; Rhee, Seung Y.

    2017-01-01

    A gene regulatory network links transcription factors to their target genes and represents a map of transcriptional regulation. Much progress has been made in deciphering gene regulatory networks computationally. However, gene regulatory network inference for most eukaryotic organisms remain challenging. To improve the accuracy of gene regulatory network inference and facilitate candidate selection for experimentation, we developed an algorithm called GRACE (Gene Regulatory network inference ACcuracy Enhancement). GRACE exploits biological a priori and heterogeneous data integration to generate high- confidence network predictions for eukaryotic organisms using Markov Random Fields in a semi-supervised fashion. GRACE uses a novel optimization scheme to integrate regulatory evidence and biological relevance. It is particularly suited for model learning with sparse regulatory gold standard data. We show GRACE’s potential to produce high confidence regulatory networks compared to state of the art approaches using Drosophila melanogaster and Arabidopsis thaliana data. In an A. thaliana developmental gene regulatory network, GRACE recovers cell cycle related regulatory mechanisms and further hypothesizes several novel regulatory links, including a putative control mechanism of vascular structure formation due to modifications in cell proliferation. PMID:28145456

  14. NRIP enhances HPV gene expression via interaction with either GR or E2

    SciTech Connect

    Chang, Szu-Wei; Lu, Pei-Yu; Guo, Jih-Huong; Tsai, Tzung-Chieh; Tsao, Yeou-Ping; Chen, Show-Li

    2012-02-05

    We previously identified a gene, nuclear receptor-interaction protein (NRIP), which functions as a transcription cofactor in glucocorticoid receptor (GR) and human papillomavirus E2 (HPV E2)-driven gene expression. Here, we comprehensively evaluated the role of NRIP in HPV-16 gene expression. NRIP acts as a transcription cofactor to enhance GR-regulated HPV-16 gene expression in the presence of hormone. NRIP also can form complex with E2 that caused NRIP-induced HPV gene expression via E2-binding sites in a hormone-independent manner. Furthermore, NRIP can associate with GR and E2 to form tri-protein complex to activate HPV gene expression via GRE, not the E2-binding site, in a hormone-dependent manner. These results indicate that NRIP and GR are viral E2-binding proteins and that NRIP regulates HPV gene expression via GRE and/or E2 binding site in the HPV promoter in a hormone-dependent or independent manner, respectively.

  15. Enhancing gene regulatory network inference through data integration with markov random fields

    DOE PAGES

    Banf, Michael; Rhee, Seung Y.

    2017-02-01

    Here, a gene regulatory network links transcription factors to their target genes and represents a map of transcriptional regulation. Much progress has been made in deciphering gene regulatory networks computationally. However, gene regulatory network inference for most eukaryotic organisms remain challenging. To improve the accuracy of gene regulatory network inference and facilitate candidate selection for experimentation, we developed an algorithm called GRACE (Gene Regulatory network inference ACcuracy Enhancement). GRACE exploits biological a priori and heterogeneous data integration to generate high- confidence network predictions for eukaryotic organisms using Markov Random Fields in a semi-supervised fashion. GRACE uses a novel optimization schememore » to integrate regulatory evidence and biological relevance. It is particularly suited for model learning with sparse regulatory gold standard data. We show GRACE’s potential to produce high confidence regulatory networks compared to state of the art approaches using Drosophila melanogaster and Arabidopsis thaliana data. In an A. thaliana developmental gene regulatory network, GRACE recovers cell cycle related regulatory mechanisms and further hypothesizes several novel regulatory links, including a putative control mechanism of vascular structure formation due to modifications in cell proliferation.« less

  16. NRIP enhances HPV gene expression via interaction with either GR or E2.

    PubMed

    Chang, Szu-Wei; Lu, Pei-Yu; Guo, Jih-Huong; Tsai, Tzung-Chieh; Tsao, Yeou-Ping; Chen, Show-Li

    2012-02-05

    We previously identified a gene, nuclear receptor-interaction protein (NRIP), which functions as a transcription cofactor in glucocorticoid receptor (GR) and human papillomavirus E2 (HPV E2)-driven gene expression. Here, we comprehensively evaluated the role of NRIP in HPV-16 gene expression. NRIP acts as a transcription cofactor to enhance GR-regulated HPV-16 gene expression in the presence of hormone. NRIP also can form complex with E2 that caused NRIP-induced HPV gene expression via E2-binding sites in a hormone-independent manner. Furthermore, NRIP can associate with GR and E2 to form tri-protein complex to activate HPV gene expression via GRE, not the E2-binding site, in a hormone-dependent manner. These results indicate that NRIP and GR are viral E2-binding proteins and that NRIP regulates HPV gene expression via GRE and/or E2 binding site in the HPV promoter in a hormone-dependent or independent manner, respectively.

  17. Enhancement of Overgrowth by Gene Interactions in Lethal(2)giant Discs Imaginal Discs from Drosophila Melanogaster

    PubMed Central

    Buratovich, M. A.; Bryant, P. J.

    1997-01-01

    Recessive lethal mutations of the lethal(2)giant discs (l(2)gd) and lethal(2)fat (l(2)ft) loci of Drosophila melanogaster cause imaginal disc hyperplasia during a prolonged larval stage. Imaginal discs from l(2)ft l(2)gd or Gl(2)gd double homozygotes show more extensive overgrowth than in either single homozygote, and double homozygous l(2)ft l(2)gd mitotic clones in adult flies show much more overgrowth than is seen in clones homozygous for either l(2)gd or l(2)ft alone. dachsous (ds) also acts as an enhancer of l(2)gd, producing dramatically overgrown discs and causing failure to pupariate in double homozygotes. The comb gap (cg) mutation, which also interacts with ds, greatly enhances the tendency of imaginal discs from l(2)gd larvae to duplicate as they overgrow. If l(2)gd homozygotes are made heterozygous for l(2)ft, then several discs duplicate, indicating that l(2)ft acts as a dominant enhancer of l(2)gd. l(2)ft also acts as a dominant enhancer of l(2)gd, and conversely l(2)gd acts as a dominant modifier of l(2)ft. The enhancement of overgrowth caused by various mutant combinations is accompanied by changes in expression of Decapentaplegic and Wingless. These results show that tumor suppressor genes act in combination to control cell proliferation, and that tissue hyperplasia can be associated with ectopic expression of genes involved in pattern formation. PMID:9335602

  18. Intron-Mediated Enhancement: A Tool for Heterologous Gene Expression in Plants?

    PubMed Central

    Laxa, Miriam

    2017-01-01

    Many plant promoters were characterized and used for transgene expression in plants. Even though these promoters drive high levels of transgene expression in plants, the expression patterns are rarely constitutive but restricted to some tissues and developmental stages. In terms of crop improvement not only the enhancement of expression per se but, in particular, tissue-specific and spatial expression of genes plays an important role. Introns were used to boost expression in transgenic plants in the field of crop improvement for a long time. However, the mechanism behind this so called intron-mediated enhancement (IME) is still largely unknown. This review highlights the complexity of IME on the levels of its regulation and modes of action and gives an overview on IME methodology, examples in fundamental research and models of proposed mechanisms. In addition, the application of IME in heterologous gene expression is discussed. PMID:28111580

  19. Enhancement of gene transactivation activity of androgen receptor by hepatitis B virus X protein

    SciTech Connect

    Zheng Yanyan; Chen Wenling; Ma, W.-L. Maverick; Chang Chawnshang; Ou, J.-H. James . E-mail: jamesou@hsc.usc.edu

    2007-07-05

    Hepatitis B virus (HBV) X protein (HBx) is a regulatory protein that is required for efficient replication of HBV in its natural host. In this report, we demonstrate by co-immunoprecipitation experiments that HBx can physically bind to the androgen receptor (AR), which is a nuclear hormone receptor that is expressed in many different tissues including the liver. This observation is further supported by confocal microscopy, which reveals that HBx can alter the subcellular localization of the AR both in the presence and in the absence of dihydrotestosterone (DHT). Further studies indicate that HBx can enhance the gene transactivation activity of AR by enhancing its DNA binding activity in a DHT-dependent manner. However, HBx does not remain associated with AR on the DNA. As AR can regulate the expression of a number of cellular genes, our results raise the possibility that HBV pathogenesis may be mediated in part via the interaction between HBx and AR.

  20. Transcription of the histone H5 gene is regulated by three differentiation-specific enhancers.

    PubMed Central

    Rousseau, S; Asselin, M; Renaud, J; Ruiz-Carrillo, A

    1993-01-01

    Histone H5, an early marker of the avian erythroid lineage, is expressed at low levels in early erythroid precursors and at higher levels in more mature cells. We show that the increase in H5 expression is due to transcriptional activation of the H5 gene following differentiation of precursor CFU(E). We have found and characterized two upstream enhancers, E1 (between -2233 and -1878 from the site of transcription initiation, +1) and E3 (between -1321 and -1163), and confirmed the presence of a downstream enhancer (C. D. Trainor, S. J. Stamler, and J. D. Engel, Nature [London] 328:827-830, 1987) E7 (between +846 and +1181) which are responsible for the increase in H5 gene transcription. The enhancers had a weak effect in nondifferentiated CFU(E) but a strong effect when the cells were induced to differentiate. Cooperation among the three enhancers, however, was not required for H5 gene activity in the differentiated cells. The enhancers contain binding sites for several ubiquitous and erythroid cell-specific nuclear proteins, including GATA-1, as demonstrated with GATA-1-specific antibodies. Although the GATA sites were required for enhancer function, the concentration of GATA-1, GATA-2, and GATA-3 decreased during cell differentiation, and overexpression of these factors had little effect on H5 transcription. Hence, the differentiation-specific effect of the enhancers is not mediated by changes in relative levels of the GATA factors. Functional analysis of the H5 promoter indicated that the requirement of several elements, including a GC box necessary for transcription enhancement, did not change during the early stages of CFU(E) differentiation. However, the UPE, a positive element in proliferating CFU(E) recognized by the transcription factor H4TF2, was dispensable in the differentiated cells. These results suggest that as the cells enter the final stages of differentiation, there is a reprogramming of the regulatory factors that control H5 transcription and that

  1. Enhancement of gene silencing potency and nuclease stability by chemically modified duplex RNA.

    PubMed

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

    2007-01-01

    In this study, we describe a development of chemically modified dsRNAs with improved biological properties. These dsRNAs possess an enhanced RNAi activity and a dramatically increased stability in cell cultured medium (containing 10% serum) in comparison with widely used 21nt siRNA. The chemically modified dsRNAs manifested a high longterm gene suppression after one week post-transfection, whereas 21nt siRNA showed a high RNAi activity only after 48 h cell transfection.

  2. Enhanced gene expression in the brain following intravenous administration of lactoferrin-bearing polypropylenimine dendriplex.

    PubMed

    Somani, Sukrut; Robb, Gillian; Pickard, Benjamin S; Dufès, Christine

    2015-11-10

    The possibility of using gene therapy for the treatment of brain diseases such as brain cancer, Alzheimer's and Parkinson's diseases, is currently hampered by the lack of gene delivery systems able to cross the blood-brain barrier and deliver DNA to the brain following intravenous administration. On the basis that lactoferrin can effectively reach the brain by using specific receptors for crossing the blood-brain barrier, we propose to investigate if a lactoferrin-bearing generation 3-diaminobutyric polypropylenimine (DAB) dendrimer would allow the transport of plasmid DNA to the brain after intravenous administration. In this work, we demonstrated that the conjugation of lactoferrin to the dendrimer led to an enhanced DNA uptake by 2.1-fold in bEnd.3 murine brain capillary endothelial cells compared to the unmodified dendriplex in vitro. In vivo, the intravenous administration of lactoferrin-bearing DAB dendriplex resulted in a significantly increased gene expression in the brain, by more than 6.4-fold compared to that of DAB dendriplex, while decreasing gene expression in the lung and the kidneys. Gene expression in the brain was significantly higher than in any other major organs of the body. Lactoferrin-bearing generation 3 polypropylenimine dendrimer is therefore a highly promising delivery system for systemic gene delivery to the brain.

  3. The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1.

    PubMed

    Zhou, Xiangjun; Sun, Tian-Hu; Wang, Ning; Ling, Hong-Qing; Lu, Shan; Li, Li

    2011-04-01

    The cauliflower (Brassica oleracea var. botrytis) Orange (Or) gene affects plant growth and development in addition to conferring β-carotene accumulation. This study was undertaken to investigate the molecular basis for the effects of the Or gene mutation in on plant growth. The OR protein was found to interact with cauliflower and Arabidopsis eukaryotic release factor 1-2 (eRF1-2), a member of the eRF1 family, by yeast two-hybrid analysis and by bimolecular fluorescence complementation (BiFC) assay. Concomitantly, the Or mutant showed reduced expression of the BoeRF1 family genes. Transgenic cauliflower plants with suppressed expression of BoeRF1-2 and BoeRF1-3 were generated by RNA interference. Like the Or mutant, the BoeRF1 RNAi lines showed increased elongation of the leaf petiole. This long-petiole phenotype was largely caused by enhanced cell elongation, which resulted from increased cell length and elevated expression of genes involved in cell-wall loosening. These findings demonstrate that the cauliflower Or gene controls petiole elongation by suppressing the expression of eRF1 genes, and provide new insights into the molecular mechanism of leaf petiole regulation.

  4. Identification of Neuronal Enhancers of the Proopiomelanocortin Gene by Transgenic Mouse Analysis and Phylogenetic Footprinting

    PubMed Central

    de Souza, Flávio S. J.; Santangelo, Andrea M.; Bumaschny, Viviana; Avale, María Elena; Smart, James L.; Low, Malcolm J.; Rubinstein, Marcelo

    2005-01-01

    The proopiomelanocortin (POMC) gene is expressed in the pituitary and arcuate neurons of the hypothalamus. POMC arcuate neurons play a central role in the control of energy homeostasis, and rare loss-of-function mutations in POMC cause obesity. Moreover, POMC is the prime candidate gene within a highly significant quantitative trait locus on chromosome 2 associated with obesity traits in several human populations. Here, we identify two phylogenetically conserved neuronal POMC enhancers designated nPE1 (600 bp) and nPE2 (150 bp) located approximately 10 to 12 kb upstream of mammalian POMC transcriptional units. We show that mouse or human genomic regions containing these enhancers are able to direct reporter gene expression to POMC hypothalamic neurons, but not the pituitary of transgenic mice. Conversely, deletion of nPE1 and nPE2 in the context of the entire transcriptional unit of POMC abolishes transgene expression in the hypothalamus without affecting pituitary expression. Our results indicate that the nPEs are necessary and sufficient for hypothalamic POMC expression and that POMC expression in the brain and pituitary is controlled by independent sets of enhancers. Our study advances the understanding of the molecular nature of hypothalamic POMC neurons and will be useful to determine whether polymorphisms in POMC regulatory regions play a role in the predisposition to obesity. PMID:15798195

  5. Overexpression of cinnamate 4-hydroxylase gene enhances biosynthesis of decursinol angelate in Angelica gigas hairy roots.

    PubMed

    Park, Nam Il; Park, Jee Hee; Park, Sang Un

    2012-02-01

    Angelica gigas is a medicinal plant that produces pyranocoumarins, including decursin (D) and decursinol angelate (DA), which have neuroprotective, anticancer, and antiandrogenic effects. In this study, the coumarin biosynthetic pathway was engineered to increase the production of DA. Specifically, a vector was constructed which contained the A. gigas phenylalanine ammonia-lyase (AgPAL) and cinnamate 4-hydroxylase (AgC4H) genes that were driven by the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic hairy roots that overexpressed AgPAL or AgC4H genes were obtained by using an Agrobacterium rhizogenes-mediated transformation system. Among them, only AgC4H-transgenic hairy root lines produced more DA than control transgenic hairy root lines. The enhanced gene expression corresponded to elevated C4H activities. This study showed the importance of C4H in the production of DA in A. gigas hairy root culture.

  6. Modeling Human Severe Combined Immunodeficiency and Correction by CRISPR/Cas9-Enhanced Gene Targeting.

    PubMed

    Chang, Chia-Wei; Lai, Yi-Shin; Westin, Erik; Khodadadi-Jamayran, Alireza; Pawlik, Kevin M; Lamb, Lawrence S; Goldman, Frederick D; Townes, Tim M

    2015-09-08

    Mutations of the Janus family kinase JAK3 gene cause severe combined immunodeficiency (SCID). JAK3 deficiency in humans is characterized by the absence of circulating T cells and natural killer (NK) cells with normal numbers of poorly functioning B cells (T(-)B(+)NK(-)). Using SCID patient-specific induced pluripotent stem cells (iPSCs) and a T cell in vitro differentiation system, we demonstrate a complete block in early T cell development of JAK3-deficient cells. Correction of the JAK3 mutation by CRISPR/Cas9-enhanced gene targeting restores normal T cell development, including the production of mature T cell populations with a broad T cell receptor (TCR) repertoire. Whole-genome sequencing of corrected cells demonstrates no CRISPR/Cas9 off-target modifications. These studies describe an approach for the study of human lymphopoiesis and provide a foundation for gene correction therapy in humans with immunodeficiencies.

  7. Immune disease-associated variants in gene enhancers point to BET epigenetic mechanisms for therapeutic intervention.

    PubMed

    Tough, David F; Prinjha, Rab K

    2016-12-07

    Genome-wide association studies have identified thousands of single nucleotide polymorphisms in the human genome that are statistically associated with particular disease traits. In this Perspective, we review emerging data suggesting that most single nucleotide polymorphisms associated with immune-mediated diseases are found in regulatory regions of the DNA - parts of the genome that control expression of the protein encoding genes - rather than causing mutations in proteins. We discuss how the emerging understanding of particular gene regulatory regions, gene enhancers and the epigenetic mechanisms by which they are regulated is opening up new opportunities for the treatment of immune-mediated diseases, focusing particularly on the BET family of epigenetic reader proteins as potential therapeutic targets.

  8. Transgenic Citrus Expressing an Arabidopsis NPR1 Gene Exhibit Enhanced Resistance against Huanglongbing (HLB; Citrus Greening)

    PubMed Central

    Dutt, Manjul; Barthe, Gary; Irey, Michael; Grosser, Jude

    2015-01-01

    Commercial sweet orange cultivars lack resistance to Huanglongbing (HLB), a serious phloem limited bacterial disease that is usually fatal. In order to develop sustained disease resistance to HLB, transgenic sweet orange cultivars ‘Hamlin’ and ‘Valencia’ expressing an Arabidopsis thaliana NPR1 gene under the control of a constitutive CaMV 35S promoter or a phloem specific Arabidopsis SUC2 (AtSUC2) promoter were produced. Overexpression of AtNPR1 resulted in trees with normal phenotypes that exhibited enhanced resistance to HLB. Phloem specific expression of NPR1 was equally effective for enhancing disease resistance. Transgenic trees exhibited reduced diseased severity and a few lines remained disease-free even after 36 months of planting in a high-disease pressure field site. Expression of the NPR1 gene induced expression of several native genes involved in the plant defense signaling pathways. The AtNPR1 gene being plant derived can serve as a component for the development of an all plant T-DNA derived consumer friendly GM tree. PMID:26398891

  9. Design and Characterization of Novel Recombinant Listeriolysin O–Protamine Fusion Proteins for Enhanced Gene Delivery

    PubMed Central

    2015-01-01

    To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO–protamine fusion protein, in a nonviral vector is a promising strategy for various nonviral gene delivery systems. PMID:25521817

  10. Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres.

    PubMed Central

    Laible, G; Wolf, A; Dorn, R; Reuter, G; Nislow, C; Lebersorger, A; Popkin, D; Pillus, L; Jenuwein, T

    1997-01-01

    Gene silencing is required to stably maintain distinct patterns of gene expression during eukaryotic development and has been correlated with the induction of chromatin domains that restrict gene activity. We describe the isolation of human (EZH2) and mouse (Ezh1) homologues of the Drosophila Polycomb-group (Pc-G) gene Enhancer of zeste [E(z)], a crucial regulator of homeotic gene expression implicated in the assembly of repressive protein complexes in chromatin. Mammalian homologues of E(z) are encoded by two distinct loci in mouse and man, and the two murine Ezh genes display complementary expression profiles during mouse development. The E(z) gene family reveals a striking functional conservation in mediating gene repression in eukaryotic chromatin: extra gene copies of human EZH2 or Drosophila E(z) in transgenic flies enhance position effect variegation of the heterochromatin-associated white gene, and expression of either human EZH2 or murine Ezh1 restores gene repression in Saccharomyces cerevisiae mutants that are impaired in telomeric silencing. Together, these data provide a functional link between Pc-G-dependent gene repression and inactive chromatin domains, and indicate that silencing mechanism(s) may be broadly conserved in eukaryotes. PMID:9214638

  11. Late gadolinium enhanced cardiovascular magnetic resonance of lamin A/C gene mutation related dilated cardiomyopathy

    PubMed Central

    2011-01-01

    Background The purpose of this study was to identify early features of lamin A/C gene mutation related dilated cardiomyopathy (DCM) with cardiovascular magnetic resonance (CMR). We characterise myocardial and functional findings in carriers of lamin A/C mutation to facilitate the recognition of these patients using this method. We also investigated the connection between myocardial fibrosis and conduction abnormalities. Methods Seventeen lamin A/C mutation carriers underwent CMR. Late gadolinium enhancement (LGE) and cine images were performed to evaluate myocardial fibrosis, regional wall motion, longitudinal myocardial function, global function and volumetry of both ventricles. The location, pattern and extent of enhancement in the left ventricle (LV) myocardium were visually estimated. Results Patients had LV myocardial fibrosis in 88% of cases. Segmental wall motion abnormalities correlated strongly with the degree of enhancement. Myocardial enhancement was associated with conduction abnormalities. Sixty-nine percent of our asymptomatic or mildly symptomatic patients showed mild ventricular dilatation, systolic failure or both in global ventricular analysis. Decreased longitudinal systolic LV function was observed in 53% of patients. Conclusions Cardiac conduction abnormalities, mildly dilated LV and depressed systolic dysfunction are common in DCM caused by a lamin A/C gene mutation. However, other cardiac diseases may produce similar symptoms. CMR is an accurate tool to determine the typical cardiac involvement in lamin A/C cardiomyopathy and may help to initiate early treatment in this malignant familiar form of DCM. PMID:21689390

  12. Identification of neuronal target genes for CCAAT/Enhancer Binding Proteins

    PubMed Central

    Kfoury, N.; Kapatos, G.

    2009-01-01

    CCAAT/Enhancer Binding Proteins (C/EBPs) play pivotal roles in development and plasticity of the nervous system. Identification of the physiological targets of C/EBPs (C/EBP target genes) should therefore provide insight into the underlying biology of these processes. We used unbiased genome-wide mapping to identify 115 C/EBPβ target genes in PC12 cells that include transcription factors, neurotransmitter receptors, ion channels, protein kinases and synaptic vesicle proteins. C/EBPβ binding sites were located primarily within introns, suggesting novel regulatory functions, and were associated with binding sites for other developmentally important transcription factors. Experiments using dominant negatives showed C/EBPβ to repress transcription of a subset of target genes. Target genes in rat brain were subsequently found to preferentially bind C/EBPα, β and δ. Analysis of the hippocampal transcriptome of C/EBPβ knockout mice revealed dysregulation of a high percentage of transcripts identified as C/EBP target genes. These results support the hypothesis that C/EBPs play non-redundant roles in the brain. PMID:19103292

  13. Enhancing hydrogen production of Enterobacter aerogenes by heterologous expression of hydrogenase genes originated from Synechocystis sp.

    PubMed

    Song, Wenlu; Cheng, Jun; Zhao, Jinfang; Zhang, Chuanxi; Zhou, Junhu; Cen, Kefa

    2016-09-01

    The hydrogenase genes (hoxEFUYH) of Synechocystis sp. PCC 6803 were cloned and heterologously expressed in Enterobacter aerogenes ATCC13408 for the first time in this study, and the hydrogen yield was significantly enhanced using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into E. aerogenes ATCC13408 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the hox genes. The hydrogenase activity of the recombinant strain is 237.6±9.3ml/(g-DW·h), which is 152% higher than the wild strain. The hydrogen yield of the recombinant strain is 298.3ml/g-glucose, which is 88% higher than the wild strain. During hydrogen fermentation, the recombinant strain produces more acetate and butyrate, but less ethanol. This is corresponding to the NADH metabolism in the cell due to the higher hydrogenase activity with the heterologous expression of hox genes.

  14. Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize.

    PubMed

    Li, Ning; Zhang, Shujuan; Zhao, Yajie; Li, Bei; Zhang, Juren

    2011-02-01

    Cereal crops accumulate starch in the seed endosperm as an energy reserve. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds. The AGPase in the maize endosperm is a heterotetramer of two small subunits, encoded by Brittle2 (Bt2) gene, and two large subunits, encoded by the Shrunken2 (Sh2) gene. The two genes (Bt2, Sh2) from maize were introduced into two elite maize inbred lines, solely and in tandem, and under the control of endosperm-specific promoters for over-expression. PCR, Southern blotting, and real-time RT-PCR analysis indicated that the transgenes were integrated into the genome of transgenic plants and were over-expressed in their progeny. The over-expression of either gene enhanced AGPase activity, seed weight and starch content compared with the WT, but the amounts were lower than plants with over-expression of both Bt2 and Sh2. Developing seeds from co-expression transgenic maize plants had higher cytoplasmic AGPase activity: the 100-grain weight increased 15% over the wild type (WT), and the starch content increased to over 74% compared with the WT of 65%. These results indicate that over-expression of the genes in transgenic maize plants could improve kernel traits. This report provides a feasible approach for increasing starch content and seed weight in maize.

  15. Stem cell-based gene therapy activated using magnetic hyperthermia to enhance the treatment of cancer.

    PubMed

    Yin, Perry T; Shah, Shreyas; Pasquale, Nicholas J; Garbuzenko, Olga B; Minko, Tamara; Lee, Ki-Bum

    2016-03-01

    Stem cell-based gene therapies, wherein stem cells are genetically engineered to express therapeutic molecules, have shown tremendous potential for cancer applications owing to their innate ability to home to tumors. However, traditional stem cell-based gene therapies are hampered by our current inability to control when the therapeutic genes are actually turned on, thereby resulting in detrimental side effects. Here, we report the novel application of magnetic core-shell nanoparticles for the dual purpose of delivering and activating a heat-inducible gene vector that encodes TNF-related apoptosis-inducing ligand (TRAIL) in adipose-derived mesenchymal stem cells (AD-MSCs). By combining the tumor tropism of the AD-MSCs with the spatiotemporal MCNP-based delivery and activation of TRAIL expression, this platform provides an attractive means with which to enhance our control over the activation of stem cell-based gene therapies. In particular, we found that these engineered AD-MSCs retained their innate ability to proliferate, differentiate, and, most importantly, home to tumors, making them ideal cellular carriers. Moreover, exposure of the engineered AD-MSCS to mild magnetic hyperthermia resulted in the selective expression of TRAIL from the engineered AD-MSCs and, as a result, induced significant ovarian cancer cell death in vitro and in vivo.

  16. Electrical stimulation and testosterone differentially enhance expression of regeneration-associated genes.

    PubMed

    Sharma, Nijee; Marzo, Sam J; Jones, Kathryn J; Foecking, Eileen M

    2010-05-01

    As functional recovery following peripheral nerve injury is dependent upon successful repair and regeneration, treatments that enhance different regenerative events may be advantageous. Using a rat facial nerve crush axotomy model, our lab has previously investigated the effects of a combinatorial treatment strategy, consisting of electrical stimulation (ES) of the proximal nerve stump and testosterone propionate (TP) administration. Results indicated that the two treatments differentially enhance facial nerve regenerative properties, whereby ES reduced the delay before sprout formation, TP accelerated the overall regeneration rate, and the combinatorial treatment had additive effects. To delineate the molecular mechanisms underlying such treatments, the present study investigated the effects of ES and TP on expression of specific regeneration-associated genes. Following a right facial nerve crush at the stylomastoid foramen, gonadectomized adult male rats were administered only ES, only TP, a combination of both, or left untreated. Real time RT-PCR analysis was used to assess fold changes in mRNA levels in the facial motor nucleus at 0 h, 6 h, 1 d, 2 d, 7 d, and 21 d post-axotomy. The candidate genes analyzed included two tubulin isoforms (alpha(1)-tubulin and beta(II)-tubulin), 43-kiloDalton growth-associated protein (GAP-43), brain derived neurotrophic factor (BDNF), pituitary adenylate cyclase-activating peptide (PACAP), and neuritin (candidate plasticity-related gene 15). The two treatments have differential effects on gene expression, with ES leading to early but transient upregulation and TP producing late but steady increases in mRNA levels. In comparison to individual treatments, the combinatorial treatment strategy has the most enhanced effects on the transcriptional program activated following injury.

  17. Methylation of tumour suppressor gene promoters in the presence and absence of transcriptional silencing in high hyperdiploid acute lymphoblastic leukaemia.

    PubMed

    Paulsson, Kajsa; An, Qian; Moorman, Anthony V; Parker, Helen; Molloy, Gael; Davies, Teresa; Griffiths, Mike; Ross, Fiona M; Irving, Julie; Harrison, Christine J; Young, Bryan D; Strefford, Jon C

    2009-03-01

    Promoter methylation is a common phenomenon in tumours, including haematological malignancies. In the present study, we investigated 36 cases of high hyperdiploid (>50 chromosomes) acute lymphoblastic leukaemia (ALL) with methylation-specific multiplex ligase-dependent probe amplification to determine the extent of aberrant methylation in this subgroup. The analysis, which comprised the promoters of 35 known tumour suppressor genes, showed that 16 genes displayed abnormal methylation in at least one case each. The highest number of methylated gene promoters seen in a single case was thirteen, with all but one case displaying methylation for at least one gene. The most common targets were ESR1 (29/36 cases; 81%), CADM1 (IGSF4, TSLC1; 25/36 cases; 69%), FHIT (24/36 cases; 67%) and RARB (22/36 cases; 61%). Interestingly, quantitative reverse transcription-polymerase chain reaction showed that although methylation of the CADM1 and RARB promoters resulted in the expected pattern of downregulation of the respective genes, no difference could be detected in FHIT expression between methylation-positive and -negative cases. Furthermore, TIMP3 was not expressed regardless of methylation status, showing that aberrant methylation does not always lead to gene expression changes. Taken together, our findings suggest that aberrant methylation of tumour suppressor gene promoters is a common phenomenon in high hyperdiploid ALL.

  18. Expression of myocyte enhancer factor-2 and downstream genes in ground squirrel skeletal muscle during hibernation.

    PubMed

    Tessier, Shannon N; Storey, Kenneth B

    2010-11-01

    Myocyte enhancer factor-2 (MEF2) transcription factors regulate the expression of a variety of genes encoding contractile proteins and other proteins associated with muscle performance. We proposed that changes in MEF2 levels and expression of selected downstream targets would aid the skeletal muscle of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) in meeting metabolic challenges associated with winter hibernation; e.g., cycles of torpor-arousal, body temperature that can fall to near 0°C, long periods of inactivity that could lead to atrophy. MEF2A protein levels were significantly elevated when animals were in torpor (maximally 2.8-fold higher than in active squirrels) and the amount of phosphorylated active MEF2A Thr312 increased during entrance into torpor. MEF2C levels also rose significantly during entrance and torpor as did the amount of phosphorylated MEF2C Ser387. Furthermore, both MEF2 members showed elevated amounts in the nuclear fraction during torpor as well as enhanced binding to DNA indicating that MEF2-mediated gene expression was up-regulated in torpid animals. Indeed, the protein products of two MEF2 downstream gene targets increased in muscle during torpor (glucose transporter isoforms 4; GLUT4) or early arousal (myogenic differentiation; MyoD). Significant increases in Glut4 and MyoD mRNA transcript levels correlated with the rise in protein product levels and provided further support for the activation of MEF2-mediated gene expression in the hibernator. Transcript levels of Mef2a and Mef2c also showed time-dependent patterns with levels of both being highest during arousal from torpor. The data suggest a significant role for MEF2-mediated gene transcription in the selective adjustment of muscle protein complement over the course of torpor-arousal cycles.

  19. Exploration of the Brn4-regulated genes enhancing adult hippocampal neurogenesis by RNA sequencing.

    PubMed

    Guo, Jingjing; Cheng, Xiang; Zhang, Lei; Wang, Linmei; Mao, Yongxin; Tian, Guixiang; Xu, Wenhao; Wu, Yuhao; Ma, Zhi; Qin, Jianbing; Tian, Meiling; Jin, Guohua; Shi, Wei; Zhang, Xinhua

    2017-02-18

    Adult hippocampal neurogenesis is essential for learning and memory, and its dysfunction is involved in neurodegenerative diseases. However, the molecular mechanisms underlying adult hippocampal neurogenesis are still largely unknown. Our previous studies indicated that the transcription factor Brn4 was upregulated and promoted neuronal differentiation of neural stem cells (NSCs) in the surgically denervated hippocampus in rats. In this study, we use high-throughput RNA sequencing to explore the molecular mechanisms underlying the enhancement of adult hippocampal neurogenesis induced by lentivirus-mediated Brn4 overexpression in vivo. After 10 days of the lentivirus injection, we found that the expression levels of genes related to neuronal development and maturation were significantly increased and the expression levels of genes related to NSC maintenance were significantly decreased, indicating enhanced neurogenesis in the hippocampus after Brn4 overexpression. Through RNA sequencing, we found that 658 genes were differentially expressed in the Brn4-overexpressed hippocampi compared with GFP-overexpressed controls. Many of these differentially expressed genes are involved in NSC division and differentiation. By using quantitative real-time PCR, we validated the expression changes of three genes, including Ctbp2, Notch2, and Gli1, all of which are reported to play key roles in neuronal differentiation of NSCs. Importantly, the expression levels of Ctbp2 and Notch2 were also significantly changed in the hippocampus of Brn4 KO mice, which indicates that the expression levels of Ctbp2 and Notch2 may be directly regulated by Brn4. Our current study provides a solid foundation for further investigation and identifies Ctbp2 and Notch2 as possible downstream targets of Brn4. © 2017 Wiley Periodicals, Inc.

  20. Addiction-Related Gene Regulation: Risks of Exposure to Cognitive Enhancers vs. Other Psychostimulants

    PubMed Central

    Steiner, Heinz; Van Waes, Vincent

    2012-01-01

    The psychostimulants methylphenidate (Ritalin, Concerta), amphetamine (Adderall), and modafinil (Provigil) are widely used in the treatment of medical conditions such as attention-deficit hyperactivity disorder and narcolepsy and, increasingly, as “cognitive enhancers” by healthy people. The long-term neuronal effects of these drugs, however, are poorly understood. A substantial amount of research over the past 2 decades has investigated the effects of psychostimulants such as cocaine and amphetamines on gene regulation in the brain because these molecular changes are considered critical for psychostimulant addiction. This work has determined in some detail the neurochemical and cellular mechanisms that mediate psychostimulant-induced gene regulation and has also identified the neuronal systems altered by these drugs. Among the most affected brain systems are corticostriatal circuits, which are part of cortico-basal ganglia-cortical loops that mediate motivated behavior. The neurotransmitters critical for such gene regulation are dopamine in interaction with glutamate, while other neurotransmitters (e.g., serotonin) play modulatory roles. This review presents (1) an overview of the main findings on cocaine- and amphetamine-induced gene regulation in corticostriatal circuits in an effort to provide a cellular framework for (2) an assessment of the molecular changes produced by methylphenidate, medical amphetamine (Adderall), and modafinil. The findings lead to the conclusion that protracted exposure to these cognitive enhancers can induce gene regulation effects in corticostriatal circuits that are qualitatively similar to those of cocaine and other amphetamines. These neuronal changes may contribute to the addiction liability of the psychostimulant cognitive enhancers. PMID:23085425

  1. Use of staurosporine, an actin-modifying agent, to enhance fibrochondrocyte matrix gene expression and synthesis.

    PubMed

    Hoben, Gwendolyn M; Athanasiou, Kyriacos A

    2008-12-01

    Modulation of the actin cytoskeleton in chondrocytes has been used to prevent or reverse dedifferentiation and to enhance protein synthesis. We have hypothesized that an actin-modifying agent, staurosporine, could be used with fibrochondrocytes to increase the gene expression and synthesis of critical fibrocartilage proteins. A range of concentrations (0.1-100 nM) was applied to fibrochondrocytes in monolayer and evaluated after 24 h and after 4 days. High-dose staurosporine treatment (10-100 nM) increased cartilage oligomeric matrix protein 60- to 500-fold and aggrecan gene expression two-fold. This effective range of staurosporine was then applied to scaffoldless tissue-engineered fibrochondrocyte constructs for 4 weeks. Whereas glycosaminoglycan synthesis was not affected, collagen content doubled, from 27.6 +/- 8.8 microg in the untreated constructs to 55.2 +/- 12.2 microg per construct with 100 nM treatment. When analyzed for specific collagens, the 10-nM group showed a significant increase in collagen type I content, whereas collagen type II was unaffected. A concomitant dose-dependent reduction was noted in construct contraction, reflecting the actin-disrupting action of staurosporine. Thus, staurosporine increases the gene expression for important matrix proteins and can be used to enhance matrix production and reduce contraction in tissue-engineered fibrocartilage constructs.

  2. A novel α/β-hydrolase gene IbMas enhances salt tolerance in transgenic sweetpotato.

    PubMed

    Liu, Degao; Wang, Lianjun; Zhai, Hong; Song, Xuejin; He, Shaozhen; Liu, Qingchang

    2014-01-01

    Salt stress is one of the major environmental stresses in agriculture worldwide and affects crop productivity and quality. The development of crops with elevated levels of salt tolerance is therefore highly desirable. In the present study, a novel maspardin gene, named IbMas, was isolated from salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line ND98. IbMas contains maspardin domain and belongs to α/β-hydrolase superfamily. Expression of IbMas was up-regulated in sweetpotato under salt stress and ABA treatment. The IbMas-overexpressing sweetpotato (cv. Shangshu 19) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content was significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbMas up-regulated the salt stress responsive genes, including pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductase, SOD, psbA and phosphoribulokinase genes, under salt stress. These findings suggest that overexpression of IbMas enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and increasing reactive oxygen species scavenging capacity.

  3. Magnetofection Enhances Adenoviral Vector-based Gene Delivery in Skeletal Muscle Cells

    PubMed Central

    Pereyra, Andrea Soledad; Mykhaylyk, Olga; Lockhart, Eugenia Falomir; Taylor, Jackson Richard; Delbono, Osvaldo; Goya, Rodolfo Gustavo; Plank, Christian; Hereñu, Claudia Beatriz

    2016-01-01

    The goal of magnetic field-assisted gene transfer is to enhance internalization of exogenous nucleic acids by association with magnetic nanoparticles (MNPs). This technique named magnetofection is particularly useful in difficult-to-transfect cells. It is well known that human, mouse, and rat skeletal muscle cells suffer a maturation-dependent loss of susceptibility to Recombinant Adenoviral vector (RAd) uptake. In postnatal, fully differentiated myofibers, the expression of the primary Coxsackie and Adenoviral membrane receptor (CAR) is severely downregulated representing a main hurdle for the use of these vectors in gene transfer/therapy. Here we demonstrate that assembling of Recombinant Adenoviral vectors with suitable iron oxide MNPs into magneto-adenovectors (RAd-MNP) and further exposure to a gradient magnetic field enables to efficiently overcome transduction resistance in skeletal muscle cells. Expression of Green Fluorescent Protein and Insulin-like Growth Factor 1 was significantly enhanced after magnetofection with RAd-MNPs complexes in C2C12 myotubes in vitro and mouse skeletal muscle in vivo when compared to transduction with naked virus. These results provide evidence that magnetofection, mainly due to its membrane-receptor independent mechanism, constitutes a simple and effective alternative to current methods for gene transfer into traditionally hard-to-transfect biological models. PMID:27274908

  4. A Novel α/β-Hydrolase Gene IbMas Enhances Salt Tolerance in Transgenic Sweetpotato

    PubMed Central

    Song, Xuejin; He, Shaozhen; Liu, Qingchang

    2014-01-01

    Salt stress is one of the major environmental stresses in agriculture worldwide and affects crop productivity and quality. The development of crops with elevated levels of salt tolerance is therefore highly desirable. In the present study, a novel maspardin gene, named IbMas, was isolated from salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line ND98. IbMas contains maspardin domain and belongs to α/β-hydrolase superfamily. Expression of IbMas was up-regulated in sweetpotato under salt stress and ABA treatment. The IbMas-overexpressing sweetpotato (cv. Shangshu 19) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content was significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbMas up-regulated the salt stress responsive genes, including pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductase, SOD, psbA and phosphoribulokinase genes, under salt stress. These findings suggest that overexpression of IbMas enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and increasing reactive oxygen species scavenging capacity. PMID:25501819

  5. Magnetofection Enhances Adenoviral Vector-based Gene Delivery in Skeletal Muscle Cells.

    PubMed

    Pereyra, Andrea Soledad; Mykhaylyk, Olga; Lockhart, Eugenia Falomir; Taylor, Jackson Richard; Delbono, Osvaldo; Goya, Rodolfo Gustavo; Plank, Christian; Hereñu, Claudia Beatriz

    2016-04-01

    The goal of magnetic field-assisted gene transfer is to enhance internalization of exogenous nucleic acids by association with magnetic nanoparticles (MNPs). This technique named magnetofection is particularly useful in difficult-to-transfect cells. It is well known that human, mouse, and rat skeletal muscle cells suffer a maturation-dependent loss of susceptibility to Recombinant Adenoviral vector (RAd) uptake. In postnatal, fully differentiated myofibers, the expression of the primary Coxsackie and Adenoviral membrane receptor (CAR) is severely downregulated representing a main hurdle for the use of these vectors in gene transfer/therapy. Here we demonstrate that assembling of Recombinant Adenoviral vectors with suitable iron oxide MNPs into magneto-adenovectors (RAd-MNP) and further exposure to a gradient magnetic field enables to efficiently overcome transduction resistance in skeletal muscle cells. Expression of Green Fluorescent Protein and Insulin-like Growth Factor 1 was significantly enhanced after magnetofection with RAd-MNPs complexes in C2C12 myotubes in vitro and mouse skeletal muscle in vivo when compared to transduction with naked virus. These results provide evidence that magnetofection, mainly due to its membrane-receptor independent mechanism, constitutes a simple and effective alternative to current methods for gene transfer into traditionally hard-to-transfect biological models.

  6. Pre-breeding for diversification of primary gene pool and genetic enhancement of grain legumes

    PubMed Central

    Sharma, Shivali; Upadhyaya, H. D.; Varshney, R. K.; Gowda, C. L. L.

    2013-01-01

    The narrow genetic base of cultivars coupled with low utilization of genetic resources are the major factors limiting grain legume production and productivity globally. Exploitation of new and diverse sources of variation is needed for the genetic enhancement of grain legumes. Wild relatives with enhanced levels of resistance/tolerance to multiple stresses provide important sources of genetic diversity for crop improvement. However, their exploitation for cultivar improvement is limited by cross-incompatibility barriers and linkage drags. Pre-breeding provides a unique opportunity, through the introgression of desirable genes from wild germplasm into genetic backgrounds readily used by the breeders with minimum linkage drag, to overcome this. Pre-breeding activities using promising landraces, wild relatives, and popular cultivars have been initiated at International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) to develop new gene pools in chickpea, pigeonpea, and groundnut with a high frequency of useful genes, wider adaptability, and a broad genetic base. The availability of molecular markers will greatly assist in reducing linkage drags and increasing the efficiency of introgression in pre-breeding programs. PMID:23970889

  7. Single-nucleotide polymorphisms and activity analysis of the promoter and enhancer of the pig lactase gene.

    PubMed

    Du, Hai-Ting; Zhu, Hong-Yan; Wang, Jia-Mei; Zhao, Wei; Tao, Xiao-Li; Ba, Cai-Feng; Tian, Yu-Min; Su, Yu-Hong

    2014-07-15

    Lactose intolerance in northern Europeans is strongly associated with a single-nucleotide polymorphism (SNP) located 14 kb upstream of the human lactase gene: -13,910 C/T. We examined whether SNPs in the 5' flanking region of the pig lactase gene are similar to those in the human gene and whether these polymorphisms play a functional role in regulating pig lactase gene expression. The 5' flanking region of the lactase gene from several different breeds of pigs was cloned and analyzed for gene regulatory activity of a luciferase reporter gene. One SNP was found in the enhancer region (-797 G/A) and two were found in the promoter region (-308G/C and -301 A/G). The promoter C-308,G-301(Pro-CG) strongly promotes the expression of the lactase gene, but the promoter G-308,A-301(Pro-GA) does not. The enhancer A-797(Enh-A) genotype for Pro-GA can significantly enhance promoter activity, but has an inhibitory effect on Pro-CG. The Enhancer G-797(Enh-G) has a significant inhibitory effect on both promoters. In conclusion, the order of effectiveness on the pig lactase gene is Enh-A+Pro-GA>Enh-A/G+Pro-CG>Enh-G+Pro-GA.

  8. Otx2 is a putative candidate to activate mice Msx1 gene from distal enhancer

    SciTech Connect

    Binato, Renata . E-mail: rebinato@biof.ufrj.br; Pizzatti, Luciana; Abdelhay, Eliana

    2007-06-29

    A comparative analysis between sequences of Msx1 promoter gene from human, mouse, and fugu allowed us to identify sequences highly conserved among these animals. One of the regions of great homology is localized between the positions -4622 and -4572, including the region described as distal enhancer. In this region putative transcription factors binding sites for Nkx2.5, CTF-CBP, Bicoid, Brn2, and Oct were found. To evaluate the functionality of these sites we performed EMSA analysis using two different regions from the distal enhancer and nuclear protein extracts from embryos. The results showed that in the presence of a Bicoid consensus binding site a DNA-protein complex can be formed. The identification of the proteins involved in this complex by mass spectrometry and Western blotting identified OTX2, a Bicoid-like protein. This protein was shown to be present in nuclear extracts of the embryonic stages analyzed by Western blot. Altogether these results suggest that OTX2 is a putative candidate to activate mice Msx1 gene from distal enhancer.

  9. Transgenic Alfalfa Plants Expressing the Sweetpotato Orange Gene Exhibit Enhanced Abiotic Stress Tolerance

    PubMed Central

    Wang, Zhi; Ke, Qingbo; Kim, Myoung Duck; Kim, Sun Ha; Ji, Chang Yoon; Jeong, Jae Cheol; Lee, Haeng-Soon; Park, Woo Sung; Ahn, Mi-Jeong; Li, Hongbing; Xu, Bingcheng; Deng, Xiping; Lee, Sang-Hoon; Lim, Yong Pyo; Kwak, Sang-Soo

    2015-01-01

    Alfalfa (Medicago sativa L.), a perennial forage crop with high nutritional content, is widely distributed in various environments worldwide. We recently demonstrated that the sweetpotato Orange gene (IbOr) is involved in increasing carotenoid accumulation and enhancing resistance to multiple abiotic stresses. In this study, in an effort to improve the nutritional quality and environmental stress tolerance of alfalfa, we transferred the IbOr gene into alfalfa (cv. Xinjiang Daye) under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter through Agrobacterium tumefaciens-mediated transformation. Among the 11 transgenic alfalfa lines (referred to as SOR plants), three lines (SOR2, SOR3, and SOR8) selected based on their IbOr transcript levels were examined for their tolerance to methyl viologen (MV)-induced oxidative stress in a leaf disc assay. The SOR plants exhibited less damage in response to MV-mediated oxidative stress and salt stress than non-transgenic plants. The SOR plants also exhibited enhanced tolerance to drought stress, along with higher total carotenoid levels. The results suggest that SOR alfalfa plants would be useful as forage crops with improved nutritional value and increased tolerance to multiple abiotic stresses, which would enhance the development of sustainable agriculture on marginal lands. PMID:25946429

  10. CCL2 enhances pluripotency of human induced pluripotent stem cells by activating hypoxia related genes.

    PubMed

    Hasegawa, Yuki; Tang, Dave; Takahashi, Naoko; Hayashizaki, Yoshihide; Forrest, Alistair R R; Suzuki, Harukazu

    2014-06-24

    Standard culture of human induced pluripotent stem cells (hiPSCs) requires basic Fibroblast Growth Factor (bFGF) to maintain the pluripotent state, whereas hiPSC more closely resemble epiblast stem cells than true naïve state ES which requires LIF to maintain pluripotency. Here we show that chemokine (C-C motif) ligand 2 (CCL2) enhances the expression of pluripotent marker genes through the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) protein. Moreover, comparison of transcriptomes between hiPSCs cultured with CCL2 versus with bFGF, we found that CCL2 activates hypoxia related genes, suggesting that CCL2 enhanced pluripotency by inducing a hypoxic-like response.Further, we show that hiPSCs cultured with CCL2 can differentiate at a higher efficiency than culturing withjust bFGF and we show CCL2 can be used in feeder-free conditions [corrected]. Taken together, our finding indicates the novel functions of CCL2 in enhancing its pluripotency in hiPSCs.

  11. Enhanced antinociceptive effects of morphine in histamine H2 receptor gene knockout mice.

    PubMed

    Mobarakeh, Jalal Izadi; Takahashi, Kazuhiro; Sakurada, Shinobu; Kuramasu, Atsuo; Yanai, Kazuhiko

    2006-09-01

    We have previously shown that antinociceptive effects of morphine are enhanced in histamine H1 receptor gene knockout mice. In the present study, involvement of supraspinal histamine H2 receptor in antinociception by morphine was examined using histamine H2 receptor gene knockout (H2KO) mice and histamine H2 receptor antagonists. Antinociception was evaluated by assays for thermal (hot-plate, tail-flick and paw-withdrawal tests), mechanical (tail-pressure test) and chemical (formalin and capsaicin tests) stimuli. Thresholds for pain perception in H2KO mice were higher than wild-type mice. Antinociceptive effects of intracerebroventricularly administered morphine were enhanced in the H2KO mice compared to wild-type mice. Intracerebroventricular co-administration of morphine and cimetidine produced significant antinociceptive effects in the wild-type mice when compared to morphine or cimetidine alone. Furthermore, zolantidine, a selective and hydrophobic H2 receptor antagonist, enhanced the effects of morphine in all nociceptive assays examined. These results suggest that histamine exerts inhibitory effects on morphine-induced antinociception through H2 receptors at the supraspinal level. Our present and previous studies suggest that H1 and H2 receptors cooperatively function to modulate pain perception in the central nervous system.

  12. All-trans retinoic acid enhances bystander effect of suicide gene therapy in the treatment of breast cancer.

    PubMed

    Kong, Heng; Liu, Xia; Yang, Liucheng; Qi, Ke; Zhang, Haoyun; Zhang, Jingwen; Huang, Zonghai; Wang, Hongxian

    2016-03-01

    All-trans retinoic acid (ATRA) has been shown to enhance the expression of connexin 43 (Cx43) and the bystander effect (BSE) in suicide gene therapy. These in turn improve effects of suicide gene therapies for several tumor types. However, whether ATRA can improve BSE remains unclear in suicide gene therapy for breast cancer. In the present study, MCF-7, human breast cancer cells were treated with ATRA in combination with a VEGFP-TK/CD gene suicide system developed by our group. We found that this combination enhances the efficiency of cell killing and apoptosis of breast cancer by strengthening the BSE in vitro. ATRA also promotes gap junction intercellular communication (GJIC) in MCF-7 cells by upregulation of the connexin 43 mRNA and protein in MCF-7 cells. These results indicate that enhancement of GJIC by ATRA in suicide gene system might serve as an attractive and cost-effective strategy of therapy for breast cancer cells.

  13. Advanced surface-enhanced Raman gene probe systems and methods thereof

    DOEpatents

    Vo-Dinh, Tuan

    2001-01-01

    The subject invention is a series of methods and systems for using the Surface-Enhanced Raman (SER)-labeled Gene Probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide material comprising the steps of immobilizing SER-labeled hybridized target oligonucleotide material on a support means, wherein the SER-labeled hybridized target oligonucleotide material comprise a SER label attached either to a target oligonucleotide of unknown sequence or to a gene probe of known sequence complementary to the target oligonucleotide sequence, the SER label is unique for the target oligonucleotide strands of a particular sequence wherein the SER-labeled oligonucleotide is hybridized to its complementary oligonucleotide strand, then the support means having the SER-labeled hybridized target oligonucleotide material adsorbed thereon is SERS activated with a SERS activating means, then the support means is analyzed.

  14. Comparation of enhanced green fluorescent protein gene transfected and wild-type porcine neural stem cells.

    PubMed

    Zheng, Yue-Mao; An, Zhi-Xing; Zhao, Xiao-E; Quan, Fu-Sheng; Zhao, Hui-Ying; Zhang, Ya-Rong; Liu, Jun; He, Xiao-Ying; He, Xiao-Ning

    2010-02-01

    The aim of this study was to transfect and express the enhanced green fluorescence protein (EGFP) gene into porcine neural stem cells (NSCs) to determine whether EGFP can be used as a marker to monitor NSCs. NSCs were isolated from embryonic day 30 fetal pig brain and transfected with EGFP gene using lipofection. Transfected and wild-type NSCs were induced to differentiate into cells of neuronal and myogenic lineages. Markers of passage three NSCs and their differentiated cells were tested by reverse transcription polymerase chain reaction. The results showed that EGFP could be expressed in NSCs and the differentiated cells. NSCs expressed Nestin, NogoA, DCX, Hes1, Oct4, CD-90 and Sox2. NSCs could differentiated into astrocyte (GFAP(+)), oligodendrocyte (GalC(+)), neuron (NF(+), NSE(+) and MAP2(+)) and myocyte (myf-6(+) and myoD(+)). We concluded that EGFP can be used as a marker in monitoring NSCs.

