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Sample records for fhit gene enhances

  1. Molecular cloning of the canine fragile histidine triad (FHIT) gene and Fhit protein expression in canine peripheral blood mononuclear cells.

    PubMed

    Hiraoka, Hiroko; Minami, Koji; Kaneko, Naoki; Shimokawa Miyama, Takako; Mizuno, Takuya; Okuda, Masaru

    2009-05-01

    A fragile histidine triad (FHIT) gene has been studied as a tumor-associated gene in humans. The aberrant FHIT gene and its protein expression have been reported in many types of human cancers. The present study explored the canine FHIT gene structure and its protein expression in the peripheral blood mononuclear cells of healthy dogs by RT-PCR, RACE and immunoblot analysis. The obtained canine FHIT gene contained nine small exons and was located on canine chromosome 20. Furthermore, we identified an alternative splicing form of the FHIT transcript. The deduced amino acid sequence was well conserved between species, and anti-human Fhit antibody could be used to detect the canine Fhit protein. These findings will be useful for future research.

  2. 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

  3. Aberration of the enzymatic activity of Fhit tumor suppressor protein enhances cancer cell death upon photodynamic therapy similarly to that driven by wild-type Fhit.

    PubMed

    Ferens, Bartosz; Kawiak, Anna; Banecki, Bogdan; Bielawski, Krzysztof P; Zawacka-Pankau, Joanna

    2009-07-18

    The tumor suppressor Fhit protein lost in many human pre-malignant tissues, possesses diadenosine triphosphate activity regulated by a photosensitizer, protoporphyrin IX (PpIX) in vitro. Interestingly, when exogenously restored, the protein suppresses the growth of human cervical carcinoma HeLa cells which is further enhanced by PpIX. Additionally, Fhit production enhances the overall response of cells to PpIX-mediated photodynamic reaction. In the present study, we have estimated, for the first time, the biological activity of two Fhit mutated forms exhibiting aberrant Ap(3)A hydrolase activity in vitro which emphasizes the recent findings that hydrolysis of Ap(3)A is not necessary for Fhit tumor suppression function. Using several biophysical methods we revealed the dynamic nature of mutant Fhit-PpIX complexes in vitro which support our previous hypothesis that Fhit-Ap(3)A-PpIX might be a signaling molecule driving apoptosis in cancer cells. Moreover, according to our findings, substitution at histidine94 in Fhit active site induces the vulnerability of HeLa cells to PpIX-PDT in a similar manner to that caused by wild-type Fhit protein. These results support the view that inhibition of Fhit hydrolase activity might be a crucial element in a Fhit-driven cancer cells death.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. Promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A genes in cervical carcinoma.

    PubMed

    Banzai, Chiaki; Nishino, Koji; Quan, Jinhua; Yoshihara, Kosuke; Sekine, Masayuki; Yahata, Tetsuro; Tanaka, Kenichi

    2014-02-01

    Aberrant DNA methylation contributes to the malignant phenotype in virtually all types of human cancer. This study explored the relationship between promoter methylation and inactivation of the DAPK1, FHIT, MGMT, and CDKN2A genes in cervical cancer. The promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A was investigated by using a methylation-specific polymerase chain reaction in 53 specimens of cervical cancer (42 squamous cell carcinoma, 11 adenocarcinoma), 22 specimens of intraepithelial neoplasia tissues, and 24 control normal cervical tissue specimens. The correlation of promoter methylation with the clinicopathological features of cervical cancer was analyzed. The expressions of DAPK1, FHIT, MGMT, and CDKN2A were detected by measuring relative mRNA levels. The promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A in cervical cancer vs. intraepithelial neoplasia vs. normal cervical tissue was 75.5 vs. 31.8 vs. 4.2 % (p < 0.0001), 66.0 vs. 59.1 vs. 25.0 % (p = 0.0033), 34.0 vs. 27.3 vs. 20.8 % (p = 0.76), and 17.0 vs. 31.8 vs. 8.3 % (p = 0.11), respectively. The methylation of the promoter region significantly decreased the expression of only DAPK1 (p = 0.03). The methylation rate of the DAPK1 gene promoter was significantly higher in cervical cancer tissues than in cervical intraepithelial neoplasia and normal cervical tissues. Promoter methylation may therefore lead to the inactivation of the DAPK1 gene, and may be related to the progression of cervical oncogenesis.

  9. 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.

  10. 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.

  11. Deletion of the FHIT gene in neoplastic and invasive cervical lesions is related to high-risk HPV infection but is independent of histopathological features.

    PubMed

    Butler, D; Collins, C; Mabruk, M; Barry Walsh, C; Leader, M B; Kay, E W

    2000-12-01

    The fragile histidine triad (FHIT) gene encompasses the common chromosomal fragile site FRA3B. Human papilloma virus (HPV), which is the main aetiological agent in cervical cancers, has been found to be able to integrate its genes into the chromosome 3 fragile site of cultured cells, deleting a piece of DNA which includes the FHIT gene. Eighty-six microdissected archival cervical LLETZ biopsies comprising cases of cervical intraepithelial neoplasia (CIN) 1 (n=27), CIN3 (n=30) and microinvasive carcinoma (n=29) were evaluated for HPV infection and FHIT gene loss of heterozygosity (LOH). FHIT gene LOH was detected by polymerase chain reaction (PCR) using fluorescently labelled intragenic microsatellite markers D3S1300 and D3S4103. PCR products were analysed on a semi-automated DNA sequencer using Fragment Manager(trade mark) software to determine allele loss. The HPV status of the lesions was determined by PCR using generic and type-specific primers in conjunction with restriction endonuclease digestion. The results were analysed using Epi-Info and SPSS-PC statistical analysis software. Haematoxylin and eosin-stained sections from the 86 cases were profiled for six histopathological features, some of which have been previously shown to be associated with microinvasive cancer. FHIT gene LOH was found in 36% of CIN1 cases, 52% of CIN3 cases and 73% of microinvasive cases (p=0.029). HPV 16 DNA was found in 68% of CIN3 cases and 93% of microinvasive cases (p<0.001). The second most prevalent HPV type found was HPV 31, which was present in only four lesions, three of which had FHIT gene LOH. When FHIT gene LOH was evaluated versus HPV 16 and 31 infection using the chi-square test, a statistically significant relationship was found (p=0.014). FHIT gene LOH was found to be independent of the histopathological features evaluated. The finding of a statistically significant relationship between FHIT gene LOH and oncogenic HPV infection suggests a link between the integration

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. Increased Sensitivity to Cisplatin in Non-Small Cell Lung Cancer Cell Lines after FHIT Gene Transfer1

    PubMed Central

    Andriani, F; Perego, P; Carenini, N; Sozzi, G; Roz, L

    2006-01-01

    Abstract To evaluate the relevance of fragile histidine triad (FHIT) status in relation to drug treatment, we analyzed the sensitivity of the Fhit-negative non-small cell lung cancer (NSCLC) cell line NCI-H460 to different drugs, after treatment with an adenoviral vector expressing the FHIT transgene. Expression of Fhit resulted in reduced sensitivity to etoposide, doxorubicin, and topotecan. This feature was associated with Fhit-induced downregulation of DNA topoisomerases I and II. In contrast, expression of Fhit did not modulate sensitivity to Taxol, but produced a slight increase in sensitivity to cisplatin, as shown by colony-forming assays. Analysis of apoptosis revealed that, after cisplatin exposure, the number of apoptotic cells was two-fold higher in Fhit-expressing H460 cells. Moreover, it appeared that wild-type p53 was required for sensitization to cisplatin because the effect was marginal in A549 and Calu-1 cells, where the p53 pathway is altered and simultaneous restoration of p53 and Fhit in Calu-1 cells increased cisplatin sensitivity. Fhit could also partially restore sensitivity to cisplatin in Bcl-2- and Bcl-xL-overexpressing H460 cells that are normally resistant to this drug. Our results support the possible relevance of FHIT in cisplatin-based chemotherapy as well as in the reversal of drug resistance in NSCLC. PMID:16533421

  19. 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.

  20. 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

  1. Specific 50'CpG island methylation signatures of FHIT and p16 genes and their potential diagnostic relevance in Indian breast cancer patients.

    PubMed

    Naqvi, Raza Ali; Hussain, Arif; Raish, Mohmammad; Noor, Afshan; Shahid, Mohammad; Sarin, Ritu; Kukreti, Himani; Khan, Nida Jameel; Ahmad, Shandar; Deo, Suryanarayan V S; Husain, Syed Akhtar; Pasha, Syed Tazeen; Basir, Seemi Farhat; Shukla, Nootan Kumar

    2008-09-01

    Even after tremendous molecular studies, early detection,more accurate and sensitive diagnosis, and prognosis of breast cancer appear to be a riddle so far. To stab the enigma, this study is designed to envisage DNA methylation signatures as cancer-specific and stage-specific biomarkers in Indian patients. Rigorous review of scattered scientific reports on aberrant DNA methylation helped us to select and analyze a potential tumor suppressor gene pair (FHIT and p16 genes) in breast cancer patients. Methylation signatures from 232 primary sporadic breast cancer patients were pinpointed by methylation-specific PCR (MSP). To increase the sensitivity, we combined both MSP and expression studies (RT-PCR and Northern blotting) in a reproducible manner. Statistical analysis illustrated that hypermethylation of FHIT gene ( p < 0.0001) and p16 gene ( p=0.04) may be used as a potential diagnostic marker to diagnose the early and locally advanced stages of breast cancer. Additionally, the study authenticates the dependency of methylation and expressional loss of p16 gene on FHIT gene silencing. This observation not only describes the severity of disease when both genes are silenced but also drives to speculate the molecular cross talk between two genes or genetic pathways dictated by them separately.

  2. Fhit Interaction with Ferredoxin Reductase Triggers Generation of Reactive Oxygen Species and Apoptosis of Cancer Cells*S⃞

    PubMed Central

    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.

    2008-01-01

    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. PMID:18319262

  3. 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.

  4. 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.

  5. 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.

  6. Reduced Fhit expression in sporadic and BRCA2-linked breast carcinomas.

    PubMed

    Ingvarsson, S; Agnarsson, B A; Sigbjornsdottir, B I; Kononen, J; Kallioniemi, O P; Barkardottir, R B; Kovatich, A J; Schwarting, R; Hauck, W W; Huebner, K; McCue, P A

    1999-06-01

    Evidence for alteration of the FHIT gene in a significant fraction of breast carcinomas has been reported, in apparent concordance with loss of heterozygosity (LOH) at chromosome region 3p14.2 in breast cancer and benign proliferative breast disease. A significantly higher frequency of LOH at the FHIT locus was reported for BRCA2-/- tumors, possibly due to misrepaired double-strand breaks at this common fragile region. To determine whether such genomic alterations lead to Fhit inactivation, we have assessed the level of Fhit expression by immunohistochemical detection in sporadic tumors and cancers occurring in BRCA2 999del5 carriers. To determine whether Fhit inactivation may have prognostic significance, we have also assessed expression of breast cancer markers and clinical features in sporadic tumors relative to Fhit expression. Of 40 consecutive sporadic breast carcinomas studied for tumor markers, 50% showed reduced Fhit expression. In these sporadic cancers, loss of Fhit expression was not correlated significantly with the presence or absence of other tumor markers. In a study of 58 sporadic and 34 BRCA2 999del5 Icelandic invasive cancers, there was a significant association of LOH at 3p14.2 with reduced expression of Fhit (P = 0.001); also the lower expression of Fhit and higher LOH at 3p14.2 in BRCA2 999del5 tumors relative to sporadic cancers was significant (P = 0.002). Thus, genetic alteration at the fragile site within the FHIT gene leads to loss of Fhit protein in a significant fraction of sporadic breast cancers and a much larger fraction of familial breast cancers with an inherited BRCA2 mutation, consistent with the idea that loss of BRCA2 function affects stability of the FHIT/FRA3B locus.

  7. 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.

  8. 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.

  9. [The biological significance of FHIT protein expression in lung cancer and precancerous tissues detected by tissue microarray].

    PubMed

    Yuan, Ling; Wang, Xinyun; Zheng, Haiyan

    2007-06-20

    Fragile histidine triad (FHIT) is a candidate tumor suppressor gene. Aberrant expression of FHIT has been observed in multiple carcinomas induced by environmental carcinogens, especially in lung cancer. In this study, the expression of FHIT protein in lung cancer progression tissue microarray was detected and their roles in oncogenesis and progression of lung cancer were discussed. The expression of FHIT protein in tissue microarray with 270 cores was detected by SP immunohistochemistry method, in which there were 89 cases of primary lung cancer, 12 cases of lymph node metastasis of lung cancer, 12 cases of precancerous lesion and 10 cases of normal lung tissue, and the clinicopathological features of lung cancer were analyzed. The expression of FHIT was localized in the cytoplasm. Loss of FHIT expression in primary cancers, precancerous lesion and lymph node metastasis of lung cancer was 46.1%, 41.7% and 50.0% respectively, while 0 in 10 cases of normal tissues. A significant difference of FHIT expression was observed among four groups (P < 0.05). Loss of FHIT expression in precancerous lesion, primary lung cancer and lymph node metastasis of lung cancer was significantly higher than that in normal lung tissue (P < 0.05). The difference among precancerous lesion, primary lung cancer and lymph node metastasis of lung cancer groups was not statistically significant (P > 0.05). Loss of FHIT expression was related to tumor histologicol types, degree of cell differentiation and the smoking history of patients (P < 0.05), but not to sex, age, gross appearance types, TNM stages, or lymph node metastasis (P > 0.05). The protein expression level of FHIT is reduced in primary cancers and precancerous tissues, especially in most squamous cell carcinomas, poorly differentiated group and the patients with a smoking history. These results indicate that loss of FHIT expression might correlate with carcinogenesis, development of lung cancer and the carcinogenesis induced by

  10. The clinicopathological significance and drug target potential of FHIT in breast cancer, a meta-analysis and literature review.