  15. Matrix immobilization enhances the tissue repair activity of growth factor gene therapy vectors.

    PubMed

    Doukas, J; Chandler, L A; Gonzalez, A M; Gu, D; Hoganson, D K; Ma, C; Nguyen, T; Printz, M A; Nesbit, M; Herlyn, M; Crombleholme, T M; Aukerman, S L; Sosnowski, B A; Pierce, G F

    2001-05-01

    Although growth factor proteins display potent tissue repair activities, difficulty in sustaining localized therapeutic concentrations limits their therapeutic activity. We reasoned that enhanced histogenesis might be achieved by combining growth factor genes with biocompatible matrices capable of immobilizing vectors at delivery sites. When delivered to subcutaneously implanted sponges, a platelet-derived growth factor B-encoding adenovirus (AdPDGF-B) formulated in a collagen matrix enhanced granulation tissue deposition 3- to 4-fold (p < or = 0.0002), whereas vectors encoding fibroblast growth factor 2 or vascular endothelial growth factor promoted primarily angiogenic responses. By day 8 posttreatment of ischemic excisional wounds, collagen-formulated AdPDGF-B enhanced granulation tissue and epithelial areas up to 13- and 6-fold (p < 0.009), respectively, and wound closure up to 2-fold (p < 0.05). At longer times, complete healing without excessive scar formation was achieved. Collagen matrices were shown to retain both vector and transgene products within delivery sites, enabling the transduction and stimulation of infiltrating repair cells. Quantitative PCR and RT-PCR demonstrated both vector DNA and transgene mRNA within wound beds as late as 28 days posttreatment. By contrast, aqueous formulations allowed vector seepage from application sites, leading to PDGF-induced hyperplasia in surrounding tissues but not wound beds. Finally, repeated applications of PDGF-BB protein were required for neotissue induction approaching equivalence to a single application of collagen-immobilized AdPDGF-B, confirming the utility of this gene transfer approach. Overall, these studies demonstrate that immobilizing matrices enable the controlled delivery and activity of tissue promoting genes for the effective regeneration of injured tissues.

  16. Triptolide T10 enhances AAV-mediated gene transfer in mice striatum.

    PubMed

    Ren, Xinmiao; Zhang, Ting; Hu, Jing; Ding, Wei; Wang, Xiaomin

    2010-08-02

    Adeno-associated virus (AAV) mediated gene transfer has been demonstrated to be an effective approach for treating Parkinson's disease (PD). Triptolide T10 is a monomeric compound isolated from tripterygium wilfordii Hook.f. (Thunder God vine), a traditional Chinese herb for anti-inflammatory medications. In the present study, we co-administered T10 with recombinant AAV2 in SH-SY5Y human neuroblastoma cells and in the striatum of C57BL/6 mice, and then evaluated the AAV-mediated gene expression levels. The results have shown that T10 significantly augmented the expression of AAV-mediated gene in a dose-dependent fashion without detectable cytotoxicity. As growing evidence indicated that inflammation contributed to the progression of PD, and the anti-inflammatory effect of T10 was shown in our previous studies, our data of T10 to enhance AAV transduction suggest that T10 might be potentially used as a facilitating reagent for the AAV gene therapy applications in neurodegenerative diseases.

  17. Magnetic nanoparticles as gene delivery agents: enhanced transfection in the presence of oscillating magnet arrays

    NASA Astrophysics Data System (ADS)

    McBain, S. C.; Griesenbach, U.; Xenariou, S.; Keramane, A.; Batich, C. D.; Alton, E. W. F. W.; Dobson, J.

    2008-10-01

    Magnetic nanoparticle-based gene transfection has been shown to be effective in combination with both viral vectors and with non-viral agents. In these systems, therapeutic or reporter genes are attached to magnetic nanoparticles which are then focused to the target site/cells via high-field/high-gradient magnets. The technique has been shown to be efficient and rapid for in vitro transfection and compares well with cationic lipid-based reagents, producing good overall transfection levels with lower doses and shorter transfection times. In spite of its potential advantages (particularly for in vivo targeting), the overall transfection levels do not generally exceed those of other non-viral agents. In order to improve the overall transfection levels while maintaining the advantages inherent in this technique, we have developed a novel, oscillating magnet array system which adds lateral motion to the particle/gene complex in order to promote transfection. Experimental results indicate that the system significantly enhances overall in vitro transfection levels in human airway epithelial cells compared to both static field techniques (p<0.005) and the cationic lipids (p<0.001) tested. In addition, it has the previously demonstrated advantages of magnetofection—rapid transfection times and requiring lower levels of DNA than cationic lipid-based transfection agents. This method shows potential for non-viral gene delivery both in vitro and in vivo.

  18. Nurr1 enhances transcription of the human dopamine transporter gene through a novel mechanism.

    PubMed

    Sacchetti, P; Mitchell, T R; Granneman, J G; Bannon, M J

    2001-03-01

    The importance of the nuclear receptor nurr1 for the appropriate development of mesencephalic dopamine-synthesizing neurons has been clearly demonstrated through the targeted disruption of the nurr1 gene. The persistence of nurr1 expression in adult tissue suggests a possible role for this transcription factor in the maintenance, as well as development, of the dopaminergic phenotype. To address this issue, we analyzed the effects of nurr1 on the transcriptional expression of the human dopamine transporter gene (hDAT), one of the most specific phenotypic markers for dopaminergic neurons. Nurr1 enhanced the transcriptional activity of hDAT gene constructs transiently transfected into a newly described cell line (SN4741) that expresses a dopaminergic phenotype, whereas other members of the NGFI-B subfamily of nuclear receptors had lesser or no effects. Nurr1 activation of hDAT was not dependent upon heterodimerization with the retinoid X receptor. Unexpectedly, functional analysis of a series of gene constructs revealed that a region of the hDAT 5'-flanking sequence devoid of NGFI-B response element (NBRE)-like sites mediated nurr1 activation. Additional experiments using a nurr1 mutant construct suggest that nurr1 activates hDAT transcription via a novel NBRE-independent mechanism.

  19. TITER AND PRODUCT AFFECTS THE DISTRIBUTION OF GENE EXPRESSION AFTER INTRAPUTAMINAL CONVECTION-ENHANCED DELIVERY

    PubMed Central

    Emborg, Marina E.; Hurley, Samuel A.; Joers, Valerie; Tromp, Do P.M.; Swanson, Christine R.; Ohshima-Hosoyama, Sachiko; Bondarenko, Viktorya; Cummisford, Kyle; Sonnemans, Marc; Hermening, Stephan; Blits, Bas; Alexander, Andrew L.

    2014-01-01

    Background Efficacy and safety of intracerebral gene therapy for brain disorders, like Parkinson’s disease, depends on appropriate distribution of gene expression. Objectives To assess if the distribution of gene expression is affected by vector titer and protein type. Methods Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received in the right and left ventral postcommisural putamen 30μl inoculation of a high or low titer suspension of AAV5 encoding glial derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Inoculations were performed using convection enhanced delivery and intraoperative MRI (IMRI). Results IMRI confirmed targeting and infusion cloud irradiating from the catheter tip into surrounding area. Postmortem analysis six weeks after surgery revealed GFP and GDNF expression ipsilateral to the injection side that had a titer-dependent distribution. GFP and GDNF expression was also observed in fibers in the Substantia Nigra (SN) pars reticulata (pr), demonstrating anterograde transport. Few GFP-positive neurons were present in the SN pars compacta (pc), possibly by direct retrograde transport of the vector. GDNF was present in many SNpc and SNpr neurons. Conclusions After controlling for target and infusate volume, intracerebral distribution of gene product is affected by vector titer and product biology. PMID:24943657

  20. Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond.

    PubMed

    Lee, Soo-Jin; Yoon, Sung-Hwa; Doh, Kyung-Oh

    2011-08-01

    Cationic liposomes have been actively used as gene delivery vehicle because of their minimal toxicity, but their relatively low efficiency of gene delivery is the major disadvantage of these vectors. Recently, cysteine residue incorporation to HIV-1 Tat peptide increased liposomemediated transfection compared with unmodified Tat peptide. Therefore, we designed a novel modified Tat peptide having a homodimeric (Tat-CTHD, Tat-NTHD) and closed structure (cyclic Tat) simply by using the disulfide bond between cysteines to develop a more efficient and safe nonviral gene delivery system. The mixing of Tat-CTHD and Tat-NTHD with DNA before mixing with lipofectamine increased the transfection efficiency compared with unmodified Tat peptide and lipofectamine only in MCF-7 breast cancer cells and rat vascular smooth muscle cells. However, cyclic Tat did not show any improvement in the transfection efficiency. In the gel retardation assay, Tat-CTHD and Tat-NTHD showed more strong binding with DNA than unmodified Tat and cyclic Tat peptide. This enhancement was only shown when Tat-CTHD and Tat-NTHD were mixed with DNA before mixing with lipofectamine. The effects of Tat- CTHD and Tat-NTHD were also valid in the experiment using DOTAP and DMRIE instead of lipofectamine. We could not find any significant cytotoxicity in the working concentration and more usage of these peptides. In conclusion, we have designed a novel transfection-enhancing peptide by easy homodimerization of Tat peptide, and the simple mix of these novel peptides with DNA increased the gene transfer of cationic lipids more efficiently with no additional cytotoxicity.

  1. Bioreducible cross-linked nanoshell enhances gene transfection of polycation/DNA polyplex in vivo.

    PubMed

    Piao, Ji-Gang; Ding, Sheng-Gang; Yang, Lu; Hong, Chun-Yan; You, Ye-Zi

    2014-08-11

    In this study, we have prepared a self-cross-linking PEG-based branched polymer, which easily forms a bioreducible nanoshell around polyplexes of cationic polymer and DNA, simply via heating the polyplex dispersions in the presence of this self-cross-linking branched polymer. This nanoshell can prevent the polyplex from dissociation and aggregation in physiological fluids without inhibiting the electrostatic interactions between the polymer and DNA. Furthermore, glutathione (GSH) can act as a stimulus to open the nanoshell after it has entered the cell. The polyplexes coated with the bioreducible nanoshell show an obvious enhancement in gene transfection in vivo compared with bare polyplexes.

  2. The pkI gene encoding pyruvate kinase I links to the luxZ gene which enhances bioluminescence of the lux operon from Photobacterium leiognathi.

    PubMed

    Lin, J W; Lu, H C; Chen, H Y; Weng, S F

    1997-10-09

    Partial 3'-end nucleotide sequence of the pkI gene (GenBank accession No. AF019143) from Photobacterium leiognathi ATCC 25521 has been determined, and the encoded pyruvate kinase I is deduced. Pyruvate kinase I is the key enzyme of glycolysis, which converts phosphoenol pyruvate to pyruvate. Alignment and comparison of pyruvate kinase Is from P. leiognathi, E. coli and Salmonella typhimurium show that they are homologous. Nucleotide sequence reveals that the pkI gene is linked to the luxZ gene that enhances bioluminescence of the lux operon from P. leiognathi. The gene order of the pkI and luxZ genes is-pk1-ter-->-R&R"-luxZ-ter"-->, whereas ter is transcriptional terminator for the pkI and related genes, and R&R" is the regulatory region and ter" is transcriptional terminator for the luxZ gene. It clearly elicits that the pkI gene and luxZ gene are divided to two operons. Functional analysis confirms that the potential hairpin loop omega T is the transcriptional terminator for the pkI and related genes. It infers that the pkI and related genes are simply linked to the luxZ gene in P. leiognathi genome.

  3. Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice.

    PubMed

    Lee, Hye Kyung; Willi, Michaela; Wang, Chaochen; Yang, Chul Min; Smith, Harold E; Liu, Chengyu; Hennighausen, Lothar

    2017-03-16

    The zinc finger protein CTCF has been invoked in establishing boundaries between genes, thereby controlling spatial and temporal enhancer activities. However, there is limited genetic evidence to support the concept that these boundaries restrict the search space of enhancers. We have addressed this question in the casein locus containing five mammary and two non-mammary genes under the control of at least seven putative enhancers. We have identified two CTCF binding sites flanking the locus and two associated with a super-enhancer. Individual deletion of these sites from the mouse genome did not alter expression of any of the genes. However, deletion of the border CTCF site separating the Csn1s1 mammary enhancer from neighboring genes resulted in the activation of Sult1d1 at a distance of more than 95 kb but not the more proximal and silent Sult1e1 gene. Loss of this CTCF site led to de novo interactions between the Sult1d1 promoter and several enhancers in the casein locus. Our study demonstrates that only one out of the four CTCF sites in the casein locus had a measurable in vivo activity. Studies on additional loci are needed to determine the biological role of CTCF sites associated with enhancers.

  4. Quantitative perturbation-based analysis of gene expression predicts enhancer activity in early Drosophila embryo

    PubMed Central

    Sayal, Rupinder; Dresch, Jacqueline M; Pushel, Irina; Taylor, Benjamin R; Arnosti, David N

    2016-01-01

    Enhancers constitute one of the major components of regulatory machinery of metazoans. Although several genome-wide studies have focused on finding and locating enhancers in the genomes, the fundamental principles governing their internal architecture and cis-regulatory grammar remain elusive. Here, we describe an extensive, quantitative perturbation analysis targeting the dorsal-ventral patterning gene regulatory network (GRN) controlled by Drosophila NF-κB homolog Dorsal. To understand transcription factor interactions on enhancers, we employed an ensemble of mathematical models, testing effects of cooperativity, repression, and factor potency. Models trained on the dataset correctly predict activity of evolutionarily divergent regulatory regions, providing insights into spatial relationships between repressor and activator binding sites. Importantly, the collective predictions of sets of models were effective at novel enhancer identification and characterization. Our study demonstrates how experimental dataset and modeling can be effectively combined to provide quantitative insights into cis-regulatory information on a genome-wide scale. DOI: http://dx.doi.org/10.7554/eLife.08445.001 PMID:27152947

  5. Enhanced angiogenesis in grafted skins by gene transfer of human hepatocyte growth factor using laser-induced stress waves

    NASA Astrophysics Data System (ADS)

    Terakawa, Mitsuhiro; Sato, Shunichi; Saitoh, Daizoh; Tsuda, Hitoshi; Ashida, Hiroshi; Okano, Hideyuki; Obara, Minoru

    2007-02-01

    We delivered a therapeutic gene, hepatocyte growth factor (HGF), to skin grafts of rats using laser-induced stress waves (LISWs) with the objective of enhancing their adhesion. The density and uniformity of neovascularities were enhanced significantly in the grafted skins that were transfected using LISWs, suggesting the efficacy of this method to improve the outcome of skin transplantation.

  6. Genetic Transformation of Artemisia carvifolia Buch with rol Genes Enhances Artemisinin Accumulation

    PubMed Central

    Dilshad, Erum; Cusido, Rosa Maria; Estrada, Karla Ramirez; Bonfill, Mercedes; Mirza, Bushra

    2015-01-01

    The potent antimalarial drug artemisinin has a high cost, since its only viable source to date is Artemisia annua (0.01–0.8% DW). There is therefore an urgent need to design new strategies to increase its production or to find alternative sources. In the current study, Artemisia carvifolia Buch was selected with the aim of detecting artemisinin and then enhancing the production of the target compound and its derivatives. These metabolites were determined by LC-MS in the shoots of A. carvifolia wild type plants at the following concentrations: artemisinin (8μg/g), artesunate (2.24μg/g), dihydroartemisinin (13.6μg/g) and artemether (12.8μg/g). Genetic transformation of A. carvifolia was carried out with Agrobacterium tumefaciens GV3101 harboring the rol B and rol C genes. Artemisinin content increased 3-7-fold in transgenics bearing the rol B gene, and 2.3-6-fold in those with the rol C gene. A similar pattern was observed for artemisinin analogues. The dynamics of artemisinin content in transgenics and wild type A.carvifolia was also correlated with the expression of genes involved in its biosynthesis. Real time qPCR analysis revealed the differential expression of genes involved in artemisinin biosynthesis, i.e. those encoding amorpha-4, 11 diene synthase (ADS), cytochrome P450 (CYP71AV1), and aldehyde dehydrogenase 1 (ALDH1), with a relatively higher transcript level found in transgenics than in the wild type plant. Also, the gene related to trichome development and sesquiterpenoid biosynthesis (TFAR1) showed an altered expression in the transgenics compared to wild type A.carvifolia, which was in accordance with the trichome density of the respective plants. The trichome index was significantly higher in the rol B and rol C gene-expressing transgenics with an increased production of artemisinin, thereby demonstrating that the rol genes are effective inducers of plant secondary metabolism. PMID:26444558

  7. Silencing the livin gene enhances the cytotoxic effects of anticancer drugs on colon cancer cells

    PubMed Central

    Oh, Bo Young; Kim, Kwang Ho; Chung, Soon Sup

    2016-01-01

    Purpose Livin is associated with drug response in several cancers. The aim of this study was to investigate the effect of silencing the livin gene expression on anticancer drug response in colorectal cancer. Methods siRNA was transfected at different concentrations (0, 10, and 30nM) into HCT116 cells, then cells were treated with either 5-fluorouracil (FU)/leucovorin (LV) or oxaliplatin (L-OHP)/5-FU/LV. Cellular viability and apoptosis were evaluated following silencing of livin gene expression combined with treatment with anticancer drugs. Results Livin gene expression was effectively suppressed by 30nM siRNA compared with control and 10nM siRNA. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed that proliferation was effectively inhibited in cells treated with a combination of both siRNA and an anticancer drug, compared to cells treated with siRNA-Livin or anticancer drug alone. In particular, the combination of 30nM siRNA and L-OHP/5-FU/LV resulted in a 93.8% and 91.4% decrease, compared to untreated control or L-OHP/5-FU/LV alone, respectively. Cellular proliferation was most effectively suppressed by a combination of 30nM of siRNA and L-OHP/5-FU/LV compared to other combinations. Conclusion siRNA-mediated down-regulation of livin gene expression could significantly suppress colon cancer growth and enhance the cytotoxic effects of anticancer drugs such as 5-FU and L-OHP. The results of this study suggest that silencing livin gene expression in combination with treatment with anticancer drugs might be a novel cancer therapy for colorectal cancer. PMID:27904848

  8. Enhanced levels of scrapie responsive gene mRNA in BSE-infected mouse brain.

    PubMed

    Dandoy-Dron, F; Benboudjema, L; Guillo, F; Jaegly, A; Jasmin, C; Dormont, D; Tovey, M G; Dron, M

    2000-03-10

    The expression of the mRNA of nine scrapie responsive genes was analyzed in the brains of FVB/N mice infected with bovine spongiform encephalopathy (BSE). The RNA transcripts of eight genes were overexpressed to a comparable extent in both BSE-infected and scrapie-infected mice, indicating a common series of pathogenic events in the two transmissible spongiform encephalopathies (TSEs). In contrast, the serine proteinase inhibitor spi 2, an analogue of the human alpha-1 antichymotrypsin gene, was overexpressed to a greater extent in the brains of scrapie-infected animals than in animals infected with BSE, reflecting either an agent specific or a mouse strain specific response. The levels of spi 2 mRNA were increased during the course of scrapie prior to the onset of clinical signs of the disease and the increase reached 11 to 45 fold relative to uninfected controls in terminally ill mice. Spi 2, in common with four of the other scrapie responsive genes studied, is known to be associated with pro-inflammatory processes. These observations underline the importance of cell reactivity in TSE. In addition, scrg2 mRNA the level of which is enhanced in TSE-infected mouse brain, was identified as a previously unrecognized long transcript of the murine aldolase C gene. However, the level of the principal aldolase C mRNA is unaffected in TSE. The increased representation of the longer transcript in the late stage of the disease may reflect changes in mRNA processing and/or stability in reactive astrocytes or in damaged Purkinje cells.

  9. Using The Corngrass1 Gene To Enhance The Biofuel Properties Of Crop Plants

    SciTech Connect

    Hake, Sarah; Chuck, George

    2015-10-29

    The development of novel plant germplasm is vital to addressing our increasing bioenergy demands. The major hurdle to digesting plant biomass is the complex structure of the cell walls, the substrate of fermentation. Plant cell walls are inaccessible matrices of macromolecules that are polymerized with lignin, making fermentation difficult. Overcoming this hurdle is a major goal toward developing usable bioenergy crop plants. Our project seeks to enhance the biofuel properties of perennial grass species using the Corngrass1 (Cg1) gene and its targets. Dominant maize Cg1 mutants produce increased biomass by continuously initiating extra axillary meristems and leaves. We cloned Cg1 and showed that its phenotype is caused by over expression of a unique miR156 microRNA gene that negatively regulates SPL transcription factors. We transferred the Cg1 phenotype to other plants by expressing the gene behind constitutive promoters in four different species, including the monocots, Brachypodium and switchgrass, and dicots, Arabidopsis and poplar. All transformants displayed a similar range of phenotypes, including increased biomass from extended leaf production, and increased vegetative branching. Field grown switchgrass transformants showed that overall lignin content was reduced, the ratio of glucans to xylans was increased, and surprisingly, that starch levels were greatly increased. The goals of this project are to control the tissue and temporal expression of Cg1 by using different promoters to drive its expression, elucidate the function of the SPL targets of Cg1 by generating gain and loss of function alleles, and isolate downstream targets of select SPL genes using deep sequencing and chromatin immunoprecipitation. We believe it is possible to control biomass accumulation, cell wall properties, and sugar levels through manipulation of either the Cg1 gene and/or its SPL targets.

  10. Enhancement of doxorubicin production by expression of structural sugar biosynthesis and glycosyltransferase genes in Streptomyces peucetius.

    PubMed

    Malla, Sailesh; Niraula, Narayan Prasad; Liou, Kwangkyoung; Sohng, Jae Kyung

    2009-08-01

    To enhance doxorubicin (DXR) production, the structural sugar biosynthesis genes desIII and desIV from Streptomyces venezuelae ATCC 15439 and the glycosyltransferase pair dnrS/dnrQ from Streptomyces peucetius ATCC 27952 were cloned into the expression vector pIBR25, which contains a strong ermE promoter. The recombinant plasmids pDnrS25 and pDnrQS25 were constructed for overexpression of dnrS and the dnrS/dnrQ pair, whereas pDesSD25 and pDesQS25 were constructed to express desIII/desIV and dnrS/dnrQ-desIII/desIV, respectively. All of these recombinant plasmids were introduced into S. peucetius ATCC 27952. The recombinant strains produced more DXR than the S. peucetius parental strain: a 1.2-fold increase with pDnrS25, a 2.8-fold increase with pDnrQS25, a 2.6-fold increase with pDesSD25, and a 5.6-fold increase with pDesQS25. This study showed that DXR production was significantly enhanced by overexpression of potential biosynthetic sugar genes and glycosyltransferase.

  11. Gene Therapy Vectors with Enhanced Transfection Based on Hydrogels Modified with Affinity Peptides

    PubMed Central

    Shepard, Jaclyn A.; Wesson, Paul J.; Wang, Christine E.; Stevans, Alyson C.; Holland, Samantha J.; Shikanov, Ariella; Grzybowski, Bartosz A.; Shea, Lonnie D.

    2011-01-01

    Regenerative strategies for damaged tissue aim to present biochemical cues that recruit and direct progenitor cell migration and differentiation. Hydrogels capable of localized gene delivery are being developed to provide a support for tissue growth, and as a versatile method to induce the expression of inductive proteins; however, the duration, level, and localization of expression isoften insufficient for regeneration. We thus investigated the modification of hydrogels with affinity peptides to enhance vector retention and increase transfection within the matrix. PEG hydrogels were modified with lysine-based repeats (K4, K8), which retained approximately 25% more vector than control peptides. Transfection increased 5- to 15-fold with K8 and K4 respectively, over the RDG control peptide. K8- and K4-modified hydrogels bound similar quantities of vector, yet the vector dissociation rate was reduced for K8, suggesting excessive binding that limited transfection. These hydrogels were subsequently applied to an in vitro co-culture model to induce NGF expression and promote neurite outgrowth. K4-modified hydrogels promoted maximal neurite outgrowth, likely due to retention of both the vector and the NGF. Thus, hydrogels modified with affinity peptides enhanced vector retention and increased gene delivery, and these hydrogels may provide a versatile scaffold for numerous regenerative medicine applications. PMID:21514659

  12. Silencing of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase gene enhances glioma radiosensitivity

    SciTech Connect

    Kim, Sung Youl; Yoo, Young Hyun; Park, Jeen-Woo

    2013-04-05

    Highlights: •Silencing of the IDPm gene enhances IR-induced autophagy in glioma cells. •Autophagy inhibition augmented apoptosis of irradiated glioma cells. •Results offer a redox-active therapeutic strategy for the treatment of cancer. -- Abstract: Reactive oxygen species (ROS) levels are elevated in organisms that have been exposed to ionizing radiation and are protagonists in the induction of cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDPm) via the supply of NADPH for antioxidant systems. In the present study, we report an autophagic response to ionizing radiation in A172 glioma cells transfected with small interfering RNA (siRNA) targeting the IDPm gene. Autophagy in A172 transfectant cells was associated with enhanced autophagolysosome formation and GFP–LC3 punctuation/aggregation. Furthermore, we found that the inhibition of autophagy by chloroquine augmented apoptotic cell death of irradiated A172 cells transfected with IDPm siRNA. Taken together, our data suggest that autophagy functions as a survival mechanism in A172 cells against ionizing radiation-induced apoptosis and the sensitizing effect of IDPm siRNA and autophagy inhibitor on the ionizing radiation-induced apoptotic cell death of glioma cells offers a novel redox-active therapeutic strategy for the treatment of cancer.

  13. Overexpression of Rab16A gene in indica rice variety for generating enhanced salt tolerance.

    PubMed

    Ganguly, Moumita; Datta, Karabi; Roychoudhury, Aryadeep; Gayen, Dipak; Sengupta, Dibyendu N; Datta, Swapan K

    2012-04-01

    We report here the overexpression of Rab16A full length gene (promoter + ORF), from the salt-tolerant indica rice Pokkali, in the salt-susceptible indica rice variety Khitish, via particle bombardment. Molecular analysis of the transgenics revealed stable integration of the transgene upto T2 generation. High level of expression of the transgene (driven by its own stress-inducible promoter), as well as the protein, was detectable in the leaves under simulated salinity stress (250 mM NaCl, 24 h); the expression level being higher than wild type (WT) plants. The Rab16A transcript also increased gradually with seed maturity, with its maximal accumulation at 30 d after pollination (DAP) i.e., fully matured seeds, explaining the protective role of Rab16A gene during seed maturation. Enhanced tolerance to salinity was observed in the plants transformed with Rab16A. The superior physiological performances of the transgenics under salt treatment were also reflected in lesser shoot or root length inhibition, reduced chlorophyll damages, lesser accumulation of Na(+) and reduced loss of K(+), increased proline content as compared with the WT plants. All these results indicated that the overproduction of RAB16A protein in the transgenics enable them to display enhanced tolerance to salinity stress with improved physiological traits. Our work demonstrates the profound potential of Group 2 LEA proteins (to which RAB16A belongs to) in conferring stress tolerance in crop plants through their genetic manipulation.

  14. Enhancement of DNA-mediated gene transfer by inhibitors of autophagic-lysosomal function.

    PubMed

    Ege, T; Reisbig, R R; Rogne, S

    1984-11-01

    A variety of compounds, known to influence the intravesicular transport and degradation of macromolecules, was studied for their effect on the efficiency of DNA-mediated gene transfer (transfection). The efficiency of transfection was measured by transformation of rat 2 thymidine kinase-deficient (tk-) cells by the cloned herpes simplex I thymidine kinase gene (pAGO). When salmon sperm DNA (average molecular weight, 6 X 10(6) D) was used as a carrier, the presence of either 20 mM NH4Cl, 1 microM carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), or 5 mM 3-methyl adenine (3-MA) in the medium during incubation of the cells with the DNA-calcium-phosphate (DNA-Ca-Pi) precipitate, enhanced the efficiency of transfection by a factor of 10. If rat thymus DNA (greater than 30 X 10(6) D) was used as a carrier, the transformation efficiency was much higher than with salmon sperm DNA. However, in this case treatment with 3-MA, NH4Cl and FCCP enhanced the transformation frequency by slightly less than a factor of two. 3-MA further increased the transfection frequency if the cells were incubated with the compound after removal of the DNA-Ca-Pi coprecipitate, whereas NH4Cl and FCCP had no such effect. Our results strongly suggest that these inhibitors of intracellular degradation can increase the frequency of transformation by increasing the cytoplasmic levels of exogenous DNA.

  15. Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells.

    PubMed

    Sapinoro, Ramil; Volcy, Ketna; Rodrigo, W W Shanaka I; Schlesinger, Jacob J; Dewhurst, Stephen

    2008-04-10

    Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcgammaRI, but not its associated gamma-chain, and was not supported by other FcgammaR family members (FcgammaRIIA, FcgammaRIIB, and FcgammaRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcgammaRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer.

  16. Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum.

    PubMed

    Li, Huan-Jun; He, Yi-Long; Zhang, De-Huai; Yue, Tong-Hui; Jiang, Lu-Xi; Li, Na; Xu, Jun-Wei

    2016-06-10

    The Vitreoscilla hemoglobin (VHb) gene was expressed in Ganoderma lucidum to enhance antitumor ganoderic acid (GA) production. The effects of VHb expression on the accumulation of GAs and lanosterol (intermediate) and the transcription of GA biosynthesis genes were also investigated. In VHb-expressing G. lucidum, the maximum concentrations of four individual GAs (GA-S, GA-T, GA-Mk and GA-Me) were 19.1±1.8, 34.6±2.1, 191.5±13.1 and 45.2±2.8μg/100mg dry weight, respectively, which were 1.4-, 2.2, 1.9- and 2.0-fold higher than those obtained in the wild-type strain. Moreover, the maximum lanosterol concentration in the strain expressing VHb was 1.28-fold lower than that in the wild-type strain. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase genes were up-regulated by 1.6-, 1.5-, and 1.6-fold, respectively, in the strain expressing VHb. This work is beneficial in developing an efficient fermentation process for the hyperproduction of GAs.

  17. In vivo analysis of mouse gastrin gene regulation in enhanced GFP-BAC transgenic mice

    PubMed Central

    Takaishi, Shigeo; Shibata, Wataru; Tomita, Hiroyuki; Jin, Guangchun; Yang, Xiangdong; Ericksen, Russell; Dubeykovskaya, Zinaida; Asfaha, Samuel; Quante, Michael; Betz, Kelly S.; Shulkes, Arthur

    2011-01-01

    Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene. PMID:21051525

  18. Importin-4 Regulates Gene Delivery by Enhancing Nuclear Retention and Chromatin Deposition by Polyplexes

    PubMed Central

    Ross, Nikki L.; Sullivan, Millicent O.

    2016-01-01

    For successful gene delivery, plasmid DNA must be able to access the nucleus in order to be transcribed. Numerous studies have shown that gene delivery occurs more readily in dividing cells, which is attributed to increased nuclear access when the nuclear envelope disassembles during mitosis; however, nonviral carriers continue to have low transfection efficiencies and require large quantities of DNA per cell to achieve reasonable gene transfer, even in dividing cells. Therefore, we hypothesized that using histone-derived nuclear localization sequences (NLS)s to target polyplexes might enhance nuclear delivery by facilitating interactions with histone effectors that mediate nuclear partitioning and retention during mitosis. We discovered a novel interaction between polyplexes linked to histone 3 (H3) N-terminal tail peptides and the histone nuclear import protein importin-4, as evidenced by strong spatial colocalization as well as significantly decreased transfection when importin-4 expression was reduced. A fraction of the histone-targeted polyplexes was also found to colocalize with the retrotranslocon of the endoplasmic reticulum, Sec61. Super resolution microscopy demonstrated a high level of polyplex binding to chromatin post-mitosis, and there also was a significant decrease in the amount of chromatin binding following importin-4 knockdown. These results provide evidence that natural histone effectors mediate both nuclear entry and deposition on chromatin by histone-targeted polyplexes, and a translocation event from the endoplasmic reticulum into the cytosol may occur before mitosis to enable the polyplexes to interact with these essential cytoplasmic proteins. PMID:26465823

  19. Minicircle DNA Provides Enhanced and Prolonged Transgene Expression Following Airway Gene Transfer

    PubMed Central

    Munye, Mustafa M.; Tagalakis, Aristides D.; Barnes, Josephine L.; Brown, Rachel E.; McAnulty, Robin J.; Howe, Steven J.; Hart, Stephen L.

    2016-01-01

    Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5–10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2–4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials. PMID:26975732

  20. In vivo analysis of mouse gastrin gene regulation in enhanced GFP-BAC transgenic mice.

    PubMed

    Takaishi, Shigeo; Shibata, Wataru; Tomita, Hiroyuki; Jin, Guangchun; Yang, Xiangdong; Ericksen, Russell; Dubeykovskaya, Zinaida; Asfaha, Samuel; Quante, Michael; Betz, Kelly S; Shulkes, Arthur; Wang, Timothy C

    2011-02-01

    Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene.

  1. Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis

    PubMed Central

    Anadón, C; Guil, S; Simó-Riudalbas, L; Moutinho, C; Setien, F; Martínez-Cardús, A; Moran, S; Villanueva, A; Calaf, M; Vidal, A; Lazo, P A; Zondervan, I; Savola, S; Kohno, T; Yokota, J; de Pouplana, L R; Esteller, M

    2016-01-01

    The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis. PMID:26640150

  2. Overexpression of cotton PYL genes in Arabidopsis enhances the transgenic plant tolerance to drought stress.

    PubMed

    Chen, Yun; Feng, Li; Wei, Ning; Liu, Zhi-Hao; Hu, Shan; Li, Xue-Bao

    2017-03-30

    PYR/PYL/RCAR proteins are putative abscisic acid (ABA) receptors that play important roles in plant responses to biotic and abiotic stresses. In this study, 27 predicted PYL proteins were identified in cotton (Gossypium hirsutum). Sequence analysis showed they are conserved in structures. Phylogenetic analysis showed that cotton PYL family could be categorized into three groups. Yeast two-hybrid assay revealed that the GhPYL proteins selectively interacted with some GhPP2C proteins. Quantitative RT-PCR analysis indicated that the most of nine GhPYL genes were down-regulated, while the other three were up-regulated in cotton under drought stress. Overexpression of GhPYL10/12/26 in Arabidopsis conferred the transgenic plants increased ABA sensitivity during seed germination and early seedling growth. On the contrary, the transgenic seedlings displayed better growth status and longer primary roots under normal conditions and mannitol stress, compared with wild type. Furthermore, the transgenic plants showed the enhanced drought tolerance, relative to wild type, when they were suffered from drought stress. Expression of some stress-related genes in transgenic plants was significant higher than that in wild type under osmotic stress. Thus, our data suggested that these cotton PYL genes may be involved in plant response and defense to drought/osmotic stress.

  3. Interactions between the nuclear matrix and an enhancer of the tryptophan oxygenase gene

    SciTech Connect

    Kaneoka, Hidenori; Miyake, Katsuhide; Iijima, Shinji

    2009-10-02

    The gene for tryptophan oxygenase (TO) is expressed in adult hepatocytes in a tissue- and differentiation-specific manner. The TO promoter has two glucocorticoid-responsive elements (GREs), and its expression is regulated by glucocorticoid hormone in the liver. We found a novel GRE in close proximity to a scaffold/matrix attachment region (S/MAR) that was located around -8.5 kb from the transcriptional start site of the TO gene by electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) assays. A combination of nuclear fractionation and quantitative PCR analysis showed that the S/MAR was tethered to the nuclear matrix in both fetal and adult hepatocytes. ChIP assay showed that, in adult hepatocytes, the S/MAR-GRE and the promoter proximal regions interacted with lamin and heterogeneous nuclear ribonucleoprotein U in a dexamethasone dependent manner, but this was not the case in fetal cells, suggesting that developmental stage-specific expression of the TO gene might rely on the binding of the enhancer (the -8.5 kb S/MAR-GRE) and the promoter to the inner nuclear matrix.

  4. Simultaneous Disruption of Mouse ASIC1a, ASIC2 and ASIC3 Genes Enhances Cutaneous Mechanosensitivity

    PubMed Central

    Kang, Sinyoung; Jang, Jun Ho; Price, Margaret P.; Gautam, Mamta; Benson, Christopher J.; Gong, Huiyu; Welsh, Michael J.; Brennan, Timothy J.

    2012-01-01

    Three observations have suggested that acid-sensing ion channels (ASICs) might be mammalian cutaneous mechanoreceptors; they are structurally related to Caenorhabditis elegans mechanoreceptors, they are localized in specialized cutaneous mechanosensory structures, and mechanical displacement generates an ASIC-dependent depolarization in some neurons. However, previous studies of mice bearing a single disrupted ASIC gene showed only subtle or no alterations in cutaneous mechanosensitivity. Because functional redundancy of ASIC subunits might explain limited phenotypic alterations, we hypothesized that disrupting multiple ASIC genes would markedly impair cutaneous mechanosensation. We found the opposite. In behavioral studies, mice with simultaneous disruptions of ASIC1a, -2 and -3 genes (triple-knockouts, TKOs) showed increased paw withdrawal frequencies when mechanically stimulated with von Frey filaments. Moreover, in single-fiber nerve recordings of cutaneous afferents, mechanical stimulation generated enhanced activity in A-mechanonociceptors of ASIC TKOs compared to wild-type mice. Responses of all other fiber types did not differ between the two genotypes. These data indicate that ASIC subunits influence cutaneous mechanosensitivity. However, it is unlikely that ASICs directly transduce mechanical stimuli. We speculate that physical and/or functional association of ASICs with other components of the mechanosensory transduction apparatus contributes to normal cutaneous mechanosensation. PMID:22506072

  5. Importin-4 Regulates Gene Delivery by Enhancing Nuclear Retention and Chromatin Deposition by Polyplexes.

    PubMed

    Ross, Nikki L; Sullivan, Millicent O

    2015-12-07

    For successful gene delivery, plasmid DNA must be able to access the nucleus in order to be transcribed. Numerous studies have shown that gene delivery occurs more readily in dividing cells, which is attributed to increased nuclear access when the nuclear envelope disassembles during mitosis; however, nonviral carriers continue to have low transfection efficiencies and require large quantities of DNA per cell to achieve reasonable gene transfer, even in dividing cells. Therefore, we hypothesized that using histone-derived nuclear localization sequences (NLS)s to target polyplexes might enhance nuclear delivery by facilitating interactions with histone effectors that mediate nuclear partitioning and retention during mitosis. We discovered a novel interaction between polyplexes linked to histone 3 (H3) N-terminal tail peptides and the histone nuclear import protein importin-4, as evidenced by strong spatial colocalization as well as significantly decreased transfection when importin-4 expression was reduced. A fraction of the histone-targeted polyplexes was also found to colocalize with the retrotranslocon of the endoplasmic reticulum, Sec61. Super resolution microscopy demonstrated a high level of polyplex binding to chromatin postmitosis, and there also was a significant decrease in the amount of chromatin binding following importin-4 knockdown. These results provide evidence that natural histone effectors mediate both nuclear entry and deposition on chromatin by histone-targeted polyplexes, and a translocation event from the endoplasmic reticulum into the cytosol may occur before mitosis to enable the polyplexes to interact with these essential cytoplasmic proteins.

  6. Human Gene-Centered Transcription Factor Networks for Enhancers and Disease Variants

    PubMed Central

    Bass, Juan I. Fuxman; Sahni, Nidhi; Shrestha, Shaleen; Garcia-Gonzalez, Aurian; Mori, Akihiro; Bhat, Numana; Yi, Song; Hill, David E.; Vidal, Marc; Walhout, Albertha J.M.

    2015-01-01

    SUMMARY Gene regulatory networks (GRNs) comprising interactions between transcription factors (TFs) and regulatory loci control development and physiology. Numerous disease-associated mutations have been identified, the vast majority residing in non-coding regions of the genome. As current GRN mapping methods test one TF at a time and require the use of cells harboring the mutation(s) of interest, they are not suitable to identify TFs that bind to wild type and mutant loci. Here, we use gene-centered yeast one-hybrid (eY1H) assays to interrogate binding of 1,086 human TFs to 246 enhancers, as well as to 109 non-coding disease mutations. We detect both loss and gain of TF interactions with mutant loci that are concordant with target gene expression changes. This work establishes eY1H assays as a powerful addition to the toolkit of mapping human GRNs and for the high-throughput characterization of genomic variants that are rapidly being identified by genome-wide association studies. PMID:25910213

  7. Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis.

    PubMed

    Anadón, C; Guil, S; Simó-Riudalbas, L; Moutinho, C; Setien, F; Martínez-Cardús, A; Moran, S; Villanueva, A; Calaf, M; Vidal, A; Lazo, P A; Zondervan, I; Savola, S; Kohno, T; Yokota, J; de Pouplana, L R; Esteller, M

    2016-08-18

    The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis.

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

    PubMed

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

    2011-11-01

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

  9. N-acetylcysteine Enhances Cystic Fibrosis Sputum Penetration and Airway Gene Transfer by Highly Compacted DNA Nanoparticles.

    PubMed

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

    2011-11-01

    For effective airway gene therapy of cystic fibrosis (CF), inhaled gene carriers must first penetrate the hyperviscoelastic sputum covering the epithelium. Whether clinically studied gene carriers can penetrate CF sputum remains unknown. Here, we measured the diffusion of a clinically tested nonviral gene carrier, composed of poly-l-lysine conjugated with a 10 kDa polyethylene glycol segment (CK30PEG10k). We found that CK30PEG10k/DNA nanoparticles were trapped in CF sputum. To improve gene carrier diffusion across sputum, we tested adjuvant regimens consisting of N-acetylcysteine (NAC), recombinant human DNase (rhDNase) or NAC together with rhDNase. While rhDNase alone did not enhance gene carrier diffusion, NAC and NAC + rhDNase increased average effective diffusivities by 6-fold and 13-fold, respectively, leading to markedly greater fractions of gene carriers that may penetrate sputum layers. We further tested the adjuvant effects of NAC in the airways of mice with Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced mucus hypersecretion. Intranasal dosing of NAC prior to CK30PEG10k/DNA nanoparticles enhanced gene expression by up to ~12-fold compared to saline control, reaching levels observed in the lungs of mice without LPS challenge. Our findings suggest that a promising synthetic nanoparticle gene carrier may transfer genes substantially more effectively to lungs of CF patients if administered following adjuvant mucolytic therapy with NAC or NAC + rhDNase.

  10. Histone H3 K27 acetylation marks a potent enhancer element on the adipogenic master regulator gene Pparg2.

    PubMed

    Ramlee, Muhammad Khairul; Zhang, Qiongyi; Idris, Muhammad; Peng, Xu; Sim, Choon Kiat; Han, Weiping; Xu, Feng

    2014-01-01

    PPARγ2 is expressed almost exclusively in adipose tissue and plays a central role in adipogenesis. Despite intensive studies over the last 2 decades, the mechanism regulating the expression of the Pparg2 gene, especially the role of cis-regulatory elements, is still not completely understood. Here, we report a comprehensive investigation of the enhancer elements within the murine Pparg2 gene. Utilizing the combined techniques of sequence conservation analysis and chromatin marker examination, we identified a potent enhancer element that augmented the expression of a reporter gene under the control of the Pparg2 promoter by 20-fold. This enhancer element was first identified as highly conserved non-coding sequence 10 (CNS10) and was later shown to be enriched with the enhancer marker H3 K27 acetylation. Further studies identified a binding site for p300 as the essential enhancer element in CNS10. Moreover, p300 physically binds to CNS10 and is required for the enhancer activity of CNS10. The depletion of p300 by siRNA resulted in significantly impaired activation of Pparg2 at the early stages of 3T3-L1 adipogenesis. In summary, our study identified a novel enhancer element on the murine Pparg2 gene and suggested a novel mechanism for the regulation of Pparg2 expression by p300 in 3T3-L1 adipogenesis.

  11. Histone H3 K27 acetylation marks a potent enhancer element on the adipogenic master regulator gene Pparg2

    PubMed Central

    Ramlee, Muhammad Khairul; Zhang, Qiongyi; Idris, Muhammad; Peng, Xu; Sim, Choon Kiat; Han, Weiping; Xu, Feng

    2014-01-01

    PPARγ2 is expressed almost exclusively in adipose tissue and plays a central role in adipogenesis. Despite intensive studies over the last 2 decades, the mechanism regulating the expression of the Pparg2 gene, especially the role of cis-regulatory elements, is still not completely understood. Here, we report a comprehensive investigation of the enhancer elements within the murine Pparg2 gene. Utilizing the combined techniques of sequence conservation analysis and chromatin marker examination, we identified a potent enhancer element that augmented the expression of a reporter gene under the control of the Pparg2 promoter by 20-fold. This enhancer element was first identified as highly conserved non-coding sequence 10 (CNS10) and was later shown to be enriched with the enhancer marker H3 K27 acetylation. Further studies identified a binding site for p300 as the essential enhancer element in CNS10. Moreover, p300 physically binds to CNS10 and is required for the enhancer activity of CNS10. The depletion of p300 by siRNA resulted in significantly impaired activation of Pparg2 at the early stages of 3T3-L1 adipogenesis. In summary, our study identified a novel enhancer element on the murine Pparg2 gene and suggested a novel mechanism for the regulation of Pparg2 expression by p300 in 3T3-L1 adipogenesis. PMID:25485585

  12. Electroporation-mediated transfer of Runx2 and Osterix genes to enhance osteogenesis of adipose stem cells.

    PubMed

    Lee, Jai-Sun; Lee, Jong-Min; Im, Gun-Il

    2011-01-01

    In the present study, we tested the hypothesis that electroporation-mediated transfer of Runx2, Osterix, or both genes enhances the in vitro and in vivo osteogenesis from adipose stem cells (ASCs). ASCs were transfected with Runx2, Osterix, or both genes using electroporation, and further cultured in monolayer or in PLGA scaffold under osteogenic medium for 14 days, then analyzed for in vitro osteogenic differentiation. Transfected ASC-PLGA scaffold hybrids were also implanted on nude mice to test for in vivo ectopic bone formation. Runx2 and Osterix genes were strongly expressed in ASCs transfected with each gene on day 7, decreasing rapidly on day 14. Runx2 protein was strongly expressed in ASCs transfected with the Runx2 gene, while Osterix protein was strongly expressed in ASCs transfected with either or both Runx2 and Osterix genes. Overexpression of Runx2 and Osterix significantly increased the gene expression of osteogenic differentiation markers (alkaline phosphatase [ALP], osteocalcin [OCN], type I collagen [COL1A1], and bone sialoprotein [BSP]) in ASCs. Transfection of Runx2 and Osterix genes enhanced the protein expression of OCN, type I collagen, and BSP, as demonstrated by Western blot analysis, and ALP activity as well as enhancing mineralization in the monolayer culture and ASC-PLGA scaffold hybrids. Runx2- or Osterix-transfected ASC-PLGA scaffold hybrids promoted bone formation in nude mice after 6 weeks of in vivo implantation.

  13. Hibiscus chlorotic ringspot virus coat protein upregulates sulfur metabolism genes for enhanced pathogen defense.

    PubMed

    Gao, Ruimin; Ng, Florence Kai Lin; Liu, Peng; Wong, Sek-Man

    2012-12-01

    In both Hibiscus chlorotic ringspot virus (HCRSV)-infected and HCRSV coat protein (CP) agroinfiltrated plant leaves, we showed that sulfur metabolism pathway related genes-namely, sulfite oxidase (SO), sulfite reductase, and adenosine 5'-phosphosulfate kinase-were upregulated. It led us to examine a plausible relationship between sulfur-enhanced resistance (SED) and HCRSV infection. We broadened an established method to include different concentrations of sulfur (0S, 1S, 2S, and 3S) to correlate them to symptom development of HCRSV-infected plants. We treated plants with glutathione and its inhibitor to verify the SED effect. Disease resistance was induced through elevated glutathione contents during HCRSV infection. The upregulation of SO was related to suppression of symptom development induced by sulfur treatment. In this study, we established that HCRSV-CP interacts with SO which, in turn, triggers SED and leads to enhanced plant resistance. Thus, we have discovered a new function of SO in the SED pathway. This is the first report to demonstrate that the interaction of a viral protein and host protein trigger SED in plants. It will be interesting if such interaction applies generally to other host-pathogen interactions that will lead to enhanced pathogen defense.

  14. Development of Patient-specific AAV Vectors After Neutralizing Antibody Selection for Enhanced Muscle Gene Transfer.

    PubMed

    Li, Chengwen; Wu, Shuqing; Albright, Blake; Hirsch, Matthew; Li, Wuping; Tseng, Yu-Shan; Agbandje-McKenna, Mavis; McPhee, Scott; Asokan, Aravind; Samulski, R Jude

    2016-02-01

    A major hindrance in gene therapy trials with adeno-associated virus (AAV) vectors is the presence of neutralizing antibodies (NAbs) that inhibit AAV transduction. In this study, we used directed evolution techniques in vitro and in mouse muscle to select novel NAb escape AAV chimeric capsid mutants in the presence of individual patient serum. AAV mutants isolated in vitro escaped broad patient-specific NAb activity but had poor transduction ability in vivo. AAV mutants isolated in vivo had enhanced NAb evasion from cognate serum and had high muscle transduction ability. More importantly, structural modeling identified a 100 amino acid motif from AAV6 in variable region (VR) III that confers this enhanced muscle tropism. In addition, a predominantly AAV8 capsid beta barrel template with a specific preference for AAV1/AAV9 in VR VII located at threefold symmetry axis facilitates NAb escape. Our data strongly support that chimeric AAV capsids composed of modular and nonoverlapping domains from various serotypes are capable of evading patient-specific NAbs and have enhanced muscle transduction.

  15. All-trans retinoic acid enhances bystander effect of suicide-gene therapy against medulloblastomas.

    PubMed

    Li, Shaoyi; Gao, Yun; Pu, Ke; Ma, Li; Song, Xiaofu; Liu, Yunhui

    2011-10-03

    In our previous study we evaluated the antitumor effect of herpes simplex virus-thymidine kinase gene (HSV-tk) on human medulloblastomas (MBs) in a therapeutic delivery system using the immortalized neural stem cell (NSC) line C17.2. However, our findings indicated that the bystander effect between C17.2tk and Daoy MB cells was weak compared to the bystander effect between NSCtk and C6 glioma cells. Gap junction intercellular communication (GJIC) is the main mechanism mediating the bystander effect in HSV-tk gene therapy. All-trans retinoic acid (ATRA) has been shown to up-regulate the expression of Connexin43 and GJIC. In this study we investigated the synergistic effect of ATRA and HSV-tk gene therapy in the treatment of MBs. We found that the expression of Connexin43 in Daoy cells was significantly increased when cells were exposed to 3μmol/l of ATRA (P<0.05). After co-culturing C17.2tk cells with Daoy cells at different ratios ranging from 1:1 to 1:16, ATRA significantly increased the bystander anti-tumor effect compared to ATRA-untreated cells (P<0.05). In intracranial co-implantation experiments, mice co-implanted with C17.2tk/Daoy cells and treated with a combination of ATRA and GCV had significantly smaller tumors compared to the animals treated with GCV alone (P<0.05). Together, our results show that ATRA enhanced the tumoricidal effect in HSVtk/GCV suicide gene therapy against Daoy MB cells by strengthening the bystander effect in vitro and in vivo.

  16. Engineering an Enhanced, Thermostable, Monomeric Bacterial Luciferase Gene As a Reporter in Plant Protoplasts

    PubMed Central

    Song, Yunhong; Wei, Jinsong; Li, Changfu; Wang, Tietao; Wang, Yao; Zhao, Tianyong; Shen, Xihui

    2014-01-01

    The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future. PMID:25271765

  17. Parkinson-associated risk variant in distal enhancer of α-synuclein modulates target gene expression.

    PubMed

    Soldner, Frank; Stelzer, Yonatan; Shivalila, Chikdu S; Abraham, Brian J; Latourelle, Jeanne C; Barrasa, M Inmaculada; Goldmann, Johanna; Myers, Richard H; Young, Richard A; Jaenisch, Rudolf

    2016-05-05

    Genome-wide association studies (GWAS) have identified numerous genetic variants associated with complex diseases, but mechanistic insights are impeded by a lack of understanding of how specific risk variants functionally contribute to the underlying pathogenesis. It has been proposed that cis-acting effects of non-coding risk variants on gene expression are a major factor for phenotypic variation of complex traits and disease susceptibility. Recent genome-scale epigenetic studies have highlighted the enrichment of GWAS-identified variants in regulatory DNA elements of disease-relevant cell types. Furthermore, single nucleotide polymorphism (SNP)-specific changes in transcription factor binding are correlated with heritable alterations in chromatin state and considered a major mediator of sequence-dependent regulation of gene expression. Here we describe a novel strategy to functionally dissect the cis-acting effect of genetic risk variants in regulatory elements on gene expression by combining genome-wide epigenetic information with clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 genome editing in human pluripotent stem cells. By generating a genetically precisely controlled experimental system, we identify a common Parkinson's disease associated risk variant in a non-coding distal enhancer element that regulates the expression of α-synuclein (SNCA), a key gene implicated in the pathogenesis of Parkinson's disease. Our data suggest that the transcriptional deregulation of SNCA is associated with sequence-dependent binding of the brain-specific transcription factors EMX2 and NKX6-1. This work establishes an experimental paradigm to functionally connect genetic variation with disease-relevant phenotypes.

  18. Non-random inactivation of large common fragile site genes in different cancers.

    PubMed

    McAvoy, S; Ganapathiraju, S C; Ducharme-Smith, A L; Pritchett, J R; Kosari, F; Perez, D S; Zhu, Y; James, C D; Smith, D I

    2007-01-01

    The common fragile sites are regions of profound genomic instability found in all individuals. The full size of each region of instability ranges from under one megabase (Mb) to greater than 10 Mbs. At least half of the CFS regions have been found to span extremely large genes that spanned from 600 kb to greater than 2.0 Mbs. The large CFS genes are also very interesting from a cancer perspective as several of them, including FHIT and WWOX, have already demonstrated the capacity to function as tumor suppressor genes, both in vitro and in vivo. We estimate that there may be 40-50 large genes localized in CFS regions. The expression of a number of the large CFS genes has been previously shown to be lost in many different cancers and this is frequently associated with a worse clinical outcome for patients. To determine if there was selection for the inactivation of different large CFS genes in different cancers, we examined the expression of 13 of the 20 known large CFS genes: FHIT, WWOX, PARK2, GRID2, NBEA, DLG2, RORA isoforms 1 and 4, DAB1, CNTNAP2, DMD, IL1RAPL1, IMMP2L and LARGE in breast, ovarian, endometrial and brain cancers using real-time RT-PCR analysis. Each cancer had a distinct profile of different large CFS genes that were inactivated. Interestingly, in breast, ovarian and endometrial cancers there were some cancers that had inactivation of expression of none or only one of the tested genes, while in other specimens there was inactivation of multiple tested genes. Brain cancers had inactivation of many of the tested genes, a number of which function in normal neurological development. We find that there is no relationship between the frequency that any specific CFS is expressed and the frequency that the gene from that region is inactivated in different cancers. Instead, it appears that different cancers select for the inactivation of different large CFS genes.

  19. Early developmental gene enhancers affect subcortical volumes in the adult human brain.

    PubMed

    Becker, Martin; Guadalupe, Tulio; Franke, Barbara; Hibar, Derrek P; Renteria, Miguel E; Stein, Jason L; Thompson, Paul M; Francks, Clyde; Vernes, Sonja C; Fisher, Simon E

    2016-05-01

    Genome-wide association screens aim to identify common genetic variants contributing to the phenotypic variability of complex traits, such as human height or brain morphology. The identified genetic variants are mostly within noncoding genomic regions and the biology of the genotype-phenotype association typically remains unclear. In this article, we propose a complementary targeted strategy to reveal the genetic underpinnings of variability in subcortical brain volumes, by specifically selecting genomic loci that are experimentally validated forebrain enhancers, active in early embryonic development. We hypothesized that genetic variation within these enhancers may affect the development and ultimately the structure of subcortical brain regions in adults. We tested whether variants in forebrain enhancer regions showed an overall enrichment of association with volumetric variation in subcortical structures of >13,000 healthy adults. We observed significant enrichment of genomic loci that affect the volume of the hippocampus within forebrain enhancers (empirical P = 0.0015), a finding which robustly passed the adjusted threshold for testing of multiple brain phenotypes (cutoff of P < 0.0083 at an alpha of 0.05). In analyses of individual single nucleotide polymorphisms (SNPs), we identified an association upstream of the ID2 gene with rs7588305 and variation in hippocampal volume. This SNP-based association survived multiple-testing correction for the number of SNPs analyzed but not for the number of subcortical structures. Targeting known regulatory regions offers a way to understand the underlying biology that connects genotypes to phenotypes, particularly in the context of neuroimaging genetics. This biology-driven approach generates testable hypotheses regarding the functional biology of identified associations. Hum Brain Mapp 37:1788-1800, 2016. © 2016 Wiley Periodicals, Inc.

  20. Multiple enhancers contribute to expression of the NK-2 homeobox gene ceh-22 in C. elegans pharyngeal muscle.

    PubMed

    Kuchenthal, C A; Chen, W; Okkema, P G

    2001-12-01

    Gene expression in the pharyngeal muscles of C. elegans is regulated in part by the NK-2 family homeodomain factor CEH-22, which is structurally and functionally related to Drosophila Tinman and the vertebrate Nkx2-5 factors. ceh-22 is expressed exclusively in the pharyngeal muscles and is the earliest gene known to be expressed in this tissue. Here we characterize the ceh-22 promoter region in transgenic C. elegans. A 1.9-kb fragment upstream of ceh-22 is sufficient to regulate reporter gene expression in a pattern identical to the endogenous gene. Within this promoter we identified two transcriptional enhancers and characterized their cell type and temporal specificity. The distal enhancer becomes active in the pharynx near the time that ceh-22 expression initiates; however, it becomes active more broadly later in development. The proximal enhancer becomes active after the onset of ceh-22 expression, but it is active specifically in the ceh-22-expressing pharyngeal muscles. We suggest these enhancers respond to distinct signals that initiate and maintain ceh-22 gene expression. Proximal enhancer activity requires a short segment containing a CEH-22 responsive element, suggesting that CEH-22 autoregulates its own expression.

  1. Identification of circadian-clock-regulated enhancers and genes of Drosophila melanogaster by transposon mobilization and luciferase reporting of cyclical gene expression.

    PubMed Central

    Stempfl, Thomas; Vogel, Marion; Szabo, Gisela; Wülbeck, Corinna; Liu, Jian; Hall, Jeffrey C; Stanewsky, Ralf

    2002-01-01

    A new way was developed to isolate rhythmically expressed genes in Drosophila by modifying the classic enhancer-trap method. We constructed a P element containing sequences that encode firefly luciferase as a reporter for oscillating gene expression in live flies. After generation of 1176 autosomal insertion lines, bioluminescence screening revealed rhythmic reporter-gene activity in 6% of these strains. Rhythmically fluctuating reporter levels were shown to be altered by clock mutations in genes that specify various circadian transcription factors or repressors. Intriguingly, rhythmic luminescence in certain lines was affected by only a subset of the pacemaker mutations. By isolating genes near 13 of the transposon insertions and determining their temporal mRNA expression pattern, we found that four of the loci adjacent to the trapped enhancers are rhythmically expressed. Therefore, this approach is suitable for identifying genetic loci regulated by the circadian clock. One transposon insert caused a mutation in the rhythmically expressed gene numb. This novel numb allele, as well as previously described ones, was shown to affect the fly's rhythm of locomotor activity. In addition to its known role in cell fate determination, this gene and the phosphotyrosine-binding protein it encodes are likely to function in the circadian system. PMID:11861563

  2. Surface-enhanced Raman spectroscopy on a surface plasmon resonance biosensor platform for gene diagnostics

    NASA Astrophysics Data System (ADS)

    Yuan, W.; Ho, H. P.; Suen, Y. K.; Kong, S. K.; Lin, Chinlon; Prasad, Paras N.; Li, J.; Ong, Daniel H. C.

    2008-02-01

    We propose to integrate the surface-enhanced Raman spectroscopy (SERS) detection capability with a surface plasmon resonance (SPR) biosensor platform. As a demonstration setup, the experimental scheme is built from a Total Internal Reflection Fluorescence (TIRF) microscope. The sample surface is a gold-coated plasmonic crystal substrate. Two oligonucleotide (ODN) probes that have been labeled with two different Raman active dyes are used to achieve a sandwich assay of target ODNs or polynucleotide. Upon complementary hybridizations between the target and probe ODNs, the target can be identified by detecting the narrow-band spectroscopic fingerprints of the Raman tags. This concept has high potential for achieving multiplexed detection of ODN targets because a very large number of probes can be incorporated to the plasmonic crystal substrate, which may find applications in gene based diseases diagnostics. We also explored the detection of single molecules and achieved some preliminary results.

  3. Seven Genes of the Enhancer of Split Complex of Drosophila Melanogaster Encode Helix-Loop-Helix Proteins

    PubMed Central

    Knust, E.; Schrons, H.; Grawe, F.; Campos-Ortega, J. A.

    1992-01-01

    Enhancer of split [E(spl)] is one of the neurogenic loci of Drosophila and, as such, is required for normal segregation of neural and epidermal cell progenitors. Genetic observations indicate that the E(spl) locus is in fact a gene complex comprising a cluster of related genes and that other genes of the region are also required for normal early neurogenesis. Three of the genes of the complex were known to encode helix-loop-helix (HLH) proteins and to be transcribed in nearly identical patterns. Here, we show that four other genes in the vicinity also encode HLH proteins and, during neuroblast segregation, three of them are expressed in the same pattern. We show by germ-line transformation that these three genes are also necessary to allow epidermal development of the neuroectodermal cells. PMID:1427040

  4. Male-enhanced expression and genetic conservation of a gene isolated with an anti-H-Y antibody.

    PubMed

    Lau, Y F; Chan, K M; Kan, Y W; Goldberg, E

    1987-01-01

    The hypothesis of the serological H-Y antigen as the inducer molecule for mammalian male sex differentiation has been considered an important working model in developmental biology. However, because of the difficulties involved in its detection, supporting evidence in molecular terms is lacking for this hypothesis. The isolation of the gene for the serological H-Y antigen is essential to the acertainment of its proposed functions. Using recombinant DNA technology and specific anti-H-Y sera we have isolated a candidate gene, the MEA gene, for the serological H-Y antigen. Molecular characterization of the MEA gene shows male-enhanced expression and genetic conservation patterns similar to those attributed to the serological H-Y antigen. The isolation of this candidate gene for the serological H-Y antigen. The isolation of this candidate gene for the serological H-Y antigen would allow further investigations to identify the functions for this molecule in molecular terms.

  5. A Gene Optimization Strategy that Enhances Production of Fully Functional P-Glycoprotein in Pichia pastoris

    PubMed Central

    Protasevich, Irina I.; Brouillette, Christie G.; Harrell, Patina M.; Hildebrandt, Ellen; Gasser, Brigitte; Mattanovich, Diethard; Ward, Andrew; Chang, Geoffrey; Urbatsch, Ina L.

    2011-01-01

    Background Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers. Methodology/Principal Findings Codon-optimized “Opti-Pgp” and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from Tm ∼40°C to 49°C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids. Conclusion The significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins. PMID:21826197

  6. Angiotensinogen gene polymorphism predicts hypertension, and iridological constitutional classification enhances the risk for hypertension in Koreans.

    PubMed

    Cho, Joo-Jang; Hwang, Woo-Jun; Hong, Seung-Heon; Jeong, Hyun-Ja; Lee, Hye-Jung; Kim, Hyung-Min; Um, Jae-Young

    2008-05-01

    This study investigated the relationship between iridological constitution and angiotensinogen (AGN) gene polymorphism in hypertensives. In addition to angiotensin converting enzyme gene, AGN genotype is also one of the most well studied genetic markers of hypertension. Furthermore, iridology, one of complementary and alternative medicine, is the diagnosis of the medical conditions through noting irregularities of the pigmentation in the iris. Iridological constitution has a strong familial aggregation and is implicated in heredity. Therefore, the study classified 87 hypertensive patients with familial history of cerebral infarction and controls (n = 88) according to Iris constitution, and determined AGN genotype. As a result, the AGN/TT genotype was associated with hypertension (chi2 = 13.413, p < .05). The frequency of T allele was 0.92 in patients and 0.76 in controls (chi2 = 13.159, p < .05). In addition, iridological constitutional classification increased the relative risk for hypertension in the subjects with AGN/T allele. These results suggest that AGN polymorphism predicts hypertension, and iridological constitutional classification enhances the risk for hypertension associated with AGN/T in a Korean population.

  7. Disruptions of Topological Chromatin Domains Cause Pathogenic Rewiring of Gene-Enhancer Interactions

    PubMed Central

    Lupiáñez, Darío G.; Kraft, Katerina; Heinrich, Verena; Krawitz, Peter; Brancati, Francesco; Klopocki, Eva; Horn, Denise; Kayserili, Hülya; Opitz, John M.; Laxova, Renata; Santos-Simarro, Fernando; Gilbert-Dussardier, Brigitte; Wittler, Lars; Borschiwer, Marina; Haas, Stefan A.; Osterwalder, Marco; Franke, Martin; Timmermann, Bernd; Hecht, Jochen; Spielmann, Malte; Visel, Axel; Mundlos, Stefan

    2016-01-01

    SUMMARY Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome. PMID:25959774

  8. Ligands located within a cholesterol domain enhance gene delivery to the target tissue

    PubMed Central

    Xu, Long; Betker, Jamie; Yin, Hao; Anchordoquy, Thomas J.

    2012-01-01

    Targeted gene delivery provides enormous potential for clinical treatment of many incurable diseases. Liposomes formulated with targeting ligands have been tested extensively both in vitro and in vivo, and many studies have strived to identify more efficacious ligands. However, the environment of the ligand within the delivery vehicle is generally not considered, and this study assesses the effect of ligand micoenvironment by utilizing a lipoplex possessing a cholesterol domain. Our recent work has shown that the presence of the targeting ligand within the cholesterol domain promotes more productive transfection in cultured cells. In the present study, lipoplexes having the identical lipid composition were formulated with different conjugates of the folate ligand such that the ligand was included in, or excluded from, the cholesterol domain. The effect of locating the ligand within the cholesterol domain was then tested in a xenograft tumor model in mice. Lipoplexes that included the ligand within the cholesterol domain showed significantly higher luciferase expression and plasmid accumulation in tumors as compared to lipoplexes in which the ligand was excluded from the domain. These results demonstrate that the microenvironment of the ligand can affect gene delivery to tumors, and show that ligand-mediated delivery can be enhanced by locating targeting ligands within a cholesterol domain. PMID:22440429

  9. Gene Replacement for the Generation of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities

    PubMed Central

    Huang, Jun; Chen, An-Liang; Zhang, Hui; Yu, Zhen; Li, Mei-Hong; Li, Na; Lin, Jia-Tan; Bai, Hua

    2015-01-01

    Avermectin (AVM) and ivermectin (IVM) are potent pesticides and acaricides which have been widely used during the past 30 years. As insect resistance to AVM and IVM is greatly increasing, alternatives are urgently needed. Here, we report two novel AVM derivatives, tenvermectin A (TVM A) and TVM B, which are considered a potential new generation of agricultural and veterinary drugs. The molecules of the TVMs were designed based on structure and pharmacological property comparisons among AVM, IVM, and milbemycin (MBM). To produce TVMs, a genetically engineered strain, MHJ1011, was constructed from Streptomyces avermitilis G8-17, an AVM industrial strain. In MHJ1011, the native aveA1 gene was seamlessly replaced with milA1 from Streptomyces hygroscopicus. The total titer of the two TVMs produced by MHJ1011 reached 3,400 mg/liter. Insecticidal tests proved that TVM had enhanced activities against Tetranychus cinnabarinus and Bursaphelenchus xylophilus, as desired. This study provides a typical example of exploration for novel active compounds through a new method of polyketide synthase (PKS) reassembly for gene replacement. The results of the insecticidal tests may be of use in elucidating the structure-activity relationship of AVMs and MBMs. PMID:26025902

  10. A dehydrin gene isolated from feral olive enhances drought tolerance in Arabidopsis transgenic plants

    PubMed Central

    Chiappetta, Adriana; Muto, Antonella; Bruno, Leonardo; Woloszynska, Magdalena; Lijsebettens, Mieke Van; Bitonti, Maria B.

    2015-01-01

    Dehydrins belong to a protein family whose expression may be induced or enhanced by developmental process and environmental stresses that lead to cell dehydration. A dehydrin gene named OesDHN was isolated and characterized from oleaster (Olea europaea L. subsp. europaea, var. sylvestris), the wild form of olive. To elucidate the contribution of OesDHN in the development of drought tolerance, its expression levels were investigated in oleaster plants during development and under drought stress condition. The involvement of OesDHN in plant stress response was also evaluated in Arabidopsis transgenic lines, engineered to overexpress this gene, and exposed to a controlled mild osmotic stress. OesDHN expression was found to be modulated during development and induced under mild drought stress in oleaster plants. In addition, the Arabidopsis transgenic plants showed a better tolerance to osmotic stress than wild-type plants. The results demonstrated that OesDHN expression is induced by drought stress and is able to confer osmotic stress tolerance. We suggest a role for OesDHN, as a putative functional marker of plant stress tolerance. PMID:26175736

  11. Enhancing Functional Robustness of Gene Regulatory Networks Based on Fitness Landscape Design

    NASA Astrophysics Data System (ADS)

    Kim, Kyung

    We aim to develop design principles for enhancing functional robustness of engineered cells using gene-network topology. We observed the effect of genetic regulation types (inhibition and activation) on robustness. Inhibition was much more stable than activation in E. coli. In the case of activation, if the upstream activator expression is shutdown by mutation, then its downstream expression is shut down as well. Without activation, the activator shutdown due to mutation will make its downstream expression ``remains`` turned off. Thus, the change in the metabolic load is higher in the activation case. Therefore, the stronger activation, the less robust the circuits are. In the inhibition case, we found that the story becomes opposite. When an inhibitor expression is shut down by mutation, the downstream expression turns on because the inhibitor is not expressed. This compensates changes in the metabolic load that might have been decreased without the inhibition. This result presents potential significant roles of network topology on the robustness of engineered cellular networks. This also emphasizes that the concept of fitness landscape, where the local slope corresponds to the fitness difference between different genotypes, can be useful to design robust gene circuits. We acknowledge the support of the NSF (MCB Award # 1515280).

  12. PETModule: a motif module based approach for enhancer target gene prediction

    PubMed Central

    Zhao, Changyong; Li, Xiaoman; Hu, Haiyan

    2016-01-01

    The identification of enhancer-target gene (ETG) pairs is vital for the understanding of gene transcriptional regulation. Experimental approaches such as Hi-C have generated valuable resources of ETG pairs. Several computational methods have also been developed to successfully predict ETG interactions. Despite these progresses, high-throughput experimental approaches are still costly and existing computational approaches are still suboptimal and not easy to apply. Here we developed a motif module based approach called PETModule that predicts ETG pairs. Tested on eight human cell types and two mouse cell types, we showed that a large number of our predictions were supported by Hi-C and/or ChIA-PET experiments. Compared with two recently developed approaches for ETG pair prediction, we shown that PETModule had a much better recall, a similar or better F1 score, and a larger area under the receiver operating characteristic curve. The PETModule tool is freely available at http://hulab.ucf.edu/research/projects/PETModule/. PMID:27436110

  13. Organic amendments enhance microbial diversity and abundance of functional genes in Australian Soils

    NASA Astrophysics Data System (ADS)

    Aldorri, Sind; McMillan, Mary; Pereg, Lily

    2016-04-01

    Food and cash crops play important roles in Australia's economy with black, grey and red clay soil, widely use for growing cotton, wheat, corn and other crops in rotation. While the majority of cotton growers use nitrogen and phosphate fertilizers only in the form of agrochemicals, a few experiment with the addition of manure or composted plant material before planting. We hypothesized that the use of such organic amendments would enhance the soil microbial function through increased microbial diversity and abundance, thus contribute to improved soil sustainability. To test the hypothesis we collected soil samples from two cotton-growing farms in close geographical proximity and with mostly similar production practices other than one grower has been using composted plants as organic amendment and the second farmer uses only agrochemicals. We applied the Biolog Ecoplate system to study the metabolic signature of microbial communities and used qPCR to estimate the abundance of functional genes in the soil. The soil treated with organic amendments clearly showed higher metabolic activity of a more diverse range of carbon sources as well as higher abundance of genes involved in the nitrogen and phosphorous cycles. Since microbes undertake a large number of soil functions, the use of organic amendments can contribute to the sustainability of agricultural soils.

  14. Gene Replacement for the Generation of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities.

    PubMed

    Huang, Jun; Chen, An-Liang; Zhang, Hui; Yu, Zhen; Li, Mei-Hong; Li, Na; Lin, Jia-Tan; Bai, Hua; Wang, Ji-Dong; Zheng, Yu-Guo

    2015-08-15

    Avermectin (AVM) and ivermectin (IVM) are potent pesticides and acaricides which have been widely used during the past 30 years. As insect resistance to AVM and IVM is greatly increasing, alternatives are urgently needed. Here, we report two novel AVM derivatives, tenvermectin A (TVM A) and TVM B, which are considered a potential new generation of agricultural and veterinary drugs. The molecules of the TVMs were designed based on structure and pharmacological property comparisons among AVM, IVM, and milbemycin (MBM). To produce TVMs, a genetically engineered strain, MHJ1011, was constructed from Streptomyces avermitilis G8-17, an AVM industrial strain. In MHJ1011, the native aveA1 gene was seamlessly replaced with milA1 from Streptomyces hygroscopicus. The total titer of the two TVMs produced by MHJ1011 reached 3,400 mg/liter. Insecticidal tests proved that TVM had enhanced activities against Tetranychus cinnabarinus and Bursaphelenchus xylophilus, as desired. This study provides a typical example of exploration for novel active compounds through a new method of polyketide synthase (PKS) reassembly for gene replacement. The results of the insecticidal tests may be of use in elucidating the structure-activity relationship of AVMs and MBMs.

  15. A heterozygous deletion in the glutamate decarboxylase 67 gene enhances maternal and fetal stress vulnerability.

    PubMed

    Uchida, Taku; Oki, Yutaka; Yanagawa, Yuchio; Fukuda, Atsuo

    2011-04-01

    Both down-regulation of glutamate decarboxylase 67 (GAD67) and maternal exposure to severe stress during pregnancy can increase the risk of schizophrenia and related psychotic disorders in the offspring. To investigate a gene-environment interaction, we performed the restraint-and-light stress to pregnant GAD67-GFP knock-in (GAD67(+/GFP)) and wild-type (GAD67(+/+)) mice three times a day for 45 min per session during gestational day (G) 15.0-17.5. The stress hormone (corticosterone) level of pregnant GAD67(+/GFP) mice (the overall GABA content is reduced because of the destruction of one allele of the endogenous GAD67 gene) was higher than that of GAD67(+/+), even without stress. The fetal body weights (GAD67(+/+)) in the GAD67(+/GFP) mothers were lower than those in the GAD67(+/+) mothers. GAD67(+/GFP) fetuses exhibited higher corticosterone (CORT) levels than GAD67(+/+) fetuses, even in non-stressed GAD67(+/+) mothers. Fetal body weight-decreases and CORT-increases by maternal stress (GAD67(+/+) mother) were significantly more in the GAD67(+/GFP) fetuses than the GAD67(+/+) fetuses. These results indicate that a GAD67 heterozygous deletion itself enhances vulnerability by many aspects, e.g., maternal stress, maternity, and being in utero. Thus, an abnormality in GAD67 could interact with environmental risk factors of psychiatric disorders, including schizophrenia.

  16. Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

    NASA Technical Reports Server (NTRS)

    Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

    1998-01-01

    Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

  17. Co-culture with periodontal ligament stem cells enhances osteogenic gene expression in de-differentiated fat cells.

    PubMed

    Tansriratanawong, Kallapat; Tamaki, Yuichi; Ishikawa, Hiroshi; Sato, Soh

    2014-10-01

    In recent decades, de-differentiated fat cells (DFAT cells) have emerged in regenerative medicine because of their trans-differentiation capability and the fact that their characteristics are similar to bone marrow mesenchymal stem cells. Even so, there is no evidence to support the osteogenic induction using DFAT cells in periodontal regeneration and also the co-culture system. Consequently, this study sought to evaluate the DFAT cells co-culture with periodontal ligament stem cells (PDLSCs) in vitro in terms of gene expression by comparing runt-related transcription factor 2 (RUNX2) and Peroxisome proliferator-activated receptor gamma 2 (PPARγ2) genes. We isolated DFAT cells from mature adipocytes and compared proliferation with PDLSCs. After co-culture with PDLSCs, we analyzed transcriptional activity implying by DNA methylation in all adipogenic gene promoters using combined bisulfite restriction analysis. We compared gene expression in RUNX2 gene with the PPARγ2 gene using quantitative RT-PCR. After being sub-cultured, DFAT cells demonstrated morphology similar to fibroblast-like cells. At the same time, PDLSCs established all stem cell characteristics. Interestingly, the co-culture system attenuated proliferation while enhancing osteogenic gene expression in RUNX2 gene. Using the co-culture system, DFAT cells could trans-differentiate into osteogenic lineage enhancing, but conversely, their adipogenic characteristic diminished. Therefore, DFAT cells and the co-culture system might be a novel cell-based therapy for promoting osteogenic differentiation in periodontal regeneration.

  18. Enhancement in motor learning through genetic manipulation of the Lynx1 gene.

    PubMed

    Miwa, Julie M; Walz, Andreas

    2012-01-01

    The cholinergic system is a neuromodulatory neurotransmitter system involved in a variety of brain processes, including learning and memory, attention, and motor processes, among others. The influence of nicotinic acetylcholine receptors of the cholinergic system are moderated by lynx proteins, which are GPI-anchored membrane proteins forming tight associations with nicotinic receptors. Previous studies indicate lynx1 inhibits nicotinic receptor function and limits neuronal plasticity. We sought to investigate the mechanism of action of lynx1 on nicotinic receptor function, through the generation of lynx mouse models, expressing a soluble version of lynx and comparing results to the full length overexpression. Using rotarod as a test for motor learning, we found that expressing a secreted variant of lynx leads to motor learning enhancements whereas overexpression of full-length lynx had no effect. Further, adult lynx1KO mice demonstrated comparable motor learning enhancements as the soluble transgenic lines, whereas previously, aged lynx1KO mice showed performance augmentation only with nicotine treatment. From this we conclude the motor learning is more sensitive to loss of lynx function, and that the GPI anchor plays a role in the normal function of the lynx protein. In addition, our data suggests that the lynx gene plays a modulatory role in the brain during aging, and that a soluble version of lynx has potential as a tool for adjusting cholinergic-dependent plasticity and learning mechanisms in the brain.

  19. Mechanisms of enhanced osteoblast gene expression in the presence of hydroxyapatite coated iron oxide magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Tran, Nhiem; Hall, Douglas; Webster, Thomas J.

    2012-11-01

    Hydroxyapatite (HA) coated iron oxide (Fe3O4) magnetic nanoparticles have been shown to enhance osteoblast (bone forming cells) proliferation and osteoblast differentiation into calcium depositing cells (through increased secretion of alkaline phosphatase, collagen and calcium deposition) compared to control samples without nanoparticles. Such nanoparticles are, thus, very promising for numerous orthopedic applications including magnetically directed osteoporosis treatment. The objective of the current study was to elucidate the mechanisms of the aforementioned improved osteoblast responses in the presence of HA coated Fe3O4 nanoparticles. Results demonstrated large amounts of fibronectin (a protein known to increase osteoblast functions) adsorption on HA coated Fe3O4 nanoparticles. Specifically, fibronectin adsorption almost doubled when HA coated Fe3O4 nanoparticle concentrations increased from 12.5 to 100 μg ml-1, and from 12.5 to 200 μg ml-1, a four fold increase was observed. Results also showed greater osteoblast gene regulation (specifically, osteocalcin, type I collagen and cbfa-1) in the presence of HA coated Fe3O4 nanoparticles. Collectively, these results provide a mechanism for the observed enhanced osteoblast functions in the presence of HA coated iron oxide nanoparticles, allowing their further investigation for a number of orthopedic applications.

  20. Cloning and sequence analysis of candidate human natural killer-enhancing factor genes

    SciTech Connect

    Shau, H.; Butterfield, L.H.; Chiu, R.; Kim, A.

    1994-12-31

    A cytosol factor from human red blood cells enhances natural killer (NK) activity. This factor, termed NK-enhancing factor (NKEF), is a protein of 44000 M{sub r} consisting of two subunits of equal size linked by disulfide bonds. NKEF is expressed in the NK-sensitive erythroleukemic cell line K562. Using an antibody specific for NKEF as a probe for immunoblot screening, we isolated several clones from a {lambda}gt11 cDNA library of K562. Additional subcloning and sequencing revealed that the candidate NKEF cDNAs fell into one of two categories of closely related but non-identical genes, referred to as NKEF A and B. They are 88% identical in amino acid sequence and 71% identical in nucleotide sequence. Southern blot analysis suggests that there are two to three NKEF family members in the genome. Analysis of predicted amino acid sequences indicates that both NKEF A and B are cytosol proteins with several phosphorylation sites each, but that they have no glycosylation sites. They are significantly homologous to several other proteins from a wide variety of organisms ranging from prokaryotes to mammals, especially with regard to several well-conserved motifs within the amino acid sequences. The biological functions of these proteins in other species are mostly unknown, but some of them were reported to be induced by oxidative stress. Therefore, as well as for immunoregulation of NK activity, NKEF may be important for cells in coping with oxidative insults. 32 refs., 3 figs.

  1. Enhanced immune response with foot and mouth disease virus VP1 and interleukin-1 fusion genes.

    PubMed

    Park, Jong Hyeon; Kim, Sun Jin; Oem, Jae Ku; Lee, Kwang Nyeong; Kim, Yong Joo; Kye, Soo Jeong; Park, Jee Yong; Joo, Yi Seok

    2006-09-01

    The capsid of the foot and mouth disease (FMD) virus carries the epitopes that are critical for inducing the immune response. In an attempt to enhance the specific immune response, plasmid DNA was constructed to express VP1/interleukin-1alpha (IL-1alpha) and precursor capsid (P1) in combination with 2A (P1-2A)/IL-1alpha under the control of the human cytomegalovirus (HCMV) immediateearly promoter and intron. After DNA transfection into MA104 (monkey kidney) cells, Western blotting and an immunofluorescence assay were used to confirm the expression of VP1 or P1-2A and IL-1alpha. Mice were inoculated with the encoding plasmids via the intradermal route, and the IgG1 and IgG2a levels were used to determine the immune responses. These results show that although the immunized groups did not carry a high level of neutralizing antibodies, the plasmids encoding the VP1/ IL-1alpha, and P1-2A /IL-1alpha fused genes were effective in inducing an enhanced immune response.

  2. Enhanced Maternal Aggression and Associated Changes in Neuropeptide Gene Expression in Multiparous Rats

    PubMed Central

    Nephew, Benjamin C.; Bridges, Robert S.; Lovelock, Dennis F.; Byrnes, Elizabeth M.

    2009-01-01

    While it has often been speculated that prior reproductive experience improves subsequent maternal care, few studies have examined specific changes in behavior during a first versus second lactation. During lactation mothers display heightened aggression toward male intruders, purportedly to protect vulnerable young. In the current study, maternal aggression was examined in primiparous and age-matched, multiparous females on postpartum days 5 (PPD5) and PPD15. Expression of oxytocin (OXT), oxytocin receptor (OXT-R), arginine vasopressin (AVP), arginine vasopressin V1a receptors (V1a), and corticotrophin releasing hormone (CRH) mRNA was measured following aggression testing at both time points using real-time quantitative PCR (qPCR) in brain regions previously implicated in the regulation of maternal aggression. Multiparity significantly enhanced maternal aggression on PPD5 but not on PPD15. In addition, this increased aggression was associated with region and gene specific changes in mRNA expression. These findings indicate that reproductive experience enhances maternal aggression, an effect that may be mediated by region specific alterations in neuropeptidergic activity. The adaptations observed in multiparous females provide an innate model for the study of neuroplasticity in the regulation of aggression. PMID:19824761

  3. Integration of differentiation signals during indirect flight muscle formation by a novel enhancer of Drosophila vestigial gene.

    PubMed

    Bernard, Frédéric; Kasherov, Petar; Grenetier, Sabrina; Dutriaux, Annie; Zider, Alain; Silber, Joël; Lalouette, Alexis

    2009-08-15

    The gene vestigial (vg) plays a key role in indirect flight muscle (IFM) development. We show here that vg is controlled by the Notch anti-myogenic signaling pathway in myoblasts and is regulated by a novel 822 bp enhancer during IFM differentiation. Interestingly, this muscle enhancer is activated in developing fibers and in a small number of myoblasts before the fusion of myoblasts with the developing muscle fibers. Moreover, we show that this enhancer is activated by Drosophila Myocyte enhancing factor 2 (MEF2), Scalloped (SD) and VG but repressed by Twist, demonstrating a sensitivity to differentiation in vivo. In vitro experiments reveal that SD can directly bind this enhancer and MEF2 can physically interact with both SD and TWI. Cumulatively, our data reveal the interplay between different myogenic factors responsible for the expression of an enhancer activated during muscle differentiation.

  4. Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

    PubMed

    Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

    2016-10-01

    We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression.

  5. Differential protein-DNA interactions at the promoter and enhancer regions of developmentally regulated U4 snRNA genes.

    PubMed

    Miyake, J H; Botros, I W; Stumph, W E

    1992-01-01

    In the chicken genome there are two closely-linked genes, U4B and U4X, that code for different sequence variants of U4 small nuclear RNA (snRNA). Both genes are expressed with nearly equal efficiency in the early embryo, but U4X gene expression is specifically down-regulated relative to U4B as development proceeds. At the present time, little is known about the mechanisms that regulate differential expression of snRNA genes. We have now identified a novel chicken factor, PPBF, that binds sequence-specifically in vitro to the proximal regulatory region of the U4X gene, but not to the proximal region of the U4B gene. PPBF is itself regulated during development and may therefore be a key factor involved in differentially regulating U4X gene transcription relative to U4B. The U4X and U4B enhancers contain distinct sequence variants of two essential motifs (octamer and SPH). The Oct-1 transcription factor binds with similar affinities to both the U4X and U4B octamer motifs. However, a second essential snRNA enhancer-binding protein, SBF, has a 20- to 30-fold lower affinity for the SPH motif in the U4X enhancer than for the homologous SPH motif in the U4B enhancer. A potential role therefore exists for SBF, as well as PPBF, in the preferential down-regulation of the U4X RNA gene during chicken development.

  6. A novel G1-specific enhancer identified in the human heat shock protein 70 gene.

    PubMed Central

    Taira, T; Narita, T; Iguchi-Ariga, S M; Ariga, H

    1997-01-01

    Expression of the human heat shock protein 70 gene (hsp70) is induced by various kinds of stress and by oncogenes. In the absence of stress, hsp70 is mainly expressed in the G1and S phases of the cell cycle, but the elements contributing to cell cycle-dependent expression from the hsp70 promoter remain elusive. We have previously reported that two elements, named HSP-MYCA and HSP-MYCB, located approximately 200 bp upstream (-200) from the transcription start site (+1) of human hsp70, are important for initiation of DNA replication at the hsp70 locus. In this report we examine the effect of these two elements on transcriptional activity from the hsp70 promoter, especially in terms of cell cycle-dependent expression. Various segments of the hsp70 promoter region (up to -300) were linked to the luciferase gene and the constructs were transfected into mouse L cells to examine their transcriptional activity. A strong enhancer activity was defined in the HSP-MYCB element, but not in HSP-MYCA. Mutations introduced within HSP-MYCB abolished the transcriptional activation. In synchronized cells, pHB-Luc (a luciferase construct containing approximately 2.4 kb of the hsp70 promoter region) as well as endogenous hsp70 showed two peaks of expression; one in G1 and the other in the S phase. Site-directed mutagenesis of HSP-MYCB in pHB-Luc abolished the expression peak in G1, but not that in the S phase. To test promoter specificity, wild-type and mutant HSP-MYCB elements were then linked to the luciferase gene in combination with the hsp70 , the cyclin A or the PCNA promoter. Both in transient experiments and established cell lines, a strong peak of expression in mid-G1phase was observed with all the constructs containing wild-type HSP-MYCB, but not with the constructs containing the mutant sequence. These results suggest that the HSP-MYCB sequence is a G1-specific enhancer and is responsible for cell cycle-dependent expression of hsp70. PMID:9115365

  7. Enhanced expression of trim14 gene suppressed Sindbis virus reproduction and modulated the transcription of a large number of genes of innate immunity.

    PubMed

    Nenasheva, V V; Kovaleva, G V; Uryvaev, L V; Ionova, K S; Dedova, A V; Vorkunova, G K; Chernyshenko, S V; Khaidarova, N V; Tarantul, V Z

    2015-07-01

    In the present research, we have studied an influence of enhanced expression TRIM14 on alphavirus Sindbis (SINV, Togaviridae family) infection. In the HEK293 cells transfected with human trim14 gene (HEK-trim14), SINV yield after infection was decreased 1000-10,000 times (3-4 lg of TCD50/ml) at 24 h p.i. and considerably less (1-2 lg of TCD50/ml) at 48 h p.i. Analysis of the expression of 43 genes directly or indirectly involved in innate immune machine in HEK-trim14 non-infected cells comparing with the control (non-transfected) HEK293 cells revealed that stable trim14 transfection in HEK293 cells caused increased transcription of 18 genes (ifna, il6 (ifnβ2), isg15, raf-1, NF-kB (nf-kb1, rela, nf-kb2, relb), grb2, grb3-3, traf3ip2, junB, c-myb, pu.1, akt1, tyk2, erk2, mek2) and lowered transcription of 3 genes (ifnγ, gata1, il-17a). The similar patterns of genes expression observe in SINV-infected non-transfected HEK293 cells. However, SINV infection of HEK-trim14 cells caused inhibition of the most interferon cascade genes as well as subunits of transcription factor NF-κB. Thus, stable enhanced expression of trim14 gene in cells activates the transcription of many immunity genes and suppresses the SINV reproduction, but SINV infection of HEK-trim14 cells promotes inhibition of some genes involved in innate immune system.

  8. The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer

    SciTech Connect

    Jaynes, J.B.; Johnson, J.E.; Buskin, J.N.; Gartside, C.L.; Hauschka, S.D.

    1988-01-01

    Muscle creatine kinase (MCK) is induced to high levels during skeletal muscle differentiation. The authors examined the upstream regulatory elements of the mouse MCK gene which specify its activation during myogenesis in culture. Fusion genes containing up to 3,300 nucleotides (nt) of MCK 5' flanking DNA in various positions and orientations relative to the bacterial chloramphenicol acetyltransferase (CAT) structural gene were transfected into cultured cells. Transient expression of CAT was compared between proliferating and differentiated MM14 mouse myoblasts and with nonmyogenic mouse L cells. The major effector of high-level expression was found to have the properties of a transcriptional enhancer. This element, located between 1,050 and 1,256 nt upstream of the transcription start site, was also found to have a major influence on the tissue and differentiation specificity of MCK expression; it activated either the MCK promoter or heterologous promoters only in differentiated muscle cells. Comparisons of viral and cellular enhancer sequences with the MCK enhancer revealed some similarities to essential regions of the simian virus 40 enhancer as well as to a region of the immunoglobulin heavy-chain enhancer, which has been implicated in tissue-specific protein binding. Even in the absence of the enhancer, low-level expression from a 776-nt MCK promoter retained differentiation specificity. In addition to positive regulatory elements, our data provide some evidence for negative regulatory elements with activity in myoblasts. These may contribute to the cell type and differentiation specificity of MCK expression.

  9. The Gene Regulatory Network of Lens Induction Is Wired through Meis-Dependent Shadow Enhancers of Pax6

    PubMed Central

    Antosova, Barbora; Smolikova, Jana; Klimova, Lucie; Lachova, Jitka; Bendova, Michaela; Kozmikova, Iryna; Machon, Ondrej; Kozmik, Zbynek

    2016-01-01

    Lens induction is a classical developmental model allowing investigation of cell specification, spatiotemporal control of gene expression, as well as how transcription factors are integrated into highly complex gene regulatory networks (GRNs). Pax6 represents a key node in the gene regulatory network governing mammalian lens induction. Meis1 and Meis2 homeoproteins are considered as essential upstream regulators of Pax6 during lens morphogenesis based on their interaction with the ectoderm enhancer (EE) located upstream of Pax6 transcription start site. Despite this generally accepted regulatory pathway, Meis1-, Meis2- and EE-deficient mice have surprisingly mild eye phenotypes at placodal stage of lens development. Here, we show that simultaneous deletion of Meis1 and Meis2 in presumptive lens ectoderm results in arrested lens development in the pre-placodal stage, and neither lens placode nor lens is formed. We found that in the presumptive lens ectoderm of Meis1/Meis2 deficient embryos Pax6 expression is absent. We demonstrate using chromatin immunoprecipitation (ChIP) that in addition to EE, Meis homeoproteins bind to a remote, ultraconserved SIMO enhancer of Pax6. We further show, using in vivo gene reporter analyses, that the lens-specific activity of SIMO enhancer is dependent on the presence of three Meis binding sites, phylogenetically conserved from man to zebrafish. Genetic ablation of EE and SIMO enhancers demostrates their requirement for lens induction and uncovers an apparent redundancy at early stages of lens development. These findings identify a genetic requirement for Meis1 and Meis2 during the early steps of mammalian eye development. Moreover, they reveal an apparent robustness in the gene regulatory mechanism whereby two independent "shadow enhancers" maintain critical levels of a dosage-sensitive gene, Pax6, during lens induction. PMID:27918583

  10. Testing the IMEter on rice introns and other aspects of intron-mediated enhancement of gene expression.

    PubMed

    Morello, Laura; Gianì, Silvia; Troina, Filippo; Breviario, Diego

    2011-01-01

    In many eukaryotes, spliceosomal introns are able to influence the level and site of gene expression. The mechanism of this Intron Mediated Enhancement (IME) has not yet been elucidated, but regulation of gene expression is likely to occur at several steps during and after transcription. Different introns have different intrinsic enhancing properties, but the determinants of these differences remain unknown. Recently, an algorithm called IMEter, which is able to predict the IME potential of introns without direct testing, has been proposed. A computer program was developed for Arabidopsis thaliana and rice (Oryza sativa L.), but was only tested experimentally in Arabidopsis by measuring the enhancement effect on GUS expression of different introns inserted within otherwise identical plasmids. To test the IMEter potential in rice, a vector bearing the upstream regulatory sequence of a rice β-tubulin gene (OsTub6) fused to the GUS reporter gene was used. The enhancing intron interrupting the OsTub6 5'-UTR was precisely replaced by seven other introns carrying different features. GUS expression level in transiently transformed rice calli does not significantly correlate with the calculated IMEter score. It was also found that enhanced GUS expression was mainly due to a strong increase in the mRNA steady-state level and that mutations at the splice recognition sites almost completely abolished the enhancing effect. Splicing also appeared to be required for IME in Arabidopsis cell cultures, where failure of the OsTub6 5' region to drive high level gene expression could be rescued by replacing the poorly spliced rice intron with one from Arabidopsis.

  11. Preservation of Gene Duplication Increases the Regulatory Spectrum of Ribosomal Protein Genes and Enhances Growth under Stress.

    PubMed

    Parenteau, Julie; Lavoie, Mathieu; Catala, Mathieu; Malik-Ghulam, Mustafa; Gagnon, Jules; Abou Elela, Sherif

    2015-12-22

    In baker's yeast, the majority of ribosomal protein genes (RPGs) are duplicated, and it was recently proposed that such duplications are preserved via the functional specialization of the duplicated genes. However, the origin and nature of duplicated RPGs' (dRPGs) functional specificity remain unclear. In this study, we show that differences in dRPG functions are generated by variations in the modality of gene expression and, to a lesser extent, by protein sequence. Analysis of the sequence and expression patterns of non-intron-containing RPGs indicates that each dRPG is controlled by specific regulatory sequences modulating its expression levels in response to changing growth conditions. Homogenization of dRPG sequences reduces cell tolerance to growth under stress without changing the number of expressed genes. Together, the data reveal a model where duplicated genes provide a means for modulating the expression of ribosomal proteins in response to stress.

  12. StAR enhances transcription of genes encoding the mitochondrial proteases involved in its own degradation.

    PubMed

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Lauria, Ines; Langer, Thomas; Orly, Joseph

    2014-02-01

    Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR.

  13. Identification of Polymorphisms in the Enhancer Region of the Bovine Prolactin Gene and Association with Fertility in Beef Cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives were to investigate the polymorphic nature of the enhancer region of the bovine prolactin (PRL) gene and determine the association of these polymorphisms with fertility in beef cows. Primers were designed to amplify a 500 base pair fragment 892 to 1392 bases upstream of the bovine PRL gen...

  14. Enhanced factor VIII heavy chain for gene therapy of hemophilia A.

    PubMed

    Chen, Lingxia; Lu, Hui; Wang, Jinhui; Sarkar, Rita; Yang, Xiao; Wang, Hongli; High, Katherine A; Xiao, Weidong

    2009-03-01

    Hemophilia A gene therapy using recombinant adenovirus-associated virus (AAV) vectors has been hampered by the size of the factor VIII (FVIII) cDNA. Previously, splitting the FVIII coding sequence into a heavy-chain (HC) fragment and a light-chain (LC) fragment for dual recombinant AAV vector delivery has been successfully explored. However, the main disadvantage of this approach is a "chain imbalance" problem in which LC secretion is approximately 1-2 logs higher than that of HC, and therefore, the majority of protein synthesized is nonfunctional. To improve HC secretion, we constructed alternate FVIII HCs based on our observation that LC facilitates HC secretion. To our surprise, most of the new HC molecules exhibited enhanced expression over the traditional HC molecule (HC(745)). The optimized HC mutein, HC(HL), including additional acidic-region-3 (ar3) sequences, exhibited three- to fivefold higher activity in both enzyme-linked immunosorbent assay (ELISA) and activated partial thromboplastin time (aPTT) assay in in vitro testing. Further characterization suggested ar3 sequences increased HC secretion, rather than promoting HC synthesis. Intravenous delivery of AAV8-HC(HL)+AAV8-LC or AAV8-HC(745)+AAV8-LC achieved phenotypic correction in hemophilia A mice. Mice receiving AAV8-HC(HL)+AAV8-LC achieved three- to fourfold higher HC expression than AAV8-HC(745)+AAV8-LC, consistent with the FVIII functional assays. HC(HL) should be substituted for HC(745) in a dual AAV vector strategy due to its enhanced expression.

  15. Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming.

    PubMed

    McClellan, Michael J; Wood, C David; Ojeniyi, Opeoluwa; Cooper, Tim J; Kanhere, Aditi; Arvey, Aaron; Webb, Helen M; Palermo, Richard D; Harth-Hertle, Marie L; Kempkes, Bettina; Jenner, Richard G; West, Michelle J

    2013-09-01

    Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of

  16. An early pharyngeal muscle enhancer from the Caenorhabditis elegans ceh-22 gene is targeted by the Forkhead factor PHA-4.

    PubMed

    Vilimas, Tomas; Abraham, Alin; Okkema, Peter G

    2004-02-15

    Caenorhabditis elegans pharyngeal muscle development involves ceh-22, an NK-2 family homeobox gene related to genes controlling heart development in other species. ceh-22 is the earliest known gene expressed in the pharyngeal muscles and is likely regulated directly by factors specifying pharyngeal muscle fate. We have previously implicated the ceh-22 distal enhancer in initiating ceh-22 expression. Here we analyze the distal enhancer using functional and comparative assays. The distal enhancer contains three subelements contributing additively to its activity, and functionally important regulatory sequences are highly conserved in Caenorhabditis briggsae. One subelement, termed DE3, is strongly active in the pharyngeal muscles, and we identified two short oligonucleotides (de199 and de209) contributing to DE3 activity. Multimerized de209 enhances transcription similarly to DE3 specifically in the pharyngeal muscles, suggesting it may be an essential site regulating ceh-22. de209 binds the pan-pharyngeal Forkhead factor PHA-4 in vitro and responds to ectopic pha-4 expression in vivo, suggesting that PHA-4 directly initiates ceh-22 expression through de209. Because de209 enhancer activity is primarily limited to the pharyngeal muscles, we hypothesize that de209 also binds factors functioning with PHA-4 to specifically activate ceh-22 expression in pharyngeal muscle.

  17. Affinity and dose of TCR engagement yield proportional enhancer and gene activity in CD4+ T cells

    PubMed Central

    Allison, Karmel A; Sajti, Eniko; Collier, Jana G; Gosselin, David; Troutman, Ty Dale; Stone, Erica L; Hedrick, Stephen M; Glass, Christopher K

    2016-01-01

    Affinity and dose of T cell receptor (TCR) interaction with antigens govern the magnitude of CD4+ T cell responses, but questions remain regarding the quantitative translation of TCR engagement into downstream signals. We find that while the response of mouse CD4+ T cells to antigenic stimulation is bimodal, activated cells exhibit analog responses proportional to signal strength. Gene expression output reflects TCR signal strength, providing a signature of T cell activation. Expression changes rely on a pre-established enhancer landscape and quantitative acetylation at AP-1 binding sites. Finally, we show that graded expression of activation genes depends on ERK pathway activation, suggesting that an ERK-AP-1 axis plays an important role in translating TCR signal strength into proportional activation of enhancers and genes essential for T cell function. DOI: http://dx.doi.org/10.7554/eLife.10134.001 PMID:27376549

  18. Expression of an isoflavone reductase-like gene enhanced by pollen tube growth in pistils of Solanum tuberosum.

    PubMed

    van Eldik, G J; Ruiter, R K; Colla, P H; van Herpen, M M; Schrauwen, J A; Wullems, G J

    1997-03-01

    Successful sexual reproduction relies on gene products delivered by the pistil to create an environment suitable for pollen tube growth. These compounds are either produced before pollination or formed during the interactions between pistil and pollen tubes. Here we describe the pollination-enhanced expression of the cp100 gene in pistils of Solanum tuberosum. Temporal analysis of gene expression revealed an enhanced expression already one hour after pollination and lasts more than 72 h. Increase in expression also occurred after touching the stigma and was not restricted to the site of touch but spread into the style. The predicted CP100 protein shows similarity to leguminous isoflavone reductases (IFRs), but belongs to a family of IFR-like NAD(P)H-dependent oxidoreductases present in various plant species.

  19. Optimized codon usage enhances the expression and immunogenicity of DNA vaccine encoding Taenia solium oncosphere TSOL18 gene.

    PubMed

    Wang, Yuan-Yuan; Chang, Xue-Lian; Tao, Zhi-Yong; Wang, Xiao-Li; Jiao, Yu-Meng; Chen, Yong; Qi, Wen-Juan; Xia, Hui; Yang, Xiao-Di; Sun, Xin; Shen, Ji-Long; Fang, Qiang

    2015-07-01

    Cysticercosis due to larval cysts of Taenia solium, is a serious public health problem affecting humans in numerous regions worldwide. The oncospheral stage-specific TSOL18 antigen is a promising candidate for an anti-cysticercosis vaccine. It has been reported that the immunogenicity of the DNA vaccine may be enhanced through codon optimization of candidate genes. The aim of the present study was to further increase the efficacy of the cysticercosis DNA vaccine; therefore, a codon optimized recombinant expression plasmid pVAX1/TSOL18 was developed in order to enhance expression and immunogenicity of TSOL18. The gene encoding TSOL18 of Taenia solium was optimized, and the resulting opt-TSOL18 gene was amplified and expressed. The results of the present study showed that the codon-optimized TSOL18 gene was successfully expressed in CHO-K1 cells, and immunized mice vaccinated with opt-TSOL18 recombinant expression plasmids demonstrated opt‑TSOL18 expression in muscle fibers, as determined by immunohistochemistry. In addition, the codon-optimized TSOL18 gene produced a significantly greater effect compared with that of TSOL18 and active spleen cells were markedly stimulated in vaccinated mice. 3H-thymidine incorporation was significantly greater in the opt-TSOL18 group compared with that of the TSOL18, pVAX and blank control groups (P<0.01). In conclusion, the eukaryotic expression vector containing the codon-optimized TSOL18 gene was successfully constructed and was confirmed to be expressed in vivo and in vitro. The expression and immunogenicity of the codon-optimized TSOL18 gene were markedly greater compared with that of the un-optimized gene. Therefore, these results may provide the basis for an optimized TSOL18 gene vaccine against cysticercosis.

  20. A novel role for the tumour suppressor Nitrilase1 modulating the Wnt/β-catenin signalling pathway

    PubMed Central

    Mittag, Sonnhild; Valenta, Tomas; Weiske, Jörg; Bloch, Laura; Klingel, Susanne; Gradl, Dietmar; Wetzel, Franziska; Chen, Yuan; Petersen, Iver; Basler, Konrad; Huber, Otmar

    2016-01-01

    Nitrilase1 was classified as a tumour suppressor in association with the fragile histidine-triad protein Fhit. However, knowledge about nitrilase1 and its tumour suppressor function is still limited. Whereas nitrilase1 and Fhit are discrete proteins in mammals, they are merged in Drosophila melanogaster and Caenorhabditis elegans. According to the Rosetta-Stone hypothesis, proteins encoded as fusion proteins in one organism and as separate proteins in another organism may act in the same signalling pathway. Although a direct interaction of human nitrilase1 and Fhit has not been shown, our previous finding that Fhit interacts with β-catenin and represses its transcriptional activity in the canonical Wnt pathway suggested that human nitrilase1 also modulates Wnt signalling. In fact, human nitrilase1 forms a complex with β-catenin and LEF-1/TCF-4, represses β-catenin-mediated transcription and shows an additive effect together with Fhit. Knockdown of human nitrilase1 enhances Wnt target gene expression. Moreover, our experiments show that β-catenin competes away human nitrilase1 from LEF-1/TCF and thereby contributes to the activation of Wnt-target gene transcription. Inhibitory activity of human nitrilase1 on vertebrate Wnt signalling was confirmed by repression of Wnt-induced double axis formation in Xenopus embryogenesis. In line with this finding, the Drosophila fusion protein Drosophila NitFhit directly binds to Armadillo and represses the Wingless pathway in reporter gene assays. Genetic experiments confirmed the repressive activity of Drosophila NitFhit on Wingless signalling in the Drosophila wing imaginal disc. In addition, colorectal tumour microarray analysis revealed a significantly reduced expression of human nitrilase1 in poorly differentiated tumours. Taken together, repression of the canonical Wnt pathway represents a new mechanism for the human nitrilase1 tumour suppressor function. PMID:27462437

  1. A novel role for the tumour suppressor Nitrilase1 modulating the Wnt/β-catenin signalling pathway.

    PubMed

    Mittag, Sonnhild; Valenta, Tomas; Weiske, Jörg; Bloch, Laura; Klingel, Susanne; Gradl, Dietmar; Wetzel, Franziska; Chen, Yuan; Petersen, Iver; Basler, Konrad; Huber, Otmar

    2016-01-01

    Nitrilase1 was classified as a tumour suppressor in association with the fragile histidine-triad protein Fhit. However, knowledge about nitrilase1 and its tumour suppressor function is still limited. Whereas nitrilase1 and Fhit are discrete proteins in mammals, they are merged in Drosophila melanogaster and Caenorhabditis elegans. According to the Rosetta-Stone hypothesis, proteins encoded as fusion proteins in one organism and as separate proteins in another organism may act in the same signalling pathway. Although a direct interaction of human nitrilase1 and Fhit has not been shown, our previous finding that Fhit interacts with β-catenin and represses its transcriptional activity in the canonical Wnt pathway suggested that human nitrilase1 also modulates Wnt signalling. In fact, human nitrilase1 forms a complex with β-catenin and LEF-1/TCF-4, represses β-catenin-mediated transcription and shows an additive effect together with Fhit. Knockdown of human nitrilase1 enhances Wnt target gene expression. Moreover, our experiments show that β-catenin competes away human nitrilase1 from LEF-1/TCF and thereby contributes to the activation of Wnt-target gene transcription. Inhibitory activity of human nitrilase1 on vertebrate Wnt signalling was confirmed by repression of Wnt-induced double axis formation in Xenopus embryogenesis. In line with this finding, the Drosophila fusion protein Drosophila NitFhit directly binds to Armadillo and represses the Wingless pathway in reporter gene assays. Genetic experiments confirmed the repressive activity of Drosophila NitFhit on Wingless signalling in the Drosophila wing imaginal disc. In addition, colorectal tumour microarray analysis revealed a significantly reduced expression of human nitrilase1 in poorly differentiated tumours. Taken together, repression of the canonical Wnt pathway represents a new mechanism for the human nitrilase1 tumour suppressor function.