    PubMed

    Su, Yunshu; Wang, Xiaoli; Li, Jun; Xu, Junming; Xu, Lijun

    2015-01-01

    FHIT is a bona fide tumor-suppressor gene and its loss contributes to tumorigenesis of epithelial cancers including breast cancer (BC). However, the association and clinicopathological significance between FHIT promoter hypermethylation and BC remains unclear. The purpose of this study is to conduct a meta-analysis and literature review to investigate the clinicopathological significance of FHIT methylation in BC. A detailed literature search was performed in PubMed, EMBASE, Web of Science, and Google Scholar databases. The data were extracted and assessed by two reviewers independently. Odds ratios with 95% corresponding confidence intervals were calculated. A total of seven relevant articles were available for meta-analysis, which included 985 patients. The frequency of FHIT hypermethylation was significantly increased in invasive ductal carcinoma compared to benign breast disease, the pooled odds ratio was 8.43, P<0.00001. The rate of FHIT hypermethylation was not significantly different between stage I/II and stage III/IV, odds ratio was 2.98, P=0.06. In addition, FHIT hypermethylation was not significantly associated with ER and PR status. FHIT hypermethylation was not significantly correlated with premenopausal and postmenopausal patients with invasive ductal carcinoma. In summary, our meta-analysis indicated that the frequency of FHIT hypermethylation was significantly increased in BC compared to benign breast disease. The rate of FHIT hypermethylation in advanced stages of BC was higher than in earlier stages; however, the difference was not statistically significant. Our data suggested that FHIT methylation could be a diagnostic biomarker of BC carcinogenesis. FHIT is a potential drug target for development of demethylation treatment for patients with BC.

  11. Loss of Fhit expression is associated with poorer survival in gastric cancer but is not an independent prognostic marker.

    PubMed

    Bragantini, Emma; Barbi, Stefano; Beghelli, Stefania; Moore, Patrick S; de Manzoni, Giovanni; Roviello, Franco; Tomezzoli, Anna; Vindigni, Carla; Baffa, Raffaele; Scarpa, Aldo

    2006-01-01

    Several studies have reported conflicting results regarding correlations of the loss of Fhit expression with clinicopathological parameters in gastric cancer. We investigated the immunohistochemical expression of Fhit in 362 cases of sporadic advanced gastric adenocarcinoma. The series included 64 cases with microsatellite instability associated with defective mismatch repair genes. Fhit expression resulted absent in 72% of the tumors analyzed. Absence of Fhit expression was more frequent in cases with diffuse and mixed histotype compared to the intestinal histotype (P=0.009). Absence of Fhit expression also correlated with tumor stage (P<0.001), lymph node involvement (P<0.001), presence of distant metastasis (P=0.033), and increasing histological grade (P=0.005). Retained Fhit expression also correlated with microsatellite instability as 61% of instable tumors had lost Fhit expression compared to 74% of microsatellite stable cancers (P=0.050). While loss of Fhit correlates with poorer survival in univariate analysis, it is not an independent prognostic factor in multivariate analysis and is thus not of clinical utility.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. The apoptotic pathway triggered by the Fhit protein in lung cancer cell lines is not affected by Bcl-2 or Bcl-x(L) overexpression.

    PubMed

    Roz, Luca; Andriani, Francesca; Ferreira, Carlos G; Giaccone, Giuseppe; Sozzi, Gabriella

    2004-12-02

    The expression of the tumour suppressor protein fragile histidine triad (Fhit) is often impaired in many human cancers and its restoration in Fhit-negative cancer cell lines suppresses tumorigenicity and induces apoptosis. Although the proapoptotic function of Fhit is well documented, little is known about its precise mechanism of action and further studies are needed in order to elucidate the putative therapeutic properties of this protein. To this end, we have engineered the lung cancer cell line NCI-H460 in order to express different molecules involved in the control of apoptotic pathways. Infection of these cells with an adenoviral vector transducing the Fhit gene (Ad-Fhit) revealed that complete protection from apoptosis was conferred by the inhibitor of caspases Cytokine response modifier A (CrmA) and by a dominant-negative form of the adapter protein Fas-associated death domain (FADD) and partial protection by a dominant-negative form of caspase-8, while cells over expressing mitochondrial mediators of the apoptotic response such as Bcl-2 or Bcl-x(L) that are resistant to treatment with cisplatin, remained highly susceptible to cell death triggered by Fhit gene transfer. In line to what was observed in H460 cells, Ad-Fhit efficacy was not affected by Bcl-2 overexpression also in two other lung cancer cell lines (A549 and Calu-1). Analysis of cytochrome c release also confirmed that in Bcl-2- or Bcl-x(L)-expressing cells apoptosis could be detected by terminal deoxynucleotidyl-transferase mediated dUTP nick-end labelling (TUNEL) assay before any evidence of mitochondrial membrane perturbation. In conclusion, our analysis indicates that the Fhit protein exerts its oncosuppressor activity through induction of an apoptotic mechanism that seems to be FADD dependent, caspase-8 mediated and independent from mitochondrial amplification.

  18. The tumour suppressor Fhit positively regulates MHC class I expression on cancer cells.

    PubMed

    Romero, Irene; Martinez, Marisol; Garrido, Cristina; Collado, Antonia; Algarra, Ignacio; Garrido, Federico; Garcia-Lora, Angel M

    2012-07-01

    MHC class I (MHC-I) molecules are ubiquitously expressed on the cells of an organism. Study of the regulation of these molecules in normal and disease conditions is important. In tumour cells, the expression of MHC-I molecules is very frequently lost, allowing these cells to evade the immune response. Cancers of different histology have shown total loss of MHC-I molecule expression, due to a coordinated transcriptional down-regulation of various antigen-processing machinery (APM) components and/or MHC-I heavy chains. The mechanisms responsible for these alterations remain unclear. We determined the possible genes involved by comparing MHC-I-positive with MHC-I-negative murine metastases derived from the same fibrosarcoma tumour clone. MHC-I-negative metastases showed transcriptional down-regulation of APM and MHC-I heavy chains. The use of microarrays and subtraction cDNA libraries revealed four candidate genes responsible for this alteration, but two of them were ruled out by real-time RT-PCR analyses. The other two genes, AP-2α and Fhit tumour suppressors, were studied by using siRNA to silence their expression in a MHC-I-positive metastatic cell line. AP-2α inhibition did not modify transcriptional expression of APM components or MHC-I heavy chains or surface expression of MHC-I. In contrast, silencing of the Fhit gene produced the transcriptional down-regulation of APM components and MHC-I heavy chains and decreased MHC-I surface expression. Moreover, transfection of Fhit in MHC-I-negative tumour cell lines restored MHC-I cell surface expression. These data indicate that defects in Fhit expression may promote MHC-I down-regulation in cancer cells and allow escape from immunosurveillance(#). Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  19. 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

  20. Influence of FHIT on benzo[a]pyrene-induced tumors and alopecia in mice: Chemoprevention by budesonide and N-acetylcysteine

    PubMed Central

    Balansky, Roumen; D'Agostini, Francesco; Ganchev, Gancho; Izzotti, Alberto; Di Marco, Barbara; Lubet, Ronald A.; Zanesi, Nicola; Croce, Carlo M.; De Flora, Silvio

    2006-01-01

    The FHIT gene has many hallmarks of a tumor-suppressor gene and is involved in a large variety of cancers. We treated A/J mice and (C57BL/6J × 129/SvJ)F1 (B6/129 F1) mice, either wild-type or FHIT+/−, with multiple doses of benzo[a]pyrene (B[a]P) by gavage. B[a]P caused a time-related increase of micronuclei in peripheral blood erythrocytes. Both A/J and B6/129 F1 mice, irrespective of their FHIT status, were sensitive to induction of forestomach tumors, whereas B[a]P induced glandular stomach hyperplasia and a high multiplicity of lung tumors in A/J mice only. Preneoplastic lesions of the uterus were more frequent in FHIT+/− mice. B6/129 F1 mice underwent spontaneous alopecia areata and hair bulb cell apoptosis, which were greatly accelerated either by FHIT heterozygosity or by B[a]P treatment, thus suggesting that FHIT plays a role in the pathogenesis of alopecia areata. The oral administration of either budesonide or N-acetyl-l-cysteine (NAC) inhibited the occurrence of this inflammatory skin disease. In addition, these agents prevented B[a]P-induced glandular stomach hyperplasia and decreased the size of both forestomach tumors and lung tumors in A/J mice. Budesonide also attenuated lung tumor multiplicity. In B6/129 F1 mice, NAC significantly decreased the proliferating cell nuclear antigen in lung tumors. Both budesonide and NAC inhibited B[a]P-induced forestomach tumors and preneoplastic lesions of the respiratory tract in B6/129 F1 mice. In conclusion, heterozygosity for FHIT affects susceptibility of mice to spontaneous alopecia areata and B[a]P-induced preneoplastic lesions of the uterus and does not alter responsiveness to budesonide and NAC. PMID:16672365

  1. Detection of FHIT and p16 mRNA deletion in biopsy specimens obtained by bronchoscopy for the diagnosis of lung cancer.

    PubMed

    Chen, Ping; Li, Jian; Wang, Yi; Zhu, Li-Rong; Hu, Yi-Ming; Tong, Xing-Ping

    2013-09-27

    The purpose of this study was to investigate the diagnostic value of the deletion of fragile histidine triad (FHIT) and p16INK4a (p16) mRNA in biopsies obtained by bronchoscopy. Biopsies were analyzed using RT-PCR in 52 patients with lung cancer and 19 patients with benign lung disease. The results showed that the detection rates of FHIT and p16 gene transcript deletion were significantly higher in lung cancer patients than in patients with benign lung disease (65.4% versus 10.5%, p=0.001 and 59.6% versus 5.3%, p<0.001, respectively). The sensitivities for detecting FHIT and p16 transcript deletion in biopsies were 65.4% and 59.6% (combined 80.8%), respectively, which were markedly better than those of histology and cytology (42.3% and 34.6%, respectively; combined 57.7%). In 22 lung cancer patients with negative histology and cytology at initial bronchoscopy, FHIT and p16 mRNA loss was detected in 40.9% (9/22) and 36.4% (8/22) cases, respectively. FHIT mRNA loss was associated with smoking status in lung cancer patients. In conclusion, deletion of FHIT and p16 mRNA can be identified in biopsies obtained during bronchoscopic procedures. FHIT and p16 mRNA deletion can be used as biomarkers in the clinical diagnosis of lung cancer and may serve as adjuncts to histology and cytology in lung cancer diagnosis.

  2. 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

  3. 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

  4. 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.

  5. Promoter hypermethylation profile of RASSF1A, FHIT, and sFRP1 in intracranial primitive neuroectodermal tumors.

    PubMed

    Chang, Qing; Pang, Jesse Chung-Sean; Li, Kay Ka Wai; Poon, Wai Sang; Zhou, Liangfu; Ng, Ho-Keung

    2005-12-01

    Medulloblastomas (MBs) and supratentorial primitive neuroectodermal tumors (SPNETs) are histologically alike intracranial PNETs found in different anatomical locations of the brain. Current evidence suggests that hypermethylation of promoter CpG islands is a common epigenetic event in a variety of human cancers. The aim of this study was to investigate whether promoter hypermethylation of putative tumor suppressor genes was involved in both types of intracranial PNETs. We examined the methylation status at promoter regions of RASSF1A, FHIT, and sFRP1 by methylation-specific polymerase chain reaction in a cohort of 25 primary MBs, 9 primary SPNETs, and 3 MB and 2 SPNET cell lines. Our results revealed no promoter hypermethylation of RASSF1A, FHIT, and sFRP1 in 2 normal cerebellar and 5 normal cerebral tissue specimens examined. In contrast, promoter hypermethylation of RASSF1A was detected in 100% of primary MBs, 67% (6/9) of primary SPNETs, and all PNET cell lines. The frequency of promoter hypermethylation of RASSF1A was significantly lower in SPNETs than in MBs (Fisher exact test, P = .014). Treatment of RASSF1A-deficient PNET cell lines with 5-aza-2'deoxycytidine, a demethylating agent, restored RASSF1A expression, providing evidence that promoter hypermethylation contributes to transcriptional silencing. In addition, promoter hypermethylation of FHIT and sFRP1 was detected in 22% (2/9) and 11% (1/9) of SPNETs, respectively, but not in any MBs studied. In conclusion, our study demonstrates that promoter hypermethylation of RASSF1A is a common event in intracranial PNETs, whereas FHIT and sFRP1 are epigenetically affected in a fraction of SPNETs.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. GeneHancer: genome-wide integration of enhancers and target genes in GeneCards