  2. An adenovirus with enhanced infectivity mediates molecular chemotherapy of ovarian cancer cells and allows imaging of gene expression.

    PubMed

    Hemminki, A; Belousova, N; Zinn, K R; Liu, B; Wang, M; Chaudhuri, T R; Rogers, B E; Buchsbaum, D J; Siegal, G P; Barnes, M N; Gomez-Navarro, J; Curiel, D T; Alvarez, R D

    2001-09-01

    The adenovirus (Ad) is a useful vector for cancer gene therapy due to its unparalleled gene transfer efficiency to dividing and quiescent cells. Primary cancer cells, however, often have highly variable or low levels of the requisite coxsackie-adenovirus receptor (CAR). Also, assessment of gene transfer and vector persistence has been logistically difficult in human clinical trials. We describe here two novel bicistronic adenoviral (Ad) vectors, AdTKSSTR and RGDTKSSTR, which contain the herpes simplex virus thymidine kinase gene (TK) for molecular chemotherapy and bystander effect. In addition, the viruses contain the human somatostatin receptor subtype-2 gene (SSTR2), the expression of which can be noninvasively imaged. We enhanced the infectivity of RGDTKSSTR by genetically incorporating the RGD-4C motif into the HI-loop of the fiber. This allows the virus to circumvent CAR deficiency by binding to alpha(v)beta(3) and alpha(v)beta(5) integrins, which are highly expressed on most ovarian cancers. The expanded tropism of RGDTKSSTR results in increased infectivity of purified primary ovarian cancer cells and allows enhanced gene transfer in the presence of malignant ascites containing anti-Ad antibodies. RGDTKSSTR may be a useful agent for treating ovarian cancer in clinical trials.

  3. CP2 binding to the promoter is essential for the enhanced transcription of globin genes in erythroid cells.

    PubMed

    Chae, Ji Hyung; Kim, Chul Geun

    2003-02-28

    We have previously reported that the reduced level of CP2 suppresses the mouse alpha- and beta-globin gene expression and hemoglobin synthesis during terminal differentiation of mouse erythroleukemia (MEL) cells in vitro [Chae et al. (1999)]. As an extension of this study, we demonstrated that human alpha-, epsilon-, and gamma- globin genes were also suppressed by the reduced expression of CP2 in K562 cells. To address how much CP2 contributes in the regulation of globin gene expression, we measured transcriptional activities of the wild type alpha-globin promoter and its various factor-binding sites mutants in erythroid and nonerythroid cells. Interestingly, CP2 site dependent transcriptional activation occurred in an erythroid-cell specific manner, even though CP2 is ubiquitously expressed. In addition, CP2 site mutation within the alpha-promoter severely suppressed promoter activity in differentiated, but not in undifferentiated MEL cells, suggesting that the CP2 binding site is needed for the enhanced transcription of globin genes during erythroid differentiation. When the human beta-globin locus control region was linked to the alpha-promoter, suppression was more severe in the CP2 site mutant in differentiated MEL cells. Overall data indicate that CP2 is a major factor in the regulation of globin expression in human and mouse erythroid cells, and CP2 binding to the globin gene promoter is essential for the enhanced transcription of globin genes in erythroid differentiation.

  4. NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly

    PubMed Central

    Gilchrist, Daniel A.; Nechaev, Sergei; Lee, Chanhyo; Ghosh, Saikat Kumar B.; Collins, Jennifer B.; Li, Leping; Gilmour, David S.; Adelman, Karen

    2008-01-01

    The Negative Elongation Factor (NELF) is a transcription regulatory complex that induces stalling of RNA polymerase II (Pol II) during early transcription elongation and represses expression of several genes studied to date, including Drosophila Hsp70, mammalian proto-oncogene junB, and HIV RNA. To determine the full spectrum of NELF target genes in Drosophila, we performed a microarray analysis of S2 cells depleted of NELF and discovered that NELF RNAi affects many rapidly inducible genes involved in cellular responses to stimuli. Surprisingly, only one-third of NELF target genes were, like Hsp70, up-regulated by NELF-depletion, whereas the majority of target genes showed decreased expression levels upon NELF RNAi. Our data reveal that the presence of stalled Pol II at this latter group of genes enhances gene expression by maintaining a permissive chromatin architecture around the promoter-proximal region, and that loss of Pol II stalling at these promoters is accompanied by a significant increase in nucleosome occupancy and a decrease in histone H3 Lys 4 trimethylation. These findings identify a novel, positive role for stalled Pol II in regulating gene expression and suggest that there is a dynamic interplay between stalled Pol II and chromatin structure. PMID:18628398

  5. Heterologous expression of transaldolase gene Tal from Saccharomyces cerevisiae in Fusarium oxysporum for enhanced bioethanol production.

    PubMed

    Fan, Jin-Xia; Yang, Xiao-Xue; Song, Jin-Zhu; Huang, Xiao-Mei; Cheng, Zhong-Xiang; Yao, Lin; Juba, Olivia S; Liang, Qing; Yang, Qian; Odeph, Margaret; Sun, Yan; Wang, Yun

    2011-08-01

    The filamentous fungus Fusarium oxysporum is known for its ability to ferment xylose-producing ethanol. However, efficiency of xylose utilization and ethanol yield was low. In this study, the transaldolase gene from Saccharomyces cerevisiae has been successfully expressed in F. oxysporum by an Agrobacterium tumefaciens-mediated transformation method. The enzymatic activity of the recombinant fungus (cs28pCAM-Sctal4) was 0.195 times higher than that of the wild-type strain (cs28). The recombinant strain also exhibited a 28.83% increase in ethanol yield on xylose media compared to the parental strain. Enhanced ethanol production and a reduction in the biomass were observed during xylose fermentation. Ethanol yield from rice straw by simultaneous saccharification and fermentation with cs28pCAM-Sctal4 was 0.25 g g⁻¹ of rice straw. The transgenic strain of F. oxysporum cs28pCAM-Sctal4 might therefore have potential applications in industrial bioenergy production.

  6. An enhancer trap screen for ecdysone-inducible genes required for Drosophila adult leg morphogenesis.

    PubMed Central

    Gates, J; Thummel, C S

    2000-01-01

    Although extensive studies of Drosophila imaginal disc development have focused on proliferation and patterning, relatively little is known about how the patterned imaginal discs are transformed into adult structures during metamorphosis. Studies focused primarily on leg development have shown that this remarkable transformation is coordinated by pulses of the steroid hormone ecdysone and requires the function of ecdysone-inducible transcription factors as well as proteases and components of the contractile cytoskeleton and adherens junctions. Here, we describe a genetic screen aimed at expanding our understanding of the hormonal regulation of Drosophila adult leg morphogenesis. We screened 1300 lethal P-element enhancer trap insertions on the second chromosome for a series of sequential parameters including pupal lethality, defects in leg morphogenesis, and ecdysone-induced lacZ reporter gene expression. From this screen we identified four mutations, one of which corresponds to bancal, which encodes the Drosophila homolog of hnRNP K. We also identified vulcan, which encodes a protein that shares sequence similarity with a family of rat SAPAP proteins. Both bancal and vulcan are inducible by ecdysone, thus linking the hormone signal with leg morphogenesis. This screen provides new directions for understanding the hormonal regulation of leg development during Drosophila metamorphosis. PMID:11102372

  7. Distribution of SR protein exonic splicing enhancer motifs in human protein-coding genes.

    PubMed

    Wang, Jinhua; Smith, Philip J; Krainer, Adrian R; Zhang, Michael Q

    2005-01-01

    Exonic splicing enhancers (ESEs) are pre-mRNA cis-acting elements required for splice-site recognition. We previously developed a web-based program called ESEfinder that scores any sequence for the presence of ESE motifs recognized by the human SR proteins SF2/ASF, SRp40, SRp55 and SC35 (http://rulai.cshl.edu/tools/ESE/). Using ESEfinder, we have undertaken a large-scale analysis of ESE motif distribution in human protein-coding genes. Significantly higher frequencies of ESE motifs were observed in constitutive internal protein-coding exons, compared with both their flanking intronic regions and with pseudo exons. Statistical analysis of ESE motif frequency distributions revealed a complex relationship between splice-site strength and increased or decreased frequencies of particular SR protein motifs. Comparison of constitutively and alternatively spliced exons demonstrated slightly weaker splice-site scores, as well as significantly fewer ESE motifs, in the alternatively spliced group. Our results underline the importance of ESE-mediated SR protein function in the process of exon definition, in the context of both constitutive splicing and regulated alternative splicing.

  8. The Or gene enhances carotenoid accumulation and stability during post-harvest storage of potato tubers.

    PubMed

    Li, Li; Yang, Yong; Xu, Qiang; Owsiany, Katherine; Welsch, Ralf; Chitchumroonchokchai, Chureeporn; Lu, Shan; Van Eck, Joyce; Deng, Xiu-Xin; Failla, Mark; Thannhauser, Theodore W

    2012-03-01

    Provitamin A carotenoids in staple crops are not very stable during storage and their loss compromises nutritional quality. To elucidate the fundamental mechanisms underlying carotenoid accumulation and stability, we investigated transgenic potato tubers that expressed the cauliflower Orange (Or) gene. We found that the Or transgene not only promoted retention of β-carotene level, but also continuously stimulated its accumulation during 5 months of cold storage. In contrast, no increased levels of carotenoids were observed in the tubers of vector-only controls or a yellow-flesh variety during the same period of storage. The increased carotenoid accumulation was found to be associated with the formation of lipoprotein-carotenoid sequestering structures, as well as with the enhanced abundance of phytoene synthase, a key enzyme in the carotenoid biosynthetic pathway. Furthermore, the provitamin A carotenoids stored were shown to be stable during simulated digestion and accessible for uptake by human intestinal absorptive cells. Proteomic analysis identified three major functional groups of proteins (i.e. heat shock proteins, glutathione-S-transferases, and carbohydrate metabolic proteins) that are potentially important in the Or-regulated carotenoid accumulation. Our results show that regulation of carotenoid sequestration capacity is an important mechanism by which carotenoid stability is regulated. Our findings suggest that induction of a proper sink structure formation in staple crops may provide the crops with a unique ability to promote and/or stabilize provitamin A accumulation during plant growth and post-harvest storage.

  9. CONVECTION-ENHANCED DELIVERY AND SYSTEMIC MANNITOL INCREASE GENE PRODUCT DISTRIBUTION OF AAV VECTORS 5, 8, AND 9 AND INCREASE GENE PRODUCT IN THE ADULT MOUSE BRAIN

    PubMed Central

    Carty, Nikisha; Lee, Daniel; Dickey, Chad; Ceballos-Diaz, Carolina; Jansen-West, Karen; Golde, Todd E.; Gordon, Marcia N.; Morgan, Dave; Nash, Kevin

    2010-01-01

    The use of recombinant adeno-associated viral (rAAV) vectors as a means of gene delivery to the central nervous system has emerged as a potentially viable method for the treatment of several types of degenerative brain diseases. However, a limitation of typical intracranial injections into the adult brain parenchyma is the relatively restricted distribution of the delivered gene to large brain regions such as the cortex, presumably due to confined dispersion of the injected particles. Optimizing the administration techniques to maximize gene distribution and gene expression is an important step in developing gene therapy studies. Here, we have found additive increases in distribution when 3 methods to increase brain distribution of rAAV were combined. The convection enhanced delivery (CED) method with the step-design cannula was used to deliver rAAV vector serotypes 5, 8 and 9 encoding GFP into the hippocampus of the mouse brain. While the CED method improved distribution of all 3 serotypes, the combination of rAAV9 and CED was particularly effective. Systemic mannitol administration, which reduces intracranial pressure, also further expanded distribution of GFP expression, in particular, increased expression on the contralateral hippocampi. These data suggest that combining advanced injection techniques with newer rAAV serotypes greatly improves viral vector distribution, which could have significant benefits for implementation of gene therapy strategies. PMID:20951738

  10. Phosphorothioate analogs of P1,P3-di(nucleosid-5'-yl) triphosphates: Synthesis, assignment of the absolute configuration at P-atoms and P-stereodependent recognition by Fhit hydrolase.

    PubMed

    Kaczmarek, Renata; Krakowiak, Agnieszka; Korczyński, Dariusz; Baraniak, Janina; Nawrot, Barbara

    2016-11-01

    Di(nucleosid-5'-yl) polyphosphates (NPnN) are involved in various biological processes, and constitute signaling molecules in the intermolecular purinergic systems. They exert tumor suppression function and are substrates for specific hydrolases (e.g., HIT proteins). Their structural analogs may serve as molecular probes and potential therapeutic agents. Three P1,P3-bis-thio-analogs of symmetrical di(nucleosid-5'-yl) triphosphates (NP3N) bearing adenosine, guanosine or ribavirin residues (6, 7 and 8, respectively), were obtained by direct condensation of corresponding base-protected nucleoside-5'-O-(2-thio-1,3,2-oxathiaphospholane) with anhydrous phosphoric acid in the presence of DBU. Deprotected products 6 and 8 were separated into individual P-diastereoisomers, whereas 7 was partially separated to yield diastereomerically enriched fractions. The absolute configuration at P-stereogenic centers in the separated diastereoisomers was assigned by RP-HPLC analysis of the products of enzymatic digestion with snake venom phosphodiesterase. The Fhit-assisted hydrolysis rates for 6 and 7 are by 2-3 orders of magnitude lower than that for the reference AP3A, and depend on the configuration of the stereogenic phosphorus atoms, while 8 occurred to be resistant to this cleavage.

  11. Identification of genes whose expressions are enhanced or reduced in baker's yeast during fed-batch culture process using molasses medium by DNA microarray analysis.

    PubMed

    Shima, Jun; Kuwazaki, Seigo; Tanaka, Fumiko; Watanabe, Hajime; Yamamoto, Hideki; Nakajima, Ryoichi; Tokashiki, Tadaaki; Tamura, Hiromi

    2005-06-25

    Genes whose expression levels are enhanced or reduced during the cultivation process that uses cane molasses in baker's yeast production were identified in this study. The results showed that baker's yeast grown in molasses medium had higher fermentation ability and stress tolerance compared with baker's yeast grown in synthetic medium. Molasses apparently provided not only sugar as a carbon source but also provided functional components that enhanced or reduced expression of genes involved in fermentation ability and stress tolerance. To identify the genes whose expression is enhanced or reduced during cultivation in molasses medium, DNA microarray analysis was then used to compare the gene expression profile of cells grown in molasses with that of cells grown in synthetic medium. To simulate the commercial baker's yeast production process, cells were cultivated using a fed-batch culture system. In molasses medium, genes involved in the synthesis or uptake of vitamins (e.g., biotin, pyridoxine and thiamine) showed enhanced expression, suggesting that vitamin concentrations in molasses medium were lower than those in synthetic medium. Genes involved in formate dehydrogenase and maltose assimilation showed enhanced expression in molasses medium. In contrast, genes involved in iron utilization (e.g., siderophore, iron transporter and ferroxidase) showed enhanced expression in synthetic medium, suggesting that iron starvation occurred. The genes involved in the metabolism of amino acids also showed enhanced expression in synthetic medium. This identification of genes provides information that will help improve the baker's yeast production process.

  12. Apoptotic genes in cancer therapy.

    PubMed

    Opalka, Bertram; Dickopp, Alexandra; Kirch, Hans-Christoph

    2002-01-01

    Induction of apoptosis in malignant cells is a major goal of cancer therapy in general and of certain cancer gene therapy strategies in particular. Numerous apoptosis-regulating genes have been evaluated for this purpose. Besides the most prominent p53 gene others include p16, p21, p27, E2F genes, FHIT, PTEN and CASPASE genes. Recently, the potential for therapy of an adenoviral gene, E1A, known for a long time for its apoptosis-inducing activity, has been discovered. In experimental settings, these genes have proven their tumor-suppressive and apoptosis-inducing activity. Clinical trials are currently being performed with selected genes. By far the most studies transfer the p53 gene using retro- or adenoviral vectors. Disease stabilization or other benefits were observed in a limited number of patients when p53 was applied alone or in combination with cytotoxic drugs. A second proapoptotic gene that has entered clinical trials is adenovirus E1A. Here, too, disease stabilization as well as/or local regression in one case have been demonstrated in selected patients. In all cases, side effects were tolerable. To further improve E1A as a therapeutic transgene, we have deleted transforming domains from the adenovirus 5 and 12 13S cDNAs. Mutants were derived which had completely lost their transforming activity in combination with the E1B oncogene but retained a pronounced tumor-suppressive activity. Cells transduced with these constructs showed a highly reduced ability to grow in soft agar, and tumor growth in nude mice could be substantially suppressed. Outgrowing tumors had lost E1A expression when analyzed in Western blots. These E1A constructs may represent valuable tools for cancer gene therapy in the future.

  13. Gene silencing of TACE enhances plaque stability and improves vascular remodeling in a rabbit model of atherosclerosis

    PubMed Central

    Zhao, Xueqiang; Kong, Jing; Zhao, Yuxia; Wang, Xuping; Bu, Peili; Zhang, Cheng; Zhang, Yun

    2015-01-01

    We aimed to test the hypothesis that gene silencing of tumor necrosis factor alpha converting enzyme (TACE) may attenuate lesion inflammation and positive vascular remodeling and enhance plaque stability in a rabbit model of atherosclerosis. Lentivirus-mediated TACE shRNA was injected into the abdominal aortic plaques of rabbits which effectively down-regulated TACE expression and activities from week 8 to week 16. TACE gene silencing reduced remodeling index and plaque burden, and diminished the content of macrophages and lipids while increased that of smooth muscle cells and collagen in the aortic plaques. In addition, TACE gene silencing attenuated the local expression of P65, iNOS, ICAM-1, VEGF and Flt-1 and activities of MMP9 and MMP2 while increased the local expression of TGF-β1 together with reduced number of neovessels in the aorta. TACE shRNA treatment resulted in down-regulated expression of TACE in macrophages and blunted ERK-P38 phosphorylation and tube formation of co-cultured mouse vascular smooth muscle cells or human umbilical vein endothelial cells. In conclusion, gene silencing of TACE enhanced plaque stability and improved vascular positive remodeling. The mechanisms may involve attenuated local inflammation, neovascularization and MMP activation, as well as enhanced collagen production probably via down-regulated ERK-NF-κB and up-regulated TGF-β1 signaling pathways. PMID:26655882

  14. Application of pulsed-magnetic field enhances non-viral gene delivery in primary cells from different origins

    NASA Astrophysics Data System (ADS)

    Kamau Chapman, Sarah W.; Hassa, Paul O.; Koch-Schneidemann, Sabine; von Rechenberg, Brigitte; Hofmann-Amtenbrink, Margarethe; Steitz, Benedikt; Petri-Fink, Alke; Hofmann, Heinrich; Hottiger, Michael O.

    Primary cell lines are more difficult to transfect when compared to immortalized/transformed cell lines, and hence new techniques are required to enhance the transfection efficiency in these cells. We isolated and established primary cultures of synoviocytes, chondrocytes, osteoblasts, melanocytes, macrophages, lung fibroblasts, and embryonic fibroblasts. These cells differed in several properties, and hence were a good representative sample of cells that would be targeted for expression and delivery of therapeutic genes in vivo. The efficiency of gene delivery in all these cells was enhanced using polyethylenimine-coated polyMAG magnetic nanoparticles, and the rates (17-84.2%) surpassed those previously achieved using other methods, especially in cells that are difficult to transfect. The application of permanent and pulsating magnetic fields significantly enhanced the transfection efficiencies in synoviocytes, chondrocytes, osteoblasts, melanocytes and lung fibroblasts, within 5 min of exposure to these magnetic fields. This is an added advantage for future in vivo applications, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.

  15. Yin Yang 1 Intronic Binding Sequences and Splicing Elicit Intron-Mediated Enhancement of Ubiquitin C Gene Expression

    PubMed Central

    Bianchi, Marzia; Crinelli, Rita; Giacomini, Elisa; Carloni, Elisa; Radici, Lucia; Magnani, Mauro

    2013-01-01

    In a number of organisms, introns affect expression of the gene in which they are contained. Our previous studies revealed that the 5′-UTR intron of human ubiquitin C (UbC) gene is responsible for the boost of reporter gene expression and is able to bind, in vitro, Yin Yang 1 (YY1) trans-acting factor. In this work, we demonstrate that intact YY1 binding sequences are required for maximal promoter activity and YY1 silencing causes downregulation of luciferase mRNA levels. However, YY1 motifs fail to enhance gene expression when the intron is moved upstream of the proximal promoter, excluding the typical enhancer hypothesis and supporting a context-dependent action, like intron-mediated enhancement (IME). Yet, almost no expression is seen in the construct containing an unspliceable version of UbC intron, indicating that splicing is essential for promoter activity. Moreover, mutagenesis of YY1 binding sites and YY1 knockdown negatively affect UbC intron removal from both endogenous and reporter transcripts. Modulation of splicing efficiency by YY1 cis-elements and protein factor may thus be part of the mechanism(s) by which YY1 controls UbC promoter activity. Our data highlight the first evidence of the involvement of a sequence-specific DNA binding factor in IME. PMID:23776572

  16. BAC transgenic zebrafish reveal hypothalamic enhancer activity around obesity associated SNP rs9939609 within the human FTO gene.

    PubMed

    Rinkwitz, Silke; Geng, Fan-Suo; Manning, Elizabeth; Suster, Maximiliano; Kawakami, Koichi; Becker, Thomas S

    2015-10-01

    Single Nucleotide Polymorphisms in FTO intron 1 have been associated with obesity risk, leading to the hypothesis that FTO is the obesity-related gene. However, other studies have shown that the FTO gene is part of the regulatory domain of the neighboring IRX3 gene and that enhancers in FTO intron 1 regulate IRX3. While Irx3 activity was shown to be necessary in the hypothalamus for the metabolic function of Irx3 in mouse, no enhancers with hypothalamic activity have been demonstrated in the risk-associated region within FTO. In order to identify potential enhancers at the human FTO locus in vivo, we tested regulatory activity in FTO intron 1 using BAC transgenesis in zebrafish. A minimal gata2 promoter-GFP cassette was inserted 1.3 kb upstream of the obesity associated SNP rs9939609 in a human FTO BAC plasmid. In addition to the previously identified expression domains in notochord and kidney, human FTO BAC:GFP transgenic zebrafish larvae expressed GFP in the ventral posterior tuberculum, the posterior hypothalamus and the anterior brainstem, which are also expression domains of zebrafish irx3a. In contrast, an in-frame insertion of a GFP cassette at the FTO start codon resulted in weak ubiquitous GFP expression indicating that the promoter of FTO does likely not react to enhancers located in the obesity risk-associated region.

  17. Phylogenetic Footprinting Reveals Evolutionarily Conserved Regions of the Gonadotropin-Releasing Hormone Gene that Enhance Cell-Specific Expression

    PubMed Central

    GIVENS, MARJORY L.; KUROTANI, REIKO; RAVE-HAREL, NAAMA; MILLER, NICHOL L. G.; MELLON, PAMELA L.

    2010-01-01

    Reproductive function is controlled by the hypothalamic neuropeptide, GnRH, which serves as the central regulator of the hypothalamic-pituitary-gonadal axis. GnRH expression is limited to a small population of neurons in the hypothalamus. Targeting this minute population of neurons (as few as 800 in the mouse) requires regulatory elements upstream of the GnRH gene that remain to be fully characterized. Previously, we have identified an evolutionarily conserved promoter region (−173 to −1) and an enhancer (−1863 to −1571) in the rat gene that targets a subset of the GnRH neurons in vivo. In the present study, we used phylogenetic sequence comparison between human and rodents and analysis of the transcription factor clusters within conserved regions in an attempt to identify additional upstream regulatory elements. This approach led to the characterization of a new upstream enhancer that regulates expression of GnRH in a cell-specific manner. Within this upstream enhancer are nine binding sites for Octamer-binding transcription factor 1 (OCT1), known to be an important transcriptional regulator of GnRH gene expression. In addition, we have identified nuclear factor I (NF1) binding to multiple elements in the GnRH-regulatory regions, each in close proximity to OCT1. We show that OCT1 and NF1 physically and functionally interact. Moreover, the OCT1 and NF1 binding sites in the regulatory regions appear to be essential for appropriate GnRH gene expression. These findings indicate a role for this upstream enhancer and novel OCT1/NF1 complexes in neuron-restricted expression of the GnRH gene. PMID:15319450

  18. Methylation of multiple genes in hepatitis C virus associated hepatocellular carcinoma

    PubMed Central

    Zekri, Abdel-Rahman N.; Bahnasy, Abeer A.; Shoeab, Fatma elzahraa M.; Mohamed, Waleed S.; El-Dahshan, Dina H.; Ali, Fahmey T.; Sabry, Gilane M.; Dasgupta, Nairajana; Daoud, Sayed S.

    2013-01-01

    We studied promoter methylation (PM) of 11 genes in Peripheral Blood Lymphocytes (PBLs) and tissues of hepatitis C virus (HCV) associated hepatocellular carcinoma (HCC) and chronic hepatitis (CH) Egyptian patients. The present study included 31 HCC with their ANT, 38 CH and 13 normal hepatic tissue (NHT) samples. In all groups, PM of APC, FHIT, p15, p73, p14, p16, DAPK1, CDH1, RARβ, RASSF1A, O6MGMT was assessed by methylation-specific PCR (MSP). APC and O6-MGMT protein expression was assessed by immunohistochemistry (IHC) in the studied HCC and CH (20 samples each) as well as in a different HCC and CH set for confirmation of MSP results. PM was associated with progression from CH to HCC. Most genes showed high methylation frequency (MF) and the methylation index (MI) increased with disease progression. MF of p14, p73, RASSF1A, CDH1 and O6MGMT was significantly higher in HCC and their ANT. MF of APC was higher in CH. We reported high concordance between MF in HCC and their ANT, MF in PBL and CH tissues as well as between PM and protein expression of APC and O6MGMT. A panel of 4 genes (APC, p73, p14, O6MGMT) classifies the cases independently into HCC and CH with high accuracy (89.9%), sensitivity (83.9%) and specificity (94.7%). HCV infection may contribute to hepatocarcinogenesis through enhancing PM of multiple genes. PM of APC occurs early in the cascade while PM of p14, p73, RASSF1A, RARB, CDH1 and O6MGMT are late changes. A panel of APC, p73, p14, O6-MGMT could be used in monitoring CH patients for early detection of HCC. Also, we found that, the methylation status is not significantly affected by whether the tissue was from the liver or PBL, indicating the possibility of use PBL as indicator to genetic profile instead of liver tissue regardless the stage of disease. PMID:25685469

  19. The dopaminergic stabilizers pridopidine and ordopidine enhance cortico-striatal Arc gene expression.

    PubMed

    Waters, Susanna; Ponten, Henrik; Edling, Malin; Svanberg, Boel; Klamer, Daniel; Waters, Nicholas

    2014-11-01

    The dopaminergic stabilizers pridopidine [4-(3-(methylsulfonyl)phenyl)-1-propylpiperidine] and ordopidine [1-ethyl-4-(2-fluoro-3-(methylsulfonyl)phenyl)piperidine] inhibit psychostimulant-induced hyperactivity, and stimulate behaviour in states of hypoactivity. While both compounds act as dopamine D2 receptor antagonists in vitro, albeit with low affinity, their specific state-dependent behavioural effect profile is not shared by D2 receptor antagonists in general. To further understand the neuropharmacological effects of pridopidine and ordopidine, and how they differ from other dopaminergic compounds in vivo, we assessed the expression of activity-regulated cytoskeleton-associated protein/activity-regulated gene 3.1 (Arc), an immediate early gene marker associated with synaptic activation, in the frontal cortex and striatum. Furthermore, monoamine neurochemistry and locomotor activity were assessed. The effects of pridopidine and ordopidine were compared to reference dopamine D1 and D2 receptor agonists and antagonists, as well as the partial dopamine D2 agonist aripiprazole. Pridopidine and ordopidine induced significant increases in cortical Arc expression, reaching 2.2- and 1.7-fold levels relative to control, respectively. In contrast, none of the reference dopamine D1 and D2 compounds tested increased cortical Arc expression. In the striatum, significant increases in Arc expression were seen with both pridopidine and ordopidine as well as the dopamine D2 receptor antagonists, remoxipride and haloperidol. Interestingly, striatal Arc expression correlated strongly and positively with striatal 3,4-dihydroxyphenylacetic acid, suggesting that antagonism of dopamine D2 receptors increases Arc expression in the striatum. In conclusion, the concurrent increase in cortical and striatal Arc expression induced by pridopidine and ordopidine appears unique for the dopaminergic stabilizers, as it was not shared by the reference compounds tested. The increase in cortical

  20. Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

    NASA Astrophysics Data System (ADS)

    Hu, Zongli; Parekh, Urvi; Maruta, Natsumi; Trusov, Yuri; Botella, Jimmy

    2015-01-01

    Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Derived RNA interference (HD-RNAi) technology to partially silence three different genes (FOW2, FRP1 and OPR) in the hemi-biotrophic fungus Fusarium oxysporum f. sp. conglutinans. Expression of double stranded RNA molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75%, 83% and 72% reduction for FOW2, FRP1 and OPR respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30-50% survival and FOW2 between 45-70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants.

  1. Enhanced Gene Detection Assays for Fumarate-Adding Enzymes Allow Uncovering of Anaerobic Hydrocarbon Degraders in Terrestrial and Marine Systems

    PubMed Central

    von Netzer, Frederick; Pilloni, Giovanni; Kleindienst, Sara; Krüger, Martin; Knittel, Katrin; Gründger, Friederike

    2013-01-01

    The detection of anaerobic hydrocarbon degrader populations via catabolic gene markers is important for the understanding of processes at contaminated sites. Fumarate-adding enzymes (FAEs; i.e., benzylsuccinate and alkylsuccinate synthases) have already been established as specific functional marker genes for anaerobic hydrocarbon degraders. Several recent studies based on pure cultures and laboratory enrichments have shown the existence of new and deeply branching FAE gene lineages, such as clostridial benzylsuccinate synthases and homologues, as well as naphthylmethylsuccinate synthases. However, established FAE gene detection assays were not designed to target these novel lineages, and consequently, their detectability in different environments remains obscure. Here, we present a new suite of parallel primer sets for detecting the comprehensive range of FAE markers known to date, including clostridial benzylsuccinate, naphthylmethylsuccinate, and alkylsuccinate synthases. It was not possible to develop one single assay spanning the complete diversity of FAE genes alone. The enhanced assays were tested with a range of hydrocarbon-degrading pure cultures, enrichments, and environmental samples of marine and terrestrial origin. They revealed the presence of several, partially unexpected FAE gene lineages not detected in these environments before: distinct deltaproteobacterial and also clostridial bssA homologues as well as environmental nmsA homologues. These findings were backed up by dual-digest terminal restriction fragment length polymorphism diagnostics to identify FAE gene populations independently of sequencing. This allows rapid insights into intrinsic degrader populations and degradation potentials established in aromatic and aliphatic hydrocarbon-impacted environmental systems. PMID:23124238

  2. Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

    PubMed Central

    Hu, Zongli; Parekh, Urvi; Maruta, Natsumi; Trusov, Yuri; Botella, Jose R.

    2015-01-01

    Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Delivered RNA interference (HD-RNAi) technology to partially silence three different genes (FOW2, FRP1, and OPR) in the hemi-biotrophic fungus F. oxysporum f. sp. conglutinans. Expression of double stranded RNA (dsRNA) molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75, 83, and 72% reduction for FOW2, FRP1, and OPR, respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30–50% survival and OPR between 45 and 70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants. PMID:25654075

  3. Development of an enhanced B-specific lentiviral vector expressing BTK: a tool for gene therapy of XLA.

    PubMed

    Moreau, T; Barlogis, V; Bardin, F; Nunes, J A; Calmels, B; Chabannon, C; Tonnelle, C

    2008-06-01

    Further development of haematopoietic stem cell (HSC) gene therapy will depend on enhancement of gene transfer safety: ad hoc improvement of vector design relating to each particular disease is thus a crucial issue for HSC gene therapy. We modified a previously described lentiviral vector by adding the Emumar B-specific enhancer to a human CD19 promoter-derived sequence (Mol Ther 2004;10:45-56). We thus significantly improved the level of expression of the green fluorescent protein (GFP) reporter gene while retaining the specificity of expression in B-cell progeny of transduced human CD34+ progenitor cells obtained from cord blood or adult bone marrow. Indeed, GFP was strongly expressed from early medullary pro-B cells to splenic mature B cells whereas transgene expression remained low in transduced immature progenitors as in myeloid and T-lymphoid progeny retrieved from xenografted NOD/SCID/gammac(null) mice. Using this lentiviral vector, we further demonstrated the possibility to express a functional human BTK protein in long-term human CD34+ cell B-lymphoid progeny. This newly designed lentiviral vector fulfils one of the pre-requisites for the development of efficient and safe gene therapy for X-linked agammaglobulinaemia, the most common primary humoral immunodeficiency disorder.

  4. Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo.

    PubMed Central

    Fasbender, A; Lee, J H; Walters, R W; Moninger, T O; Zabner, J; Welsh, M J

    1998-01-01

    Adenovirus (Ad)-mediated gene transfer to airway epithelia is inefficient because the apical membrane lacks the receptor activity to bind adenovirus fiber protein. Calcium phosphate (CaPi) precipitates have been used to deliver plasmid DNA to cultured cell lines. However, such precipitates are not effective in many primary cultures or in vivo. Here we show that incorporating recombinant adenovirus into a CaPi coprecipitate markedly enhances transgene expression in cells that are resistant to adenovirus infection. Enhancement requires that the virus be contained in the precipitate and viral proteins are required to increase expression. Ad: CaPi coprecipitates increase gene transfer by increasing fiber-independent binding of virus to cells. With differentiated cystic fibrosis (CF) airway epithelia in vitro, a 20-min application of Ad:CaPi coprecipitates that encode CF transmembrane conductance regulator produced as much CF transmembrane conductance regulator Cl- current as a 24-h application of adenovirus alone. We found that Ad:CaPi coprecipitates also increased transgene expression in mouse lung in vivo; importantly, expression was particularly prominent in airway epithelia. These results suggest a new mechanism for gene transfer that may be applicable to a number of different gene transfer applications and could be of value in gene transfer to CF airway epithelia in vivo. PMID:9649572

  5. Increased Production of Fatty Acids and Triglycerides in Aspergillus oryzae by Enhancing Expressions of Fatty Acid Synthesis-Related Genes

    SciTech Connect

    Tamano, Koichi; Bruno, Kenneth S.; Karagiosis, Sue A.; Culley, David E.; Deng, Shuang; Collett, James R.; Umemura, Myco; Koike, Hideaki; Baker, Scott E.; Machida, Masa

    2013-01-01

    Microbial production of fats and oils is being developedas a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillusoryzae. Examination of the A.oryzaegenome demonstrates that it contains twofatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhancedthe expressionof fatty acid synthesis-related genes by replacing their promoters with thepromoter fromthe constitutively highly expressedgene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthasegenes we successfullyincreasedtheproduction of fatty acids and triglyceridesby more than two fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesteraseincreasedproductivity to a lesser extent.Increasing expression ofacetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored usingquantitative real-time RT-PCR. Our data demonstrates that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.

  6. Identification and characterization of a neuroretina-specific enhancer element in the quail Pax-6 (Pax-QNR) gene.

    PubMed Central

    Plaza, S; Dozier, C; Langlois, M C; Saule, S

    1995-01-01

    Using nuclear run-on assays, we showed that the tissue-specific expression of quail Pax-6 (Pax-QNR) P0-initiated mRNAs is due in part to regulation of the gene at the transcriptional level. Regulatory sequences governing neuroretina-specific expression of the P0-initiated mRNAs were investigated. By using reporter-based expression assays, we characterized a region within the Pax-QNR gene, located 7.5 kbp downstream from the P0 promoter, that functions as an enhancer in neuroretina cells but not in nonexpressing P0-initiated mRNA cells (quail embryo cells and quail retinal pigment epithelial cells). This enhancer element functioned in a position- and orientation-independent manner both on the Pax-QNR P0 promoter and the heterologous thymidine kinase promoter. Moreover, this enhancer element exhibited a developmental stage-specific activity during embryonic neuroretina development: in contrast to activity at day E7, the enhancer activity was very weak at day E5. This paralleled the level of expression of P0-initiated mRNAs observed at the same stages. Using footprinting, gel retardation, and Southwestern (DNA-protein) analysis, we demonstrated the existence of four neuroretina-specific nuclear protein-binding sites, involving multiple unknown factors. In addition we showed that the quail enhancer element is structurally and functionally conserved in mice. All of these results strongly suggest that this enhancer element may contribute to the neuroretina-specific transcriptional regulation of the Pax-6 gene in vivo. PMID:7529875

  7. Salmeterol enhances the cardiac response to gene therapy in Pompe disease.

    PubMed

    Han, Sang-Oh; Li, Songtao; Koeberl, Dwight D

    2016-05-01

    Enzyme replacement therapy (ERT) with recombinant human (rh) acid α-glucosidase (GAA) has prolonged the survival of patients. However, the paucity of cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle, where it is needed to take up rhGAA, correlated with a poor response to ERT by muscle in Pompe disease. Clenbuterol, a selective β2 receptor agonist, enhanced the CI-MPR expression in striated muscle through Igf-1 mediated muscle hypertrophy, which correlated with increased CI-MPR (also the Igf-2 receptor) expression. In this study we have evaluated 4 new drugs in GAA knockout (KO) mice in combination with an adeno-associated virus (AAV) vector encoding human GAA, 3 alternative β2 agonists and dehydroepiandrosterone (DHEA). Mice were injected with AAV2/9-CBhGAA (1E+11 vector particles) at a dose that was not effective at clearing glycogen storage from the heart. Heart GAA activity was significantly increased by either salmeterol (p<0.01) or DHEA (p<0.05), in comparison with untreated mice. Furthermore, glycogen content was reduced in the heart by treatment with DHEA (p<0.001), salmeterol (p<0.05), formoterol (p<0.01), or clenbuterol (p<0.01) in combination with the AAV vector, in comparison with untreated GAA-KO mice. Wirehang testing revealed that salmeterol and the AAV vector significantly increased performance, in comparison with the AAV vector alone (p<0.001). Similarly, salmeterol with the vector increased performance significantly more than any of the other drugs. The most effective individual drugs had no significant effect in absence of vector, in comparison with untreated mice. Thus, salmeterol should be further developed as adjunctive therapy in combination with either ERT or gene therapy for Pompe disease.

  8. A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy

    PubMed Central

    Hassan, Sally; Ward, John

    2016-01-01

    ABSTRACT With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase‐mediated plasmid supercoiling. SGS from three different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57‐SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total percentage supercoiled monomer was maintained to 85–90%. A twofold increase in plasmid yield was also observed for pUC57‐SGS in comparison to pUC57. pUC57‐SGS displayed greater segregational stability than pUC57‐cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. Biotechnol. Bioeng. 2016;113: 2064–2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26928284

  9. Overexpression of acetylcholinesterase gene in rice results in enhancement of shoot gravitropism.

    PubMed

    Yamamoto, Kosuke; Shida, Satoshi; Honda, Yoshihiro; Shono, Mariko; Miyake, Hiroshi; Oguri, Suguru; Sakamoto, Hikaru; Momonoki, Yoshie S

    2015-09-25

    Acetylcholine (ACh), a known neurotransmitter in animals and acetylcholinesterase (AChE) exists widely in plants, although its role in plant signal transduction is unclear. We previously reported AChE in Zea mays L. might be related to gravitropism based on pharmacological study using an AChE inhibitor. Here we clearly demonstrate plant AChE play an important role as a positive regulator in the gravity response of plants based on a genetic study. First, the gene encoding a second component of the ACh-mediated signal transduction system, AChE was cloned from rice, Oryza sativa L. ssp. Japonica cv. Nipponbare. The rice AChE shared high homology with maize, siratro and Salicornia AChEs. Similar to animal and other plant AChEs, the rice AChE hydrolyzed acetylthiocholine and propionylthiocholine, but not butyrylthiocholine. Thus, the rice AChE might be characterized as an AChE (E.C.3.1.1.7). Similar to maize and siratro AChEs, the rice AChE exhibited low sensitivity to the AChE inhibitor, neostigmine bromide, compared with the electric eel AChE. Next, the functionality of rice AChE was proved by overexpression in rice plants. The rice AChE was localized in extracellular spaces of rice plants. Further, the rice AChE mRNA and its activity were mainly detected during early developmental stages (2 d-10 d after sowing). Finally, by comparing AChE up-regulated plants with wild-type, we found that AChE overexpression causes an enhanced gravitropic response. This result clearly suggests that the function of the rice AChE relate to positive regulation of gravitropic response in rice seedlings.

  10. Low-Dose Irradiation Enhances Gene Targeting in Human Pluripotent Stem Cells

    PubMed Central

    Hatada, Seigo; Subramanian, Aparna; Mandefro, Berhan; Ren, Songyang; Kim, Ho Won; Tang, Jie; Funari, Vincent; Baloh, Robert H.; Sareen, Dhruv

    2015-01-01

    Human pluripotent stem cells (hPSCs) are now being used for both disease modeling and cell therapy; however, efficient homologous recombination (HR) is often crucial to develop isogenic control or reporter lines. We showed that limited low-dose irradiation (LDI) using either γ-ray or x-ray exposure (0.4 Gy) significantly enhanced HR frequency, possibly through induction of DNA repair/recombination machinery including ataxia-telangiectasia mutated, histone H2A.X and RAD51 proteins. LDI could also increase HR efficiency by more than 30-fold when combined with the targeting tools zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats. Whole-exome sequencing confirmed that the LDI administered to hPSCs did not induce gross genomic alterations or affect cellular viability. Irradiated and targeted lines were karyotypically normal and made all differentiated lineages that continued to express green fluorescent protein targeted at the AAVS1 locus. This simple method allows higher throughput of new, targeted hPSC lines that are crucial to expand the use of disease modeling and to develop novel avenues of cell therapy. Significance The simple and relevant technique described in this report uses a low level of radiation to increase desired gene modifications in human pluripotent stem cells by an order of magnitude. This higher efficiency permits greater throughput with reduced time and cost. The low level of radiation also greatly increased the recombination frequency when combined with developed engineered nucleases. Critically, the radiation did not lead to increases in DNA mutations or to reductions in overall cellular viability. This novel technique enables not only the rapid production of disease models using human stem cells but also the possibility of treating genetically based diseases by correcting patient-derived cells. PMID:26185257

  11. Role of endogenous insulin gene enhancer protein ISL-1 in angiogenesis

    PubMed Central

    Xiong, Si-qi; Jiang, Hai-bo; Li, Yan-xiu; Li, Hai-bo; Xu, Hui-zhuo; Wu, Zhen-kai; Zheng, Wei

    2016-01-01

    Objective To elucidate the role of insulin gene enhancer protein ISL-1 (Islet-1) in angiogenesis and regulation of vascular endothelial growth factor (VEGF) expression in vitro and in vivo. Methods siRNA targeting Islet-1 was transfected to human umbilical vein endothelial cell lines (HUVECs). The expression of Islet-1 and VEGF in the cultured cells was measured using real-time PCR and immunoblotting. 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue (MTT) assay was used to analyze the proliferation of HUVECs affected by Islet-1. Wound healing and Transwell assays were conducted to assess the motility of HUVECs. The formation of capillary-like structures was examined using growth factor–reduced Matrigel. siRNA targeting Islet-1 was intravitreally injected into the murine model of oxygen-induced retinopathy (OIR). Retinal neovascularization was evaluated with angiography using fluorescein-labeled dextran and then quantified histologically. Real-time PCR and immunoblotting were used to determine whether local Islet-1 silencing affected the expression of Islet-1 and VEGF in murine retinas. Results The expression of Islet-1 and VEGF in HUVECs was knocked down by siRNA. Reduced endogenous Islet-1 levels in cultured cells greatly inhibited the proliferation, migration, and tube formation in HUVECs in vitro. Retinal neovascularization following injection of Islet-1 siRNA was significantly reduced compared with that of the contralateral control eye. Histological analysis indicated that the neovascular nuclei protruding into the vitreous cavity were decreased. Furthermore, the Islet-1 and VEGF expression levels were downregulated in murine retinas treated with siRNA against Islet-1. Conclusions Reducing the expression of endogenous Islet-1 inhibits proliferation, migration, and tube formation in vascular endothelial cells in vitro and suppresses retinal angiogenesis in vivo. Endogenous Islet-1 regulates angiogenesis via VEGF. PMID:27994436

  12. Common Fragile Site Tumor Suppressor Genes and Corresponding Mouse Models of Cancer

    PubMed Central

    Drusco, Alessandra; Pekarsky, Yuri; Costinean, Stefan; Antenucci, Anna; Conti, Laura; Volinia, Stefano; Aqeilan, Rami I.; Huebner, Kay; Zanesi, Nicola

    2011-01-01

    Chromosomal common fragile sites (CFSs) are specific mammalian genomic regions that show an increased frequency of gaps and breaks when cells are exposed to replication stress in vitro. CFSs are also consistently involved in chromosomal abnormalities in vivo related to cancer. Interestingly, several CFSs contain one or more tumor suppressor genes whose structure and function are often affected by chromosomal fragility. The two most active fragile sites in the human genome are FRA3B and FRA16D where the tumor suppressor genes FHIT and WWOX are located, respectively. The best approach to study tumorigenic effects of altered tumor suppressors located at CFSs in vivo is to generate mouse models in which these genes are inactivated. This paper summarizes our present knowledge on mouse models of cancer generated by knocking out tumor suppressors of CFS. PMID:21318118

  13. Common fragile site tumor suppressor genes and corresponding mouse models of cancer.

    PubMed

    Drusco, Alessandra; Pekarsky, Yuri; Costinean, Stefan; Antenucci, Anna; Conti, Laura; Volinia, Stefano; Aqeilan, Rami I; Huebner, Kay; Zanesi, Nicola

    2011-01-01

    Chromosomal common fragile sites (CFSs) are specific mammalian genomic regions that show an increased frequency of gaps and breaks when cells are exposed to replication stress in vitro. CFSs are also consistently involved in chromosomal abnormalities in vivo related to cancer. Interestingly, several CFSs contain one or more tumor suppressor genes whose structure and function are often affected by chromosomal fragility. The two most active fragile sites in the human genome are FRA3B and FRA16D where the tumor suppressor genes FHIT and WWOX are located, respectively. The best approach to study tumorigenic effects of altered tumor suppressors located at CFSs in vivo is to generate mouse models in which these genes are inactivated. This paper summarizes our present knowledge on mouse models of cancer generated by knocking out tumor suppressors of CFS.

  14. Enhancement of heterologous production of eicosapentaenoic acid in Escherichia coli by substitution of promoter sequences within the biosynthesis gene cluster.

    PubMed

    Lee, Su-Jin; Kim, Chul Ho; Seo, Pil-Soo; Kwon, Ohsuk; Hur, Byung-Ki; Seo, Jeong-Woo

    2008-12-01

    To enhance the heterologous production of eicosapentaenoic acid (EPA) in Escherichia coli, the EPA biosynthesis gene cluster from Shewanella oneidensis MR-1 was cloned under the lacZ promoter on a high-copy number plasmid, pBluescript SK+. The production of EPA was remarkably enhanced yielding levels of up to 7.5% of the total fatty acid content in the recombinant E. coli strain by induction with IPTG, whereas the stimulation of EPA production was abolished by adding glucose into the culture medium, probably due to glucose repression acting on the promoter activity.

  15. Plant defense gene promoter enhances the reliability of shiva-1 gene-induced resistance to soft rot disease in potato.

    PubMed

    Yi, Jung Yoon; Seo, Hyo Won; Yang, Moon Sik; Robb, E Jane; Nazar, Ross N; Lee, Shin Woo

    2004-11-01

    PAL5, a tomato (Lycopersicon esculentum Mill.) plant defense gene that encodes phenylalanine ammonia-lyase, is known to respond to a variety of environmental stresses including pathogen infection and wounding. A shiva-1 gene recombinant that encodes a small synthetic antibacterial peptide under the PAL5 gene promoter was transformed into potato (Solanum tuberosum L.) and its ability to induce resistance to Erwinia carotovora was compared with a construct under the control of the constitutive and widely used cauliflower mosaic virus (CaMV) 35S promoter. The shiva-1 peptide, an analog of natural cecropin B, was shown previously to have high bactericidal activity in vitro, but when expressed in vivo under the control of the CaMV 35S promoter, the effects were very inconsistent. As observed previously, in the present studies a few transformants with the CaMV 35S promoter were highly resistant when assayed for susceptibility to soft rot disease. In marked contrast the majority of transformants with the PAL5 gene promoter were highly resistant. More-detailed analyses of the incorporated DNA indicated that most of the transformants with the CaMV 35S promoter contained multiple copies of the transforming DNA while all of the PAL5 recombinants contained single copies. The highly resistant CaMV 35S recombinant also was present as a single copy. The results indicate that, at least in this instance, a constitutive promoter may not be ideal for the effective expression of a foreign gene and suggest that multiple insertions may have negative consequences.

  16. Ultrasound-targeted HSVtk and Timp3 gene delivery for synergistically enhanced antitumor effects in hepatoma.

    PubMed

    Yu, B-F; Wu, J; Zhang, Y; Sung, H-W; Xie, J; Li, R-K

    2013-05-01

    Cancer gene therapy has great potential for decreasing tumor-induced mortality but has been clinically limited by non-targeted and insufficient gene transfer. We evaluated gene therapy targeting hepatocellular carcinoma (HCC) using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system and the tissue inhibitor of metalloproteinase 3 (Timp3) gene. Ultrasound-targeted microbubble destruction (UTMD) targeted gene delivery to the tumor tissue, and the α-fetoprotein promoter targeted HSVtk expression to the HCC cells. Human HepG2 cells transfected with the HSVtk or Timp3 gene demonstrated a reduction in cell viability by >40% compared with the vector control. Cell viability was further inhibited by over 50% with co-transfection of the genes. HepG2 cells were inoculated subcutaneously into athymic mice to induce tumors. UTMD-mediated delivery of HSVtk or Timp3 suppressed tumor growth by >45% and increased survival of tumor-bearing animals (P<0.01 vs vector control). Co-delivery of the genes resulted in a further 30% improvement in tumor suppression and significant extension of animal survival (P<0.01 vs vector control). Targeted gene delivery increased the number of apoptotic cells and decreased the vascular density of the tumors. Targeted co-delivery of the genes synergistically improved the antitumor effects and may provide an effective therapy for HCC.

  17. A ras-dependent pathway abolishes activity of a muscle-specific enhancer upstream from the muscle creatine kinase gene.

    PubMed Central

    Sternberg, E A; Spizz, G; Perry, M E; Olson, E N

    1989-01-01

    Differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific genes whose products are required for the specialized functions of the mature muscle fiber. The program for myogenic differentiation is subject to negative control by several peptide growth factors and by the products of mutationally activated ras oncogenes, which persistently activate intracellular cascades normally triggered by specific growth factors. Previously, we reported that induction of the muscle creatine kinase (mck) gene during myogenesis was dependent on a distal upstream enhancer that cooperated with a proximal promoter to direct high levels of expression in developing muscle cells (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909). To investigate the mechanisms whereby ras blocks the induction of muscle-specific genes, we have examined the ability of mck 5' regulatory elements to direct expression of the linked reporter gene for chloramphenicol acetyltransferase (cat) in C2 myoblasts bearing mutant N-ras and H-ras oncogenes. In this paper we report that expression of activated ras alleles abolishes activity of the mck upstream enhancer but does not affect the activity of the mck promoter. The ability of ras to repress the expression of mck-cat fusion genes that have been transfected either transiently or stably into myoblasts suggests that ras may exert its effects on muscle-specific genes through mechanisms independent of chromatin configurations or DNA methylation. These results also suggest that ras blocks establishment of the myogenic phenotype by preventing the accumulation of regulatory factors required for transcriptional induction of muscle-specific genes. Images PMID:2651901

  18. Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish.

    PubMed

    Asakawa, Kazuhide; Suster, Maximiliano L; Mizusawa, Kanta; Nagayoshi, Saori; Kotani, Tomoya; Urasaki, Akihiro; Kishimoto, Yasuyuki; Hibi, Masahiko; Kawakami, Koichi

    2008-01-29

    Targeted gene expression is a powerful approach to study the function of genes and cells in vivo. In Drosophila, the P element-mediated Gal4-UAS method has been successfully used for this purpose. However, similar methods have not been established in vertebrates. Here we report the development of a targeted gene expression methodology in zebrafish based on the Tol2 transposable element and its application to the functional study of neural circuits. First, we developed gene trap and enhancer trap constructs carrying an engineered yeast Gal4 transcription activator (Gal4FF) and transgenic reporter fish carrying the GFP or the RFP gene downstream of the Gal4 recognition sequence (UAS) and showed that the Gal4FF can activate transcription through UAS in zebrafish. Second, by using this Gal4FF-UAS system, we performed large-scale screens and generated a large collection of fish lines that expressed Gal4FF in specific tissues, cells, and organs. Finally, we developed transgenic effector fish carrying the tetanus toxin light chain (TeTxLC) gene downstream of UAS, which is known to block synaptic transmission. We crossed the Gal4FF fish with the UAS:TeTxLC fish and analyzed double transgenic embryos for defects in touch response. From this analysis, we discovered that targeted expression of TeTxLC in distinct populations of neurons in the brain and the spinal cord caused distinct abnormalities in the touch response behavior. These studies illustrate that our Gal4FF gene trap and enhancer trap methods should be an important resource for genetic analysis of neuronal functions and behavior in vertebrates.

  19. Enhancement of welan gum production in Sphingomonas sp. HT-1 via heterologous expression of Vitreoscilla hemoglobin gene.

    PubMed

    Liu, Xiaoliu; Zhu, Ping; Jiang, Ruifan; Wu, Lingtian; Feng, Xiaohai; Li, Sha; Xu, Hong

    2017-01-20

    Welan gum is a microbial polysaccharide produced by Sphingomonas sp. Its production is limited by the dissolved oxygen levels in the highly viscous fermentation. A strategy of heterologous expression of the Vitreoscilla hemoglobin gene in Sphingomonas sp. HT-1 was investigated to alleviate oxygen limitation and improve the yield of welan gum. Ultimately, the welan gum production increased from 25.3g/L to 34.6g/L, whereas the rheological behavior of welan gum solutions remained virtually unchanged. The transcriptional levels of the key genes in the electron transfer chain, TCA cycle and welan gum synthesis pathway, as well as ATP level revealed that the VHb expression in Sphingomonas sp. HT-1 enhanced welan gum biosynthesis by improving respiration and ATP supply. This study would pave the genetic manipulation way for enhancing welan gum yield, and it's also of great importance for the industrial applications of welan gum under harsh conditions.

  20. DMD and IL1RAPL1: two large adjacent genes localized within a common fragile site (FRAXC) have reduced expression in cultured brain tumors.

    PubMed

    McAvoy, S; Ganapathiraju, S; Perez, D S; James, C D; Smith, D I

    2007-01-01

    Common fragile sites (CFSs) are large regions of profound genomic instability found in all individuals. Spanning the center of the two most frequently expressed CFS regions, FRA3B (3p14.3) and FRA16D (16q23.2), are the 1.5 Mb FHIT gene and the 1.0 Mb WWOX gene. These genes are frequently deleted and/or altered in many different cancers. Both FHIT and WWOX have been demonstrated to function as tumor suppressors, both in vitro and in vivo. A number of other large CFS genes have been identified and are also frequently inactivated in multiple cancers. Based on these data, several additional very large genes were tested to determine if they were derived from within CFS regions, but DCC and RAD51L1 were not. However, the 2.0 Mb DMD gene and its immediately distal neighbor, the 1.8 Mb IL1RAPL1 gene are CFS genes contained within the FRAXC CFS region (Xp21.2-->p21.1). They are abundantly expressed in normal brain but were dramatically underexpressed in every brain tumor cell line and xenograft (derived from an intracranial model of glioblastoma multiforme) examined. We studied the expression of eleven other large CFS genes in the same panel of brain tumor cell lines and xenografts and found reduced expression of multiple large CFS genes in these samples. In this report we show that there is selective loss of specific large CFS genes in different cancers that does not appear to be mediated by the relative instability within different CFS regions. Further, the inactivation of multiple large CFS genes in xenografts and brain tumor cell lines may help to explain why this type of cancer is highly aggressive and associated with a poor clinical outcome.

  1. ChimerDB 3.0: an enhanced database for fusion genes from cancer transcriptome and literature data mining.

    PubMed

    Lee, Myunggyo; Lee, Kyubum; Yu, Namhee; Jang, Insu; Choi, Ikjung; Kim, Pora; Jang, Ye Eun; Kim, Byounggun; Kim, Sunkyu; Lee, Byungwook; Kang, Jaewoo; Lee, Sanghyuk

    2017-01-04

    Fusion gene is an important class of therapeutic targets and prognostic markers in cancer. ChimerDB is a comprehensive database of fusion genes encompassing analysis of deep sequencing data and manual curations. In this update, the database coverage was enhanced considerably by adding two new modules of The Cancer Genome Atlas (TCGA) RNA-Seq analysis and PubMed abstract mining. ChimerDB 3.0 is composed of three modules of ChimerKB, ChimerPub and ChimerSeq. ChimerKB represents a knowledgebase including 1066 fusion genes with manual curation that were compiled from public resources of fusion genes with experimental evidences. ChimerPub includes 2767 fusion genes obtained from text mining of PubMed abstracts. ChimerSeq module is designed to archive the fusion candidates from deep sequencing data. Importantly, we have analyzed RNA-Seq data of the TCGA project covering 4569 patients in 23 cancer types using two reliable programs of FusionScan and TopHat-Fusion. The new user interface supports diverse search options and graphic representation of fusion gene structure. ChimerDB 3.0 is available at http://ercsb.ewha.ac.kr/fusiongene/.

  2. Genes That Enhance the Ecological Fitness of Shewanella oneidensis MR-1 in Sediments Reveal the Value of Antibiotic Resistance▿ †

    PubMed Central

    Groh, Jennifer L.; Luo, Qingwei; Ballard, Jimmy D.; Krumholz, Lee R.

    2007-01-01

    Environmental bacteria persist in various habitats, yet little is known about the genes that contribute to growth and survival in their respective ecological niches. Signature-tagged mutagenesis (STM) of Shewanella oneidensis MR-1 coupled with a screen involving incubations of mutant strains in anoxic aquifer sediments allowed us to identify 47 genes that enhance fitness in sediments. Gene functions inferred from annotations provide us with insight into physiological and ecological processes that environmental bacteria use while growing in sediment ecosystems. Identification of the mexF gene and other potential membrane efflux components by STM demonstrated that homologues of multidrug resistance genes present in pathogens are required for sediment fitness of nonpathogenic bacteria. Further studies with a mexF deletion mutant demonstrated that the multidrug resistance pump encoded by mexF is required for resistance to antibiotics, including chloramphenicol and tetracycline. Chloramphenicol-adapted cultures exhibited mutations in the gene encoding a TetR family regulatory protein, indicating a role for this protein in regulating expression of the mexEF operon. The relative importance of mexF for sediment fitness suggests that antibiotic efflux may be a required process for bacteria living in sediment systems. PMID:17114320

  3. Enhanced blood-brain barrier penetration and glioma therapy mediated by a new peptide modified gene delivery system.

    PubMed

    Yao, Hui; Wang, Kaiyuan; Wang, Yi; Wang, Shanshan; Li, Jianfeng; Lou, Jinning; Ye, Liya; Yan, Xueying; Lu, Weiyue; Huang, Rongqin

    2015-01-01

    Successful glioma gene therapy lays on two important factors, the therapeutic genes and efficient delivery vehicles to cross the blood-brain barrier (BBB) and reach gliomas. In this work, a new gene vector was constructed based on dendrigraft poly-l-lysines (DGL) and polyethyleneglycol (PEG), conjugated with a cell-penetrating peptide, the nucleolar translocation signal (NoLS) sequence of the LIM Kinase 2 (LIMK2) protein (LIMK2 NoLS peptide, LNP), yielding DGL-PEG-LNP. Plasmid DNA encoding inhibitor of growth 4 (ING4) was applied as the therapeutic gene. DGL-PEG-LNP/DNA nanoparticles (NPs) were monodispersed, with a mean diameter of 90.6 ± 8.9 nm. The conjugation of LNP significantly enhanced the BBB-crossing efficiency, cellular uptake and gene expression within tumor cells. Mechanism studies suggested the involvement of energy, caveolae-mediated endocytosis and macropinocytosis in cellular uptake of LNP-modified NPs. MTT results showed that no apparent cytotoxicity was observed when cells were treated with synthesized vectors. Furthermore, LNP-modified NPs mediated strongest and most intensive apoptosis on the tumor site, and the longest median survival time of glioma-bearing mice. All the results demonstrated that LNP is a kind of efficient CPPs especially for BBB-crossing application, and DGL-PEG-LNP/DNA is a potential non-viral platform for glioma gene therapy via intravenous administration.

  4. PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements.

    PubMed

    Mi, Huaiyu; Huang, Xiaosong; Muruganujan, Anushya; Tang, Haiming; Mills, Caitlin; Kang, Diane; Thomas, Paul D

    2017-01-04

    The PANTHER database (Protein ANalysis THrough Evolutionary Relationships, http://pantherdb.org) contains comprehensive information on the evolution and function of protein-coding genes from 104 completely sequenced genomes. PANTHER software tools allow users to classify new protein sequences, and to analyze gene lists obtained from large-scale genomics experiments. In the past year, major improvements include a large expansion of classification information available in PANTHER, as well as significant enhancements to the analysis tools. Protein subfamily functional classifications have more than doubled due to progress of the Gene Ontology Phylogenetic Annotation Project. For human genes (as well as a few other organisms), PANTHER now also supports enrichment analysis using pathway classifications from the Reactome resource. The gene list enrichment tools include a new 'hierarchical view' of results, enabling users to leverage the structure of the classifications/ontologies; the tools also allow users to upload genetic variant data directly, rather than requiring prior conversion to a gene list. The updated coding single-nucleotide polymorphisms (SNP) scoring tool uses an improved algorithm. The hidden Markov model (HMM) search tools now use HMMER3, dramatically reducing search times and improving accuracy of E-value statistics. Finally, the PANTHER Tree-Attribute Viewer has been implemented in JavaScript, with new views for exploring protein sequence evolution.

  5. ChimerDB 3.0: an enhanced database for fusion genes from cancer transcriptome and literature data mining

    PubMed Central

    Lee, Myunggyo; Lee, Kyubum; Yu, Namhee; Jang, Insu; Choi, Ikjung; Kim, Pora; Jang, Ye Eun; Kim, Byounggun; Kim, Sunkyu; Lee, Byungwook; Kang, Jaewoo; Lee, Sanghyuk

    2017-01-01

    Fusion gene is an important class of therapeutic targets and prognostic markers in cancer. ChimerDB is a comprehensive database of fusion genes encompassing analysis of deep sequencing data and manual curations. In this update, the database coverage was enhanced considerably by adding two new modules of The Cancer Genome Atlas (TCGA) RNA-Seq analysis and PubMed abstract mining. ChimerDB 3.0 is composed of three modules of ChimerKB, ChimerPub and ChimerSeq. ChimerKB represents a knowledgebase including 1066 fusion genes with manual curation that were compiled from public resources of fusion genes with experimental evidences. ChimerPub includes 2767 fusion genes obtained from text mining of PubMed abstracts. ChimerSeq module is designed to archive the fusion candidates from deep sequencing data. Importantly, we have analyzed RNA-Seq data of the TCGA project covering 4569 patients in 23 cancer types using two reliable programs of FusionScan and TopHat-Fusion. The new user interface supports diverse search options and graphic representation of fusion gene structure. ChimerDB 3.0 is available at http://ercsb.ewha.ac.kr/fusiongene/. PMID:27899563

  6. PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements

    PubMed Central

    Mi, Huaiyu; Huang, Xiaosong; Muruganujan, Anushya; Tang, Haiming; Mills, Caitlin; Kang, Diane; Thomas, Paul D.

    2017-01-01

    The PANTHER database (Protein ANalysis THrough Evolutionary Relationships, http://pantherdb.org) contains comprehensive information on the evolution and function of protein-coding genes from 104 completely sequenced genomes. PANTHER software tools allow users to classify new protein sequences, and to analyze gene lists obtained from large-scale genomics experiments. In the past year, major improvements include a large expansion of classification information available in PANTHER, as well as significant enhancements to the analysis tools. Protein subfamily functional classifications have more than doubled due to progress of the Gene Ontology Phylogenetic Annotation Project. For human genes (as well as a few other organisms), PANTHER now also supports enrichment analysis using pathway classifications from the Reactome resource. The gene list enrichment tools include a new ‘hierarchical view’ of results, enabling users to leverage the structure of the classifications/ontologies; the tools also allow users to upload genetic variant data directly, rather than requiring prior conversion to a gene list. The updated coding single-nucleotide polymorphisms (SNP) scoring tool uses an improved algorithm. The hidden Markov model (HMM) search tools now use HMMER3, dramatically reducing search times and improving accuracy of E-value statistics. Finally, the PANTHER Tree-Attribute Viewer has been implemented in JavaScript, with new views for exploring protein sequence evolution. PMID:27899595

  7. Translational initiation frequency of atp genes from Escherichia coli: identification of an intercistronic sequence that enhances translation.

    PubMed Central

    McCarthy, J E; Schairer, H U; Sebald, W

    1985-01-01

    The c, b and delta subunit genes of the Escherichia coli atp operon were cloned individually in an expression vector between the tac fusion promoter and the galK gene. The relative rates of subunit synthesis directed by the cloned genes were similar in vitro and in vivo and compared favourably with the subunit stoichiometry of the assembled proton-translocating ATP synthase of E. coli in vivo. The rate of synthesis of subunit c was at least six times that of subunit b and 18 times that of subunit delta. Progressive shortening of the long intercistronic sequence lying upstream of the subunit c gene showed that maximal expression of this gene is dependent upon the presence of a sequence stretching greater than 20 bp upstream of the Shine-Dalgarno site. This sequence thus acts to enhance the rate of translational initiation. The possibility that similar sequences might perform the same function in other operons of E. coli and bacteriophage lambda is also discussed. Translation of the subunit b cistron is partially coupled to translation of the preceding subunit c cistron. In conclusion, the expression of all the atp operon genes could be adjusted to accommodate the subunit requirements of ATP synthase assembly primarily by means of mechanisms which control the efficiency of translational initiation and re-initiation at the respective cistron start codons. Images Fig. 3. Fig. 4. PMID:2862030

  8. Enhanced gene transfer to arthritic joints using adeno-associated virus type 5: implications for intra-articular gene therapy

    PubMed Central

    Adriaansen, J; Tas, S; Klarenbeek, P; Bakker, A; Apparailly, F; Firestein, G; Jorgensen, C; Vervoordeldonk, M; Tak, P

    2005-01-01

    Background: Gene therapy of the joint has great potential as a new therapeutic approach for the treatment of rheumatoid arthritis (RA). The vector chosen is of crucial importance for clinical success. Objective: To investigate the tropism and transduction efficiency in arthritic joints in vivo, and in synovial cells in vitro, using five different serotypes of recombinant adeno-associated virus (rAAV) encoding ß-galactosidase or green fluorescent protein genes. Methods: rAAV was injected into the ankle joints of rats with adjuvant arthritis after the onset of disease. Synovial tissue was examined at different time points for ß-galactosidase protein and gene expression by in situ staining and polymerase chain reaction (PCR) analysis, respectively. In addition, the ability of rAAV to transduce primary human fibroblast-like synoviocytes from patients with RA was investigated in vitro. Results: Intra-articular injection of the rAAV5 serotype resulted in the highest synovial transduction, followed by much lower expression using rAAV2. Expression of the transgene was already detectable 7 days after injection and lasted for at least 4 weeks. Only background staining was seen for serotypes 1, 3, and 4. Importantly, there was a minimal humoral immune response to rAAV5 compared with rAAV2. Additionally, it was found that both rAAV2 and rAAV5 can efficiently transduce human fibroblast-like synoviocytes obtained from patients with RA. Conclusion: Intra-articular rAAV mediated gene therapy in RA might be improved by using rAAV5 rather than other serotypes. PMID:15878906

  9. Electroactive biodegradable polyurethane significantly enhanced Schwann cells myelin gene expression and neurotrophin secretion for peripheral nerve tissue engineering.

    PubMed

    Wu, Yaobin; Wang, Ling; Guo, Baolin; Shao, Yongpin; Ma, Peter X

    2016-05-01

    Myelination of Schwann cells (SCs) is critical for the success of peripheral nerve regeneration, and biomaterials that can promote SCs' neurotrophin secretion as scaffolds are beneficial for nerve repair. Here we present a biomaterials-approach, specifically, a highly tunable conductive biodegradable flexible polyurethane by polycondensation of poly(glycerol sebacate) and aniline pentamer, to significantly enhance SCs' myelin gene expression and neurotrophin secretion for peripheral nerve tissue engineering. SCs are cultured on these conductive polymer films, and the biocompatibility of these films and their ability to enhance myelin gene expressions and sustained neurotrophin secretion are successfully demonstrated. The mechanism of SCs' neurotrophin secretion on conductive films is demonstrated by investigating the relationship between intracellular Ca(2+) level and SCs' myelination. Furthermore, the neurite growth and elongation of PC12 cells are induced by adding the neurotrophin medium suspension produced from SCs-laden conductive films. These data suggest that these conductive degradable polyurethanes that enhance SCs' myelin gene expressions and sustained neurotrophin secretion perform great potential for nerve regeneration applications.

  10. SH2B1 modulates chromatin state and MyoD occupancy to enhance expressions of myogenic genes.

    PubMed

    Chen, Kuan-Wei; Chang, Yu-Jung; Yeh, Chia-Ming; Lian, Yen-Ling; Chan, Michael W Y; Kao, Cheng-Fu; Chen, Linyi

    2017-02-01

    As mesoderm-derived cell lineage commits to myogenesis, a spectrum of signaling molecules, including insulin growth factor (IGF), activate signaling pathways and ultimately instruct chromatin remodeling and the transcription of myogenic genes. MyoD is a key transcription factor during myogenesis. In this study, we have identified and characterized a novel myogenic regulator, SH2B1. Knocking down SH2B1 delays global chromatin condensation and decreases the formation of myotubes. SH2B1 interacts with histone H1 and is required for the removal of histone H1 from active transcription sites, allowing for the expressions of myogenic genes, IGF2 and MYOG. Chromatin immunoprecipitation assays suggest the requirement of SH2B1 for the induction of histone H3 lysine 4 trimethylation as well as the reduction of histone H3 lysine 9 trimethylation at the promoters and/or enhancers of IGF2 and MYOG genes during myogenesis. Furthermore, SH2B1 is required for the transcriptional activity of MyoD and MyoD occupancy at the enhancer/promoter regions of IGF2 and MYOG during myogenesis. Together, this study demonstrates that SH2B1 fine-tunes global-local chromatin states, expressions of myogenic genes and ultimately promotes myogenesis.

  11. Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis

    PubMed Central

    Pandey, Shiv S.; Singh, Sucheta; Babu, C. S. Vivek; Shanker, Karuna; Srivastava, N. K.; Shukla, Ashutosh K.; Kalra, Alok

    2016-01-01

    Not much is known about the mechanism of endophyte-mediated induction of secondary metabolite production in Catharanthus roseus. In the present study two fungal endophytes, Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6 were isolated from the leaves of the plant that were found to enhance vindoline content by 229–403%. The isolated endophytes did not affect the primary metabolism of the plant as the maximum quantum efficiency of PSII, net CO2 assimilation, plant biomass and starch content of endophyte-inoculated plants was similar to endophyte-free control plants. Expression of terpenoid indole alkaloid (TIA) pathway genes, geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR), 16-hydoxytabersonine-O-methyltransferase (16OMT), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in endophyte-inoculated plants. Endophyte inoculation upregulated the expression of the gene for transcriptional activator octadecanoid-responsive Catharanthus AP2-domain protein (ORCA3) and downregulated the expression of Cys2/His2-type zinc finger protein family transcriptional repressors (ZCTs). The gene for the vacuolar class III peroxidase (PRX1), responsible for coupling vindoline and catharanthine, was upregulated in endophyte-inoculated plants. These endophytes may enhance vindoline production by modulating the expression of key structural and regulatory genes of vindoline biosynthesis without affecting the primary metabolism of the host plant. PMID:27220774

  12. ATHB17 enhances stress tolerance by coordinating photosynthesis associated nuclear gene and ATSIG5 expression in response to abiotic stress

    PubMed Central

    Zhao, Ping; Cui, Rong; Xu, Ping; Wu, Jie; Mao, Jie-Li; Chen, Yu; Zhou, Cong-Zhao; Yu, Lin-Hui; Xiang, Cheng-Bin

    2017-01-01

    Photosynthesis is sensitive to environmental stress and must be efficiently modulated in response to abiotic stress. However, the underlying mechanisms are not well understood. Here we report that ARABIDOPSIS THALIANA HOMEOBOX 17 (ATHB17), an Arabidopsis HD-Zip transcription factor, regulated the expression of a number of photosynthesis associated nuclear genes (PhANGs) involved in the light reaction and ATSIG5 in response to abiotic stress. ATHB17 was responsive to ABA and multiple stress treatments. ATHB17-overexpressing plants displayed enhanced stress tolerance, whereas its knockout mutant was more sensitive compared to the wild type. Through RNA-seq and quantitative real-time reverse transcription PCR (qRT-PCR) analysis, we found that ATHB17 did not affect the expression of many known stress-responsive marker genes. Interestingly, we found that ATHB17 down-regulated many PhANGs and could directly modulate the expression of several PhANGs by binding to their promoters. Moreover, we identified ATSIG5, encoding a plastid sigma factor, as one of the target genes of ATHB17. Loss of ATSIG5 reduced salt tolerance while overexpression of ATSIG5 enhanced salt tolerance, similar to that of ATHB17. ATHB17 can positively modulate the expression of many plastid encoded genes (PEGs) through regulation of ATSIG5. Taken together, our results suggest that ATHB17 may play an important role in protecting plants by adjusting expression of PhANGs and PEGs in response to abiotic stresses. PMID:28358040

  13. Polymorphism of chicken myocyte-specific enhancer-binding factor 2A gene and its association with chicken carcass traits.

    PubMed

    Zhou, Yan; Liu, Yiping; Jiang, Xiaosong; Du, Huarui; Li, Xiaocheng; Zhu, Qing

    2010-01-01

    Myocyte-specific enhancer-binding factor 2A (MEF2A) gene is a member of the myocyte-specific enhancer-binding factor 2 (MEF2) protein family which involved in vertebrate skeletal muscle development and differentiation. The aim of the current study is to investigate the potential associations between MEF2A gene SNPs (single nucleotide polymorphisms) and the carcass traits in 471 chicken samples from four populations. Three new SNPs (T46023C, A72626G, and T89232G) were detected in the chicken MEF2A gene. The T46023C genotypes were associated with live body weight (BW), carcass weight (CW), eviscerated weight, semi-eviscerated weight (SEW), and leg muscle weight (LMW) (P < 0.05); the A72626G genotypes were associated with BW, CW, LMW (P < 0.01) and breast muscle weight (BMW), leg muscle percentage (LMP) (P < 0.05); whereas the T89232G genotypes were associated with carcass percentage (CP) and semi-eviscerated percentage (SEP) (P < 0.05). The haplotypes constructed on the three SNPs were associated with BW, CW, LMW (P < 0.01), SEW, BMW, CP (P < 0.05). Significantly and suggestive dominant effects of diplotype H1H2 were observed for BW, CW, SEW, BMW and CP, whereas diplotype H5H5 had a negative effect on BW, CW, SEW, BMW and LMW. Our results suggest that the MEF2A gene may be a potential marker affecting the muscle trait of chickens.

  14. Combining Selective Pressures to Enhance the Durability of Disease Resistance Genes

    PubMed Central

    Bourguet, Denis

    2016-01-01

    The efficacy of disease resistance genes in plants decreases over time because of the selection of virulent pathogen genotypes. A key goal of crop protection programs is to increase the durability of the resistance conferred by these genes. The spatial and temporal deployment of plant disease resistance genes is considered to be a major factor determining their durability. In the literature, four principal strategies combining resistance genes over time and space have been considered to delay the evolution of virulent pathogen genotypes. We reviewed this literature with the aim of determining which deployment strategy results in the greatest durability of resistance genes. Although theoretical and empirical studies comparing deployment strategies of more than one resistance gene are very scarce, they suggest that the overall durability of disease resistance genes can be increased by combining their presence in the same plant (pyramiding). Retrospective analyses of field monitoring data also suggest that the pyramiding of disease resistance genes within a plant is the most durable strategy. By extension, we suggest that the combination of disease resistance genes with other practices for pathogen control (pesticides, farming practices) may be a relevant management strategy to slow down the evolution of virulent pathogen genotypes. PMID:28066472

  15. In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance.

    PubMed

    Pandey, Sonika; Patel, Manish Kumar; Mishra, Avinash; Jha, Bhavanath

    2016-01-01

    Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.

  16. Hexanoic acid and polyethylene glycol double grafted amphiphilic chitosan for enhanced gene delivery: influence of hydrophobic and hydrophilic substitution degree.

    PubMed

    Layek, Buddhadev; Haldar, Manas K; Sharma, Gitanjali; Lipp, Lindsey; Mallik, Sanku; Singh, Jagdish

    2014-03-03

    Gene therapy holds immense potential as a future therapeutic strategy for the treatment of numerous genetic diseases which are incurable to date. Nevertheless, safe and efficient gene delivery remains the most challenging aspects of gene therapy. To overcome this difficulty a series of hexanoic acid (HA) and monomethoxy poly(ethylene glycol) (mPEG) double grafted chitosan-based (HPC) nanomicelles were developed as nonviral gene carrier. HPC polymers with various HA and mPEG substitution degrees were synthesized, and their chemical structures were confirmed by (1)H NMR spectroscopy. HPC nanomicelles exhibited excellent blood compatibility and cell viability, as demonstrated by in vitro hemolysis and MTT assay, respectively. The cationic HPC nanomicelles retained the plasmid DNA (pDNA) binding capacity of chitosan and formed stable HPC/pDNA polyplexes with diameters below 200 nm. Both hydrophobic and hydrophilic substitution resulted in suppressed nonspecific protein adsorption on HPC/pDNA polyplexes and increased pDNA dissociation. However, resistance against DNase I degradation was enhanced by HA conjugation while being inhibited by mPEG substitution. Amphiphilic modification resulted in 3-4.5-fold higher cellular uptake in human embryonic kidney 293 cells (HEK 293) mainly through clathrin-mediated pathway. The optimal HPC/pDNA polyplexes displayed 50-fold and 1.2-fold higher gene transfection compared to unmodified chitosan and Fugene, respectively, in HEK 293 cells. Moreover, both the cellular uptake and in vitro transfection study suggested a clear dependence of gene expression on the extent of HA and mPEG substitution. These findings demonstrate that amphiphilic HPC nanomicelles with the proper combination of HA and mPEG substitution could be used as a promising gene carrier for efficient gene therapy.

  17. In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance

    PubMed Central

    Pandey, Sonika; Patel, Manish Kumar; Jha, Bhavanath

    2016-01-01

    Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas. PMID:27411057

  18. Activation of mu opioid receptors in the striatum differentially augments methamphetamine-induced gene expression and enhances stereotypic behavior.

    PubMed

    Horner, Kristen A; Hebbard, John C; Logan, Anna S; Vanchipurakel, Golda A; Gilbert, Yamiece E

    2012-03-01

    Mu opioid receptors are densely expressed in the patch compartment of striatum and contribute to methamphetamine-induced patch-enhanced gene expression and stereotypy. To further elucidate the role of mu opioid receptor activation in these phenomena, we examined whether activation of mu opioid receptors would enhance methamphetamine-induced stereotypy and prodynorphin, c-fos, arc and zif/268 expression in the patch and/or matrix compartments of striatum, as well as the impact of mu opioid receptor activation on the relationship between patch-enhanced gene expression and stereotypy. Male Sprague-Dawley rats were intrastriatally infused with d-Ala(2)-N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO; 1 μg/μL), treated with methamphetamine (0.5 mg/kg) and killed at 45 min or 2 h later. DAMGO augmented methamphetamine-induced zif/268 mRNA expression in the patch and matrix compartments, while prodynorphin expression was increased in the dorsolateral patch compartment. DAMGO pre-treatment did not affect methamphetamine-induced arc and c-fos expression. DAMGO enhanced methamphetamine-induced stereotypy and resulted in greater patch versus matrix expression of prodynorphin in the dorsolateral striatum, leading to a negative correlation between the two. These findings indicate that mu opioid receptors contribute to methamphetamine-induced stereotypy, but can differentially influence the genomic responses to methamphetamine. These data also suggest that prodynorphin may offset the overstimulation of striatal neurons by methamphetamine.

  19. DNA replication origin and transcriptional enhancer in c-myc gene share the c-myc protein binding sequences.

    PubMed Central

    Ariga, H; Imamura, Y; Iguchi-Ariga, S M

    1989-01-01

    We have previously reported that c-myc protein, or protein(s) complexed with c-myc protein, binds to the region upstream of the first exon of the c-myc gene and that this region contains an origin of cellular DNA replication (ori) and also a transcriptional enhancer. Here we show by Southwestern blotting that c-myc protein binds directly to a 7 bp sequence within the above region. Furthermore, we show that the c-myc protein binding sequences are indispensable for both ori and enhancer functions, but that additional sequences are required for maximal ori and enhancer activities. Thus, c-myc protein is a sequence specific factor which is apparently used both in initiation of DNA replication and in regulation of RNA transcription. Images PMID:2686984

  20. Owning genetic information and gene enhancement techniques: why privacy and property rights may undermine social control of the human genome.

    PubMed

    Moore, A D

    2000-04-01

    In this article I argue that the proper subjects of intangible property claims include medical records, genetic profiles, and gene enhancement techniques. Coupled with a right to privacy these intangible property rights allow individuals a zone of control that will, in most cases, justifiably exclude governmental or societal invasions into private domains. I argue that the threshold for overriding privacy rights and intangible property rights is higher, in relation to genetic enhancement techniques and sensitive personal information, than is commonly suggested. Once the bar is raised, so-to-speak, the burden of overriding it is formidable. Thus many policy decisions that have been recently proposed or enacted--citywide audio and video surveillance, law enforcement DNA sweeps, genetic profiling, national bans on genetic testing and enhancement of humans, to name a few--will have to be backed by very strong arguments.

  1. Adenoviral gene transfer of Akt enhances myocardial contractility and intracellular calcium handling

    PubMed Central

    Cittadini, A; Monti, MG; Iaccarino, G; Di Rella, F; Tsichlis, PN; Di Gianni, A; Strömer, H; Sorriento, D; Peschle, C; Trimarco, B; Saccà, L; Condorelli, G

    2010-01-01

    The serine-threonine kinase Akt/PKB mediates stimuli from different classes of cardiomyocyte receptors, including the growth hormone/insulin like growth factor and the β-adrenergic receptors. Whereas the growth-promoting and antiapoptotic properties of Akt activation are well established, little is known about the effects of Akt on myocardial contractility, intracellular calcium (Ca2+) handling, oxygen consumption, and β-adrenergic pathway. To this aim, Sprague–Dawley rats were subjected to a wild-type Akt in vivo adenoviral gene transfer using a catheter-based technique combined with aortopulmonary crossclamping. Left ventricular (LV) contractility and intracellular Ca2+ handling were evaluated in an isolated isovolumic buffer-perfused, aequorin-loaded whole heart preparations 10 days after the surgery. The Ca2+–force relationship was obtained under steady-state conditions in tetanized muscles. No significant hypertrophy was detected in adenovirus with wild-type Akt (Ad.Akt) versus controls rats (LV-to-body weight ratio 2.6±0.2 versus 2.7±0.1 mg/g, controls versus Ad.Akt, P, NS). LV contractility, measured as developed pressure, increased by 41% in Ad.Akt. This was accounted for by both more systolic Ca2+ available to the contractile machinery (+19% versus controls) and by enhanced myofilament Ca2+ responsiveness, documented by an increased maximal Ca2+-activated pressure (+19% versus controls) and a shift to the left of the Ca2+–force relationship. Such increased contractility was paralleled by a slight increase of myocardial oxygen consumption (14%), while titrated dose of dobutamine providing similar inotropic effect augmented oxygen consumption by 39% (P<0.01). Phospholamban, calsequestrin, and ryanodine receptor LV mRNA and protein content were not different among the study groups, while sarcoplasmic reticulum Ca2+ ATPase protein levels were significantly increased in Ad.Akt rats. β-Adrenergic receptor density, affinity, kinase-1 levels, and

  2. Analysis of Dengue Virus Enhancing Epitopes Using Peptide Antigens Derived from the Envelope Glycoprotein Gene Sequence

    DTIC Science & Technology

    1990-11-27

    AD________ AD-A230 976 ARMY PROJECT NO: 89PP9961 TITLE: ANALYSIS OF DENGUE VIRUS ENHANCING EPITOPES USING PEPTIDE ANTIGENS DERIVED FROM THE ENVELOPE...INO. INO r CCESSION NO I1I TITLE (Include Security Classification) Analysis of Dengue Virus Enhancing Epitopes Using Peptide Antigens Derived From the...necessary and identify by block number) Antibody-dependent enhancement (ADE) ot dengue (DEN) virus infection in human mononuclear cells in vitro has been

  3. The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cauliflower Or gene affects plant growth and development in addition to conferring beta-carotene accumulation. This study was undertaken to investigate the molecular basis of the Or gene mutation in controlling plant growth. The OR protein was found to interact with cauliflower and Arabidopsis e...

  4. Identification of disease resistance genes for enhancement of existing potato cultivars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A plant’s ability to defend itself against host-specific microbes is specified by disease resistance (R) genes. Upon recognition of an invading pathogen, R proteins are responsible for the activation of a multitude of responses ultimately leading to resistance. The majority of R genes are dominant a...

  5. Temporally controlled multiple-gene delivery in scaffolds: A promising strategy to enhance bone regeneration.

    PubMed

    Liu, Jinsong; Xu, Lihua; Li, Yiming; Ma, Jianfeng

    2011-02-01

    Bone defects sometimes require more effective repair regimens than conventional clinical therapies can provide. On account of this, tissue-engineered scaffolds have emerged as a promising alternative. Scaffolds that release genes encoding growth factors (GFs) offer additional benefits for bone regeneration in comparison with scaffolds providing protein delivery. The present gene delivery systems focus on unitary or dual genes delivery without controlled release. In the meantime, evidences indicate that bone formation is a complex cascade of events, in which time-dependent expression of multiple growth factors is involved. In our hypothesis, a temporally controlled, multi-gene delivery system embedded in a scaffold matrix can be fabricated; such a system is capable of mimicking the expression of growth factor profile in osteogenesis. Consequently, bone regeneration can be promoted by sequential gene expression of multiple growth factors.

  6. Tumor specific ultrasound enhanced gene transfer in vivo with novel liposomal bubbles.

    PubMed

    Suzuki, Ryo; Takizawa, Tomoko; Negishi, Yoichi; Utoguchi, Naoki; Sawamura, Kaori; Tanaka, Kumiko; Namai, Eisuke; Oda, Yusuke; Matsumura, Yasuhiro; Maruyama, Kazuo

    2008-01-22

    Bubble liposomes (liposomes which entrap an ultrasound imaging gas) may constitute a unique system for delivering various molecules efficiently into mammalian cells in vitro. In this study, Bubble liposomes were compared with cationic lipid (CL)-DNA complexes as potential gene delivery carriers into tumor in vivo. The delivery of genes by Bubble liposomes depended on the intensity of the applied ultrasound. Transfection efficiency plateaued at 0.7 W/cm(2) ultrasound intensity. Bubble liposomes efficiently transferred genes into cultured cells even when the cells were exposed to ultrasound for only 1 s. In addition, Bubble liposomes could introduce the luciferase gene more effectively than CL-DNA complexes into mouse ascites tumor cells and solid tumor tissue. We conclude that the combination of Bubble liposomes and ultrasound is a minimally-invasive and tumor specific gene transfer method in vivo.

  7. Screening and Analysis of Janelia FlyLight Project Enhancer-Gal4 Strains Identifies Multiple Gene Enhancers Active During Hematopoiesis in Normal and Wasp-Challenged Drosophila Larvae

    PubMed Central

    Tokusumi, Tsuyoshi; Tokusumi, Yumiko; Brahier, Mark S.; Lam, Victoria; Stoller-Conrad, Jessica R.; Kroeger, Paul T.; Schulz, Robert A.

    2016-01-01

    A GFP expression screen has been conducted on >1000 Janelia FlyLight Project enhancer-Gal4 lines to identify transcriptional enhancers active in the larval hematopoietic system. A total of 190 enhancers associated with 87 distinct genes showed activity in cells of the third instar larval lymph gland and hemolymph. That is, gene enhancers were active in cells of the lymph gland posterior signaling center (PSC), medullary zone (MZ), and/or cortical zone (CZ), while certain of the transcriptional control regions were active in circulating hemocytes. Phenotypic analyses were undertaken on 81 of these hematopoietic-expressed genes, with nine genes characterized in detail as to gain- and loss-of-function phenotypes in larval hematopoietic tissues and blood cells. These studies demonstrated the functional requirement of the cut gene for proper PSC niche formation, the hairy, Btk29A, and E2F1 genes for blood cell progenitor production in the MZ domain, and the longitudinals lacking, dFOXO, kayak, cap-n-collar, and delilah genes for lamellocyte induction and/or differentiation in response to parasitic wasp challenge and infestation of larvae. Together, these findings contribute substantial information to our knowledge of genes expressed during the larval stage of Drosophila hematopoiesis and newly identify multiple genes required for this developmental process. PMID:27913635

  8. Screening and Analysis of Janelia FlyLight Project Enhancer-Gal4 Strains Identifies Multiple Gene Enhancers Active During Hematopoiesis in Normal and Wasp-Challenged Drosophila Larvae.

    PubMed

    Tokusumi, Tsuyoshi; Tokusumi, Yumiko; Brahier, Mark S; Lam, Victoria; Stoller-Conrad, Jessica R; Kroeger, Paul T; Schulz, Robert A

    2017-02-09

    A GFP expression screen has been conducted on >1000 Janelia FlyLight Project enhancer-Gal4 lines to identify transcriptional enhancers active in the larval hematopoietic system. A total of 190 enhancers associated with 87 distinct genes showed activity in cells of the third instar larval lymph gland and hemolymph. That is, gene enhancers were active in cells of the lymph gland posterior signaling center (PSC), medullary zone (MZ), and/or cortical zone (CZ), while certain of the transcriptional control regions were active in circulating hemocytes. Phenotypic analyses were undertaken on 81 of these hematopoietic-expressed genes, with nine genes characterized in detail as to gain- and loss-of-function phenotypes in larval hematopoietic tissues and blood cells. These studies demonstrated the functional requirement of the cut gene for proper PSC niche formation, the hairy, Btk29A, and E2F1 genes for blood cell progenitor production in the MZ domain, and the longitudinals lacking, dFOXO, kayak, cap-n-collar, and delilah genes for lamellocyte induction and/or differentiation in response to parasitic wasp challenge and infestation of larvae. Together, these findings contribute substantial information to our knowledge of genes expressed during the larval stage of Drosophila hematopoiesis and newly identify multiple genes required for this developmental process.

  9. Construction of Saccharomyces cerevisiae strains with enhanced ethanol tolerance by mutagenesis of the TATA-binding protein gene and identification of novel genes associated with ethanol tolerance.

    PubMed

    Yang, Jungwoo; Bae, Ju Yun; Lee, Young Mi; Kwon, Hyeji; Moon, Hye-Yun; Kang, Hyun Ah; Yee, Su-Bog; Kim, Wankee; Choi, Wonja

    2011-08-01

    Since elevated ethanol is a major stress during ethanol fermentation, yeast strains tolerant to ethanol are highly desirable for the industrial scale ethanol production. A technology called global transcriptional machinery engineering (gTME), which exploits a mutant library of SPT15 encoding the TATA-binding protein of Saccharomyces cerevisiae (Alper et al., 2006; Science 314: 1565-1568), seems to a powerful tool for creating ethanol-tolerant strains. However, the ability of created strains to tolerate high ethanol on rich media remains unproven. In this study, a similar strategy was used to obtain five strains with enhanced ethanol tolerance (ETS1-5) of S. cerevisiae. Comparing global transcriptional profiles of two selected strains ETS2 and ETS3 with that of the control identified 42 genes that were commonly regulated with twofold change. Out of 34 deletion mutants available from a gene knockout library, 18 were ethanol sensitive, suggesting that these genes were closely associated with ethanol tolerance. Eight of them were novel with most being functionally unknown. To establish a basis for future industrial applications, strains iETS2 and iETS3 were created by integrating the SPT15 mutant alleles of ETS2 and ETS3 into the chromosomes, which also exhibited enhanced ethanol tolerance and survival upon ethanol shock on a rich medium. Fermentation with 20% glucose for 24 h in a bioreactor revealed that iETS2 and iETS3 grew better and produced approximately 25% more ethanol than a control strain. The ethanol yield and productivity were also substantially enhanced: 0.31 g/g and 2.6 g/L/h, respectively, for control and 0.39 g/g and 3.2 g/L/h, respectively, for iETS2 and iETS3. Thus, our study demonstrates the utility of gTME in generating strains with enhanced ethanol tolerance that resulted in increase of ethanol production. Strains with enhanced tolerance to other stresses such as heat, fermentation inhibitors, osmotic pressure, and so on, may be further created by

  10. Not4 enhances JAK/STAT pathway-dependent gene expression in Drosophila and in human cells.

    PubMed

    Grönholm, Juha; Kaustio, Meri; Myllymäki, Henna; Kallio, Jenni; Saarikettu, Juha; Kronhamn, Jesper; Valanne, Susanna; Silvennoinen, Olli; Rämet, Mika

    2012-03-01

    The JAK/STAT pathway is essential for organogenesis, innate immunity, and stress responses in Drosophila melanogaster. The JAK/STAT pathway and its associated regulators have been highly conserved in evolution from flies to humans. We have used a genome-wide RNAi screen in Drosophila S2 cells to identify regulators of the JAK/STAT pathway, and here we report the characterization of Not4 as a positive regulator of the JAK/STAT pathway. Overexpression of Not4 enhanced Stat92E-mediated gene responses in vitro and in vivo in Drosophila. Specifically, Not4 increased Stat92E-mediated reporter gene activation in S2 cells; and in flies, Not4 overexpression resulted in an 8-fold increase in Turandot M (TotM) and in a 4-fold increase in Turandot A (TotA) stress gene activation when compared to wild-type flies. Drosophila Not4 is structurally related to human CNOT4, which was found to regulate interferon-γ- and interleukin-4-induced STAT-mediated gene responses in human HeLa cells. Not4 was found to coimmunoprecipitate with Stat92E but not to affect tyrosine phosphorylation of Stat92E in Drosophila cells. However, Not4 is required for binding of Stat92E to its DNA recognition sequence in the TotM gene promoter. In summary, Not4/CNOT4 is a novel positive regulator of the JAK/STAT pathway in Drosophila and in humans.

  11. Evaluation of Vascular Delivery Methodologies to Enhance rAAV6-mediated Gene Transfer to Canine Striated Musculature

    PubMed Central

    Gregorevic, Paul; Schultz, Brian R; Allen, James M; Halldorson, Jeffrey B; Blankinship, Michael J; Meznarich, Norman A; Kuhr, Christian S; Doremus, Caitlin; Finn, Eric; Liggitt, Denny; Chamberlain, Jeffrey S

    2009-01-01

    A growing body of research supports the development of recombinant adeno-associated viral (rAAV) vectors for delivery of gene expression cassettes to striated musculature as a method of treating severe neuromuscular conditions. However, it is unclear whether delivery protocols that achieve extensive gene transfer in mice can be adapted to produce similarly extensive gene transfer in larger mammals and ultimately patients. Consequently, we sought to investigate methodological modifications that would facilitate rAAV-mediated gene transfer to the striated musculature of canines. A simple procedure incorporating acute (i) occlusion of limb blood flow, (ii) exsanguination via compression bandage, and (iii) vector “dwell” time of <20 minutes, markedly enhanced the transduction of limb muscles, compared with a simple bolus limb infusion of vector. A complementary method whereby vector was infused into the jugular vein led to efficient transduction of cardiomyocytes and to a lesser degree the diaphragm. Together these methods can be used to achieve transgene expression in heart, diaphragm, and limb muscles of juvenile dogs using rAAV6 vectors. These results establish that rAAV-mediated gene delivery is a viable approach to achieving systemic transduction of striated musculature in mammals approaching the dimensions of newborn humans. PMID:19471246

  12. Tracking of dendritic cell migration into lymph nodes using molecular imaging with sodium iodide symporter and enhanced firefly luciferase genes.

    PubMed

    Lee, Ho Won; Yoon, Seung Yun; Singh, Thoudam Debraj; Choi, Yoon Ju; Lee, Hong Je; Park, Ji Young; Jeong, Shin Young; Lee, Sang-Woo; Ha, Jeoung-Hee; Ahn, Byeong-Cheol; Jeon, Yong Hyun; Lee, Jaetae

    2015-05-14

    We sought to evaluate the feasibility of molecular imaging using the human sodium iodide symporter (hNIS) gene as a reporter, in addition to the enhanced firefly luciferase (effluc) gene, for tracking dendritic cell (DCs) migration in living mice. A murine dendritic cell line (DC2.4) co-expressing hNIS and effluc genes (DC/NF) was established. For the DC-tracking study, mice received either parental DCs or DC/NF cells in the left or right footpad, respectively, and combined I-124 PET/CT and bioluminescence imaging (BLI) were performed. In vivo PET/CT imaging with I-124 revealed higher activity of the radiotracer in the draining popliteal lymph nodes (DPLN) of the DC/NF injection site at day 1 than DC injection site (p < 0.05). The uptake value further increased at day 4 (p < 0.005). BLI also demonstrated migration of DC/NF cells to the DPLNs at day 1 post-injection, and signals at the DPLNs were much higher at day 4. These data support the feasibility of hNIS reporter gene imaging in the tracking of DC migration to lymphoid organs in living mice. DCs expressing the NIS reporter gene could be a useful tool to optimize various strategies of cell-based immunotherapy.

  13. Mutations in a delta9-Stearoyl-ACP-Desaturase Gene Are Associated with Enhanced Stearic Acid Levels in Soybean Seeds

    SciTech Connect

    Zhang, P.; Shanklin, J.; Burton, J. W.; Upchurch, R. G.; Whittle, E.; Dewey, R. E.

    2008-11-01

    Stearic acid (18:0) is typically a minor component of soybean [Glycine max (L.) Merr.] oil, accounting for only 2 to 4% of the total fatty acid content. Increasing stearic acid levels of soybean oil would lead to enhanced oxidative stability, potentially reducing the need for hydrogenation, a process leading to the formation of undesirable trans fatty acids. Although mutagenesis strategies have been successful in developing soybean germplasm with elevated 18:0 levels in the seed oil, the specific gene mutations responsible for this phenotype were not known. We report a newly identified soybean gene, designated SACPD-C, that encodes a unique isoform of {Delta}{sup 9}-stearoyl-ACP-desaturase, the enzyme responsible for converting stearic acid to oleic acid (18:1). High levels of SACPD-C transcript were only detected in developing seed tissue, suggesting that the encoded desaturase functions to enhance oleic acid biosynthetic capacity as the immature seed is actively engaged in triacylglycerol production and storage. The participation of SACPD-C in storage triacylglycerol synthesis is further supported by the observation of mutations in this gene in two independent sources of elevated 18:0 soybean germplasm, A6 (30% 18:0) and FAM94-41 (9% 18:0). A molecular marker diagnostic for the FAM94-41 SACPD-C gene mutation strictly associates with the elevated 18:0 phenotype in a segregating population, and could thus serve as a useful tool in the development of cultivars with oils possessing enhanced oxidative stability.

  14. Elastin-like polypeptide matrices for enhancing adeno-associated virus-mediated gene delivery to human neural stem cells.

    PubMed

    Kim, J-S; Chu, H S; Park, K I; Won, J-I; Jang, J-H

    2012-03-01

    The successful development of efficient and safe gene delivery vectors continues to be a major obstacle to gene delivery in stem cells. In this study, we have developed an elastin-like polypeptide (ELP)-mediated adeno-associated virus (AAV) delivery system for transducing fibroblasts and human neural stem cells (hNSCs). AAVs have significant promise as therapeutic vectors because of their safety and potential for use in gene targeting in stem cell research. ELP has been recently employed as a biologically inspired 'smart' biomaterial that exhibits an inverse temperature phase transition, thereby demonstrating promise as a novel drug carrier. The ELP that was investigated in this study was composed of a repetitive penta-peptide with [Val-Pro-Gly-Val-Gly]. A novel AAV variant, AAV r3.45, which was previously engineered by directed evolution to enhance transduction in rat NSCs, was nonspecifically immobilized onto ELPs that were adsorbed beforehand on a tissue culture polystyrene surface (TCPS). The presence of different ELP quantities on the TCPS led to variations in surface morphology, roughness and wettability, which were ultimately key factors in the modulation of cellular transduction. Importantly, with substantially reduced viral quantities compared with bolus delivery, ELP-mediated AAV delivery significantly enhanced delivery efficiency in fibroblasts and hNSCs, which have great potential for use in tissue engineering applications and neurodegenerative disorder treatments, respectively. The enhancement of cellular transduction in stem cells, as well as the feasibility of ELPs for utilization in three-dimensional scaffolds, will contribute to the advancement of gene therapy for stem cell research and tissue regenerative medicine.

  15. The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation.

    PubMed

    Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

    2014-01-01

    Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than

  16. The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation

    PubMed Central

    Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

    2014-01-01

    Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than

  17. A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus.

    PubMed Central

    Dorsch-Häsler, K; Keil, G M; Weber, F; Jasin, M; Schaffner, W; Koszinowski, U H

    1985-01-01

    Using the simian virus 40 "enhancer trap" approach, we have identified a transcription enhancer located just upstream of the major immediate early gene of murine cytomegalovirus. This enhancer has several striking properties. (i) Together with the enhancer of human cytomegalovirus, it is the strongest transcription enhancer found to date. (ii) It is an extremely long enhancer, spanning greater than 700 base pairs. (iii) It consists of a rather complex pattern of sequence repeats, the longest of which is 181 base pairs. Also, several types of short sequence motifs are scattered throughout the enhancer in monomeric, heterodimeric, or homodimeric (palindromic) form. These motifs have been identified to be components of other enhancers and promoters, and they are presumably binding sites for specific nuclear factors. Our analysis suggests that enhancers are composed of a modular arrangement of short conserved sequence motifs and that enhancer strength is correlated with the redundancy of these motifs. Images PMID:3001696

  18. A Ve homologous gene from Gossypium barbadense, Gbvdr3, enhances the defense response against Verticillium dahliae.

    PubMed

    Chen, Tianzi; Kan, Jialiang; Yang, Yuwen; Ling, Xitie; Chang, Youhong; Zhang, Baolong

    2016-01-01

    The tomato Ve1 gene and several Ve1 homologues are involved in the resistance to Verticillium dahliae. Here, we report on another Ve homologous gene, Gbvdr3, from a Verticillium wilt-resistant cotton cultivar, Gossypium barbadense Hai7124, which has a 3207-bp region that encodes a predicted receptor-like protein. Transient expression analyses indicated that Gbvdr3 is localized in the plasma membrane, and virus-induced gene silencing of Gbvdr3 compromised the resistance of Hai7124 cotton to a defoliating strain of V. dahliae, V991, but not to a non-defoliating strain, BP2. This resistance pattern was further confirmed by over-expression of Gbvdr3 in transgenic Arabidopsis, which significantly elevated the expression of the ethylene-regulated gene GST2, the ethylene- and jasmonic acid-regulated defense-related genes PR3 and PDF1.2, and the salicylic acid-regulated genes PR1 and PR5, but not the PR2 gene. It also triggered the accumulation of hydrogen peroxide and callose at early time points during infection by the V991 defoliating strain. In contrast, elevated accumulation of hydrogen peroxide or callose in Gbvdr3-expressed Arabidopsis leaves was not apparent under infection by the non-defoliating strain, BP2. These results suggested that Gbvdr3 is involved in the resistance to a unique spectrum of defoliating V. dahliae strains.

  19. Overexpression of bacterial ethylene-forming enzyme gene in Trichoderma reesei enhanced the production of ethylene

    PubMed Central

    Chen, Xi; Liang, Yong; Hua, Jing; Tao, Li; Qin, Wensheng; Chen, Sanfeng

    2010-01-01

    In order to efficiently utilize natural cellulose materials to produce ethylene, three expression vectors containing the ethylene-forming enzyme (efe) gene from Pseudomonas syringae pv. glycinea were constructed. The target gene was respectively controlled by different promoters: cbh I promoter from Trichoderma reesei cellobiohydrolases I gene, gpd promoter from Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene and pgk I promoter from T. reesei 3-phosphoglycerate kinase I gene. After transforming into T. reesei QM9414, 43 stable transformants were obtained by PCR amplification and ethylene determination. Southern blot analysis of 14 transformants demonstrated that the efe gene was integrated into chromosomal DNA with copy numbers from 1 to 4. Reverse transcription polymerase chain reaction (RT-PCR) analysis of 6 transformants showed that the heterologous gene was transcribed. By using wheat straw as a carbon source, the ethylene production rates of aforementioned 14 transformants were measured. Transformant C30-3 with pgk I promoter had the highest ethylene production (4,012 nl h-1 l-1). This indicates that agricultural wastes could be used to produce ethylene in recombinant filamentous fungus T. reesei. PMID:20150979

  20. Peripheral viral infection induced microglial sensome genes and enhanced microglial cell activity in the hippocampus of neonatal piglets.