    PubMed Central

    Rappaport, Noa; Hadar, Rotem; Plaschkes, Inbar; Iny Stein, Tsippi; Rosen, Naomi; Kohn, Asher; Twik, Michal; Safran, Marilyn

    2017-01-01

    Abstract A major challenge in understanding gene regulation is the unequivocal identification of enhancer elements and uncovering their connections to genes. We present GeneHancer, a novel database of human enhancers and their inferred target genes, in the framework of GeneCards. First, we integrated a total of 434 000 reported enhancers from four different genome-wide databases: the Encyclopedia of DNA Elements (ENCODE), the Ensembl regulatory build, the functional annotation of the mammalian genome (FANTOM) project and the VISTA Enhancer Browser. Employing an integration algorithm that aims to remove redundancy, GeneHancer portrays 285 000 integrated candidate enhancers (covering 12.4% of the genome), 94 000 of which are derived from more than one source, and each assigned an annotation-derived confidence score. GeneHancer subsequently links enhancers to genes, using: tissue co-expression correlation between genes and enhancer RNAs, as well as enhancer-targeted transcription factor genes; expression quantitative trait loci for variants within enhancers; and capture Hi-C, a promoter-specific genome conformation assay. The individual scores based on each of these four methods, along with gene–enhancer genomic distances, form the basis for GeneHancer’s combinatorial likelihood-based scores for enhancer–gene pairing. Finally, we define ‘elite’ enhancer–gene relations reflecting both a high-likelihood enhancer definition and a strong enhancer–gene association. GeneHancer predictions are fully integrated in the widely used GeneCards Suite, whereby candidate enhancers and their annotations are displayed on every relevant GeneCard. This assists in the mapping of non-coding variants to enhancers, and via the linked genes, forms a basis for variant–phenotype interpretation of whole-genome sequences in health and disease. Database URL: http://www.genecards.org/ PMID:28605766

  16. Ultrasound enhances retrovirus-mediated gene transfer.

    PubMed

    Naka, Toshio; Sakoda, Tsuyoshi; Doi, Takashi; Tsujino, Takeshi; Masuyama, Tohru; Kawashima, Seinosuke; Iwasaki, Tadaaki; Ohyanagi, Mitsumasa

    2007-01-01

    Viral vector systems are efficient for transfection of foreign genes into many tissues. Especially, retrovirus based vectors integrate the transgene into the genome of the target cells, which can sustain long term expression. However, it has been demonstrated that the transduction efficiency using retrovirus is relatively lower than those of other viruses. Ultrasound was recently reported to increase gene expression using plasmid DNA, with or without, a delivery vehicle. However, there are no reports, which show an ultrasound effect to retrovirus-mediated gene transfer efficiency. Retrovirus-mediated gene transfer systems were used for transfection of 293T cells, bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and rat skeletal muscle myoblasts (L6 cells) with beta-galactosidase (beta-Gal) genes. Transduction efficiency and cell viability assay were performed on 293T cells that were exposed to varying durations (5 to 30 seconds) and power levels (1.0 watts/cm(2) to 4.0 watts/cm(2)) of ultrasound after being transduced by a retrovirus. Effects of ultrasound to the retrovirus itself was evaluated by transduction efficiency of 293T cells. After exposure to varying power levels of ultrasound to a retrovirus for 5 seconds, 293T cells were transduced by a retrovirus, and transduction efficiency was evaluated. Below 1.0 watts/cm(2) and 5 seconds exposure, ultrasound showed increased transduction efficiency and no cytotoxicity to 293T cells transduced by a retrovirus. Also, ultrasound showed no toxicity to the virus itself at the same condition. Exposure of 5 seconds at the power of 1.0 watts/cm(2) of an ultrasound resulted in significant increases in retrovirus-mediated gene expression in all four cell types tested in this experiment. Transduction efficiencies by ultrasound were enhanced 6.6-fold, 4.8-fold, 2.3-fold, and 3.2-fold in 293T cells, BAECs, RASMCs, and L6 cells, respectively. Furthermore, beta-Gal activities were also increased

  17. 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.

  18. Quantitative assessment of lung cancer associated with genes methylation in the peripheral blood.

    PubMed

    Tan, Shanjuan; Sun, Changqing; Wei, Xiaoling; Li, Yanqiang; Wu, Yongjun; Yan, Zhen; Feng, Feifei; Wang, Jing; Wu, Yiming

    2013-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide due mainly to late diagnosis and poor prognosis. Aberrant promoter methylation is an important mechanism for silencing of tumor suppressor genes during carcinogenesis and a promising tool for the development of molecular biomarkers. We evaluated the p16, RASSF1A, and FHIT genes promoter methylation status in peripheral blood DNA between 200 lung cancer patients and 200 normal controls by using SYBR green-based quantitative methylation-specific PCR (qMSP). There were statistically significant differences in the methylation status of p16, RASSF1A, and FHIT between the cancer cases and controls (p16: P = .008, RASSF1A: P = .038, FHIT: P = .002). When the subjects were categorized into quartiles based on the genes methylation status, the risk of lung cancer was found to increase as methylation status increased (p16: Ptrend = .002, RASSF1A: Ptrend = .014, FHIT: Ptrend = .001). When the median of methylation status was used as the cutoff between high and low methylation status, individuals with high methylation status were at a significantly higher risk of lung cancer than those with low methylation status (p16: adjusted odds ratio = 1.597, P = .028; RASSF1A: adjusted odds ratio = 1.551, P = .039; FHIT: adjusted odds ratio = 1.763, P = .008). In addition, there were no significant correlations between p16, RASSF1A, or FHIT methylation status and gender (P > .05), age (P > .05), smoking history (P > .05), histological type (P > .05), or clinical stage (P > .05). These results suggest that the high methylation statuses of p16, RASSF1A, or FHIT genes were associated with a significantly increased risk of lung cancer; the risk of lung cancer increased as the methylation status increased. Further investigation of their definitive usefulness in clinical practice is warranted.

  19. 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.

  20. Gene and enhancer trap tagging of vascular-expressed genes in poplar trees

    Treesearch

    Andrew Groover; Joseph R. Fontana; Gayle Dupper; Caiping Ma; Robert Martienssen; Steven Strauss; Richard Meilan

    2004-01-01

    We report a gene discovery system for poplar trees based on gene and enhancer traps. Gene and enhancer trap vectors carrying the β-glucuronidase (GUS) reporter gene were inserted into the poplar genome via Agrobacterium tumefaciens transformation, where they reveal the expression pattern of genes at or near the insertion sites. Because GUS...