    PubMed

    Ji, Peng; Schachtschneider, Kyle M; Schook, Lawrence B; Walker, Frederick R; Johnson, Rodney W

    2016-05-01

    Although poorly understood, early-life infection is predicted to affect brain microglial cells, making them hypersensitive to subsequent stimuli. To investigate this, we assessed gene expression in hippocampal tissue obtained from a previously published study reporting increased microglial cell activity and reduced hippocampal-dependent learning in neonatal piglets infected with porcine reproductive and respiratory syndrome virus (PRRSV), a virus that induces interstitial pneumonia. Infection altered expression of 455 genes, of which 334 were up-regulated and 121 were down-regulated. Functional annotation revealed that immune function genes were enriched among the up-regulated differentially expressed genes (DEGs), whereas calcium binding and synaptic vesicle genes were enriched among the down-regulated DEGs. Twenty-six genes encoding part of the microglia sensory apparatus (i.e., the sensome) were up-regulated (e.g., IL1R1, TLR2, and TLR4), whereas 15 genes associated with the synaptosome and synaptic receptors (e.g., NPTX2, GABRA2, and SLC5A7) were down-regulated. As the sensome may foretell microglia reactivity, we next inoculated piglets with culture medium or PRRSV at PD 7 and assessed hippocampal microglia morphology and function at PD 28 when signs of infection were waning. Consistent with amplification of the sensome, microglia from PRRSV piglets had enhanced responsiveness to chemoattractants, increased phagocytic activity, and secreted more TNFα in response to lipopolysaccharide and Poly I:C. Immunohistochemical staining indicated PRRSV infection increased microglia soma length and length-to-width ratio. Bipolar rod-like microglia not evident in hippocampus of control piglets, were present in infected piglets. Collectively, this study suggests early-life infection alters the microglia sensome as well as microglial cell morphology and function.

  1. Enhanced production of ε-caprolactone by coexpression of bacterial hemoglobin gene in recombinant Escherichia coli expressing cyclohexanone monooxygenase gene.

    PubMed

    Lee, Won-Heong; Park, Eun-Hee; Kim, Myoung-Dong

    2014-12-28

    Baeyer-Villiger (BV) oxidation of cyclohexanone to epsilon-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E. coli strain to produce a maximum epsilon-caprolactone concentration of 15.7 g/l in a fed-batch BV oxidation of cyclohexanone, which corresponded to a 43% improvement compared with the control strain expressing CHMO only under the same conditions.

  2. Augmenting Microarray Data with Literature-Based Knowledge to Enhance Gene Regulatory Network Inference

    PubMed Central

    Kilicoglu, Halil; Shin, Dongwook; Rindflesch, Thomas C.

    2014-01-01

    Gene regulatory networks are a crucial aspect of systems biology in describing molecular mechanisms of the cell. Various computational models rely on random gene selection to infer such networks from microarray data. While incorporation of prior knowledge into data analysis has been deemed important, in practice, it has generally been limited to referencing genes in probe sets and using curated knowledge bases. We investigate the impact of augmenting microarray data with semantic relations automatically extracted from the literature, with the view that relations encoding gene/protein interactions eliminate the need for random selection of components in non-exhaustive approaches, producing a more accurate model of cellular behavior. A genetic algorithm is then used to optimize the strength of interactions using microarray data and an artificial neural network fitness function. The result is a directed and weighted network providing the individual contribution of each gene to its target. For testing, we used invasive ductile carcinoma of the breast to query the literature and a microarray set containing gene expression changes in these cells over several time points. Our model demonstrates significantly better fitness than the state-of-the-art model, which relies on an initial random selection of genes. Comparison to the component pathways of the KEGG Pathways in Cancer map reveals that the resulting networks contain both known and novel relationships. The p53 pathway results were manually validated in the literature. 60% of non-KEGG relationships were supported (74% for highly weighted interactions). The method was then applied to yeast data and our model again outperformed the comparison model. Our results demonstrate the advantage of combining gene interactions extracted from the literature in the form of semantic relations with microarray analysis in generating contribution-weighted gene regulatory networks. This methodology can make a significant contribution to

  3. Immune deficiency enhances expression of recombinant human antibody in mice after nonviral in vivo gene transfer.

    PubMed

    Kitaguchi, Kohji; Toda, Mikako; Takekoshi, Masataka; Maeda, Fumiko; Muramatsu, Tatsuo; Murai, Atsushi

    2005-10-01

    A cDNA encoding human antibody against hepatitis B virus was expressed in normal and severe combined immune deficiency (SCID) mice to clarify whether or not host immune status affects circulating levels of the recombinant human antibody (RhAb) after nonviral in vivo gene transfer. For transferring genes, either electroporation (EP) or hydrodynamics-based transfection (HD) was employed. The former was applied to the leg muscle to express the gene, while the latter primarily targeted foreign gene expression in the liver. The expressed RhAb was secreted into the blood circulation, and its existence was assayed by ELISA. Prior to the investigation of host immune status, suitable forms of plasmid expression vectors and types of electrodes were determined in normal mice. Results showed that the vector encoding both the light and heavy chains driven by the CMV promoter had the highest plasma RhAb concentrations, and a pair of pincette-type electrodes conferred the best performance. In both EP and HD, the SCID state showed an increased and prolonged RhAb production in the blood circulation due probably to suppressed recognition of RhAb as a foreign protein to the host animal. The difference in gene transfer methods demonstrated a characteristic pattern: an early and sharp rise followed by a relatively rapid decrease in HD, in contrast to a gradual rise followed by a plateau level maintained in EP. As a result, with the same amount of gene transferred, the plasma RhAb concentrations for the first 7 or 8 weeks were higher in HD than EP, while the reverse was true for the latter period. Multiple gene transfer contributed to maintaining and prolonging high RhAb concentrations in plasma by both methods with similar characteristic patterns accompanying the respective gene transfer method. These results suggest the importance of host immunological potency for maintaining plasma RhAb concentrations if these gene transfer technologies are used for clinical and therapeutic purposes.

  4. Proteomic Analyses Reveal the Mechanism of Dunaliella salina Ds-26-16 Gene Enhancing Salt Tolerance in Escherichia coli

    PubMed Central

    Wang, Yanlong; Hu, Bin; Du, Shipeng; Gao, Shan; Chen, Xiwen; Chen, Defu

    2016-01-01

    We previously screened the novel gene Ds-26-16 from a 4 M salt-stressed Dunaliella salina cDNA library and discovered that this gene conferred salt tolerance to broad-spectrum organisms, including E. coli (Escherichia coli), Haematococcus pluvialis and tobacco. To determine the mechanism of this gene conferring salt tolerance, we studied the proteome of E. coli overexpressing the full-length cDNA of Ds-26-16 using the iTRAQ (isobaric tags for relative and absolute quantification) approach. A total of 1,610 proteins were identified, which comprised 39.4% of the whole proteome. Of the 559 differential proteins, 259 were up-regulated and 300 were down-regulated. GO (gene ontology) and KEGG (Kyoto encyclopedia of genes and genomes) enrichment analyses identified 202 major proteins, including those involved in amino acid and organic acid metabolism, energy metabolism, carbon metabolism, ROS (reactive oxygen species) scavenging, membrane proteins and ABC (ATP binding cassette) transporters, and peptidoglycan synthesis, as well as 5 up-regulated transcription factors. Our iTRAQ data suggest that Ds-26-16 up-regulates the transcription factors in E. coli to enhance salt resistance through osmotic balance, energy metabolism, and oxidative stress protection. Changes in the proteome were also observed in E. coli overexpressing the ORF (open reading frame) of Ds-26-16. Furthermore, pH, nitric oxide and glycerol content analyses indicated that Ds-26-16 overexpression increases nitric oxide content but has no effect on glycerol content, thus confirming that enhanced nitric oxide synthesis via lower intercellular pH was one of the mechanisms by which Ds-26-16 confers salt tolerance to E. coli. PMID:27135411

  5. Enhanced freeze tolerance of baker's yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough.

    PubMed

    Tan, Haigang; Dong, Jian; Wang, Guanglu; Xu, Haiyan; Zhang, Cuiying; Xiao, Dongguang

    2014-08-01

    Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker's yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301(TPS1) overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301(TPS1) were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301(TPS1) was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker's yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker's yeast.

  6. Biological delivery approaches for gene therapy: strategies to potentiate efficacy and enhance specificity.

    PubMed

    Mohit, Elham; Rafati, Sima

    2013-12-01

    Nowadays many therapeutic agents such as suicide genes, anti-angiogenesis agents, cytokines, chemokines and other therapeutic genes were delivered to cancer cells. Various biological delivery systems have been applied for directing therapeutic gene to target cells. Some of these successful preclinical studies, steps forward to clinical trials and a few are examined in phase III clinical trials. In this review, the biological gene delivery systems were categorized into microorganism and cell based delivery systems. Viral, bacterial, yeast and parasite are among microorganism based delivery systems which are expanded in this review. In cell based approach, different strategies such as tumor cells, stem cells, dendritic cells and sertoli cells will be discussed. Different drawbacks are associated with each delivery system; therefore, many strategies have been improved and potentiated their direction toward specific target cells. Herein, further to the principle of each delivery system, the progresses of these approaches for development of newer generation are discussed.

  7. The Or gene enhances carotenoid accumulation and stability during post-harvest storage of potato tubers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Provitamin A carotenoids in staple crops are not very stable during storage and their loss compromises nutritional quality. To elucidate the fundamental mechanisms underlying carotenoid accumulation and stability, we investigated transgenic potato tubers that express the cauliflower Orange (Or) gene...

  8. Overexpression of the ascorbate peroxidase gene from eggplant and sponge gourd enhances flood tolerance in transgenic Arabidopsis.

    PubMed

    Chiang, Chih-Ming; Chen, Chiu-Chen; Chen, Shi-Peng; Lin, Kuan-Hung; Chen, Li-Ru; Su, Yu-Huei; Yen, His-Cheng

    2017-03-01

    Previously, we found that the flood resistance of eggplant (Solanum melongena) and sponge gourd (Luffa cylindrica) enhanced ascorbate peroxidase (APX) activity under flooding, and consequently, both the SmAPX and LcAPX genes were cloned. In this study, the SmAPX and LcAPX genes were transferred under a ubiquitin promoter to Arabidopsis (At) via Agrobacterium tumefaciens. The expression and amount of APX and APX activities of the SmAPX and LcAPX transgenic lines were significantly higher than those of non-transgenic (NT) plants under a waterlogged condition. Furthermore, the SmAPX, LcAPX, At-sucrose synthases (SUS)-1, phosphoenolpyruvate carboxylase (PEPC), and lactate dehydrogenase (LDH) genes were overexpressed in all transgenic Arabidopsis lines after flooding treatment. Compared to NT plants, the malondialdehyde (MDA) contents and H2O2 accumulation were significantly lower, but germination rates were significantly higher in all transgenic lines with higher APX activity, indicating that the overexpression of SmAPX and LcAPX in Arabidopsis could enhance flood tolerance by eliminating H2O2. Moreover, Arabidopsis seedlings overexpressing SmAPX and LcAPX also displayed greater resistance to flooding and less oxidative injury than NT plants subjected to flooding condition.

  9. Enhancing the Stress Tolerance and Virulence of an Entomopathogen by Metabolic Engineering of Dihydroxynaphthalene Melanin Biosynthesis Genes ▿ †

    PubMed Central

    Tseng, Min N.; Chung, Pei C.; Tzean, Shean S.

    2011-01-01

    Entomopathogenic fungi have been used for biocontrol of insect pests for many decades. However, the efficacy of such fungi in field trials is often inconsistent, mainly due to environmental stresses, such as UV radiation, temperature extremes, and desiccation. To circumvent these hurdles, metabolic engineering of dihydroxynaphthalene (DHN) melanin biosynthetic genes (polyketide synthase, scytalone dehydratase, and 1,3,8-trihydroxynaphthalene reductase genes) cloned from Alternaria alternata were transformed into the amelanotic entomopathogenic fungus Metarhizium anisopliae via Agrobacterium-mediated transformation. Melanin expression in the transformant of M. anisopliae was verified by spectrophotometric methods, liquid chromatography/mass spectrometry (LC/MS), and confocal microscopy. The transformant, especially under stresses, showed notably enhanced antistress capacity and virulence, in terms of germination and survival rate, infectivity, and reduced median time to death (LT50) in killing diamondback moth (Plutella xylostella) larvae compared with the wild type. The possible mechanisms in enhancing the stress tolerance and virulence, and the significance and potential for engineering melanin biosynthesis genes in other biocontrol agents and crops to improve antistress fitness are discussed. PMID:21571888

  10. Aminoclay as a highly effective cationic vehicle for enhancing adenovirus-mediated gene transfer through nanobiohybrid complex formation.

    PubMed

    Kim, Soo-Yeon; Lee, Sang-Jin; Han, Hyo-Kyung; Lim, Soo-Jeong

    2017-02-01

    Electrostatic complexation of adenovirus (Ad) with cationic lipids or polymers has been shown to be an effective means for overcoming the limitations of adenoviral vectors and enhancing gene-transfer efficacy. However, such complexation causes cytotoxicity, limiting the use of this strategy. The present study explored the potential of 3-aminopropyl functionalized magnesium phyllosilicate (aminoclay) as a cationic vehicle for improving Ad-mediated gene transfer without inducing cytotoxicity. Aminoclay complexation produced a dose-dependent increase in Ad-mediated transgene expression in both Ad infection-sensitive and -refractory cells, thereby greatly lowering the Ad dose required for transgene expression. Unlike the case for cationic lipids (Lipofectamine) or polymers (Polybrene), the enhancement effect of aminoclay was not accompanied by significant cytotoxicity regardless of cell lines and it was not observed for nonviral plasmid vectors. Physical characterization studies revealed that nanobiohybrid complexes formed between aminoclay and Ad particles through electrostatic interactions, creating aggregates of Ad particles whose surface was shielded with aminoclay nanosheet oligomers. It appears that aminoclay complexation changes the surface charge of Ad particles from a negative to a highly positive value and thus increases Ad binding to cellular membranes, thereby providing an additional cellular entry mechanism, namely caveolae-dependent endocytosis. Aminoclay-Ad nanobiohybrids may serve as a next-generation efficient, versatile and biocompatible gene-delivery carrier.

  11. Coupling of enhancer and insulator properties identified in two retrotransposons modulates their mutagenic impact on nearby genes.

    PubMed

    Conte, Caroline; Dastugue, Bernard; Vaury, Chantal

    2002-03-01

    We recently reported a novel transposition system in which two retroelements from Drosophila melanogaster, ZAM and Idefix, are highly mobilized and preferentially insert within intergenic regions. Among the loci where new copies are detected, a hot spot for their insertion was identified at the white locus, where up to three elements occurred within a 3-kb fragment upstream of the transcriptional start site of white. We have used these insertions as molecular entry points to throw light on the mutagenic effect exerted by multiple insertions of retrotransposons within intergenic regions of a genome. Analysis of the molecular mechanisms by which ZAM and Idefix elements interfere with the regulation of the white gene has shown that ZAM bears cis-acting regulatory sequences able to enhance transcription of the white gene in the eyes of the flies. This activation may be counteracted by Idefix, which acts as an insulator able to isolate the white gene from the upstream ZAM enhancer. In addition to revealing a novel insulator sequence with its own specific features, our data clearly illustrate how retroelements can act as epigenetic factors able to interfere with the transcriptional regulation of their host.

  12. Heterozygous inactivation of the Nf1 gene in myeloid cells enhances neointima formation via a rosuvastatin-sensitive cellular pathway.

    PubMed

    Stansfield, Brian K; Bessler, Waylan K; Mali, Raghuveer; Mund, Julie A; Downing, Brandon; Li, Fang; Sarchet, Kara N; DiStasi, Matthew R; Conway, Simon J; Kapur, Reuben; Ingram, David A

    2013-03-01

    Mutations in the NF1 tumor suppressor gene cause Neurofibromatosis type 1 (NF1). Neurofibromin, the protein product of NF1, functions as a negative regulator of Ras activity. Some NF1 patients develop cardiovascular disease, which represents an underrecognized disease complication and contributes to excess morbidity and mortality. Specifically, NF1 patients develop arterial occlusion resulting in tissue ischemia and sudden death. Murine studies demonstrate that heterozygous inactivation of Nf1 (Nf1(+/-)) in bone marrow cells enhances neointima formation following arterial injury. Macrophages infiltrate Nf1(+/-) neointimas, and NF1 patients have increased circulating inflammatory monocytes in their peripheral blood. Therefore, we tested the hypothesis that heterozygous inactivation of Nf1 in myeloid cells is sufficient for neointima formation. Specific ablation of a single copy of the Nf1 gene in myeloid cells alone mobilizes a discrete pro-inflammatory murine monocyte population via a cell autonomous and gene-dosage dependent mechanism. Furthermore, lineage-restricted heterozygous inactivation of Nf1 in myeloid cells is sufficient to reproduce the enhanced neointima formation observed in Nf1(+/-) mice when compared with wild-type controls, and homozygous inactivation of Nf1 in myeloid cells amplified the degree of arterial stenosis after arterial injury. Treatment of Nf1(+/-) mice with rosuvastatin, a stain with anti-inflammatory properties, significantly reduced neointima formation when compared with control. These studies identify neurofibromin-deficient myeloid cells as critical cellular effectors of Nf1(+/-) neointima formation and propose a potential therapeutic for NF1 cardiovascular disease.

  13. High hydrostatic pressure activates gene expression that leads to ethanol production enhancement in a Saccharomyces cerevisiae distillery strain

    PubMed Central

    Bravim, Fernanda; Lippman, Soyeon I.; da Silva, Lucas F.; Souza, Diego T.; Fernandes, A. Alberto R.; Masuda, Claudio A.; Broach, James R.

    2016-01-01

    High hydrostatic pressure (HHP) is a stress that exerts broad effects on microorganisms with characteristics similar to those of common environmental stresses. In this study, we aimed to identify genetic mechanisms that can enhance alcoholic fermentation of wild Saccharomyces cerevisiae isolated from Brazilian spirit fermentation vats. Accordingly, we performed a time course microarray analysis on a S. cerevisiae strain submitted to mild sublethal pressure treatment of 50 MPa for 30 min at room temperature, followed by incubation for 5, 10 and 15 min without pressure treatment. The obtained transcriptional profiles demonstrate the importance of post-pressurisation period on the activation of several genes related to cell recovery and stress tolerance. Based on these results, we over-expressed genes strongly induced by HHP in the same wild yeast strain and identified genes, particularly SYM1, whose over-expression results in enhanced ethanol production and stress tolerance upon fermentation. The present study validates the use of HHP as a biotechnological tool for the fermentative industries. PMID:22915193

  14. [Enhancement of photoassimilate utilization by manipulation of ADP-glucose pyrophosphorylase gene]. Final progress report

    SciTech Connect

    Okita, T.W.

    1999-04-01

    Part 1 of this research focuses on patterns of gene expression of ADPG-pyrophosphorylase in native and transgenic potato plants. To elucidate the mechanism controlling AGP expression during plant development, the expression of the potato tuber AGP small subunit (sAGP) gene was analyzed in transgenic potato plants using a promoter-{beta}-glucuronidase expression system. Part II evaluated the structure-function relationships of AGP.

  15. Gene correction of a duchenne muscular dystrophy mutation by meganuclease-enhanced exon knock-in.

    PubMed

    Popplewell, Linda; Koo, Taeyoung; Leclerc, Xavier; Duclert, Aymeric; Mamchaoui, Kamel; Gouble, Agnés; Mouly, Vincent; Voit, Thomas; Pâques, Frédéric; Cédrone, Frédéric; Isman, Olga; Yáñez-Muñoz, Rafael J; Dickson, George

    2013-07-01

    Duchenne muscular dystrophy (DMD) is a severe inherited, muscle-wasting disorder caused by mutations in the DMD gene. Gene therapy development for DMD has concentrated on vector-based DMD minigene transfer, cell-based gene therapy using genetically modified adult muscle stem cells or healthy wild-type donor cells, and antisense oligonucleotide-induced exon-skipping therapy to restore the reading frame of the mutated DMD gene. This study is an investigation into DMD gene targeting-mediated correction of deletions in human patient myoblasts using a target-specific meganuclease (MN) and a homologous recombination repair matrix. The MN was designed to cleave within DMD intron 44, upstream of a deletion hotspot, and integration-competent lentiviral vectors expressing the nuclease (LVcMN) were generated. MN western blotting and deep gene sequencing for LVcMN-induced non-homologous end-joining InDels (microdeletions or microinsertions) confirmed efficient MN expression and activity in transduced DMD myoblasts. A homologous repair matrix carrying exons 45-52 (RM45-52) was designed and packaged into integration-deficient lentiviral vectors (IDLVs; LVdRM45-52). After cotransduction of DMD myoblasts harboring a deletion of exons 45 to 52 with LVcMN and LVdRM45-52 vectors, targeted knock-in of the RM45-52 region in the correct location in DMD intron 44, and expression of full-length, correctly spliced wild-type dystrophin mRNA containing exons 45-52 were observed. This work demonstrates that genome surgery on human DMD gene mutations can be achieved by MN-induced locus-specific genome cleavage and homologous recombination knock-in of deleted exons. The feasibility of human DMD gene repair in patient myoblasts has exciting therapeutic potential.

  16. Enhanced gene expression in epithelial cells transfected with amino acid-substituted gemini nanoparticles.

    PubMed

    Yang, Peng; Singh, Jagbir; Wettig, Shawn; Foldvari, Marianna; Verrall, Ronald E; Badea, Ildiko

    2010-08-01

    Gemini surfactants are versatile gene delivery agents because of their ability to bind and compact DNA and their low cellular toxicity. Through modification of the alkyl tail length and the chemical nature of the spacer, new compounds can be generated with the potential to improve the efficiency of gene delivery. Amino acid (glycine and lysine) and dipeptide (glycyl-lysine and lysyl-lysine) substituted spacers of gemini surfactants were synthesized, and their efficiency of gene delivery was assessed in epithelial cells for topical cutaneous and mucosal applications. Three different epithelial cell lines, COS-7, PAM212 and Sf 1Ep cells, were transfected with plasmid DNA encoding for interferon gamma and green fluorescent protein complexed with the amino acid-substituted gemini compounds in the presence of 1,2 dioleyl-sn-glycero-phosphatidyl-ethanolamine as a helper lipid. Gene expression was quantified by ELISA. Size, zeta potential and circular dichroism measurements were used to characterize the plasmid-gemini (PG) and plasmid-gemini surfactant-helper lipid (PGL) complexes. Gene expression was found to increase up to 72h and then declined by the 7th day. In general, the glycine-substituted surfactant showed consistently high gene expression in all three cell lines. Results of physicochemical and spectroscopic studies of the complexes indicate that substitution of the gemini spacer does not interfere with compaction of the DNA. The superior performance of these spacer-substituted gemini surfactants might be attributed to their better biocompatibility compared to the surfactants possessing unsubstituted spacers.

  17. Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.

    PubMed

    Cervera, Laura; Gutiérrez-Granados, Sonia; Berrow, Nicholas Simon; Segura, Maria Mercedes; Gòdia, Francesc

    2015-05-01

    Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96 h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered.

  18. The upstream muscle-specific enhancer of the rat muscle creatine kinase gene is composed of multiple elements.

    PubMed Central

    Horlick, R A; Benfield, P A

    1989-01-01

    A series of constructs that links the rat muscle creatine kinase promoter to the bacterial chloramphenicol acetyltransferase gene was generated. These constructs were introduced into differentiating mouse C2C12 myogenic cells to localize sequences that are important for up-regulation of the creatine kinase gene during myogenic differentiation. A muscle-specific enhancer element responsible for induction of chloramphenicol acetyltransferase expression during myogenesis was localized to a 159-base-pair region from 1,031 to 1,190 base pairs upstream of the transcription start site. Analysis of transient expression experiments using promoters mutated by deletion indicated the presence of multiple functional domains within this muscle-specific regulatory element. A DNA fragment spanning this region was used in DNase I protection experiments. Nuclear extracts derived from C2 myotubes protected three regions (designated E1, E2, and E3) on this fragment from digestion, which indicated there may be three or more trans-acting factors that interact with the creatine kinase muscle enhancer. Gel retardation assays revealed that factors able to bind specifically to E1, E2, and E3 are present in a wide variety of tissues and cell types. Transient expression assays demonstrated that elements in regions E1 and E3, but not necessarily E2, are required for full enhancer activity. Images PMID:2761536

  19. Elevated levels of CYP94 family gene expression alleviate the jasmonate response and enhance salt tolerance in rice.

    PubMed

    Kurotani, Ken-ichi; Hayashi, Kenji; Hatanaka, Saki; Toda, Yosuke; Ogawa, Daisuke; Ichikawa, Hiroaki; Ishimaru, Yasuhiro; Tashita, Ryo; Suzuki, Takeshi; Ueda, Minoru; Hattori, Tsukaho; Takeda, Shin

    2015-04-01

    The plant hormone jasmonate and its conjugates (JAs) have important roles in growth control, leaf senescence and defense responses against insects and microbial attacks. JA biosynthesis is induced by several stresses, including mechanical wounding, pathogen attacks, drought and salinity stresses. However, the roles of JAs under abiotic stress conditions are unclear. Here we report that increased expression of the Cyt P450 family gene CYP94C2b enhanced viability of rice plants under saline conditions. This gene encodes an enzyme closely related to CYP94C1 that catalyzes conversion of bioactive jasmonate-isoleucine (JA-Ile) into 12OH-JA-Ile and 12COOH-JA-Ile. Inactivation of JA was facilitated in a rice line with enhanced CYP94C2b expression, and responses to exogenous JA and wounding were alleviated. Moreover, salt stress-induced leaf senescence but not natural senescence was delayed in the transgenic rice. These results suggest that bioactive JAs have a negative effect on viability under salt stress conditions and demonstrate that manipulating JA metabolism confers enhanced salt tolerance in rice.

  20. Evolution of Bacillus subtilis to enhanced hypobaric growth: global alterations in gene expression

    NASA Astrophysics Data System (ADS)

    Nicholson, Wayne; Robles-Martinez, Jose; Rivas-Castillo, Andrea; Schuerger, Andrew

    Much astrobiology research is concerned with defining the environmental limits for life in the universe. Because Mars currently is the primary target for life detection missions, it is important to understand how terrestrial microbes might survive, proliferate, and evolve in martian envi-ronments. This issue is relevant in three distinct but related contexts: (i) testing panspermia hypotheses [1], (ii) mitigating the forward contamination of Mars [2], and (iii) understanding the molecular mechanisms leading to microbial growth in extreme extraterrestrial environments [3]. Prime candidates for Earth-to-Mars transfer include bacteria of the genus Bacillus, spores of which are significant contaminants of Mars-bound spacecraft and which are considered good candidates for lithopanspermia [1-4]. It is thus relevant to assess the potential for such microbes to survive and proliferate in the martian environment. The martian atmosphere poses a significant barrier to growth of terrestrial microbes, due to its low pressure (1-10 mbar; average 7 mbar) and anoxic (˜95% CO2) composition. In an earlier study [5] we showed that low pressures approaching those found on the surface of Mars exhibited an inhibitory effect on the germination and vegetative growth of several Bacillus spp. isolated from spacecraft or their assembly facilities. Even in an Earth-like 80%N2/20%O2 atmosphere, growth of B. subtilis cells was nearly completely inhibited at pressures below 35 mbar, well above the highest pressure on the martian surface [5]. The purpose of the present investigation was to use low pressure as a selective agent to test the hypothesis that a terrestrial microorganism, Bacillus subtilis, could evolve the ability for enhanced growth under hypobaric conditions approaching those of Mars. B. subtilis wild-type strains WN624 (SpcR) and WN628 (CmR) have been described previously [6] and were used as ancestral strains. Strains were propagated in LB liquid medium containing the appropriate

  1. Knockdown of ICB-1 gene enhanced estrogen responsiveness of ovarian and breast cancer cells.

    PubMed

    Konwisorz, Anna; Springwald, Anette; Haselberger, Martina; Goerse, Regina; Ortmann, Olaf; Treeck, Oliver

    2010-03-01

    ICB-1 chromosome 1 open reading frame 38 (C1orf38) is a human gene initially described by our group to be involved in differentiation processes of cancer cells. Recently, we have reported ICB-1 as a novel estrogen target gene and identified an estrogen response element in its promoter. In this study, we examined the role of ICB-1 in regulation of proliferation of breast and ovarian cancer cells. We knocked down its expression in estrogen-dependent MCF-7 breast cancer cells and hormone-unresponsive SK-OV-3 ovarian cancer cells by stable transfection with a specific shRNA plasmid followed by G-418 selection. Knockdown of ICB-1 enabled a considerable estrogen response of SK-OV-3 cells in terms of proliferation. This transformation of SK-OV-3 cells into an estrogen-responsive phenotype was accompanied by upregulation of estrogen receptor alpha (ERalpha) expression and a significant decrease of ERbeta expression on the mRNA level. Expression of ERalpha-dependent genes progesterone receptor, pS2, fibulin 1c, and c-fos was elevated in SK-OV-3 cells stably expressing ICB-1 shRNA. In MCF-7 cells, ICB-1 knockdown exerted similar effects on gene expression, supporting a general role of ICB-1 in estrogen responsiveness. Our data suggest that differentiation-associated gene ICB-1 might exert antagonistic actions on cellular estrogen response, which can result in inhibition of estradiol-triggered proliferation. The molecular mechanisms mediating this inhibitory effect of ICB-1 on estrogen signaling are suggested to be limitation of ERalpha transcript levels but sustaining high levels of ERbeta, reducing both activation of ERalpha target genes and cellular proliferation. The identification of ICB-1 as a new player in endocrine-related cancer encourages further studies on the significance of this gene in cancer development and therapy.

  2. Multifunctional non-viral gene vectors with enhanced stability, improved cellular and nuclear uptake capability, and increased transfection efficiency

    NASA Astrophysics Data System (ADS)

    Yang, Zhe; Jiang, Zhaozhong; Cao, Zhong; Zhang, Chao; Gao, Di; Luo, Xingen; Zhang, Xiaofang; Luo, Huiyan; Jiang, Qing; Liu, Jie

    2014-08-01

    We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors.We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell

  3. Overexpression of a MADS-Box Gene from Birch (Betula platyphylla) Promotes Flowering and Enhances Chloroplast Development in Transgenic Tobacco

    PubMed Central

    Qu, Guan-Zheng; Zheng, Tangchun; Liu, Guifeng; Wang, Wenjie; Zang, Lina; Liu, Huanzhen; Yang, Chuanping

    2013-01-01

    In this study, a MADS-box gene (BpMADS), which is an ortholog of AP1 from Arabidopsis, was isolated from birch (Betula platyphylla). Transgenic Arabidopsis containing a BpMADS promoter::GUS construct was produced, which exhibited strong GUS staining in sepal tissues. Ectopic expression of BpMADS significantly enhanced the flowering of tobacco (35S::BpMADS). In addition, the chloroplasts of transgenic tobacco exhibited much higher growth and division rates, as well rates of photosynthesis, than wild-type. A grafting experiment demonstrated that the flowering time of the scion was not affected by stock that overexpressed BpMADS. In addition, the overexpression of BpMADS resulted in the upregulation of some flowering-related genes in tobacco. PMID:23691043

  4. IgD cross-linking induces gene expression profiling changes and enhances apoptosis in chronic lymphocytic leukemia cells.

    PubMed

    Tavolaro, Simona; Peragine, Nadia; Chiaretti, Sabina; Ricciardi, Maria Rosaria; Raponi, Sara; Messina, Monica; Santangelo, Simona; Marinelli, Marilisa; Di Maio, Valeria; Mauro, Francesca Romana; Del Giudice, Ilaria; Foà, Robin; Guarini, Anna

    2013-04-01

    Gene profile and functional changes upon IgD cross-linking were evaluated in chronic lymphocytic leukemia (CLL). Microarrays highlighted responsiveness to IgD in all cases, independently of clinico-biological characteristics. Stimulated samples exhibited the down-regulation of transcripts of B-cell receptor signaling and cell-adhesion at 24h and the up-modulation of differentiation and apoptosis genes at 48 h. A significant increase in apoptosis upon ligation was also documented. Furthermore, comparison between IgD and IgM stimulation displayed a differential transcriptional/functional response. In conclusion, CLL respond to IgD displaying expression changes and cell-death enhancement, indicating the apoptosis induction via-IgD as an alternative approach for CLL management.

  5. Tph2 gene deletion enhances amphetamine-induced hypermotility: effect of 5-HT restoration and role of striatal noradrenaline release.

    PubMed

    Carli, Mirjana; Kostoula, Chrysaugi; Sacchetti, Giuseppina; Mainolfi, Pierangela; Anastasia, Alessia; Villani, Claudia; Invernizzi, Roberto William

    2015-11-01

    Variants of tryptophan hydroxylase-2 (Tph2), the gene encoding enzyme responsible for the synthesis of brain serotonin (5-HT), have been associated with neuropsychiatric disorders, substance abuse and addiction. This study assessed the effect of Tph2 gene deletion on motor behavior and found that motor activity induced by 2.5 and 5 mg/kg amphetamine was enhanced in Tph2(-/-) mice. Using the in vivo microdialysis technique we found that the ability of amphetamine to stimulate noradrenaline (NA) release in the striatum was reduced by about 50% in Tph2(-/-) mice while the release of dopamine (DA) was not affected. Tph2 deletion did not affect the release of NA and DA in the prefrontal cortex. The role of endogenous 5-HT in enhancing the effect of amphetamine was confirmed showing that treatment with the 5-HT precursor 5-hydroxytryptophan (10 mg/kg) restored tissue and extracellular levels of brain 5-HT and the effects of amphetamine on striatal NA release and motor activity in Tph2(-/-) mice. Treatment with the NA precursor dihydroxyphenylserine (400 mg/kg) was sufficient to restore the effect of amphetamine on striatal NA release and motor activity in Tph2(-/-) mice. These findings indicate that amphetamine-induced hyperactivity is attenuated by endogenous 5-HT through the inhibition of striatal NA release. Tph2(-/-) mice may be a useful preclinical model to assess the role of 5-HT-dependent mechanisms in the action of psychostimulants. Acute sensitivity to the motor effects of amphetamine has been associated to increased risk of psychostimulant abuse. Here, we show that deletion of Tph2, the gene responsible for brain 5-HT synthesis, enhances the motor effect of amphetamine in mice through the inhibition of striatal NA release. This suggests that Tph2(-/-) mice is a useful preclinical model to assess the role of 5-HT-dependent mechanisms in psychostimulants action. Tph2, tryptophan hydroxylase-2.

  6. Enhanced production of steviol glycosides in mycorrhizal plants: a concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes.

    PubMed

    Mandal, Shantanu; Upadhyay, Shivangi; Singh, Ved Pal; Kapoor, Rupam

    2015-04-01

    Stevia rebaudiana (Bertoni) produces steviol glycosides (SGs)--stevioside (stev) and rebaudioside-A (reb-A) that are valued as low calorie sweeteners. Inoculation with arbuscular mycorrhizal fungi (AMF) augments SGs production, though the effect of this interaction on SGs biosynthesis has not been studied at molecular level. In this study transcription profiles of eleven key genes grouped under three stages of the SGs biosynthesis pathway were compared. The transcript analysis showed upregulation of genes encoding 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes viz.,1-deoxy-D-xylulose 5-phospate synthase (DXS), 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR) and 2-C-methyl-D-erytrithol 2,4-cyclodiphosphate synthase (MDS) in mycorrhizal (M) plants. Zn and Mn are imperative for the expression of MDS and their enhanced uptake in M plants could be responsible for the increased transcription of MDS. Furthermore, in the second stage of SGs biosynthesis pathway, mycorrhization enhanced the transcription of copalyl diphosphate synthase (CPPS) and kaurenoic acid hydroxylase (KAH). Their expression is decisive for SGs biosynthesis as CPPS regulates flow of metabolites towards synthesis of kaurenoid precursors and KAH directs these towards steviol synthesis instead of gibberellins. In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. While higher transcription of all the three characterized UGTs in M plants explains augmented production of SGs; higher transcript levels of UGT76G1, specifically improved reb-A to stev ratio implying increased sweetness. The work signifies that AM symbiosis upregulates the transcription of all eleven SGs biosynthesis genes as a result of improved nutrition and enhanced sugar concentration due to increased photosynthesis in M plants.

  7. Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication

    PubMed Central

    De Leo, A; Colavita, F; Ciccosanti, F; Fimia, G M; Lieberman, P M; Mattia, E

    2015-01-01

    Autophagy, an important degradation system involved in maintaining cellular homeostasis, serves also to eliminate pathogens and process their fragments for presentation to the immune system. Several viruses have been shown to interact with the host autophagic machinery to suppress or make use of this cellular catabolic pathway to enhance their survival and replication. Epstein Barr virus (EBV) is a γ-herpes virus associated with a number of malignancies of epithelial and lymphoid origin in which establishes a predominantly latent infection. Latent EBV can periodically reactivate to produce infectious particles that allow the virus to spread and can lead to the death of the infected cell. In this study, we analyzed the relationship between autophagy and EBV reactivation in Burkitt's lymphoma cells. By monitoring autophagy markers and EBV lytic genes expression, we demonstrate that autophagy is enhanced in the early phases of EBV lytic activation but decreases thereafter concomitantly with increased levels of EBV lytic proteins. In a cell line defective for late antigens expression, we found an inverse correlation between EBV early antigens expression and autophagosomes formation, suggesting that early after activation, the virus is able to suppress autophagy. We report here for the first time that inhibition of autophagy by Bafilomycin A1 or shRNA knockdown of Beclin1 gene, highly incremented EBV lytic genes expression as well as intracellular viral DNA and viral progeny yield. Taken together, these findings indicate that EBV activation induces the autophagic response, which is soon inhibited by the expression of EBV early lytic products. Moreover, our findings open the possibility that pharmacological inhibitors of autophagy may be used to enhance oncolytic viral therapy of EBV-related lymphomas. PMID:26335716

  8. EDGAR 2.0: an enhanced software platform for comparative gene content analyses

    PubMed Central

    Blom, Jochen; Kreis, Julian; Spänig, Sebastian; Juhre, Tobias; Bertelli, Claire; Ernst, Corinna; Goesmann, Alexander

    2016-01-01

    The rapidly increasing availability of microbial genome sequences has led to a growing demand for bioinformatics software tools that support the functional analysis based on the comparison of closely related genomes. By utilizing comparative approaches on gene level it is possible to gain insights into the core genes which represent the set of shared features for a set of organisms under study. Vice versa singleton genes can be identified to elucidate the specific properties of an individual genome. Since initial publication, the EDGAR platform has become one of the most established software tools in the field of comparative genomics. Over the last years, the software has been continuously improved and a large number of new analysis features have been added. For the new version, EDGAR 2.0, the gene orthology estimation approach was newly designed and completely re-implemented. Among other new features, EDGAR 2.0 provides extended phylogenetic analysis features like AAI (Average Amino Acid Identity) and ANI (Average Nucleotide Identity) matrices, genome set size statistics and modernized visualizations like interactive synteny plots or Venn diagrams. Thereby, the software supports a quick and user-friendly survey of evolutionary relationships between microbial genomes and simplifies the process of obtaining new biological insights into their differential gene content. All features are offered to the scientific community via a web-based and therefore platform-independent user interface, which allows easy browsing of precomputed datasets. The web server is accessible at http://edgar.computational.bio. PMID:27098043

  9. Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte, Trichophyton mentagrophytes.

    PubMed

    Yamada, Tsuyoshi; Makimura, Koichi; Hisajima, Tatsuya; Ishihara, Yumiko; Umeda, Yoshiko; Abe, Shigeru

    2009-09-01

    The frequency of targeted gene disruption via homologous recombination is low in the clinically important dermatophyte, Trichophyton mentagrophytes. The Ku genes, Ku70 and Ku80, encode key components of the nonhomologous end-joining pathway involved in DNA double-strand break repair. Their deletion increases the homologous recombination frequency, facilitating targeted gene disruption. To improve the homologous recombination frequency in T. mentagrophytes, the Ku80 ortholog was inactivated. The nucleotide sequence of the Ku80 locus containing a 2788-bp ORF encoding a predicted product of 728 amino acids was identified, and designated as TmKu80. The predicted TmKu80 product showed a high degree of amino acid sequence similarity to known fungal Ku80 proteins. Ku80 disruption mutant strains of T. mentagrophytes were constructed by Agrobacterium tumefaciens-mediated genetic transformation. The average homologous recombination frequency was 73.3 +/- 25.2% for the areA/nit-2-like nitrogen regulatory gene (tnr) in Ku80(-) mutants, about 33-fold higher than that in wild-type controls. A high frequency (c. 67%) was also obtained for the Tri m4 gene encoding a putative serine protease. Ku80(-) mutant strains will be useful for large-scale reverse genetics studies of dermatophytes, including T. mentagrophytes, providing valuable information on the basic mechanisms of host invasion.

  10. An omp gene enhances cell tolerance of Cu(II) in Sinorhizobium meliloti CCNWSX0020.

    PubMed

    Li, Zhefei; Lu, Mingmei; Wei, Gehong

    2013-09-01

    The main aim of this work was to study molecular characterization of a DNA fragment conferring resistance to Cu(II) in Sinorhizobium meliloti CCNWSX0020. The strain CCNWSX0020, resistant to 1.4 mmol l(-1) Cu(II) in tryptone-yeast extract medium was isolated from Medicago lupulina growing in mine tailings of Fengxian County, China. The availability of the complete genome sequence of S. meliloti CCNWSX0020 provides an opportunity for investigating genes that play significant roles in Cu(II) resistance. A copper resistance gene, with a length of 1,445 bp, encoding 481 amino acids, designated omp, was identified by cDNA-amplified fragment length polymorphism from S. meliloti CCNWSX0020. The expression of omp gene strongly increased in the presence of Cu(II). The omp-defective mutants display sensitivities to Cu(II) compared with their wild types. The Cu(II)-sensitive phenotype of the mutant was complemented by a 1.5-kb DNA fragment containing omp gene. BLAST analysis revealed that this gene encoded a hypothetical outer membrane protein with 75 % similarity to outer membrane efflux protein in Rhizobium leguminosarum bv. viciae 3841. These studies suggested that the omp product was involved in the Cu(II) tolerance of S. meliloti CCNWSX0020.

  11. Mechanical properties and structure-function relationships in articular cartilage repaired using IGF-I gene-enhanced chondrocytes.

    PubMed

    Griffin, Darvin J; Ortved, Kyla F; Nixon, Alan J; Bonassar, Lawrence J

    2016-01-01

    Several studies have demonstrated the benefits of IGF-I gene therapy in enhancing the histologic and biochemical content of cartilage repaired by chondrocyte transplantation. However, there is little to no data on the mechanical performance of IGF-I augmented cartilage grafts. This study evaluated the compressive properties of full-thickness chondral defects in the equine femur repaired with and without IGF-I gene therapy. Animals were randomly assigned to one of three study cohorts based on chondrocyte treatment provided in each defect: (i) IGF-I gene delivered by recombinant adeno-associated virus (rAAV)-5; (ii) AAV-5 delivering GFP as a reporter; (iii) naïve cells without virus. In each case, the opposite limb was implanted with a fibrin carrier without cells. Samples were prepared for confined compression testing to measure the aggregate modulus and hydraulic permeability. All treatment groups, regardless of cell content or transduction, had mechanical properties inferior to native cartilage. Overexpression of IGF-I increased modulus and lowered permeability relative to other treatments. Investigation of structure-property relationships revealed that Ha and k were linearly correlated with GAG content but logarithmically correlated with collagen content. This provides evidence that IGF-I gene therapy can improve healing of articular cartilage and can greatly increase the mechanical properties of repaired grafts.

  12. Methyl-Cytosine-Driven Structural Changes Enhance Adduction Kinetics of an Exon 7 fragment of the p53 Gene

    NASA Astrophysics Data System (ADS)

    Malla, Spundana; Kadimisetty, Karteek; Fu, You-Jun; Choudhary, Dharamainder; Schenkman, John B.; Rusling, James F.

    2017-01-01

    Methylation of cytosine (C) at C-phosphate-guanine (CpG) sites enhances reactivity of DNA towards electrophiles. Mutations at CpG sites on the p53 tumor suppressor gene that can result from these adductions are in turn correlated with specific cancers. Here we describe the first restriction-enzyme-assisted LC-MS/MS sequencing study of the influence of methyl cytosines (MeC) on kinetics of p53 gene adduction by model metabolite benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), using methodology applicable to correlate gene damage sites for drug and pollutant metabolites with mutation sites. This method allows direct kinetic measurements by LC-MS/MS sequencing for oligonucleotides longer than 20 base pairs (bp). We used MeC and non-MeC (C) versions of a 32 bp exon 7 fragment of the p53 gene. Methylation of 19 cytosines increased the rate constant 3-fold for adduction on G at the major reactive CpG in codon 248 vs. the non-MeC fragment. Rate constants for non-CpG codons 244 and 243 were not influenced significantly by MeC. Conformational and hydrophobicity changes in the MeC-p53 exon 7 fragment revealed by CD spectra and molecular modeling increase the BPDE binding constant to G in codon 248 consistent with a pathway in which preceding reactant binding greatly facilitates the rate of covalent SN2 coupling.

  13. Association with Amino Acids Does Not Enhance Efficacy of Polymerized Liposomes As a System for Lung Gene Delivery

    PubMed Central

    Bandeira, Elga; Lopes-Pacheco, Miquéias; Chiaramoni, Nadia; Ferreira, Débora; Fernandez-Ruocco, Maria J.; Prieto, Maria J.; Maron-Gutierrez, Tatiana; Perrotta, Ramiro M.; de Castro-Faria-Neto, Hugo C.; Rocco, Patricia R. M.; Alonso, Silvia del Valle; Morales, Marcelo M.

    2016-01-01

    Development of improved drug and gene delivery systems directly into the lungs is highly desirable given the important burden of respiratory diseases. We aimed to evaluate the safety and efficacy of liposomes composed of photopolymerized lipids [1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine] associated with amino acids as vectors for gene delivery into the lungs of healthy animals. Lipopolymer vesicles, in particular, are more stable than other types of liposomes. In this study, lipopolymers were associated with l-arginine, l-tryptophan, or l-cysteine. We hypothesized that the addition of these amino acids would enhance the efficacy of gene delivery to the lungs by the lipopolymers. l-Arginine showed the highest association efficiency due to its positive charge and better surface interactions. None of the formulations caused inflammation or altered lung mechanics, suggesting that these lipopolymers can be safely administered as aerosols. All formulations were able to induce eGFP mRNA expression in lung tissue, but the addition of amino acids reduced delivery efficacy when compared with the simple lipopolymer particle. These results indicate that this system could be further explored for gene or drug delivery targeting lung diseases. PMID:27199766

  14. Enhanced adenoviral gene delivery to motor and dorsal root ganglion neurons following injection into demyelinated peripheral nerves.

    PubMed

    Zhang, Yongjie; Zheng, Yiyan; Zhang, Yi Ping; Shields, Lisa B E; Hu, Xiaoling; Yu, Panpan; Burke, Darlene A; Wang, Heming; Jun, Cai; Byers, Jonathan; Whittemore, Scott R; Shields, Christopher B

    2010-08-15

    Injection of viral vectors into peripheral nerves may transfer specific genes into their dorsal root ganglion (DRG) neurons and motoneurons. However, myelin sheaths of peripheral axons block the entry of viral particles into nerves. We studied whether mild, transient peripheral nerve demyelination prior to intraneural viral vector injection would enhance gene transfer to target DRG neurons and motoneurons. The right sciatic nerve of C57BL/6 mice was focally demyelinated with 1% lysolecithin, and the left sciatic nerve was similarly injected with saline (control). Five days after demyelination, 0.5 microl of Ad5-GFP was injected into both sciatic nerves at the site of previous injection. The effectiveness of gene transfer was evaluated by counting GFP(+) neurons in the DRGs and ventral horns. After peripheral nerve demyelination, there was a fivefold increase in the number of infected DRG neurons and almost a 15-fold increase in the number of infected motoneurons compared with the control, nondemyelinated side. Focal demyelination reduced the myelin sheath barrier, allowing greater virus-axon contact. Increased CXADR expression on the demyelinated axons facilitated axoplasmic viral entry. No animals sustained any prolonged neurological deficits. Increased gene delivery into DRG neurons and motoneurons may provide effective treatment for amyotrophic lateral sclerosis, pain, and spinal cord injury.

  15. Methyl-Cytosine-Driven Structural Changes Enhance Adduction Kinetics of an Exon 7 fragment of the p53 Gene

    PubMed Central

    Malla, Spundana; Kadimisetty, Karteek; Fu, You-Jun; Choudhary, Dharamainder; Schenkman, John B.; Rusling, James F.

    2017-01-01

    Methylation of cytosine (C) at C-phosphate-guanine (CpG) sites enhances reactivity of DNA towards electrophiles. Mutations at CpG sites on the p53 tumor suppressor gene that can result from these adductions are in turn correlated with specific cancers. Here we describe the first restriction-enzyme-assisted LC-MS/MS sequencing study of the influence of methyl cytosines (MeC) on kinetics of p53 gene adduction by model metabolite benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), using methodology applicable to correlate gene damage sites for drug and pollutant metabolites with mutation sites. This method allows direct kinetic measurements by LC-MS/MS sequencing for oligonucleotides longer than 20 base pairs (bp). We used MeC and non-MeC (C) versions of a 32 bp exon 7 fragment of the p53 gene. Methylation of 19 cytosines increased the rate constant 3-fold for adduction on G at the major reactive CpG in codon 248 vs. the non-MeC fragment. Rate constants for non-CpG codons 244 and 243 were not influenced significantly by MeC. Conformational and hydrophobicity changes in the MeC-p53 exon 7 fragment revealed by CD spectra and molecular modeling increase the BPDE binding constant to G in codon 248 consistent with a pathway in which preceding reactant binding greatly facilitates the rate of covalent SN2 coupling. PMID:28102315

  16. Transformation of tomato with a bacterial codA gene enhances tolerance to salt and water stresses.

    PubMed

    Goel, Deepa; Singh, Ajay K; Yadav, Vichita; Babbar, Shashi B; Murata, Norio; Bansal, Kailash C

    2011-07-15

    Genetically engineered tomato (Lycopersicon esculentum) with the ability to synthesize glycinebetaine was generated by introducing the codA gene encoding choline oxidase from Arthrobacter globiformis. Integration of the codA gene in transgenic tomato plants was verified by PCR analysis and DNA blot hybridization. Transgenic expression of gene was verified by RT-PCR analysis and RNA blot hybridization. The codA-transgenic plants showed higher tolerance to salt stress during seed germination, and subsequent growth of young seedlings than wild-type plants. The codA transgene enhanced the salt tolerance of whole plants and leaves. Mature leaves of codA-transgenic plants revealed higher levels of relative water content, chlorophyll content, and proline content than those of wild-type plants under salt and water stresses. Results from the current study suggest that the expression of the codA gene in transgenic tomato plants induces the synthesis of glycinebetaine and improves the tolerance of plants to salt and water stresses.

  17. Immunosuppressive Myeloid Cells' Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy.

    PubMed

    Kamran, Neha; Kadiyala, Padma; Saxena, Meghna; Candolfi, Marianela; Li, Youping; Moreno-Ayala, Mariela A; Raja, Nicholas; Shah, Diana; Lowenstein, Pedro R; Castro, Maria G

    2017-01-04

    Survival of glioma (GBM) patients treated with the current standard of care remains dismal. Immunotherapeutic approaches that harness the cytotoxic and memory potential of the host immune system have shown great benefit in other cancers. GBMs have developed multiple strategies, including the accumulation of myeloid-derived suppressor cells (MDSCs) to induce immunosuppression. It is therefore imperative to develop multipronged approaches when aiming to generate a robust anti-tumor immune response. Herein, we tested whether combining MDSC depletion or checkpoint blockade would augment the efficacy of immune-stimulatory herpes simplex type-I thymidine kinase (TK) plus Fms-like tyrosine kinase ligand (Flt3L)-mediated immune stimulatory gene therapy. Our results show that MDSCs constitute >40% of the tumor-infiltrating immune cells. These cells express IL-4Rα, inducible nitric oxide synthase (iNOS), arginase, programmed death ligand 1 (PDL1), and CD80, molecules that are critically involved in antigen-specific T cell suppression. Depletion of MDSCs strongly enhanced the TK/Flt3L gene therapy-induced tumor-specific CD8 T cell response, which lead to increased median survival and percentage of long-term survivors. Also, combining PDL1 or CTLA-4 immune checkpoint blockade greatly improved the efficacy of TK/Flt3L gene therapy. Our results, therefore, indicate that blocking MDSC-mediated immunosuppression holds great promise for increasing the efficacy of gene therapy-mediated immunotherapies for GBM.

  18. Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product.

    PubMed

    Romanczuk, H; Wormington, W M

    1989-02-01

    Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.

  19. Impaired spatial memory and enhanced long-term potentiation in mice with forebrain-specific ablation of the Stim genes

    PubMed Central

    Garcia-Alvarez, Gisela; Shetty, Mahesh S.; Lu, Bo; Yap, Kenrick An Fu; Oh-Hora, Masatsugu; Sajikumar, Sreedharan; Bichler, Zoë; Fivaz, Marc

    2015-01-01

    Recent findings point to a central role of the endoplasmic reticulum-resident STIM (Stromal Interaction Molecule) proteins in shaping the structure and function of excitatory synapses in the mammalian brain. The impact of the Stim genes on cognitive functions remains, however, poorly understood. To explore the function of the Stim genes in learning and memory, we generated three mouse strains with conditional deletion (cKO) of Stim1 and/or Stim2 in the forebrain. Stim1, Stim2, and double Stim1/Stim2 cKO mice show no obvious brain structural defects or locomotor impairment. Analysis of spatial reference memory in the Morris water maze revealed a mild learning delay in Stim1 cKO mice, while learning and memory in Stim2 cKO mice was indistinguishable from their control littermates. Deletion of both Stim genes in the forebrain resulted, however, in a pronounced impairment in spatial learning and memory reflecting a synergistic effect of the Stim genes on the underlying neural circuits. Notably, long-term potentiation (LTP) at CA3-CA1 hippocampal synapses was markedly enhanced in Stim1/Stim2 cKO mice and was associated with increased phosphorylation of the AMPA receptor subunit GluA1, the transcriptional regulator CREB and the L-type Voltage-dependent Ca2+ channel Cav1.2 on protein kinase A (PKA) sites. We conclude that STIM1 and STIM2 are key regulators of PKA signaling and synaptic plasticity in neural circuits encoding spatial memory. Our findings also reveal an inverse correlation between LTP and spatial learning/memory and suggest that abnormal enhancement of cAMP/PKA signaling and synaptic efficacy disrupts the formation of new memories. PMID:26236206

  20. Proteasome inhibition enhances resistance to DNA damage via upregulation of Rpn4-dependent DNA repair genes.

    PubMed

    Karpov, Dmitry S; Spasskaya, Daria S; Tutyaeva, Vera V; Mironov, Alexander S; Karpov, Vadim L

    2013-09-17

    The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

  1. Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing.

    PubMed

    Wu, Hong; Watanabe, Tomoko; Araki, Yoshio; Kitagaki, Hiroshi; Akao, Takeshi; Takagi, Hiroshi; Shimoi, Hitoshi

    2009-06-01

    Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.

  2. Peroxisome Proliferator-activated Receptor γ Regulates Genes Involved in Insulin/Insulin-like Growth Factor Signaling and Lipid Metabolism during Adipogenesis through Functionally Distinct Enhancer Classes*

    PubMed Central

    Oger, Frédérik; Dubois-Chevalier, Julie; Gheeraert, Céline; Avner, Stéphane; Durand, Emmanuelle; Froguel, Philippe; Salbert, Gilles; Staels, Bart; Lefebvre, Philippe; Eeckhoute, Jérôme

    2014-01-01

    The nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ is a transcription factor whose expression is induced during adipogenesis and that is required for the acquisition and control of mature adipocyte functions. Indeed, PPARγ induces the expression of genes involved in lipid synthesis and storage through enhancers activated during adipocyte differentiation. Here, we show that PPARγ also binds to enhancers already active in preadipocytes as evidenced by an active chromatin state including lower DNA methylation levels despite higher CpG content. These constitutive enhancers are linked to genes involved in the insulin/insulin-like growth factor signaling pathway that are transcriptionally induced during adipogenesis but to a lower extent than lipid metabolism genes, because of stronger basal expression levels in preadipocytes. This is consistent with the sequential involvement of hormonal sensitivity and lipid handling during adipocyte maturation and correlates with the chromatin structure dynamics at constitutive and activated enhancers. Interestingly, constitutive enhancers are evolutionary conserved and can be activated in other tissues, in contrast to enhancers controlling lipid handling genes whose activation is more restricted to adipocytes. Thus, PPARγ utilizes both broadly active and cell type-specific enhancers to modulate the dynamic range of activation of genes involved in the adipogenic process. PMID:24288131

  3. Cloning and mapping of a novel human cDNA homologous to DROER, the enhancer of the Drosophila melanogaster rudimentary gene

    SciTech Connect

    Isomura, Minoru; Okui, Keiko; Nakamura, Yusuke

    1996-02-15

    This article reports on the isolation and localization to human chromosome 7q34 of a human cDNA clone that encodes a protein which is homologous to DROER, the enhancer of the Drosophila melanogaster rudimentary gene. The structure and expression of this gene is also discussed. 12 refs., 3 figs.

  4. Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene

    PubMed Central

    Himeda, Charis L.; Debarnot, Céline; Homma, Sachiko; Beermann, Mary Lou; Miller, Jeffrey B.

    2014-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is linked to epigenetic dysregulation of the chromosome 4q35 D4Z4 macrosatellite. However, this does not account for the tissue specificity of FSHD pathology, which requires stable expression of an alternative full-length mRNA splice form of DUX4 (DUX4-fl) from the D4Z4 array in skeletal muscle. Here, we describe the identification of two enhancers, DUX4 myogenic enhancer 1 (DME1) and DME2 which activate DUX4-fl expression in skeletal myocytes but not fibroblasts. Analysis of the chromatin revealed histone modifications and RNA polymerase II occupancy consistent with DME1 and DME2 being functional enhancers. Chromosome conformation capture analysis confirmed association of DME1 and DME2 with the DUX4 promoter in vivo. The strong interaction between DME2 and the DUX4 promoter in both FSHD and unaffected primary myocytes was greatly reduced in fibroblasts, suggesting a muscle-specific interaction. Nucleosome occupancy and methylome sequencing analysis indicated that in most FSHD myocytes, both enhancers are associated with nucleosomes but have hypomethylated DNA, consistent with a permissive transcriptional state, sporadic occupancy, and the observed DUX4 expression in rare myonuclei. Our data support a model in which these myogenic enhancers associate with the DUX4 promoter in skeletal myocytes and activate transcription when epigenetically derepressed in FSHD, resulting in the pathological misexpression of DUX4-fl. PMID:24636994

  5. A gene-type-specific enhancer regulates the carbamyl phosphate synthetase I promoter by cooperating with the proximal GAG activating element.

    PubMed Central

    Goping, I S; Lamontagne, S; Shore, G C; Nguyen, M

    1995-01-01

    The rat carbamyl phosphate synthetase I gene is expressed in two cell types: hepatocytes and epithelial cells of the intestinal mucosa. The proximal promoter contains a single activating element, GAG, two repressor elements (sites I and III) and an anti-repressor element (site II). Although these elements together exhibit the potential for complex regulation, they are unable to confer tissue-specific promoter activity. Here we have identified a cell-type-specific enhancer that lies 10 kilobases upstream of the promoter. Unexpectedly, the enhancer also functioned in a gene-type-specific manner. The enhancer stimulated promoter activity exclusively through the proximal GAG element. Abrogation of GAG, either directly by mutation of GAG or indirectly by sites I and III repressors, abolished enhancer activation. Conversely, activation of the heterologous thymidine kinase promoter by the enhancer required the introduction of GAG. The requirement for GAG, therefore, functions to constrain the enhancer to a specific target promoter. PMID:7784176

  6. A gene expression atlas of developing oat seeds for enhancing nutritional composition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oat (Avena sativa L.) genome resources are less abundant than for wheat and barley, but next generation sequencing (NGS) technologies have great potential to accelerate new genome information for oat in a cost-effective manner. We are employing RNA-Seq to develop a gene expression atlas of developin...

  7. Artificial citrate operon and Vitreoscilla hemoglobin gene enhanced mineral phosphate solubilizing ability of Enterobacter hormaechei DHRSS.

    PubMed

    Yadav, Kavita; Kumar, Chanchal; Archana, G; Kumar, G Naresh

    2014-10-01

    Mineral phosphate solubilization by bacteria is mediated through secretion of organic acids, among which citrate is one of the most effective. To overproduce citrate in bacterial systems, an artificial citrate operon comprising of genes encoding NADH-insensitive citrate synthase of E. coli and Salmonella typhimurium sodium-dependent citrate transporter was constructed. In order to improve its mineral phosphate solubilizing (MPS) ability, the citrate operon was incorporated into E. hormaechei DHRSS. The artificial citrate operon transformant secreted 7.2 mM citric acid whereas in the native strain, it was undetectable. The transformant released 0.82 mM phosphate in flask studies in buffered medium containing rock phosphate as sole P source. In fermenter studies, similar phenotype was observed under aerobic conditions. However, under microaerobic conditions, no citrate was detected and P release was not observed. Therefore, an artificial citrate gene cluster containing Vitreoscilla hemoglobin (vgb) gene under its native promoter, along with artificial citrate operon under constitutive tac promoter, was constructed and transformed into E. hormaechei DHRSS. This transformant secreted 9 mM citric acid under microaerobic conditions and released 1.0 mM P. Thus, incorporation of citrate operon along with vgb gene improves MPS ability of E. hormaechei DHRSS under buffered, microaerobic conditions mimicking rhizospheric environment.

  8. The cytoskeleton enhances gene expression in the response to the Harpin elicitor in grapevine.

    PubMed

    Qiao, Fei; Chang, Xiao-Li; Nick, Peter

    2010-09-01

    The cytoskeleton undergoes dramatic reorganization during plant defence. This response is generally interpreted as part of the cellular repolarization establishing physical barriers against the invading pathogen. To gain insight into the functional significance of cytoskeletal responses for defence, two Vitis cell cultures that differ in their microtubular dynamics were used, and the cytoskeletal response to the elicitor Harpin in parallel to alkalinization of the medium as a fast response, and the activation of defence-related genes were followed. In one cell line derived from the grapevine cultivar 'Pinot Noir', microtubules contained mostly tyrosinylated alpha-tubulin, indicating high microtubular turnover, whereas in another cell line derived from the wild grapevine V. rupestris, the alpha-tubulin was strongly detyrosinated, indicating low microtubular turnover. The cortical microtubules were disrupted and actin filaments were bundled in both cell lines, but the responses were elevated in V. rupestris as compared with V. vinifera cv. 'Pinot Noir'. The cytoskeletal responsiveness correlated with elicitor-induced alkalinization and the expression of defence genes. Using resveratrol synthase and stilbene synthase as examples, it could be shown that pharmacological manipulation of microtubules could induce gene expression in the absence of elicitor. These findings are discussed with respect to a role for microtubules as positive regulators of defence-induced gene expression.

  9. Over-expression of OsDREB genes lead to enhanced drought tolerance in rice.

    PubMed

    Chen, Jian-Qiang; Meng, Xiu-Ping; Zhang, Yun; Xia, Mian; Wang, Xi-Ping

    2008-12-01

    The DREB transcription factors, which specifically interact with C-repeat/DRE (A/GCCGAC), play an important role in plant abiotic stress tolerance by controlling the expression of many cold or/and drought-inducible genes in an ABA-independent pathway. We have isolated three novel rice DREB genes, OsDREB1E, OsDREB1G, and OsDREB2B, which are homologous to Arabidopsis DREB genes. The yeast one-hybrid assay indicated that OsDREB1E, OsDREB1G, and OsDREB2B can specifically bind to the C-repeat/DRE element. To elucidate the function of respective OsDREB genes, we have stably introduced these to rice by Agrobacterium-mediated transformation. Transgenic rice plants analysis revealed that over-expression of OsDREB1G and OsDREB2B in rice significantly improved their tolerance to water deficit stress, while over-expression of OsDREB1E could only slightly improved the tolerance to water deficit stress, suggesting that the OsDREBs might participate in the stress response pathway in different manners.

  10. Overexpression of PYL5 in rice enhances drought tolerance, inhibits growth, and modulates gene expression

    PubMed Central

    Kim, Hyunmi; Lee, Kyeyoon; Hwang, Hyunsik; Kim, Beom-Gi

    2014-01-01

    Abscisic acid (ABA) is a phytohormone that plays important roles in the regulation of seed dormancy and adaptation to abiotic stresses. Previous work identified OsPYL/RCARs as functional ABA receptors regulating ABA-dependent gene expression in Oryza sativa. OsPYL/RCARs thus are considered to be good candidate genes for improvement of abiotic stress tolerance in crops. This work demonstrates that the cytosolic ABA receptor OsPYL/RCAR5 in O. sativa functions as a positive regulator of abiotic stress-responsive gene expression. The constitutive expression of OsPYL/RCAR5 in rice driven by the Zea mays ubiquitin promoter induced the expression of many stress-responsive genes even under normal growth conditions and resulted in improved drought and salt stress tolerance in rice. However, it slightly reduced plant height under paddy field conditions and severely reduced total seed yield. This suggests that, although exogenous expression of OsPYL/RCAR5 is able to improve abiotic stress tolerance in rice, fine regulation of its expression will be required to avoid deleterious effects on agricultural traits. PMID:24474809

  11. Autophagy Genes Enhance Murine Gammaherpesvirus 68 Reactivation From Latency by Preventing Virus-induced Systemic Inflammation

    PubMed Central

    Park, Sunmin; Buck, Michael D.; Desai, Chandni; Zhang, Xin; Loginicheva, Ekaterina; Martinez, Jennifer; Freeman, Michael L.; Saitoh, Tatsuya; Akira, Shizuo; Guan, Jun-Lin; He, You-Wen; Blackman, Marcia A.; Handley, Scott A.; Levine, Beth; Green, Douglas R.; Reese, Tiffany A.; Artyomov, Maxim N.; Virgin, Herbert W.

    2016-01-01

    SUMMARY Host genes that regulate systemic inflammation upon chronic viral infection are incompletely understood. Murine γ-herpesvirus 68 (MHV68) infection is characterized by latency in macrophages, and reactivation is inhibited by Interferon-γ (IFN-γ). Using a Lysozyme-M-cre (LysMcre) expression system, we show that deletion of autophagy-related (Atg) genes Fip200, beclin 1, Atg14, Atg16L1, Atg7, Atg3, and Atg5, in the myeloid compartment, inhibited MHV68 reactivation in macrophages. Atg5-deficiency did not alter reactivation from B cells, and effects on reactivation from macrophages were not explained by alterations in productive viral replication or the establishment of latency. Rather, chronic MHV68 infection triggered increased systemic inflammation, increased T cell production of IFN-γ and an IFN-γ-induced transcriptional signature in macrophages from Atg gene-deficient mice. The Atg5-related reactivation defect was partially reversed by neutralization of IFN-γ. Thus Atg genes in myeloid cells dampen virus-induced systemic inflammation, creating an environment that fosters efficient MHV68 reactivation from latency. PMID:26764599

  12. The National Institutes of Health Oversight of Human Gene Transfer Research: Enhancing Science and Safety.

    PubMed

    O'Reilly, Marina; Jambou, Robert; Rosenthal, Eugene; Montgomery, Maureen; Hassani, Morad; Gargiulo, Linda; Corrigan-Curay, Jacqueline

    2015-01-01

    The National Institutes of Health (NIH) oversight of human gene transfer research, which is defined as the deliberate transfer of recombinant and/or synthetic nucleic acid molecules to humans, originates with the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines). The NIH Guidelines, which were first published in the Federal Register almost 40 years ago, have been amended numerous times to remain responsive to scientific progress and to clearly define the responsibilities of NIH, the Recombinant DNA Advisory Committee (RAC), investigators, and institutions. Human gene transfer trials conducted at clinical sites in the United States (USA) are subject to the NIH Guidelines if they are conducted at, or sponsored by, an institution that receives any support for recombinant or synthetic nucleic acid research from the NIH. Human gene transfer trials conducted either in the USA or abroad are also subject to the NIH Guidelines if the investigational agent was developed with NIH funds and the institution that developed the investigational materials sponsors or participates in these projects. Trials are registered with the NIH Office Biotechnology Activities (OBA) and there are ongoing reporting requirements. Each new trial is reviewed by the RAC, and those that are novel or raise unique ethical or social issues are selected for review at quarterly public RAC meetings. The RAC also advises the NIH on policy and other matters relating to clinical gene transfer research and biosafety.

  13. The application of powerful promoters to enhance gene expression in industrial microorganisms.

    PubMed

    Zhou, Shenghu; Du, Guocheng; Kang, Zhen; Li, Jianghua; Chen, Jian; Li, Huazhong; Zhou, Jingwen

    2017-02-01

    Production of useful chemicals by industrial microorganisms has been attracting more and more attention. Microorganisms screened from their natural environment usually suffer from low productivity, low stress resistance, and accumulation of by-products. In order to overcome these disadvantages, rational engineering of microorganisms to achieve specific industrial goals has become routine. Rapid development of metabolic engineering and synthetic biology strategies provide novel methods to improve the performance of industrial microorganisms. Rational regulation of gene expression by specific promoters is essential to engineer industrial microorganisms for high-efficiency production of target chemicals. Identification, modification, and application of suitable promoters could provide powerful switches at the transcriptional level for fine-tuning of a single gene or a group of genes, which are essential for the reconstruction of pathways. In this review, the characteristics of promoters from eukaryotic, prokaryotic, and archaea microorganisms are briefly introduced. Identification of promoters based on both traditional biochemical and systems biology routes are summarized. Besides rational modification, de novo design of promoters to achieve gradient, dynamic, and logic gate regulation are also introduced. Furthermore, flexible application of static and dynamic promoters for the rational engineering of industrial microorganisms is highlighted. From the perspective of powerful promoters in industrial microorganisms, this review will provide an extensive description of how to regulate gene expression in industrial microorganisms to achieve more useful goals.

  14. Transgenic tobacco plants overexpressing a grass PpEXP1 gene exhibit enhanced tolerance to heat stress.

    PubMed

    Xu, Qian; Xu, Xiao; Shi, Yang; Xu, Jichen; Huang, Bingru

    2014-01-01

    Heat stress is a detrimental abiotic stress limiting the growth of many plant species and is associated with various cellular and physiological damages. Expansins are a family of proteins which are known to play roles in regulating cell wall elongation and expansion, as well as other growth and developmental processes. The in vitro roles of expansins regulating plant heat tolerance are not well understood. The objectives of this study were to isolate and clone an expansin gene in a perennial grass species (Poa pratensis) and to determine whether over-expression of expansin may improve plant heat tolerance. Tobacco (Nicotiana tabacum) was used as the model plant for gene transformation and an expansin gene PpEXP1 from Poa pratensis was cloned. Sequence analysis showed PpEXP1 belonged to α-expansins and was closely related to two expansin genes in other perennial grass species (Festuca pratensis and Agrostis stolonifera) as well as Triticum aestivum, Oryza sativa, and Brachypodium distachyon. Transgenic tobacco plants over-expressing PpEXP1 were generated through Agrobacterium-mediated transformation. Under heat stress (42°C) in growth chambers, transgenic tobacco plants over-expressing the PpEXP1 gene exhibited a less structural damage to cells, lower electrolyte leakage, lower levels of membrane lipid peroxidation, and lower content of hydrogen peroxide, as well as higher chlorophyll content, net photosynthetic rate, relative water content, activity of antioxidant enzyme, and seed germination rates, compared to the wild-type plants. These results demonstrated the positive roles of PpEXP1 in enhancing plant tolerance to heat stress and the possibility of using expansins for genetic modification of cool-season perennial grasses in the development of heat-tolerant germplasm and cultivars.

  15. PLGA-based gene delivering nanoparticle enhance suppression effect of miRNA in HePG2 cells

    NASA Astrophysics Data System (ADS)

    Feng Liang, Gao; Zhu, Yan Liang; Sun, Bo; Hu, Fei Hu; Tian, Tian; Li, Shu Chun; Xiao, Zhong Dang

    2011-07-01

    The biggest challenge in the field of gene therapy is how to effectively deliver target genes to special cells. This study aimed to develop a new type of poly( D, L-lactide-co-glycolide) (PLGA)-based nanoparticles for gene delivery, which are capable of overcoming the disadvantages of polyethylenimine (PEI)- or cationic liposome-based gene carrier, such as the cytotoxicity induced by excess positive charge, as well as the aggregation on the cell surface. The PLGA-based nanoparticles presented in this study were synthesized by emulsion evaporation method and characterized by transmission electron microscopy, dynamic light scattering, and energy dispersive spectroscopy. The size of PLGA/PEI nanoparticles in phosphate-buffered saline (PBS) was about 60 nm at the optimal charge ratio. Without observable aggregation, the nanoparticles showed a better monodispersity. The PLGA-based nanoparticles were used as vector carrier for miRNA transfection in HepG2 cells. It exhibited a higher transfection efficiency and lower cytotoxicity in HepG2 cells compared to the PEI/DNA complex. The N/P ratio (ratio of the polymer nitrogen to the DNA phosphate) 6 of the PLGA/PEI/DNA nanocomplex displays the best property among various N/P proportions, yielding similar transfection efficiency when compared to Lipofectamine/DNA lipoplexes. Moreover, nanocomplex shows better serum compatibility than commercial liposome. PLGA nanocomplexes obviously accumulate in tumor cells after transfection, which indicate that the complexes contribute to cellular uptake of pDNA and pronouncedly enhance the treatment effect of miR-26a by inducing cell cycle arrest. Therefore, these results demonstrate that PLGA/PEI nanoparticles are promising non-viral vectors for gene delivery.

  16. Reduced expression of CTR1 gene modulated by mitochondria causes enhanced ethylene response in cytoplasmic male-sterile Brassica juncea.

    PubMed

    Liu, Xunyan; Yang, Xiaodong; Zhao, Xuan; Yang, Jinghua; Zhang, Mingfang

    2012-06-01

    We studied how mitochondria affect ethylene response via modulation of CTR1 expression in cytoplasmic male-sterile (CMS) Brassica juncea. The expression of CTR1 gene was reduced in CMS compared with male-fertile (MF) lines. We observed that hypocotyl and root lengths were shorter than in the MF line during germination in the dark. An enhanced ethylene response was observed in CMS plants as shown by the CMS and maintainer line phenotypes treated with 1-aminocyclopropane-1-carboxylic acid. The phenotype in CMS plants could be recovered to the maintainer line when treated with Ag(+) . One ethylene response gene, plant defensin gene, was detected to be induced in CMS. The behavior of this phenotype could be mimicked by treating the maintainer line with antimycin A that disturbs mitochondrial function, which showed reduced length of hypocotyl and roots, and resulted in similar expression patterns of ethylene-related genes as in CMS. The reduced length of hypocotyl and roots could be recovered to the maintainer line by treatment with gibberellic acid (GA(3) ). In addition, the GA(3) content was reduced in CMS plants and in the MF line treated with antimycin A. Ethylene treatment markedly affects GA(3) content; however, GA(3) did not significantly affect ethylene-related gene expression in regards to regulation of hypocotyl and root length, which suggests that ethylene acts upstream via gibberellin to regulate hypocotyls and root development. Taken together, our results suggest a link between mitochondrial modulation of the ethylene and gibberellin pathway that regulates the development of hypocotyl and roots.

  17. Formation of LID vector complexes in water alters physicochemical properties and enhances pulmonary gene expression in vivo.

    PubMed

    Jenkins, R G; Meng, Q-H; Hodges, R J; Lee, L K; Bottoms, S E W; Laurent, G J; Willis, D; Ayazi Shamlou, P; McAnulty, R J; Hart, S L

    2003-06-01

    There is currently an urgent need to develop efficient gene-delivery systems for the lung that are free of inflammatory effects. The LID vector is a synthetic gene delivery system, comprised of lipofectin (L), an integrin-targeting peptide (I) and DNA (D) that has previously been shown to have high transfection efficiency in the lung. We have assessed the effect of alternative methods of complex preparation on structural features of the complex, levels and duration of reporter gene expression and the host response to the LID vector. We have demonstrated that making the complex in water affects the structure of the LID complexes making them smaller and more stable with a more cationic surface charge than complexes prepared in phosphate-buffered saline (PBS). When the LID vector was constituted in water and instilled intratracheally into the lungs of mice there was a 10-fold increase in luciferase activity compared with preparation in PBS. Furthermore, luciferase activity was still evident 1 week following vector instillation. This enhancement may be because of altered complex structure, although effects of the hypotonic vector solution on the lung cannot be excluded. The inflammatory effects of instilling the LID vector in water were minimal, even after three administrations of the LID vector, with only mild alterations in cytokine and broncho-alveolar lavage fluid (BALF) cell profiles. These results demonstrate that the LID vector can generate high, and prolonged, levels of gene expression in the lung from small quantities of DNA and that careful attention to synthetic polyplex structure may be important to optimize efficiency of gene expression in vivo.

  18. ENHANCED DISEASE SUSCEPTIBILITY 1 and SALICYLIC ACID act redundantly to regulate resistance gene-mediated signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Resistance (R) protein–associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non–race-specific disease resistance 1 (NDR1), ...

  19. Ultraviolet Radiation-Elicited Enhancement of Isoflavonoid Accumulation, Biosynthetic Gene Expression, and Antioxidant Activity in Astragalus membranaceus Hairy Root Cultures.

    PubMed

    Jiao, Jiao; Gai, Qing-Yan; Wang, Wei; Luo, Meng; Gu, Cheng-Bo; Fu, Yu-Jie; Ma, Wei

    2015-09-23

    In this work, Astragalus membranaceus hairy root cultures (AMHRCs) were exposed to ultraviolet radiation (UV-A, UV-B, and UV-C) for promoting isoflavonoid accumulation. The optimum enhancement for isoflavonoid production was achieved in 34-day-old AMHRCs elicited by 86.4 kJ/m(2) of UV-B. The resulting isoflavonoid yield was 533.54 ± 13.61 μg/g dry weight (DW), which was 2.29-fold higher relative to control (232.93 ± 3.08 μg/g DW). UV-B up-regulated the transcriptional expressions of all investigated genes involved in isoflavonoid biosynthetic pathway. PAL and C4H were found to be two potential key genes that controlled isoflavonoid biosynthesis. Moreover, a significant increase was noted in antioxidant activity of extracts from UV-B-elicited AMHRCs (IC50 values = 0.85 and 1.08 mg/mL) in comparison with control (1.38 and 1.71 mg/mL). Overall, this study offered a feasible elicitation strategy to enhance isoflavonoid accumulation in AMHRCs and also provided a basis for metabolic engineering of isoflavonoid biosynthesis in the future.

  20. Polymer nanopillar-gold arrays as surface-enhanced Raman spectroscopy substrate for the simultaneous detection of multiple genes.

    PubMed

    Picciolini, Silvia; Mehn, Dora; Morasso, Carlo; Vanna, Renzo; Bedoni, Marzia; Pellacani, Paola; Marchesini, Gerardo; Valsesia, Andrea; Prosperi, Davide; Tresoldi, Cristina; Ciceri, Fabio; Gramatica, Furio

    2014-10-28

    In our study, 2D nanopillar arrays with plasmonic crystal properties are optimized for surface-enhanced Raman spectroscopy (SERS) application and tested in a biochemical assay for the simultaneous detection of multiple genetic leukemia biomarkers. The special fabrication process combining soft lithography and plasma deposition techniques allows tailoring of the structural and chemical parameters of the crystal surfaces. In this way, it has been possible to tune the plasmonic resonance spectral position close to the excitation wavelength of the monochromatic laser light source in order to maximize the enhancing properties of the substrate. Samples are characterized by scanning electron microscopy and reflectance measurements and tested for SERS activity using malachite green. Besides, as the developed substrate had been prepared on a simple glass slide, SERS detection from the support side is also demonstrated. The optimized substrate is functionalized with thiol-modified capture oligonucleotides, and concentration-dependent signal of the target nucleotide is detected in a sandwich assay with labeled gold nanoparticles. Gold nanoparticles functionalized with different DNA and various Raman reporters are applied in a microarray-based assay recognizing a disease biomarker (Wilms tumor gene) and housekeeping gene expressions in the same time on spatially separated microspots. The multiplexing performance of the SERS-based bioassay is illustrated by distinguishing Raman dyes based on their complex spectral fingerprints.

  1. Feeding hydroalcoholic extract powder of Lepidium meyenii (maca) enhances testicular gene expression of 3β-hydroxysteroid dehydrogenase in rats.

    PubMed

    Ohta, Y; Kawate, N; Inaba, T; Morii, H; Takahashi, K; Tamada, H

    2017-03-06

    Although feeding diets containing the extract powder of Lepidium meyenii (maca), a plant growing in Peru's Central Andes, increases serum testosterone concentration associated with enhanced ability of testosterone production by Leydig cells in male rats, changes in testicular steroidogenesis-related factors by the maca treatment are not known. This study examined the effects of maca on testicular gene expressions for luteinizing hormone receptor, steroidogenic acute regulatory protein and steroidogenic enzymes. Eight-week-old male rats were given the diets with or without (control) the maca extract powder (2%) for 6 weeks, and mRNA levels were determined by reverse transcription quantitative real-time PCR. The results showed that the testicular mRNA level of HSD3B1 (3β-hydroxysteroid dehydrogenase; 3β-HSD) increased by the treatment, whereas the levels of the other factors examined did not change. These results suggest that increased expression of 3β-HSD gene may be involved in the enhanced steroidogenic ability by the maca treatment in rat testes.

  2. Combination of Interleukin-27 and MicroRNA for Enhancing Expression of Anti-Inflammatory and Proosteogenic Genes

    PubMed Central

    Figueiredo Neto, Manoel

    2017-01-01

    Remission of inflammation has become an achievable goal in inflammatory or rheumatoid arthritis (RA); however, bone erosion continues in many patients. Interleukin- (IL-) 27 regulates immune and bone cell balance and also suppresses activities of several inflammatory cell types in RA. Despite its promise, challenges to clinical translation of IL-27 have been its partial effects in vivo. Due to their ability to modulate plasticity of bone and immune cell differentiation, we examined the potential for several microRNA (miR) candidates in enhancing the effects of IL-27. Using differentiation, luciferase, and real time quantitative PCR assays, we show that IL-27 promotes osteoblast differentiation, reduces expression of osteoblast inhibitory genes, and reduces osteoclast differentiation, and results suggest a potential coordination with TGFβ/BMP/SMAD and JAK/STAT pathways. We selected miRNA regulators of these and related pathways to examine whether the effects of IL-27 could be augmented for therapeutic applications. miR-29b and miR-21 augmented IL-27 proosteogenic while downregulating osteoclastogenic signals and also worked to reduce inflammatory signaling in activated macrophages, while miR-21 and miR-20b worked with IL-27 to reduce inflammatory gene expression in fibroblasts and T cells. It appears that several miRNAs can be utilized to enhance IL-27's impact on modulating osteogenesis and reducing proinflammatory signaling. PMID:28265470

  3. Trichostatin A enhances vascular repair by injected human endothelial progenitors through increasing the expression of TAL1-dependent genes.

    PubMed

    Palii, Carmen G; Vulesevic, Branka; Fraineau, Sylvain; Pranckeviciene, Erinija; Griffith, Alexander J; Chu, Alphonse; Faralli, Hervé; Li, Yuhua; McNeill, Brian; Sun, Jie; Perkins, Theodore J; Dilworth, F Jeffrey; Perez-Iratxeta, Carol; Suuronen, Erik J; Allan, David S; Brand, Marjorie

    2014-05-01

    A major goal of cell therapy for vascular diseases is to promote revascularization through the injection of endothelial stem/progenitor cells. The gene regulatory mechanisms that underlie endothelial progenitor-mediated vascular repair, however, remain elusive. Here, we identify the transcription factor TAL1/SCL as a key mediator of the vascular repair function of primary human endothelial colony-forming cells (ECFCs). Genome-wide analyses in ECFCs demonstrate that TAL1 activates a transcriptional program that promotes cell adhesion and migration. At the mechanistic level, we show that TAL1 upregulates the expression of migratory and adhesion genes through recruitment of the histone acetyltransferase p300. Based on these findings, we establish a strategy that enhances the revascularization efficiency of ECFCs after ischemia through ex vivo priming with the histone deacetylase inhibitor TSA. Thus, small molecule epigenetics drugs are effective tools for modifying the epigenome of stem/progenitor cells prior to transplantation as a means to enhance their therapeutic potential.

  4. Enhancement of the immunogenicity of DNA replicon vaccine of Clostridium botulinum neurotoxin serotype A by GM-CSF gene adjuvant.

    PubMed

    Li, Na; Yu, Yun-Zhou; Yu, Wei-Yuan; Sun, Zhi-Wei

    2011-03-01

    Granulocyte-macrophage clony-stimulating factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells to the site of antigen synthesis as well as stimulate the maturation of dendritic cells.This study evaluated the utility of GM-CSF as a plasmid DNA replicon vaccine adjuvants for botulinum neurotoxin serotype A (BoNT/A) in mouse model. In balb/c mice that received the plasmid DNA replicon vaccines derived from Semliki Forest virus (SFV) carrying the Hc gene of BoNT/A (AHc), both antibody and lymphoproliferative response specific to AHc were induced, the immunogenicity was enhanced by co-delivery or coexpress of the GM-CSF gene. In particular, when AHc and GM-CSF were coexpressed within the SFV based DNA vaccine, the anti-AHc antibody titers and survival rates of immunized mice after challenged with BoNT/A were significantly increased, and further enhanced by coimmunization with aluminum phosphate adjuvant.

  5. Gene duplication confers enhanced expression of 27-kDa γ-zein for endosperm modification in quality protein maize.

    PubMed

    Liu, Hongjun; Shi, Junpeng; Sun, Chuanlong; Gong, Hao; Fan, Xingming; Qiu, Fazhan; Huang, Xuehui; Feng, Qi; Zheng, Xixi; Yuan, Ningning; Li, Changsheng; Zhang, Zhiyong; Deng, Yiting; Wang, Jiechen; Pan, Guangtang; Han, Bin; Lai, Jinsheng; Wu, Yongrui

    2016-05-03

    The maize opaque2 (o2) mutant has a high nutritional value but it develops a chalky endosperm that limits its practical use. Genetic selection for o2 modifiers can convert the normally chalky endosperm of the mutant into a hard, vitreous phenotype, yielding what is known as quality protein maize (QPM). Previous studies have shown that enhanced expression of 27-kDa γ-zein in QPM is essential for endosperm modification. Taking advantage of genome-wide association study analysis of a natural population, linkage mapping analysis of a recombinant inbred line population, and map-based cloning, we identified a quantitative trait locus (qγ27) affecting expression of 27-kDa γ-zein. qγ27 was mapped to the same region as the major o2 modifier (o2 modifier1) on chromosome 7 near the 27-kDa γ-zein locus. qγ27 resulted from a 15.26-kb duplication at the 27-kDa γ-zein locus, which increases the level of gene expression. This duplication occurred before maize domestication; however, the gene structure of qγ27 appears to be unstable and the DNA rearrangement frequently occurs at this locus. Because enhanced expression of 27-kDa γ-zein is critical for endosperm modification in QPM, qγ27 is expected to be under artificial selection. This discovery provides a useful molecular marker that can be used to accelerate QPM breeding.

  6. Chronic morphine treatment enhances sciatic nerve stimulation-induced immediate early gene expression in the rat dorsal horn.

    PubMed

    Bojovic, Ognjen; Bramham, Clive R; Tjølsen, Arne

    2015-01-01

    Synaptic plasticity is a property of neurons that can be induced by conditioning electrical stimulation (CS) of afferent fibers in the spinal cord. This is a widely studied property of spinal cord and hippocampal neurons. CS has been shown to trigger enhanced expression of immediate early gene proteins (IEGPs), with peak increases observed 2 hour post stimulation. Chronic morphine treatment has been shown to promoteinduce opioid-induced hyperalgesia, and also to increase CS-induced central sensitization in the dorsal horn. As IEGP expression may contribute to development of chronic pain states, we aimed to determine whether chronic morphine treatment affects the expression of IEGPs following sciatic nerve CS. Changes in expression of the IEGPs Arc, c-Fos or Zif268 were determined in cells of the lumbar dorsal horn of the spinal cord. Chronic Morphine pretreatment over 7 days led to a significant increase in the number of IEGP positive cells observed at both 2 h and 6 h after CS. The same pattern of immunoreactivity was obtained for all IEGPs, with peak increases occurring at 2 h post CS. In contrast, morphine treatment alone in sham operated animals had no effect on IEGP expression. We conclude that chronic morphine treatment enhances stimulus-induced expression of IEGPs in the lumbar dorsal horn. These data support the notion that morphine alters gene expression responses linked to nociceptive stimulation and plasticity.

  7. Drosophila TDP-43 RNA-Binding Protein Facilitates Association of Sister Chromatid Cohesion Proteins with Genes, Enhancers and Polycomb Response Elements

    PubMed Central

    Misulovin, Ziva; Gause, Maria; Rickels, Ryan A; Shilatifard, Ali

    2016-01-01

    The cohesin protein complex mediates sister chromatid cohesion and participates in transcriptional control of genes that regulate growth and development. Substantial reduction of cohesin activity alters transcription of many genes without disrupting chromosome segregation. Drosophila Nipped-B protein loads cohesin onto chromosomes, and together Nipped-B and cohesin occupy essentially all active transcriptional enhancers and a large fraction of active genes. It is unknown why some active genes bind high levels of cohesin and some do not. Here we show that the TBPH and Lark RNA-binding proteins influence association of Nipped-B and cohesin with genes and gene regulatory sequences. In vitro, TBPH and Lark proteins specifically bind RNAs produced by genes occupied by Nipped-B and cohesin. By genomic chromatin immunoprecipitation these RNA-binding proteins also bind to chromosomes at cohesin-binding genes, enhancers, and Polycomb response elements (PREs). RNAi depletion reveals that TBPH facilitates association of Nipped-B and cohesin with genes and regulatory sequences. Lark reduces binding of Nipped-B and cohesin at many promoters and aids their association with several large enhancers. Conversely, Nipped-B facilitates TBPH and Lark association with genes and regulatory sequences, and interacts with TBPH and Lark in affinity chromatography and immunoprecipitation experiments. Blocking transcription does not ablate binding of Nipped-B and the RNA-binding proteins to chromosomes, indicating transcription is not required to maintain binding once established. These findings demonstrate that RNA-binding proteins help govern association of sister chromatid cohesion proteins with genes and enhancers. PMID:27662615

  8. Overexpression of Prunus mume Dehydrin Genes in Tobacco Enhances Tolerance to Cold and Drought.

    PubMed

    Bao, Fei; Du, Dongliang; An, Yang; Yang, Weiru; Wang, Jia; Cheng, Tangren; Zhang, Qixiang

    2017-01-01

    Dehydrins, known as group 2 or D-11 family late-embryogenesis-abundant (LEA) proteins, play important roles in plant growth and stress tolerance. Six dehydrin genes were previously identified from the genome of Prunus mume. In this study, five of them (PmLEA8, PmLEA10, PmLEA19, PmLEA20, and PmLEA29) were cloned from cold-resistant P. mume 'Beijingyudie'. Real-time RT-PCR analysis indicated that all these genes could be up-regulated by one or several treatments (ABA, SA, low temperature, high temperature, PEG, and NaCl treatments). The results of spot assay demonstrated that the expression of all these dehydrins, except PmLEA8, conferred improved osmotic and freezing-resistance to the recombinant Escherichia coli. So four dehydrin genes, PmLEA10, PmLEA19, PmLEA20 and PmLEA29 were chosen for individual over-expression in tobacco plants. The transgenic tobacco plants showed lower relative content of malondialdehyde, relative electrolyte leakage and higher relative content of water than control plants when exposed to cold and drought stress. These results demonstrated that PmLEAs were involved in plant responses to cold and drought.

  9. Close-field electroporation gene delivery using the cochlear implant electrode array enhances the bionic ear.

    PubMed

    Pinyon, Jeremy L; Tadros, Sherif F; Froud, Kristina E; Y Wong, Ann C; Tompson, Isabella T; Crawford, Edward N; Ko, Myungseo; Morris, Renée; Klugmann, Matthias; Housley, Gary D

    2014-04-23

    The cochlear implant is the most successful bionic prosthesis and has transformed the lives of people with profound hearing loss. However, the performance of the "bionic ear" is still largely constrained by the neural interface itself. Current spread inherent to broad monopolar stimulation of the spiral ganglion neuron somata obviates the intrinsic tonotopic mapping of the cochlear nerve. We show in the guinea pig that neurotrophin gene therapy integrated into the cochlear implant improves its performance by stimulating spiral ganglion neurite regeneration. We used the cochlear implant electrode array for novel "close-field" electroporation to transduce mesenchymal cells lining the cochlear perilymphatic canals with a naked complementary DNA gene construct driving expression of brain-derived neurotrophic factor (BDNF) and a green fluorescent protein (GFP) reporter. The focusing of electric fields by particular cochlear implant electrode configurations led to surprisingly efficient gene delivery to adjacent mesenchymal cells. The resulting BDNF expression stimulated regeneration of spiral ganglion neurites, which had atrophied 2 weeks after ototoxic treatment, in a bilateral sensorineural deafness model. In this model, delivery of a control GFP-only vector failed to restore neuron structure, with atrophied neurons indistinguishable from unimplanted cochleae. With BDNF therapy, the regenerated spiral ganglion neurites extended close to the cochlear implant electrodes, with localized ectopic branching. This neural remodeling enabled bipolar stimulation via the cochlear implant array, with low stimulus thresholds and expanded dynamic range of the cochlear nerve, determined via electrically evoked auditory brainstem responses. This development may broadly improve neural interfaces and extend molecular medicine applications.

  10. Enhanced gene delivery to the lung using biodegradable polyunsaturated cationic phosphatidylcholine-detergent conjugates.

    PubMed

    Pierrat, Philippe; Kereselidze, Dimitri; Lux, Marie; Lebeau, Luc; Pons, Françoise

    2016-09-10

    Lung diseases are among the more representative causes of mortality and morbidity worldwide and gene therapy is considered as a promising therapeutic approach for their treatment. However the design of efficient nucleic acid carriers for airway administration still is a challenge and there is a pressing need for new developments in this field. Herein, new synthetic DNA carriers based on the conjugation of a phospholipid and C12E4, a nonionic detergent, are developed. DNA complexes with phosphatidylcholine-detergent conjugates are administered in mouse airways, and transgene expression and inflammatory activity as an index of toxicity are investigated as a function of time, DNA dose, and presence of helper and stealth lipids. Introduction of a biodegradable linker between the phosphatidylcholine and detergent moieties significantly attenuates the severity of inflammatory response that characterizes cationic lipid-mediated gene transfer. Concurrent introduction of polyunsaturated fatty acid chains in the carrier scaffold improves transgene expression and further reduces airway inflammation. Finally, the biodegradable phosphatidylcholine-detergent conjugates favorably compare to GL67A, the gold standard for DNA delivery to the airway that is currently under clinical evaluation. Our findings indicate that the lipid formulations described herein may have great potential as nucleic acid carriers for gene therapy.

  11. Overexpression of Prunus mume Dehydrin Genes in Tobacco Enhances Tolerance to Cold and Drought

    PubMed Central

    Bao, Fei; Du, Dongliang; An, Yang; Yang, Weiru; Wang, Jia; Cheng, Tangren; Zhang, Qixiang

    2017-01-01

    Dehydrins, known as group 2 or D-11 family late-embryogenesis-abundant (LEA) proteins, play important roles in plant growth and stress tolerance. Six dehydrin genes were previously identified from the genome of Prunus mume. In this study, five of them (PmLEA8, PmLEA10, PmLEA19, PmLEA20, and PmLEA29) were cloned from cold-resistant P. mume ‘Beijingyudie’. Real-time RT-PCR analysis indicated that all these genes could be up-regulated by one or several treatments (ABA, SA, low temperature, high temperature, PEG, and NaCl treatments). The results of spot assay demonstrated that the expression of all these dehydrins, except PmLEA8, conferred improved osmotic and freezing-resistance to the recombinant Escherichia coli. So four dehydrin genes, PmLEA10, PmLEA19, PmLEA20 and PmLEA29 were chosen for individual over-expression in tobacco plants. The transgenic tobacco plants showed lower relative content of malondialdehyde, relative electrolyte leakage and higher relative content of water than control plants when exposed to cold and drought stress. These results demonstrated that PmLEAs were involved in plant responses to cold and drought. PMID:28224001

  12. Use of Hydroxyapatite Doping to Enhance Responsiveness of Heat-Inducible Gene Switches to Focused Ultrasound.

    PubMed

    Fabiilli, Mario L; Phanse, Rahul A; Moncion, Alexander; Fowlkes, J Brian; Franceschi, Renny T

    2016-03-01

    Recently, we demonstrated that ultrasound-based hyperthermia can activate cells containing a heat-activated and ligand-inducible gene switch in a spatio-temporally controlled manner. These engineered cells can be incorporated into hydrogel scaffolds (e.g., fibrin) for in vivo implantation, where ultrasound can be used to non-invasively pattern transgene expression. Due to their high water content, the acoustic attenuation of fibrin scaffolds is low. Thus, long ultrasound exposures and high acoustic intensities are needed to generate sufficient hyperthermia for gene activation. Here, we demonstrate that the attenuation of fibrin scaffolds and the resulting hyperthermia achievable with ultrasound can be increased significantly by doping the fibrin with hydroxyapatite (HA) nanopowder. The attenuation of a 1% (w/v) fibrin scaffold with 5% (w/v) HA was similar to soft tissue. Transgene activation of cells harboring the gene switch occurred at lower acoustic intensities and shorter exposures when the cells were encapsulated in HA-doped fibrin scaffolds versus undoped scaffolds. Inclusion of HA in the fibrin scaffold did not affect the viability of the encapsulated cells.

  13. Satellite panicum mosaic virus coat protein enhances the performance of plant virus gene vectors.

    PubMed

    Everett, Anthany L; Scholthof, Herman B; Scholthof, Karen-Beth G

    2010-01-05

    The coat protein of satellite panicum mosaic virus (SPCP) is known to effectively protect its cognate RNA from deleterious events, and here, we tested its stabilizing potential for heterologous virus-based gene vectors in planta. In support of this, a Potato virus X (PVX) vector carrying the SPMV capsid protein (PVX-SPCP) gene was stable for at least three serial systemic passages through Nicotiana benthamiana. To test the effect of SPCP in trans, PVX-SPCP was co-inoculated onto N. benthamiana together with a Tomato bushy stunt virus (TBSV) vector carrying a green fluorescent protein (GFP) gene that normally does not support systemic GFP expression. In contrast, co-inoculation of TBSV-GFP plus PVX-SPCP resulted in GFP accumulation and concomitant green fluorescent spots in upper, non-inoculated leaves in a temperature-responsive manner. These results suggest that the multifaceted SPMV CP has intriguing effects on virus-host interactions that surface in heterologous systems.

  14. Use of Hydroxyapatite Doping to Enhance Responsiveness of Heat-Inducible Gene Switches to Focused Ultrasound

    PubMed Central

    Fabiilli, Mario L.; Phanse, Rahul A.; Moncion, Alexander; Fowlkes, J. Brian; Franceschi, Renny T.

    2015-01-01

    Recently, we demonstrated that ultrasound-based hyperthermia can activate cells containing a heat-activated and ligand-inducible gene switch in a spatio-temporally controlled manner. These engineered cells can be incorporated into hydrogel scaffolds (e.g., fibrin) for in vivo implantation, where ultrasound can be used to non-invasively pattern transgene expression. Due to their high water content, the acoustic attenuation of fibrin scaffolds is low. Thus, long ultrasound exposures and high acoustic intensities are needed to generate sufficient hyperthermia for gene activation. Here, we demonstrate that the attenuation of fibrin scaffolds and the resulting hyperthermia achievable with ultrasound can be increased significantly by doping the fibrin with hydroxyapatite (HA) nanopowder. The attenuation of a 1% (w/v) fibrin scaffold with 5% (w/v) HA was similar to soft tissue. Transgene activation of cells harboring the gene switch occurred at lower acoustic intensities and shorter exposures when the cells were encapsulated in HA-doped fibrin scaffolds versus undoped scaffolds. Inclusion of HA in the fibrin scaffold did not affect the viability of the encapsulated cells. PMID:26712417

  15. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

    PubMed Central

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-01-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma. PMID:27625116

  16. Overexpression of the Maize psbA Gene Enhances Drought Tolerance Through Regulating Antioxidant System, Photosynthetic Capability, and Stress Defense Gene Expression in Tobacco.

    PubMed

    Huo, Yongjin; Wang, Meiping; Wei, Yangyang; Xia, Zongliang

    2015-01-01

    The psbA (encoding D1 protein) plays an important role in protecting photosystem II (PSII) from oxidative damage in higher plants. In our previous study, the role of the psbA from maize (Zea mays. L) in response to SO2 stress was characterized. To date, information about the involvement of the psbA gene in drought response is scarce. Here we found that overexpression (OE) of ZmpsbA showed increased D1 protein abundance and enhanced drought stress tolerance in tobacco. The drought-tolerant phenotypes of the OE lines were accompanied by increases of key antioxidant enzymes SOD, CAT, and POD activities, but decreases of hydrogen peroxide, malondialdehyde, and ion leakage. Further investigation showed that the OE plants had much less reductions than the wild-type in the net photosynthesis rate (Pn), stomatal conductance (Gs), and the maximal photochemical efficiency of PSII (Fv/Fm) during drought stress; indicating that OE of ZmpsbA may alleviate photosynthesis inhibition during drought. qRT-PCR analysis revealed that there was significantly increased expression of NtLEA5, NtERD10C, NtAREB, and NtCDPK2 in ZmpsbA-OE lines. Together, our results indicate that ZmpsbA improves drought tolerance in tobacco possibly by alleviating photosynthesis reduction, reducing reactive oxygen species accumulation and membrane damage, and modulating stress defense gene expression. ZmpsbA could be exploited for engineering drought-tolerant plants in molecular breeding of crops.

  17. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

    NASA Astrophysics Data System (ADS)

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-09-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

  18. Characterization of the 2,3-Oxidosqualene Cyclase Gene from Antrodia cinnamomea and Enhancement of Cytotoxic Triterpenoid Compound Production.

    PubMed

    Lin, Yan-Liang; Lee, Yi-Ru; Tsao, Nai-Wen; Wang, Sheng-Yang; Shaw, Jei-Fu; Chu, Fang-Hua

    2015-07-24

    Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.

  19. Transgenic Brassica chinensis plants expressing a bacterial codA gene exhibit enhanced tolerance to extreme temperature and high salinity.

    PubMed

    Wang, Qing-bin; Xu, Wen; Xue, Qing-zhong; Su, Wei-ai

    2010-11-01

    Transgenic Brassica compestris L. spp. chinensis plants expressing a choline oxidase (codA) gene from Arthrobacter globiformis were obtained through Agrobacterium tumefaciens-mediated transformation. In the transgenic plants, codA gene expression and its product transportation to chloroplasts were detected by the enzyme-linked immunosorbent assay (ELISA) examination, immunogold localization, and (1)H-nuclear magnetic resonance ((1)H-NMR). Stress tolerance was evaluated in the T(3) plants under extreme temperature and salinity conditions. The plants of transgenic line 1 (L1) showed significantly higher net photosynthetic rate (P(n)) and P(n) recovery rate under high (45 °C, 4 h) and low temperature (1 °C, 48 h) treatments, and higher photosynthetic rate under high salinity conditions (100, 200, and 300 mmol/L NaCl, respectively) than the wild-type plants. The enhanced tolerance to high temperature and high salinity stresses in transgenic plants is associated with the accumulation of betaine, which is not found in the wild-type plants. Our results indicate that the introduction of codA gene from Arthrobacter globiformis into Brassica compestris L. spp. chinensis could be a potential strategy for improving the plant tolerance to multiple stresses.

  20. Bex3 Dimerization Regulates NGF-Dependent Neuronal Survival and Differentiation by Enhancing trkA Gene Transcription.

    PubMed

    Calvo, Laura; Anta, Begoña; López-Benito, Saray; Martín-Rodriguez, Carlos; Lee, Francis S; Pérez, Pilar; Martín-Zanca, Dionisio; Arévalo, Juan C

    2015-05-06

    The development of the nervous system is a temporally and spatially coordinated process that relies on the proper regulation of the genes involved. Neurotrophins and their receptors are directly responsible for the survival and differentiation of sensory and sympathetic neurons; however, it is not fully understood how genes encoding Trk neurotrophin receptors are regulated. Here, we show that rat Bex3 protein specifically regulates TrkA expression by acting at the trkA gene promoter level. Bex3 dimerization and shuttling to the nucleus regulate the transcription of the trkA promoter under basal conditions and also enhance nerve growth factor (NGF)-mediated trkA promoter activation. Moreover, qChIP assays indicate that Bex3 associates with the trkA promoter within a 150 bp sequence, immediately upstream from the transcription start site, which is sufficient to mediate the effects of Bex3. Consequently, the downregulation of Bex3 using shRNA increases neuronal apoptosis in NGF-dependent sensory neurons deprived of NGF and compromises PC12 cell differentiation in response to NGF. Our results support an important role for Bex3 in the regulation of TrkA expression and in NGF-mediated functions through modulation of the trkA promoter.

  1. Transcription of a donor enhances its use during double-strand break-induced gene conversion in human cells.

    PubMed

    Schildkraut, Ezra; Miller, Cheryl A; Nickoloff, Jac A

    2006-04-01

    Homologous recombination (HR) mediates accurate repair of double-strand breaks (DSBs) but carries the risk of large-scale genetic change, including loss of heterozygosity, deletions, inversions, and translocations. Nearly one-third of the human genome consists of repetitive sequences, and DSB repair by HR often requires choices among several homologous repair templates, including homologous chromosomes, sister chromatids, and linked or unlinked repeats. Donor preference during DSB-induced gene conversion was analyzed by using several HR substrates with three copies of neo targeted to a human chromosome. Repair of I-SceI nuclease-induced DSBs in one neo (the recipient) required a choice between two donor neo genes. When both donors were downstream, there was no significant bias for proximal or distal donors. When donors flanked the recipient, we observed a marked (85%) preference for the downstream donor. Reversing the HR substrate in the chromosome eliminated this preference, indicating that donor choice is influenced by factors extrinsic to the HR substrate. Prior indirect evidence suggested that transcription might increase donor use. We tested this question directly and found that increased transcription of a donor enhances its use during gene conversion. A preference for transcribed donors would minimize the use of nontranscribed (i.e., pseudogene) templates during repair and thus help maintain genome stability.

  2. Overexpression of snakin-1 gene enhances resistance to Rhizoctonia solani and Erwinia carotovora in transgenic potato plants.

    PubMed

    Almasia, Natalia I; Bazzini, Ariel A; Hopp, H Esteban; Vazquez-Rovere, Cecilia

    2008-05-01

    Snakin-1 (SN1), a cysteine-rich peptide with broad-spectrum antimicrobial activity in vitro, was evaluated for its ability to confer resistance to pathogens in transgenic potatoes. Genetic variants of this gene were cloned from wild and cultivated Solanum species. Nucleotide sequences revealed highly evolutionary conservation with 91-98% identity values. Potato plants (S. tuberosum subsp. tuberosum cv. Kennebec) were transformed via Agrobacterium tumefaciens with a construct encoding the S. chacoense SN1 gene under the regulation of the ubiquitous CaMV 35S promoter. Transgenic lines were molecularly characterized and challenged with either Rhizoctonia solani or Erwinia carotovora to analyse whether constitutive in vivo overexpression of the SN1 gene may lead to disease resistance. Only transgenic lines that accumulated high levels of SN1 mRNA exhibited significant symptom reductions of R. solani infection such as stem cankers and damping-off. Furthermore, these overexpressing lines showed significantly higher survival rates throughout the fungal resistance bioassays. In addition, the same lines showed significant protection against E. carotovora measured as: a reduction of lesion areas (from 46.5 to 88.1% with respect to the wild-type), number of fallen leaves and thickened or necrotic stems. Enhanced resistance to these two important potato pathogens suggests in vivo antifungal and antibacterial activity of SN1 and thus its possible biotechnological application.