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. The use of genes for performance enhancement: doping or therapy?

    PubMed

    Oliveira, R S; Collares, T F; Smith, K R; Collares, T V; Seixas, F K

    2011-12-01

    Recent biotechnological advances have permitted the manipulation of genetic sequences to treat several diseases in a process called gene therapy. However, the advance of gene therapy has opened the door to the possibility of using genetic manipulation (GM) to enhance athletic performance. In such 'gene doping', exogenous genetic sequences are inserted into a specific tissue, altering cellular gene activity or leading to the expression of a protein product. The exogenous genes most likely to be utilized for gene doping include erythropoietin (EPO), vascular endothelial growth factor (VEGF), insulin-like growth factor type 1 (IGF-1), myostatin antagonists, and endorphin. However, many other genes could also be used, such as those involved in glucose metabolic pathways. Because gene doping would be very difficult to detect, it is inherently very attractive for those involved in sports who are prepared to cheat. Moreover, the field of gene therapy is constantly and rapidly progressing, and this is likely to generate many new possibilities for gene doping. Thus, as part of the general fight against all forms of doping, it will be necessary to develop and continually improve means of detecting exogenous gene sequences (or their products) in athletes. Nevertheless, some bioethicists have argued for a liberal approach to gene doping.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

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

    PubMed

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

    2014-02-01

    In Drosophila, the 330 kb bithorax complex regulates cellular differentiation along the anterior–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.

  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. [Gene angora enhances the effects of gene waved alopecia in mice].

    PubMed

    Malinina, N A; Koniukhova, B V; Nesterova, A P

    2007-11-01

    Interaction between the mutant gene angora-Y (Fgf5(go-Y)) and the mutant gene waved alopecia (wal) in mice has been studied. Gene Fgf5(go-Y) in a homozygous state increases the length of hair of all types, whereas the homozygotes at wal gene display a waved hair with subsequent development of partial alopecia. Crosses between Fgf5(go-Y)/Fgf5(go-Y) and wal/wal mice gave the animals displaying the genotypes +/Fgf5(go-Y) wal/wal and Fgf5(go-Y)/Fgf5(go-Y) wal/walas well as F2 +/+ wal/wal mice. The first signs of alopecia in F2 +/+ wal/wal appear at the same time as in the mutant wal/wal BALB/c mice. This demonstrates that the genetic background has no effect on the expression of mutant gene wal. A single dose of gene Fgf5(go-Y) in +/Fgf5(go-Y) wal/wal mice causes a considerably earlier appearance of the first signs of alopecia compared with the +/+ wal/wal single homozygotes. The signs of alopecia in double homozygotes Fgf5(go-Y)/Fgf5(go-Y) wal/wal appear even earlier than in the mice +/Fgf5(go-Y) wal/wal. By the end of the first month after birth, the majority of double homozygotes have a virtually bold back with preserved scarce long hairs, guard hairs. Alopecia covers also the sides and belly. However, the head retains its hair and the regions of thinned long hairs remain on the limbs and near the tail base. The data obtained demonstrate that gene Fgf5(go-Y) is a modifier of gene wal, as it enhances considerably the effect of gene wal. This appears in an earlier development of alopecia and its more pronounced progress in the mice with genotypes +/Fgf5(go-Y) wal/wal and, particularly, Fgf5(go-Y)/Fgf5(go-Y) wal/wal.

  4. Epigenetic signature and enhancer activity of the human APOE gene

    PubMed Central

    Yu, Chang-En; Cudaback, Eiron; Foraker, Jessica; Thomson, Zachary; Leong, Lesley; Lutz, Franziska; Gill, James Anthony; Saxton, Aleen; Kraemer, Brian; Navas, Patrick; Keene, C. Dirk; Montine, Thomas; Bekris, Lynn M.

    2013-01-01

    The human apolipoprotein E (APOE) gene plays an important role in lipid metabolism. It has three common genetic variants, alleles ɛ2/ɛ3/ɛ4, which translate into three protein isoforms of apoE2, E3 and E4. These isoforms can differentially influence total serum cholesterol levels; therefore, APOE has been linked with cardiovascular disease. Additionally, its ɛ4 allele is strongly associated with the risk of Alzheimer's disease (AD), whereas the ɛ2 allele appears to have a modest protective effect for AD. Despite decades of research having illuminated multiple functional differences among the three apoE isoforms, the precise mechanisms through which different APOE alleles modify diseases risk remain incompletely understood. In this study, we examined the genomic structure of APOE in search for properties that may contribute novel biological consequences to the risk of disease. We identify one such element in the ɛ2/ɛ3/ɛ4 allele-carrying 3′-exon of APOE. We show that this exon is imbedded in a well-defined CpG island (CGI) that is highly methylated in the human postmortem brain. We demonstrate that this APOE CGI exhibits transcriptional enhancer/silencer activity. We provide evidence that this APOE CGI differentially modulates expression of genes at the APOE locus in a cell type-, DNA methylation- and ɛ2/ɛ3/ɛ4 allele-specific manner. These findings implicate a novel functional role for a 3′-exon CGI and support a modified mechanism of action for APOE in disease risk, involving not only the protein isoforms but also an epigenetically regulated transcriptional program at the APOE locus driven by the APOE CGI. PMID:23892237

  5. Postexercise muscle cooling enhances gene expression of PGC-1α.

    PubMed

    Ihsan, Mohammed; Watson, Greig; Choo, Hui Cheng; Lewandowski, Paul; Papazzo, Annateresa; Cameron-Smith, David; Abbiss, Chris R

    2014-10-01

    This study aimed to investigate the influence of localized muscle cooling on postexercise vascular, metabolic, and mitochondrial-related gene expression. Nine physically active males performed 30 min of continuous running at 70% of their maximal aerobic velocity, followed by intermittent running to exhaustion at 100% maximal aerobic velocity. After exercise, subjects immersed one leg in a cold water bath (10°C, COLD) to the level of their gluteal fold for 15 min. The contralateral leg remained outside the water bath and served as control (CON). Core body temperature was monitored throughout the experiment, whereas muscle biopsies and muscle temperature (Tm) measurements were obtained from the vastus lateralis before exercise (PRE), immediately postexercise (POST-EX, Tm only), immediately after cooling, and 3 h postexercise (POST-3H). Exercise significantly increased core body temperature (PRE, 37.1°C ± 0.4°C vs POST-EX, 39.3°C ± 0.5°C, P < 0.001) and Tm in both CON (PRE, 33.9°C ± 0.7°C vs POST-EX, 39.1°C ± 0.5°C) and COLD legs (PRE, 34.2°C ± 0.9°C vs POST-EX, 39.4°C ± 0.3°C), respectively (P < 0.001). After cooling, Tm was significantly lower in COLD (28.9°C ± 2.3°C vs 37.0°C ± 0.8°C, P < 0.001) whereas PGC-1α messenger RNA expression was significantly higher in COLD at POST-3H (P = 0.014). Significant time effects were evident for changes in vascular endothelial growth factor (P = 0.038) and neuronal nitric oxide synthase (P = 0.019) expression. However, no significant condition effects between COLD and CON were evident for changes in both vascular endothelial growth factor and neuronal nitric oxide synthase expressions. These data indicate that an acute postexercise cooling intervention enhances the gene expression of PGC-1α and may therefore provide a valuable strategy to enhance exercise-induced mitochondrial biogenesis.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. Characterization of the enhancer element of the Danio rerio minor globin gene locus.