  3. Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains.

    PubMed

    Di Bernardo, Giovanni; Del Gaudio, Stefania; Cammarota, Marcella; Galderisi, Umberto; Cascino, Antonino; Cipollaro, Marilena

    2002-02-15

    Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (epsilon globin, p53 and gamma interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to alpha(lambda)mu(omicron)sigma(tau) 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed.

  4. Enhancer and promoter elements directing activation and glucocorticoid repression of the. cap alpha. /sub 1/-fetoprotein gene in hepatocytes

    SciTech Connect

    Guertin, M.; La Rue, H.; Bernier, D.; Wrange, O.; Chevrette, M.; Gingras, M.C.; Belanger, L.

    1988-04-01

    Mutations were introduced in 7 kilobases of 5'-flanking rat ..cap alpha../sub 1/-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.

  5. Regulation of natural killer enhancing factor (NKEF) genes in teleost fish, gilthead seabream and European sea bass.

    PubMed

    Esteban, María A; Chaves-Pozo, Elena; Arizcun, Marta; Meseguer, José; Cuesta, Alberto

    2013-10-01

    Peroxiredoxins (Prx) are a family of antioxidant proteins also involved in inflammation and innate immunity. Prx1 and Prx2 are also known as natural killer enhancing factor (NKEF)-A and NKEF-B, respectively, by their ability to prime the mammalian NK-cells activity. In teleost fish, NKEF genes have been isolated but their regulation has been scarcely evaluated. We have identified orthologues of the NKEF-A and NKEF-B genes in the teleost European sea bass (Dicentrarchus labrax) which showed constitutive expression and wide distribution in their tissues. In vitro, the gilthead seabream (Sparus aurata) and sea bass NKEFs were slightly up-regulated in head-kidney leucocytes after stimulation with unmethylated CpG oligodeoxynucleotides, poly I:C or pathogenic bacteria. In vivo, seabream and sea bass infection with nodavirus up-regulated the expression of NKEF genes in the brain (target tissue for nodavirus) and head-kidney at different infection times. Although further studies are necessary to ascertain their role as antioxidant proteins and in the immune response in teleost fish, our results suggest a primary role of seabream and sea bass NKEFs in the innate immune response against bacterial and viral agents.

  6. Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing.

    PubMed

    Jiang, Wen Zhi; Henry, Isabelle M; Lynagh, Peter G; Comai, Luca; Cahoon, Edgar B; Weeks, Donald P

    2016-11-11

    The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. thaliana T2 and T3 generation seeds exhibiting these types of altered fatty acid profiles were homozygous for disrupted FAD2 alleles. In the allohexaploid, Camelina, guide RNAs were designed that simultaneously targeted all three homoeologous FAD2 genes. This strategy that significantly enhanced oil composition in T3 and T4 generation Camelina seeds was associated with a combination of germ-line mutations and somatic cell mutations in FAD2 genes in each of the three Camelina subgenomes.

  7. Expression of Rice Chitinase Gene in Genetically Engineered Tomato Confers Enhanced Resistance to Fusarium Wilt and Early Blight

    PubMed Central

    Jabeen, Nyla; Chaudhary, Zubeda; Gulfraz, Muhammad; Rashid, Hamid; Mirza, Bushra

    2015-01-01

    This is the first study reporting the evaluation of transgenic lines of tomato harboring rice chitinase (RCG3) gene for resistance to two important fungal pathogens Fusarium oxysporum f. sp. lycopersici (Fol) causing fusarium wilt and Alternaria solani causing early blight (EB). In this study, three transgenic lines TL1, TL2 and TL3 of tomato Solanum lycopersicum Mill. cv. Riogrande genetically engineered with rice chitinase (RCG 3) gene and their R1 progeny was tested for resistance to Fol by root dip method and A. solani by detached leaf assay. All the R0 transgenic lines were highly resistant to these fungal pathogens compared to non-transgenic control plants. The pattern of segregation of three independent transformant for Fol and A. solani was also studied. Mendelian segregation was observed in transgenic lines 2 and 3 while it was not observed in transgenic line 1. It was concluded that introduction of chitinase gene in susceptible cultivar of tomato not only enhanced the resistance but was stably inherited in transgenic lines 2 and 3. PMID:26361473

  8. Expression of auxin synthesis gene tms1 under control of tuber-specific promoter enhances potato tuberization in vitro.

    PubMed

    Kolachevskaya, Oksana O; Alekseeva, Valeriya V; Sergeeva, Lidiya I; Rukavtsova, Elena B; Getman, Irina A; Vreugdenhil, Dick; Buryanov, Yaroslav I; Romanov, Georgy A

    2015-09-01

    Phytohormones, auxins in particular, play an important role in plant development and productivity. Earlier data showed positive impact of exogenous auxin on potato (Solanum tuberosum L.) tuberization. The aim of this study was to generate potato plants with increased auxin level predominantly in tubers. To this end, a pBinB33-tms1 vector was constructed harboring the Agrobacterium auxin biosynthesis gene tms1 fused to tuber-specific promoter of the class I patatin gene (B33-promoter) of potato. Among numerous independently generated B33:tms1 lines, those without visible differences from control were selected for detailed studies. In the majority of transgenic lines, tms1 gene transcription was detected, mostly in tubers rather than in shoots. Indoleacetic acid (IAA) content in tubers and the auxin tuber-to-shoot ratio were increased in tms1-expressing transformants. The organ-specific increase in auxin synthesis in B33:tms1-transformants accelerated and intensified the process of tuber formation, reduced the dose of carbohydrate supply required for in vitro tuberization, and decreased the photoperiodic dependence of tuber initiation. Overall, a positive correlation was observed between tms1 expression, IAA content in tubers, and stimulation of tuber formation. The revealed properties of B33:tms1 transformants imply an important role for auxin in potato tuberization and offer prospects to magnify potato productivity by a moderate organ-specific enhancement of auxin content.

  9. Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene.

    PubMed

    Buch, Aditi D; Archana, G; Kumar, G Naresh

    2009-08-01

    Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coli citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant approximately 2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2- and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coli gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3- and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525

  10. A Novel Gene, OZONE-RESPONSIVE APOPLASTIC PROTEIN1, Enhances Cell Death in Ozone Stress in Rice1

    PubMed Central

    Ueda, Yoshiaki; Siddique, Shahid; Frei, Michael

    2015-01-01

    A novel protein, OZONE-RESPONSIVE APOPLASTIC PROTEIN1 (OsORAP1), was characterized, which was previously suggested as a candidate gene underlying OzT9, a quantitative trait locus for ozone stress tolerance in rice (Oryza sativa). The sequence of OsORAP1 was similar to that of ASCORBATE OXIDASE (AO) proteins. It was localized in the apoplast, as shown by transient expression of an OsORAP1/green fluorescent protein fusion construct in Nicotiana benthamiana leaf epidermal and mesophyll cells, but did not possess AO activity, as shown by heterologous expression of OsORAP1 in Arabidopsis (Arabidopsis thaliana) mutants with reduced background AO activity. A knockout rice line of OsORAP1 showed enhanced tolerance to ozone stress (120 nL L−1 average daytime concentration, 20 d), as demonstrated by less formation of leaf visible symptoms (i.e. cell death), less lipid peroxidation, and lower NADPH oxidase activity, indicating reduced active production of reactive oxygen species. In contrast, the effect of ozone on chlorophyll content was not significantly different among the lines. These observations suggested that OsORAP1 specifically induced cell death in ozone stress. Significantly enhanced expression of jasmonic acid-responsive genes in the knockout line implied the involvement of the jasmonic acid pathway in symptom mitigation. Sequence analysis revealed extensive polymorphisms in the promoter region of OsORAP1 between the ozone-susceptible cv Nipponbare and the ozone-tolerant cv Kasalath, the OzT9 donor variety, which could be responsible for the differential regulation of OsORAP1 reported earlier. These pieces of evidence suggested that OsORAP1 enhanced cell death in ozone stress, and its expression levels could explain the effect of a previously reported quantitative trait locus. PMID:26220952

  11. Adenoviral mediated gene transfer of PDGF-B enhances wound healing in type I and type II diabetic wounds.

    PubMed

    Keswani, Sundeep G; Katz, Anna B; Lim, Foong-Yen; Zoltick, Philip; Radu, Antoneta; Alaee, Datis; Herlyn, Meenhard; Crombleholme, Timothy M

    2004-01-01

    We have shown that the genetically diabetic mouse (C57BLKS/J-m+/+Lepr(db)) has a wound healing and neovascularization deficit associated with an inability to recruit endothelial precursor cells (EPCs) to the wound. This may account for a fundamental mechanism in impaired diabetic wound healing. We hypothesized that the adenoviral mediated overexpression of platelet-derived growth factor-B (PDGF-B) would enhance wound healing, improve neovascularization, and recruit EPCs to the epithelial wound in three diabetic mouse models. Eight-mm full-thickness flank wounds were made in db/db, nonobese NOD/Ltj, streptozotocin, and C57BLKS/J mice. Wounds were treated with either 1 x 10(8) PFU Ad-PDGF-B or Ad LacZ or phosphate buffered saline solution. Wounds harvested at seven days were analyzed for epithelial gap, blood vessel density, granulation tissue area, and EPCs per high powered field. All three diabetic models have a significant wound healing and neovascularization defect compared to C57BLKS/J controls. Adenoviral-PDGF-B treatment significantly enhanced epithelial gap closure in db/db, streptozotocin, and nonobese NOD/Ltj mice as compared to diabetic phosphate buffered saline solution or Ad LacZ controls. A similar increase in the formation of granulation tissue and vessel density was also observed. All three models had reduced levels of GATA-2 positive EPCs in the wound bed that was corrected by the adenoviral mediated gene transfer of PDGF. EPC recruitment was positively correlated with neovascularization and wound healing. Three different diabetic models have a wound healing impairment and a decreased ability to recruit EPCs. The vulnerary effect of adenoviral mediated gene therapy with PDGF-B significantly enhanced wound healing and neovascularization in diabetic wounds. The PDGF-B mediated augmentation of EPC recruitment to the wound bed may be a fundamental mechanism of these results.

  12. A bacteriocin gene cluster able to enhance plasmid maintenance in Lactococcus lactis

    PubMed Central

    2014-01-01

    Background Lactococcus lactis is widely used as a dairy starter and has been extensively studied. Based on the acquired knowledge on its physiology and metabolism, new applications have been envisaged and there is an increasing interest of using L. lactis as a cell factory. Plasmids constitute the main toolbox for L. lactis genetic engineering and most rely on antibiotic resistant markers for plasmid selection and maintenance. In this work, we have assessed the ability of the bacteriocin Lactococcin 972 (Lcn972) gene cluster to behave as a food-grade post-segregational killing system to stabilize recombinant plasmids in L. lactis in the absence of antibiotics. Lcn972 is a non-lantibiotic bacteriocin encoded by the 11-kbp plasmid pBL1 with a potent antimicrobial activity against Lactococcus. Results Attempts to clone the full lcn972 operon with its own promoter (P972), the structural gene lcn972 and the immunity genes orf2-orf3 in the unstable plasmid pIL252 failed and only plasmids with a mutated promoter were recovered. Alternatively, cloning under other constitutive promoters was approached and achieved, but bacteriocin production levels were lower than those provided by pBL1. Segregational stability studies revealed that the recombinant plasmids that yielded high bacteriocin titers were maintained for at least 200 generations without antibiotic selection. In the case of expression vectors such as pTRL1, the Lcn972 gene cluster also contributed to plasmid maintenance without compromising the production of the fluorescent mCherry protein. Furthermore, unstable Lcn972 recombinant plasmids became integrated into the chromosome through the activity of insertion sequences, supporting the notion that Lcn972 does apply a strong selective pressure against susceptible cells. Despite of it, the Lcn972 gene cluster was not enough to avoid the use of antibiotics to select plasmid-bearing cells right after transformation. Conclusions Inserting the Lcn972 cluster into

  13. Analysis of Dengue Virus Enhancing Epitopes Using Peptide Antigens Derived From the Envelope Glycoprotein Gene Sequence.

    DTIC Science & Technology

    1991-11-29

    AD-A261 707 AD____ ARMY PROJECT ORDER NO: 89PP9961 TITLE: ANALYSIS OF DENGUE VIRUS ENHANCING EPITOPES USING PEPTIDE ANTIGENS DERIVED FROM THE...DATES COVERED 29 Nov 91 Final Report (9/1/89 - 11/30/91) 4. TITLE AND SUBTITLE Ana ysis or Dengue Vnrus nancing 5. FUNDING NUMBERS Epitopes Using...biological events leading to the development of severe disease manifestations of dengue infections ( dengue hemorrhagic fever/ dengue shock syndrome

  14. Safety of Pulsed High Intensity Focused Ultrasound for Enhanced Drug and Gene Delivery

    NASA Astrophysics Data System (ADS)

    Kam, Anthony W.; Wang, Honghui; Farahani, Keyvan; Thomasson, David; O'Neill, Brian; Angstadt, Mary; Jesson, Johnny; Li, King C. P.

    2007-05-01

    For a limited range of exposure parameters, pulsed high intensity focused ultrasound (HIFU) has been shown to increase the delivery of certain systemically administered macromolecular diagnostic and therapeutic agents in mice. The mechanism for the enhanced delivery has not been demonstrated definitively and, in principle, can include thermal, cavitational, and non-cavitation mechanical effects. The sonicated tissue has no damage on histology. As a step towards clinical translation, the safety of this technique needs to be assessed in a clinically relevant manner. In this study, the safety of pulsed HIFU is evaluated with near real-time phase shift magnetic resonance (MR) thermometry and anatomic MR imaging using rabbits as subjects. MR guidance enables pulsed HIFU enhanced delivery to be implemented safely from a thermal standpoint. Although the effects of pulsed HIFU are not seen on anatomic MR images, they may be detected on MR sequences sensitive to permeability, diffusion, and elasticity. Such work that may optimize pulsed HIFU enhanced delivery is in progress.

  15. An initiation site of DNA replication with transcriptional enhancer activity present upstream of the c-myc gene.

    PubMed Central

    Iguchi-Ariga, S M; Okazaki, T; Itani, T; Ogata, M; Sato, Y; Ariga, H

    1988-01-01

    We have previously reported that c-myc protein may promote cellular DNA replication by binding to initiation sites of replication. Here we report that a putative origin of human cellular DNA replication (ori) is present at approximately 2 kb upstream of the coding region of the c-myc gene itself. The c-myc protein, or protein(s) complexed with c-myc protein, bind to the upstream region (approximately 200 bp in length) which has transcriptional enhancer activity as well as autonomously replicating activity in human cells, suggesting that the c-myc protein may be an enhancer binding protein as well as a DNA replication protein. Results with deletion mutants suggest that the sequence essential to the origin of DNA replication may be adjacent to, but cannot be clearly separated from, the sequence responsible for enhancer activity. Furthermore, when cloned DNA containing putative c-myc protein binding sequences was transfected as competitor into HL-60 cells, expression of c-myc was inhibited, suggesting that c-myc protein itself may be necessary for c-myc expression. Images PMID:3053161

  16. Photochemical internalization (PCI) enhanced nonviral transfection of tumor suppressor and pro-drug activating genes; a potential treatment modality for gliomas

    NASA Astrophysics Data System (ADS)

    Wang, Frederick; Zamora, Genesis; Sun, Chung-Ho; Trinidad, Anthony; Berg, Kristian; Madsen, Steen; Kwon, Young Jik; Hirschberg, Henry

    2014-03-01

    The overall objective of the research is to investigate the utility of photochemical internalization for the enhanced nonviral transfection of genes into cells. We have examined, in detail, the evaluation of photochemical internalization (PCI) as a method for the non-viral introduction of the tumor suppressor gene PTEN and the PCI mediated transfection of the cytosine deaminase (CD) pro drug activating gene into glioma cell monolayers and multi-cell tumor spheroids. Expression of the CD gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-fluorocytosine (5-FC), to the toxic metabolite, 5-fluorouracil (5-FU).

  17. Enhanced gene expression of systemically administered plasmid DNA in the liver with therapeutic ultrasound and microbubbles.

    PubMed

    Raju, Balasundar I; Leyvi, Evgeniy; Seip, Ralf; Sethuraman, Shriram; Luo, Xiaoyan; Bird, Andrew; Li, Songtao; Koeberl, Dwight

    2013-01-01

    Ultrasound-mediated delivery (USMD) of novel therapeutic agents in the presence of microbubbles is a potentially safe and effective method for gene therapy offering many desired characteristics, such as low toxicity, potential for repeated treatment, and organ specificity. In this study, we tested the capability of USMD to improve gene expression in mice livers using glycogen storage disease Type Ia as a model disease under systemic administration of naked plasmid DNA. Image-guided therapeutic ultrasound was used in two studies to provide therapeutic ultrasound to mice livers. In the first study, involving wild-type mice, control animals received naked plasmid DNA (pG6Pase 150 μg) via the tail vein, followed by an infusion of microbubbles; the treated animals additionally received therapeutic ultrasound (1 MHz). Following the procedure, the animals were left to recover and were subsequently euthanized after 2 d and liver samples were extracted. Reverse transcription polymerase chain reaction (RT-PCR) assays were performed on the samples to quantify mRNA expression. In addition, Western blot assays of FLAG-tagged glucose-6-phosphatase (G6Pase) were performed to evaluate protein expression. Ultrasound-exposed animals showed a 4-fold increase in G6Pase RNA in the liver, in comparison with control animals. Furthermore, results from Western blot analysis demonstrated a 2-fold increased protein expression in ultrasound-exposed animals after two days ( p < 0.05). A second pilot study was performed with G6Pase knockout mice, and the animals were monitored for correction of hypoglycemia over a period of 3 weeks before tissue analysis. The RT-PCR assays of samples from these animals demonstrated increased G6Pase RNA in the liver following ultrasound treatment. These results demonstrate that USMD can increase gene expression of systemically injected naked pDNA in the liver and also provide insight into the development of realistic approaches that can be translated into

  18. Gene therapy with IL-12 induced enhanced anti-tumor activity in fibrosarcoma mouse model.

    PubMed

    Razi Soofiyani, Saiedeh; Kazemi, Tohid; Lotfipour, Farzaneh; Mohammad Hosseini, Akbar; Shanehbandi, Dariush; Hallaj-Nezhadi, Somayeh; Baradaran, Behzad

    2016-12-01

    Context Immunotherapy is among the most promising modalities for treatment of cancer. Recently, interleukin 12 (IL-12) has been used as an immunotherapeutic agent in cancer gene therapy. IL-12 can activate dendritic cells (DCs) and boost anti-tumor immune responses. Objective In the current study, we have investigated if IL-12 gene therapy can lead to the regression of tumor mass in a mouse model of fibrosarcoma. Material and methods To investigate the therapeutic efficacy of IL-12, WEHI-164 tumor cells were transfected with murine-IL12 plasmids using Lipofectamine. Enzyme linked immunosorbent assay (ELISA) was used to confirm IL-12 expression in transfected cells. The fibrosarcoma mouse model was established by subcutaneous injection of transfected cells to Balb/C mice. Mice were sacrificed and the tumors were extracted. Tumor sizes were measured by caliper. The expression of IL-12 and IFN-γ was studied with real-time PCR and western blotting. The expression of Ki-67(a tumor proliferation marker) in tumor mass was studied by immunohistochemistry staining. Results and discussion The group treated with IL-12 showed a significant decrease in tumor mass volume (P: 0.000). The results of real-time PCR and western blotting showed that IL-12 and IFN-γ expression increased in the group treated with IL-12 (relative expression of IL-12: 1.9 and relative expression of IFN-γ: 1.766). Immunohistochemistry staining showed that Ki-67 expression was reduced in the group treated with IL-12. Conclusion IL-12 gene therapy successfully led to regress of tumor mass in the fibrosarcoma mouse model. This may serve as a candidate therapeutic approach for treatment of cancer.

  19. Low plant density enhances gene dispersal in the Amazonian understory herb Heliconia acuminata.

    PubMed

    Côrtes, Marina C; Uriarte, María; Lemes, Maristerra R; Gribel, Rogério; Kress, W John; Smouse, Peter E; Bruna, Emilio M

    2013-11-01

    In theory, conservation genetics predicts that forest fragmentation will reduce gene dispersal, but in practice, genetic and ecological processes are also dependent on other population characteristics. We used Bayesian genetic analyses to characterize parentage and propagule dispersal in Heliconia acuminata L. C. Richard (Heliconiaceae), a common Amazonian understory plant that is pollinated and dispersed by birds. We studied these processes in two continuous forest sites and three 1-ha fragments in Brazil's Biological Dynamics of Forest Fragments Project. These sites showed variation in the density of H. acuminata. Ten microsatellite markers were used to genotype flowering adults and seedling recruits and to quantify realized pollen and seed dispersal distances, immigration of propagules from outside populations, and reproductive dominance among parents. We tested whether gene dispersal is more dependent on fragmentation or density of reproductive plants. Low plant densities were associated with elevated immigration rates and greater propagule dispersal distances. Reproductive dominance among inside-plot parents was higher for low-density than for high-density populations. Elevated local flower and fruit availability is probably leading to spatially more proximal bird foraging and propagule dispersal in areas with high density of reproductive plants. Nevertheless, genetic diversity, inbreeding coefficients and fine-scale spatial genetic structure were similar across populations, despite differences in gene dispersal. This result may indicate that the opposing processes of longer dispersal events in low-density populations vs. higher diversity of contributing parents in high-density populations balance the resulting genetic outcomes and prevent genetic erosion in small populations and fragments.

  20. Overexpression of stress-related genes enhances cell viability and velum formation in Sherry wine yeasts.

    PubMed

    Fierro-Risco, Jesús; Rincón, Ana María; Benítez, Tahía; Codón, Antonio C

    2013-08-01

    Flor formation and flor endurance have been related to ability by Saccharomyces cerevisiae flor yeasts to resist hostile conditions such as oxidative stress and the presence of acetaldehyde and ethanol. Ethanol and acetaldehyde toxicity give rise to formation of reactive oxygen species (ROS) and loss of cell viability. Superoxide dismutases Sod1p and Sod2p and other proteins such as Hsp12p are involved in oxidative stress tolerance. In this study, genes SOD1, SOD2, and HSP12 were overexpressed in flor yeast strains FJF206, FJF414 and B16. In the SOD1 and SOD2 transformant strains superoxide dismutases encoded by genes SOD1 and SOD2 increased their specific activity considerably as a direct result of overexpression of genes SOD1 and SOD2, indirectly, catalase, glutathione reductase, and glutathione peroxidase activities increased too. The HSP12 transformant strains showed higher levels of glutathione peroxidase and reductase activities. These transformant strains showed an increase in intracellular glutathione content, a reduction in peroxidized lipid concentration, and higher resistance to oxidative stress conditions. As a result, flor formation by these strains took place more rapidly than by their parental strains, velum being thicker and with higher percentages of viable cells. In addition, a slight decrease in ethanol and glycerol concentrations, and an increase in acetaldehyde were detected in wines matured under velum formed by transformant strains, as compared to their parental strains. In the industry, velum formed by transformant strains with increased viability may result in acceleration of both metabolism and wine aging, thus reducing time needed for wine maturation.

  1. Nanogyroids Incorporating Multivalent Lipids: Enhanced Membrane Charge Density and Pore Forming Ability for Gene Silencing

    PubMed Central

    Leal, Cecília; Ewert, Kai K.; Shirazi, Rahau S.; Bouxsein, Nathan F.; Safinya, Cyrus R.

    2011-01-01

    The self-assembly of a custom-synthesized pentavalent cationic lipid (MVL5) and glycerol monooleate (GMO) with small interfering RNA (siRNA) results in the formation of a double-gyroid bicontinuous inverted cubic phase with co-localized lipid/siRNA domains as shown by synchrotron X-ray scattering and fluorescence microscopy. The high charge density (due to MVL5) and positive Gaussian modulus of the GMO-containing membranes confer optimal electrostatic and elastic properties for endosomal escape, enabling efficient siRNA delivery and effective, specific gene silencing. PMID:21612245

  2. A Soybean C2H2-Type Zinc Finger Gene GmZF1 Enhanced Cold Tolerance in Transgenic Arabidopsis

    PubMed Central

    Ma, Xue-Feng; Xu, Zhao-Shi; Liu, Meng-Meng; Shan, Shu-Guang; Cheng, Xian-Guo

    2014-01-01

    Zinc finger proteins were involved in response to different environmental stresses in plant species. A typical Cys2/His2-type (C2H2-type) zinc finger gene GmZF1 from soybean was isolated and was composed of 172 amino acids containing two conserved C2H2-type zinc finger domains. Phylogenetic analysis showed that GmZF1 was clustered on the same branch with six C2H2-type ZFPs from dicotyledonous plants excepting for GsZFP1, and distinguished those from monocotyledon species. The GmZF1 protein was localized at the nucleus, and has specific binding activity with EP1S core sequence, and nucleotide mutation in the core sequence of EPSPS promoter changed the binding ability between GmZF1 protein and core DNA element, implying that two amino acid residues, G and C boxed in core sequence TGACAGTGTCA possibly play positive regulation role in recognizing DNA-binding sites in GmZF1 proteins. High accumulation of GmZF1 mRNA induced by exogenous ABA suggested that GmZF1 was involved in an ABA-dependent signal transduction pathway. Over-expression of GmZF1 significantly improved the contents of proline and soluble sugar and decreased the MDA contents in the transgenic lines exposed to cold stress, indicating that transgenic Arabidopsis carrying GmZF1 gene have adaptive mechanisms to cold stress. Over-expression of GmZF1 also increased the expression of cold-regulated cor6.6 gene by probably recognizing protein-DNA binding sites, suggesting that GmZF1 from soybean could enhance the tolerance of Arabidopsis to cold stress by regulating expression of cold-regulation gene in the transgenic Arabidopsis. PMID:25286048

  3. Inducible and constitutive expression of an elicitor gene Hrip1 from Alternaria tenuissima enhances stress tolerance in Arabidopsis.

    PubMed

    Peng, Xue-Cong; Qiu, De-Wen; Zeng, Hong-Mei; Guo, Li-Hua; Yang, Xiu-Fen; Liu, Zheng

    2015-02-01

    Hrip1 is a novel hypersensitive response-inducing protein secreted by Alternaria tenuissima that activates defense responses and systemic acquired resistance in tobacco. This study investigates the role that Hrip1 plays in responses to abiotic and biotic stress using transgenic Arabidopsis thaliana expressing the Hrip1 gene under the control of the stress-inducible rd29A promoter or constitutive cauliflower mosaic virus 35S promoter. Bioassays showed that inducible Hrip1 expression in rd29A∷Hrip1 transgenic lines had a significantly higher effect on plant height, silique length, plant dry weight, seed germination and root length under salt and drought stress compared to expression in 35S∷Hrip1 lines and wild type plants. The level of enhancement of resistance to Botrytis cinerea by the 35S∷Hrip1 lines was higher than in the rd29A∷Hrip1 lines. Moreover, stress-related gene expression in the transgenic Arabidopsis lines was significantly increased by 200 mM NaCl and 200 mM mannitol treatments, and defense genes in the jasmonic acid and ethylene signaling pathway were significantly up-regulated after Botrytis inoculation in the Hrip1 transgenic plants. Furthermore, the activity of some antioxidant enzymes, such as peroxidase and catalase increased after salt and drought stress and Botrytis infection. These results suggested that the Hrip1 protein contributes to abiotic and biotic resistance in transgenic Arabidopsis and may be used as a useful gene for resistance breeding in crops. Although the constitutive expression of Hrip1 is suitable for biotic resistance, inducible Hrip1 expression is more responsive for abiotic resistance.

  4. Involvement of elevated proline accumulation in enhanced osmotic stress tolerance in Arabidopsis conferred by chimeric repressor gene silencing technology.

    PubMed

    Kazama, Daisuke; Kurusu, Takamitsu; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Tada, Yuichi

    2014-01-01

    Arabidopsis plants transformed with a chimeric repressor for 6 transcription factors (TFs), including ADA2b, Msantd, DDF1, DREB26, AtGeBP, and ATHB23, that were converted by Chimeric REpressor gene Silencing Technology (CRES-T), show elevated salt and osmotic stress tolerance compared with wild type (WT) plants. However, the roles of TFs in salt and osmotic signaling remain largely unknown. Their hyper-osmotic stress tolerance was evaluated using 3 criteria: germination rate, root length, and rate of seedlings with visible cotyledons at the germination stage. All CRES-T lines tested exhibited better performance than WT, at least for one criterion under stress conditions. Under 600 mM mannitol stress, 3-week-old CRES-T lines accumulated proline, which is a major compatible solute involved in osmoregulation, at higher levels than WT. Expression levels of the delta 1-pyrroline-5-carboxylate synthase gene in CRES-T lines were similar to or lower than those in WT. In contrast, expression of the proline dehydrogenase (PHD) gene in DREB26-SRDX was significantly downregulated and that in ADA2b-SRDX and AtGeBP-SRDX was also rather downregulated compared with that in WT. Although plants at different stages were used for stress tolerance test and proline measurement in this study, we previously reported that 4 out of the 6 CRES-T lines showed better growth than WT after 4 weeks of incubation under 400 mM mannitol. These results suggest that proline accumulation caused by PHD gene suppression may be involved in enhanced osmotic stress tolerance in the CRES-T lines, and that these TFs may be involved in regulating proline metabolism in Arabidopsis.

  5. Salmonella plasmid virulence gene spvB enhances bacterial virulence by inhibiting autophagy in a zebrafish infection model.

    PubMed

    Li, Yuan-Yuan; Wang, Ting; Gao, Song; Xu, Guang-Mei; Niu, Hua; Huang, Rui; Wu, Shu-Yan

    2016-02-01

    Salmonella enterica serovar typhimurium (S. typhimurium) is a facultative intracellular pathogen that can cause gastroenteritis and systemic infection in a wide range of hosts. Salmonella plasmid virulence gene spvB is closely related to bacterial virulence in different cells and animal models, and the encoded protein acts as an intracellular toxin required for ADP-ribosyl transferase activity. However, until now there is no report about the pathogenecity of spvB gene on zebrafish. Due to the outstanding advantages of zebrafish in analyzing bacteria-host interactions, a S. typhimurium infected zebrafish model was set up here to study the effect of spvB on autophagy and intestinal pathogenesis in vivo. We found that spvB gene could decrease the LD50 of S. typhimurium, and the strain carrying spvB promoted bacterial proliferation and aggravated the intestinal damage manifested by the narrowed intestines, fallen microvilli, blurred epithelium cell structure and infiltration of inflammatory cells. Results demonstrated the enhanced virulence induced by spvB in zebrafish. In spvB-mutant strain infected zebrafish, the levels of Lc3 turnover and Beclin1 expression increased, and the double-membraned autophagosome structures were observed, suggesting that spvB can inhibit autophagy activity. In summary, our results indicate that S. typhimurium strain containing spvB displays more virulence, triggering an increase in bacterial survival and intestine injuries by suppressing autophagy for the first time. This model provides novel insights into the role of Salmonella plasmid virulence gene in bacterial pathogenesis, and can help to further elucidate the relationship between bacteria and host immune response.

  6. Cytokine-enhanced vaccine and suicide gene therapy as surgery adjuvant treatments for spontaneous canine melanoma.

    PubMed

    Finocchiaro, L M E; Glikin, G C

    2008-02-01

    We evaluated the safety, efficacy and anti-tumor effects of a surgery adjuvant treatment on canine patients with malignant melanoma. This approach combined suicide gene therapy with a subcutaneous vaccine composed by formolized tumor cells and irradiated xenogeneic cells producing human interleukin-2 and granulocyte-macrophage colony-stimulating factor. The post-surgical margin of the cavity was infiltrated with lipid-complexed thymidine kinase suicide gene coadministrated with ganciclovir. Toxicity was minimal or absent in all patients. With respect to surgery-treated controls (SC), this combined treatment (CT) significantly increased the fraction of patients local disease-free from 6 to 58% and distant metastases-free from 43 to 78% (Fisher's Exact test). In addition, CT significantly improved both SC overall 78 (23-540) and metastasis-free survival 112 (0-467) days to more than 1312 days (respective ranges: 43-1312 and 0-1312) (Kaplan-Meier analysis). In those patients subjected to partial surgery or presenting local recurrence, the efficacy of CT was verified by a 49% of objective responses that averaged 85% of tumor mass loss, while 22% displayed tumor progression as 94% of SC did. Therefore, surgery adjuvant CT controlled tumor growth, delaying or preventing post-surgical recurrence and distant metastasis, significantly extending survival and recovering the quality of life.

  7. Chemokine-enhanced chemotaxis of lymphangioleiomyomatosis cells with mutations in the tumor suppressor TSC2 gene.

    PubMed

    Pacheco-Rodriguez, Gustavo; Kumaki, Fumiyuki; Steagall, Wendy K; Zhang, Yi; Ikeda, Yoshihiko; Lin, Jing-Ping; Billings, Eric M; Moss, Joel

    2009-02-01

    Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction caused by LAM cells (smooth-muscle-like cells) that have mutations in the tumor suppressor genes tuberous sclerosis complex (TSC) 1 or 2 and have the capacity to metastasize. Since chemokines and their receptors function in chemotaxis of metastatic cells, we hypothesized that LAM cells may be recruited by chemokine(s) in the lung. Quantification of 25 chemokines in bronchoalveolar lavage fluid from LAM patients and healthy volunteers revealed that concentrations of CCL2, CXCL1, and CXCL5 were significantly higher in samples from LAM patients than those from healthy volunteers. In vitro, CCL2 or MCP-1 induced selective migration of cells, showing loss of heterozygosity of TSC2 from a heterogeneous population of cells grown from explanted LAM lungs. Additionally, the frequencies of single-nucleotide polymorphisms in the CCL2 gene promoter region differed significantly in LAM patients and healthy volunteers (p = 0.018), and one polymorphism was associated significantly more frequently with the decline of lung function. The presence (i.e., potential functionality) of chemokine receptors was evaluated using immunohistochemistry in lung sections from 30 LAM patients. Expression of chemokines and these receptors varied among LAM patients and differed from that seen in some cancers (e.g., breast cancer and melanoma cells). These observations are consistent with the notion that chemokines such as CCL2 may serve to determine mobility and specify the site of metastasis of the LAM cell.

  8. Sugar beet M14 glyoxalase I gene can enhance plant tolerance to abiotic stresses.

    PubMed

    Wu, Chuan; Ma, Chunquan; Pan, Yu; Gong, Shilong; Zhao, Chenxi; Chen, Sixue; Li, Haiying

    2013-05-01

    Glyoxalase I is the first enzyme of the glyoxalase system that can detoxify methylglyoxal, a cytotoxic compound increased rapidly under stress conditions. Here we report cloning and characterization of a glyoxalase I from sugar beet M14 line (an interspecific hybrid between a wild species Beta corolliflora Zoss and a cultivated species B. vulgaris L). The full-length gene BvM14-glyoxalase I has 1,449 bp in length with an open reading frame of 1,065 bp encoding 354 amino acids. Sequence analysis shows the conserved glyoxalase I domains, metal and glutathione binding sites and secondary structure (α-helixes and β-sheets). The BvM14-glyoxalase I gene was ubiquitously expressed in different tissues of sugar beet M14 line and up-regulated in response to salt, mannitol and oxidative stresses. Heterologous expression of BvM14-glyoxalase I could increase E. coli tolerance to methylglyoxal. Transgenic tobacco plants constitutively expressing BvM14-glyoxalase I were generated. Both leaf discs and seedlings showed significant tolerance to methylglyoxal, salt, mannitol and H2O2. These results suggest an important role of BvM14-glyoxalase I in cellular detoxification and tolerance to abiotic stresses.

  9. Polyethylene glycol-grafted polyethylenimine used to enhance adenovirus gene delivery.

    PubMed

    Singarapu, Kumar; Pal, Ivy; Ramsey, Joshua D

    2013-07-01

    An improved adenoviral-based gene delivery vector was developed by complexing adenovirus (Ad) with a biocompatible, grafted copolymer PEG-g-PEI composed of polyethylene glycol (PEG) and polyethylenimine (PEI). Although an Ad-based gene vector is considered relatively safe, its native tropism, tendency to elicit an immune response, and susceptibility to inactivating antibodies makes the virus less than ideal. The goal of the current study was to determine whether Ad could be complexed with a PEG-g-PEI copolymer that would enable the virus to transduce cells lacking the Ad receptor, while avoiding the issues commonly associated with PEI. A copolymer library was synthesized using 2 kDa PEG and either linear or branched PEI (25 kDa) with a PEG to PEI grafting ratio of 10, 20, or 30. The results of the study indicate that PEG-g-PEI/Ad complexes are indeed able to transduce CAR-negative NIH 3T3 cells. The results also demonstrate that the PEG-g-PEI/Ad complexes are less toxic, less hemolytic, and more appropriately sized than PEI/Ad complexes.

  10. PPARα gene variants as predicted performance-enhancing polymorphisms in professional Italian soccer players

    PubMed Central

    Proia, Patrizia; Bianco, Antonino; Schiera, Gabriella; Saladino, Patrizia; Contrò, Valentina; Caramazza, Giovanni; Traina, Marcello; Grimaldi, Keith A; Palma, Antonio; Paoli, Antonio

    2014-01-01

    Background The PPARα gene encodes the peroxisome proliferator-activator receptor alpha, a central regulator of expression of other genes involved in fatty acid metabolism. The purpose of this study was to determine the prevalence of G allele of the PPARα intron 7 G/C polymorphism (rs4253778) in professional Italian soccer players. Methods Sixty professional soccer players and 30 sedentary volunteers were enrolled in the study. Samples of venous blood were obtained at rest, in the morning, by conventional clinical procedures; blood serum was collected and total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides were measured. An aliquot of anticoagulant-treated blood was used to prepare genomic DNA from whole blood. The G/C polymorphic site in PPARα intron 7 was scanned by using the PCR-RFLP (polymerase chain reaction restriction fragment length polymorphism) protocol with TaqI enzyme. Results We found variations in genotype distribution of PPARα polymorphism between professional soccer players and sedentary volunteers. Particularly, G alleles and the GG genotype were significantly more frequent in soccer players compared with healthy controls (64% versus 48%). No significant correlations were found between lipid profile and genotype background. Conclusion Previous results demonstrated an association of intron 7 G allele as well as the GG genotype in endurance athletes. Our result suggests that this is the case also in professional soccer players. PMID:25525399

  11. Enhancing VSV oncolytic activity with an improved cytosine deaminase suicide gene strategy.

    PubMed

    Leveille, S; Samuel, S; Goulet, M-L; Hiscott, J

    2011-06-01

    Oncolytic viruses (OVs) are promising therapeutic agents for cancer treatment, with recent studies emphasizing the combined use of chemotherapeutic compounds and prodrug suicide gene strategies to improve OV efficacy. In the present study, the synergistic activity of recombinant vesicular stomatitis virus (VSV)-MΔ51 virus expressing the cytosine deaminase/uracil phosphoribosyltransferase (CD::UPRT) suicide gene and 5-fluorocytosine (5FC) prodrug was investigated in triggering tumor cell oncolysis. In a panel of VSV-sensitive and -resistant cells-prostate PC3, breast MCF7 and TSA, B-lymphoma Karpas and melanoma B16-F10-the combination treatment increased killing of non-infected bystander cells in vitro via the release of 5FC toxic derivatives. In addition, we showed a synergistic effect on cancer cell killing with VSV-MΔ51 and the active form of the drug 5-fluorouracil. Furthermore, by monitoring VSV replication at the tumor site and maximizing 5FC bioavailability, we optimized the treatment regimen and improved survival of animals bearing TSA mammary adenocarcinoma. Altogether, this study emphasizes the potency of the VSV-CD::UPRT and 5FC combination, and demonstrates the necessity of optimizing each step of a multicomponent therapy to design efficient treatment.

  12. Multiscale Simulation as a Framework for the Enhanced Design of Nanodiamond-Polyethylenimine-based Gene Delivery

    PubMed Central

    Kim, Hansung; Man, Han Bin; Saha, Biswajit; Kopacz, Adrian M.; Lee, One-Sun; Schatz, George C.; Ho, Dean; Liu, Wing Kam

    2012-01-01

    Nanodiamonds (NDs) are emerging carbon platforms with promise as gene/drug delivery vectors for cancer therapy. Specifically, NDs functionalized with the polymer polyethylenimine (PEI) can transfect small interfering RNAs (siRNA) in vitro with high efficiency and low cytotoxicity. Here we present a modeling framework to accurately guide the design of ND-PEI gene platforms and elucidate binding mechanisms between ND, PEI, and siRNA. This is among the first ND simulations to comprehensively account for ND size, charge distribution, surface functionalization, and graphitization. The simulation results are compared with our experimental results both for PEI loading onto NDs and for siRNA (C-myc) loading onto ND-PEI for various mixing ratios. Remarkably, the model is able to predict loading trends and saturation limits for PEI and siRNA, while confirming the essential role of ND surface functionalization in mediating ND-PEI interactions. These results demonstrate that this robust framework can be a powerful tool in ND platform development, with the capacity to realistically treat other nanoparticle systems. PMID:23304428

  13. Enhanced osteoclastogenesis by mitochondrial retrograde signaling through transcriptional activation of the cathepsin K gene.

    PubMed

    Guha, Manti; Srinivasan, Satish; Koenigstein, Alexander; Zaidi, Mone; Avadhani, Narayan G

    2016-01-01

    Mitochondrial dysfunction has emerged as an important factor in wide ranging human pathologies. We have previously defined a retrograde signaling pathway that originates from dysfunctional mitochondria (Mt-RS) and causes a global nuclear transcriptional reprograming as its end point. Mitochondrial dysfunction causing disruption of mitochondrial membrane potential and consequent increase in cytosolic calcium [Ca(2) ](c) activates calcineurin and the transcription factors NF-κB, NFAT, CREB, and C/EBPδ. In macrophages, this signaling complements receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastic differentiation. Here, we show that the Mt-RS activated transcriptional coactivator heterogeneous ribonucleoprotein A2 (hnRNP A2) is induced by hypoxia in murine macrophages. We demonstrate that the cathepsin K gene (Ctsk), one of the key genes upregulated during osteoclast differentiation, is transcriptionally activated by Mt-RS factors. HnRNP A2 acts as a coactivator with nuclear transcription factors, cRel, and C/EBPδ for Ctsk promoter activation under hypoxic conditions. Notably, our study shows that hypoxia-induced activation of the stress target factors mediates effects similar to that of RANKL with regard to Ctsk activation. We therefore suggest that mitochondrial dysfunction and activation of Mt-RS, induced by various pathophysiologic conditions, is a potential risk factor for osteoclastogenesis and bone loss.

  14. Transcriptional activation of TFEB/ZKSCAN3 target genes underlies enhanced autophagy in spinobulbar muscular atrophy.

    PubMed

    Chua, Jason P; Reddy, Satya L; Merry, Diane E; Adachi, Hiroaki; Katsuno, Masahisa; Sobue, Gen; Robins, Diane M; Lieberman, Andrew P

    2014-03-01

    Spinobulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in exon 1 of the androgen receptor (AR) gene. SBMA demonstrates androgen-dependent toxicity due to unfolding and aggregation of the mutant protein. There are currently no disease-modifying therapies, but of increasing interest for therapeutic targeting is autophagy, a highly conserved cellular process mediating protein quality control. We have previously shown that genetic manipulations inhibiting autophagy diminish skeletal muscle atrophy and extend the lifespan of AR113Q knock-in mice. In contrast, manipulations inducing autophagy worsen muscle atrophy, suggesting that chronic, aberrant upregulation of autophagy contributes to pathogenesis. Since the degree to which autophagy is altered in SBMA and the mechanisms responsible for such alterations are incompletely defined, we sought to delineate autophagic status in SBMA using both cellular and mouse models. Here, we confirm that autophagy is induced in cellular and knock-in mouse models of SBMA and show that the transcription factors transcription factor EB (TFEB) and ZKSCAN3 operate in opposing roles to underlie these changes. We demonstrate upregulation of TFEB target genes in skeletal muscle from AR113Q male mice and SBMA patients. Furthermore, we observe a greater response in AR113Q mice to physiological stimulation of autophagy by both nutrient starvation and exercise. Taken together, our results indicate that transcriptional signaling contributes to autophagic dysregulation and provides a mechanistic framework for the pathologic increase of autophagic responsiveness in SBMA.

  15. Asymmetric 1-Alkyl-2-acyl Phosphatidylcholine: a Helper Lipid for Enhanced Non-viral Gene Delivery

    PubMed Central

    Huang, Zhaohua; Li, Weijun; Szoka, Francis C.

    2011-01-01

    Rationally designed asymmetrical alkylacyl phosphatidylcholines (APC) have been synthesized and evaluated as helper lipids for non-viral gene delivery. A long aliphatic chain (C22~C24) was introduced at the 1-position of glycerol backbone, a branched lipid chain (C18) at the 2-position, and a phosphocholine head group at the 3-position. The fusogenicity of APC depends on the length and degree of saturation of the alkyl chain. Cationic lipids were formulated with APC as either lipoplexes or nanolipoparticles, and evaluated for their stability, transfection efficiency, and cytotoxicity. APC mediated high in vitro transfection efficiency, and had low cytotoxicity. Small nanolipoparticles (less than 100 nm) can be obtained with APC by applying as low as 0.1% PEG-lipid. Our study extends the type of helper lipids that are suitable for gene transfer and points the way to improve non-viral nucleic acid delivery system other than the traditional cationic lipids optimization. This work is supported by NIH grant EB003008. PMID:21718766

  16. Asian Sand Dust Enhances the Inflammatory Response and Mucin Gene Expression in the Middle Ear

    PubMed Central

    Chang, Jiwon; Go, Yoon Young; Park, Moo Kyun; Chae, Sung-Won; Lee, Seon-Heui; Song, Jae-Jun

    2016-01-01

    Objectives. Asia sand dust (ASD) is known to cause various human diseases including respiratory infection. The aim of this study was to examine the effect of ASD on inflammatory response in human middle ear epithelial cells (HMEECs) in vitro and in vivo. Methods. Cell viability was assessed using the cell counting kit-8 assay. The mRNA levels of various genes including COX-2, TNF-a, MUC 5AC, MUC 5B, TP53, BAX, BCL-2, NOX4, and SOD1 were analyzed using semiquantitative realtime polymerase chain reaction. COX-2 protein levels were determined by western blot analysis. Sprague Dawley rats were used for in vivo investigations of inflammatory reactions in the middle ear epithelium as a result of ASD injection. Results. We observed dose-dependent decrease in HMEEC viability. ASD exposure significantly increased COX-2, TNF-a, MUC5AC, and MUC5B mRNA expression. Also, ASD affected the mRNA levels of apoptosis- and oxidative stress-related genes. Western blot analysis revealed a dose-dependent increase in COX-2 production. Animal studies also demonstrated an ASD-induced inflammatory response in the middle ear epithelium. Conclusion. Environmental ASD exposure can result in the development of otitis media. PMID:27095518

  17. The role of proximal-enhancer elements in the glucocorticoid regulation of carbamoylphosphate synthetase gene transcription from the upstream response unit.

    PubMed

    Schoneveld, Onard J L M; Gaemers, Ingrid C; Hoogenkamp, Maarten; Lamers, Wouter H

    2005-11-01

    As part of the urea cycle, carbamoylphosphate synthetase (CPS) converts toxic ammonia resulting from amino-acid catabolism into urea. Liver-specific and glucocorticoid-dependent expression of the gene involves a distal enhancer, a promoter-proximal enhancer, and the minimal promoter itself. When challenged with glucocorticoids, the glucocorticoid-responsive unit (GRU) in the distal enhancer of the carbamoylphosphate-synthetase gene can only activate gene expression if, in addition to the minimal promoter, the proximal enhancer is present. Here, we identify and characterise two elements in the proximal CPS enhancer that are involved in glucocorticoid-dependent gene activation mediated by the GRU. A purine-rich stretch forming a so-called GAGA-box and a glucocorticoid-response element (GRE) are both crucial for the efficacy of the GRU and appear to constitute a promoter-proximal response unit that activates the promoter. The glucocorticoid response of the CPS gene is, therefore, dependent on the combined action of a distal and a promoter-proximal response unit.

  18. Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells

    SciTech Connect

    Tsujimoto, Yoshihide )

    1989-03-01

    The biological activity of the human BCL-2 gene product was analyzed in an Epstein-Barr virus (EBV)-infected human lymphoblastoid B-cell line transfected with BCL-2 sequences driven by the simian virus 40 promoter and enhancer. Overproduction of the BCL-2 protein conferred a selective growth advantage to the EBV-infected B cells as compared with control transfectants in low-serum medium and also after seeding at limiting dilution but did not render the cells tumorigenic in athymic nude mice. This growth enhancement was also seen in cells transfected with the BCL-2 gene with its own promoter juxtaposed to the immunoglobulin heavy chain gene enhancer, which represents the translocated form of the BCL-2 gene observed in follicular lymphomas with the t(14;18) translocation. The growth advantage of EBV-infected B cells overproducing the BCL-2 protein is neither due to the enhanced growth factor production nor due to an enhanced sensitivity of the BCL-2 transfectants to interleukins 1 or 6, although both lymphokines are known to stimulate proliferation of EBV-infected B-cell lines. The growth advantage of EBV-infected B-cell lines. The growth advantage of EBV-infected B cells by overproduction of the BCL-2 protein suggests the direct involvement of the BCL-2 gene product in the pathogenesis of follicular lymphoma.

  19. Pronounced enhancement of glucocorticoid-induced gene expression following severe heat shock of heat-conditioned cells hints to intricate cell survival tactics.

    PubMed

    Mitsiou, Dimitra J; Florentin, Ida; Baki, Lia; Georgakopoulos, Anastasios; Alexis, Michael N

    2005-02-01

    We have previously reported that severe heat shock of HeLa cells stably transfected with a chloramphenicol acetyltransferase (CAT) gene, transcription of which is controlled by two glucocorticoid-responsive elements and a minimal promoter, pronouncedly enhanced glucocorticoid-induced CAT expression compared to that of non-heated cells, in spite of the glucocorticoid-receptor-mediated transcription of the gene being temporarily compromised by the shock. We now report that prolonged severe heat shock of properly heat-conditioned cells resulted in far more pronounced enhancement of glucocorticoid-induced CAT mRNA and protein expressions, in spite of a similar heat-induced loss of receptor-mediated CAT gene transcription. During recovery from the shock the hormonal activation of transcription exceeded that of non-heated cells. While CAT mRNA translation was restored appreciably later than CAT gene transcription, mRNA and protein expressions were thermally enhanced to a comparable extent, consistent with the integrity of CAT mRNA being preserved during recovery. CAT mRNA turnover was fully impaired during early recovery, suggesting that stabilisation of CAT mRNA as well as stimulation of the hormonal activation of CAT gene transcription account for the thermal enhancement of glucocorticoid-induced CAT expression. This data hint to cell survival tactics designed to safeguard high expression of genes of stress-enduring function.

  20. Stable transfection of extrinsic Smac gene enhances apoptosis-inducing effects of chemotherapeutic drugs on gastric cancer cells

    PubMed Central

    Zheng, Li-Duan; Tong, Qiang-Song; Wang, Liang; Liu, Jun; Qian, Wei

    2005-01-01

    AIM: To explore the feasibility of enhancing apoptosis-inducing effects of chemotherapeutic drugs on human gastric cancer cells by stable transfection of extrinsic Smac gene. METHODS: After Smac gene was transferred into gastric cancer cell line MKN-45, subclone cells were obtained by persistent G418 selection. Cellular Smac gene expression was determined by RT-PCR and Western blotting. After treatment with mitomycin (MMC) as an apoptotic inducer, in vitro cell growth activities were investigated by trypan blue-staining method and MTT colorimetry. Cell apoptosis and its rates were determined by electronic microscopy, annexin V-FITC and propidium iodide staining flow cytometry. Cellular caspase-3 protein expression and its activities were assayed by Western blotting and colorimetry. RESULTS: When compared with MKN-45 cells, the selected subclone cell line MKN-45/Smac had significantly higher Smac mRNA (3.12±0.21 vs 0.82±0.14, t = 7.52, P<0.01) and protein levels (4.02±0.24 vs 0.98±0.11, t = 8.32, P<0.01). After treatment with 10 μg/mL MMC for 6-24 h, growth inhibition rate of MKN-45/Smac (15.8±1.2-54.8±2.9%) was significantly higher than that of MKN-45 (5.8±0.4- 24.0±1.5%, t = 6.42, P<0.01). Partial MKN-45/Smac cancer cells presented characteristic morphological changes of apoptosis under the electronic microscope with an apoptosis rate of 36.4±2.1%, which was significantly higher than that of MKN-45 (15.2±0.8%, t = 9.25, P<0.01). Compared with MKN-45, caspase-3 expression levels in MKN-45/Smac were improved significantly (3.39±0.42 vs 0.96±0.14, t = 8.63, P<0.01), while its activities were 3.25 times as many as those of MKN-45 (0.364±0.010 vs 0.112±0.007, t = 6.34, P<0.01). CONCLUSION: Stable transfection of extrinsic Smac gene and its over-expression in gastric cancer cell line can significantly enhance cellular caspase-3 expression and activities, ameliorate apoptosis-inducing effects of mitomycin C on cancer cells, which is a novel strategy to

  1. 5'-Hydroxymethylcytosine Precedes Loss of CpG Methylation in Enhancers and Genes Undergoing Activation in Cardiomyocyte Maturation

    PubMed Central

    Kranzhöfer, David K.; Gilsbach, Ralf; Grüning, Björn A.; Backofen, Rolf; Nührenberg, Thomas G.; Hein, Lutz

    2016-01-01

    Background Cardiomyocytes undergo major changes in DNA methylation during maturation and transition to a non-proliferative state after birth. 5’-hydroxylation of methylated cytosines (5hmC) is not only involved in DNA loss of CpG methylation but is also thought to b