    PubMed

    Nefedochkina, Anastasia V; Petrova, Natalia V; Ioudinkova, Elena S; Kovina, Anastasia P; Iarovaia, Olga V; Razin, Sergey V

    2016-04-01

    In Danio rerio, the alpha- and beta-globin genes are present in two clusters: a major cluster located on chromosome 3 and a minor cluster located on chromosome 12. In contrast to the segregated alpha- and beta-globin gene domains of warm-blooded animals, in Danio rerio, each cluster contains both alpha- and beta-globin genes. Expression of globin genes present in the major cluster is controlled by an erythroid-specific enhancer similar to the major regulatory element of mammalian and avian alpha-globin gene domains. The enhancer controlling expression of the globin genes present in the minor locus has not been identified yet. Based on the distribution of epigenetic marks, we have selected two genomic regions that might harbor an enhancer of the minor locus. Using transient transfection of constructs with a reporter gene, we have demonstrated that a ~500-bp DNA fragment located ~1.7 Kb upstream of the αe4 gene possesses an erythroid-specific enhancer active with respect to promoters present in both the major and the minor globin gene loci of Danio rerio. The identified enhancer element harbors clustered binding sites for GATA-1, NF-E2, and EKLF similar to the enhancer of the major globin locus on chromosome 3. Both enhancers appear to have emerged as a result of independent evolution of a duplicated regulatory element present in an ancestral single alpha-/beta-globin locus that existed before teleost-specific genome duplication.

  12. 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.

  13. Standardized Plant Disease Evaluations will Enhance Resistance Gene Discovery

    USDA-ARS?s Scientific Manuscript database

    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...

  14. 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

  15. Master transcription factors and mediator establish super-enhancers at key cell identity genes.

    PubMed

    Whyte, Warren A; Orlando, David A; Hnisz, Denes; Abraham, Brian J; Lin, Charles Y; Kagey, Michael H; Rahl, Peter B; Lee, Tong Ihn; Young, Richard A

    2013-04-11

    Master transcription factors Oct4, Sox2, and Nanog bind enhancer elements and recruit Mediator to activate much of the gene expression program of pluripotent embryonic stem cells (ESCs). We report here that the ESC master transcription factors form unusual enhancer domains at most genes that control the pluripotent state. These domains, which we call super-enhancers, consist of clusters of enhancers that are densely occupied by the master regulators and Mediator. Super-enhancers differ from typical enhancers in size, transcription factor density and content, ability to activate transcription, and sensitivity to perturbation. Reduced levels of Oct4 or Mediator cause preferential loss of expression of super-enhancer-associated genes relative to other genes, suggesting how changes in gene expression programs might be accomplished during development. In other more differentiated cells, super-enhancers containing cell-type-specific master transcription factors are also found at genes that define cell identity. Super-enhancers thus play key roles in the control of mammalian cell identity. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. 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

  17. 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

  18. 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.

  19. [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.

  20. 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.

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

    PubMed

    Smallwood, Andrea; Ren, Bing

    2013-06-01

    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. Copyright © 2013. Published by Elsevier Ltd.

  2. 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

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

    DOEpatents

    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.

  4. 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.

  5. 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.

  6. 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, ...

  7. 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, ...

  8. 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

  9. 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).

  10. The coupling between enhancer activity and hypomethylation of kappa immunoglobulin genes is developmentally regulated

    SciTech Connect

    Kelley, D.E.; Pollok, B.A.; Atchison, M.L.; Perry, R.P.

    1988-02-01

    Previous studies have indicated that immunoglobulin enhancers are essential for establishing transcriptional competence but not for maintaining the activity of constitutively transcribed genes. To understand the basis for this developmental shift away from dependence on enhancer function, the authors investigated the relationship between transcriptional activity and methylation status of the immunoglobulin kappa-light-chain genes (kappa genes) in mouse cell lines representing different stages of B-cell maturation. Using pre-B-cell lines in which the level of a critical kappa enhancer-binding factor, NF-kappaB, was controlled by the administration of withdrawal of lipopolysaccharide and plasmacytoma lines that either contain or lack this factor, they studied the properties of endogenous kappa genes and of transfected kappa genes which were stably integrated into the genomes of these cells. In the pre-B cells, the exogenous (originally unmethylated) kappa genes, as well as the endogenous kappa genes, were fully methylated and persistently dependent on enhancer function, even after more than 30 generations in a transcriptionally active state. In plasmacytoma cells, the endogenous kappa genes were invariably hypomethylated, whereas exogenous kappa genes were hypomethylated only in cells that contain NF-kappaB and are thus permissive for kappa enhancer function. These results indicate that the linkage of hypomethylation to enhancer-dependent activation of kappa transcription occurs after the pre-B-cell stage of development. The change in methylation status, together with associated changes in chromatin structure, may suffice to eliminate or lessen the importance of the enhancer for the maintenance of the transcriptionally active state.

  11. Dual gene expression cassette is superior than single gene cassette for enhancing sheath blight tolerance in transgenic rice.

    PubMed

    Karmakar, Subhasis; Molla, Kutubuddin A; Das, Kaushik; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

    2017-08-11

    Sheath blight, caused by the necrotrophic fungal pathogen Rhizoctonia solani, is a serious and destructive disease of the rice. In order to improve sheath blight resistance, we developed three different kinds of transgenic rice lines. The first transgenic line overexpresses the rice chitinase gene (OsCHI11); the second contains the Arabidopsis NPR1 (AtNPR1) gene and, the third has pyramided constructs with both the genes (OsCHI11 and AtNPR1). This is a comparative study between the single-gene transgenic lines and the double gene transgenic in terms of their ability to activate the plant defense system. Rice plants of each individual construct were screened via PCR, Southern hybridization, activity assays, and expression analysis. The best transgenic lines of each construct were chosen for comparative study. The fold change in qRT-PCR and activity assays revealed that the pyramided transgenic rice plants show a significant upregulation of defense-related genes, PR genes, and antioxidant marker genes as compared to the single transgene. Simultaneous co-expression of both the genes was found to be more efficient in tolerating oxidative stress. In R. solani (RS) toxin assay, mycelial agar disc bioassay, and in vivo plant bioassay, pyramided transgenic plant lines were more competent at restricting the pathogen development and enhancing sheath blight tolerance as compared to single gene transformants.

  12. SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression.

    PubMed

    Cao, Kaixiang; Collings, Clayton K; Marshall, Stacy A; Morgan, Marc A; Rendleman, Emily J; Wang, Lu; Sze, Christie C; Sun, Tianjiao; Bartom, Elizabeth T; Shilatifard, Ali

    2017-04-15

    The homeotic (Hox) genes are highly conserved in metazoans, where they are required for various processes in development, and misregulation of their expression is associated with human cancer. In the developing embryo, Hox genes are activated sequentially in time and space according to their genomic position within Hox gene clusters. Accumulating evidence implicates both enhancer elements and noncoding RNAs in controlling this spatiotemporal expression of Hox genes, but disentangling their relative contributions is challenging. Here, we identify two cis-regulatory elements (E1 and E2) functioning as shadow enhancers to regulate the early expression of the HoxA genes. Simultaneous deletion of these shadow enhancers in embryonic stem cells leads to impaired activation of HoxA genes upon differentiation, while knockdown of a long noncoding RNA overlapping E1 has no detectable effect on their expression. Although MLL/COMPASS (complex of proteins associated with Set1) family of histone methyltransferases is known to activate transcription of Hox genes in other contexts, we found that individual inactivation of the MLL1-4/COMPASS family members has little effect on early Hox gene activation. Instead, we demonstrate that SET1A/COMPASS is required for full transcriptional activation of multiple Hox genes but functions independently of the E1 and E2 cis-regulatory elements. Our results reveal multiple regulatory layers for Hox genes to fine-tune transcriptional programs essential for development. © 2017 Cao et al.; Published by Cold Spring Harbor Laboratory Press.

  13. 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

  14. Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements.

    PubMed

    Mumbach, Maxwell R; Satpathy, Ansuman T; Boyle, Evan A; Dai, Chao; Gowen, Benjamin G; Cho, Seung Woo; Nguyen, Michelle L; Rubin, Adam J; Granja, Jeffrey M; Kazane, Katelynn R; Wei, Yuning; Nguyen, Trieu; Greenside, Peyton G; Corces, M Ryan; Tycko, Josh; Simeonov, Dimitre R; Suliman, Nabeela; Li, Rui; Xu, Jin; Flynn, Ryan A; Kundaje, Anshul; Khavari, Paul A; Marson, Alexander; Corn, Jacob E; Quertermous, Thomas; Greenleaf, William J; Chang, Howard Y

    2017-09-25

    The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.

  15. 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.

  16. Surface modification of nonviral nanocarriers for enhanced gene delivery.

    PubMed

    Fortier, Charles; Durocher, Yves; De Crescenzo, Gregory

    2014-01-01

    Biomedical nanotechnology has given a new lease of life to gene therapy with the ever-developing and ever-diversifying nonviral gene delivery nanocarriers. These are designed to pass a series of barriers in order to bring their nucleic acid cargo to the right subcellular location of particular cells. For a given application, each barrier has its dedicated strategy, which translates into a physicochemical, biological and temporal identity of the nanocarrier surface. Different strategies have thus been explored to implement adequate surface identities on nanocarriers over time for systemic delivery. In that context, this review will mainly focus on organic nanocarriers, for which these strategies will be described and discussed.

  17. Standardized plant disease evaluations will enhance resistance gene discovery

    USDA-ARS?s Scientific Manuscript database

    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...

  18. 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.

  19. Finding genes that enhance the nutritional value of rice

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  1. 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

  2. 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

  3. Overcoming Redundancy: An RNAi Enhancer Screen for Morphogenesis Genes in Caenorhabditis elegans

    PubMed Central

    Sawyer, Jacob M.; Glass, Stephanie; Li, Trudy; Shemer, Gidi; White, Noor D.; Starostina, Natalia G.; Kipreos, Edward T.; Jones, Corbin D.; Goldstein, Bob

    2011-01-01

    Morphogenesis is an important component of animal development. Genetic redundancy has been proposed to be common among morphogenesis genes, posing a challenge to the genetic dissection of morphogenesis mechanisms. Genetic redundancy is more generally a challenge in biology, as large proportions of the genes in diverse organisms have no apparent loss of function phenotypes. Here, we present a screen designed to uncover redundant and partially redundant genes that function in an example of morphogenesis, gastrulation in Caenorhabditis elegans. We performed an RNA interference (RNAi) enhancer screen in a gastrulation-sensitized double-mutant background, targeting genes likely to be expressed in gastrulating cells or their neighbors. Secondary screening identified 16 new genes whose functions contribute to normal gastrulation in a nonsensitized background. We observed that for most new genes found, the closest known homologs were multiple other C. elegans genes, suggesting that some may have derived from rounds of recent gene duplication events. We predict that such genes are more likely than single copy genes to comprise redundant or partially redundant gene families. We explored this prediction for one gene that we identified and confirmed that this gene and five close relatives, which encode predicted substrate recognition subunits (SRSs) for a CUL-2 ubiquitin ligase, do indeed function partially redundantly with each other in gastrulation. Our results implicate new genes in C. elegans gastrulation, and they show that an RNAi-based enhancer screen in C. elegans can be used as an efficient means to identify important but redundant or partially redundant developmental genes. PMID:21527776

  4. 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.

  5. 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

  6. Identification of the HIT-45 protein from Trypanosoma brucei as an FHIT protein/dinucleoside triphosphatase: substrate specificity studies on the recombinant and endogenous proteins.

    PubMed

    Banerjee, Hiren; Palenchar, Jennifer B; Lukaszewicz, Maciej; Bojarska, Elzbieta; Stepinski, Janusz; Jemielity, Jacek; Guranowski, Andrzej; Ng, Stephanie; Wah, David A; Darzynkiewicz, Edward; Bellofatto, Vivian

    2009-08-01

    A new member of the FHIT protein family, designated HIT-45, has been identified in the African trypanosome Trypanosoma brucei. Recombinant HIT-45 proteins were purified from trypanosomal and bacterial protein expression systems and analyzed for substrate specificity using various dinucleoside polyphosphates, including those that contain the 5'-mRNA cap, i.e., m(7)GMP. This enzyme exhibited typical dinucleoside triphosphatase activity (EC 3.6.1.29), having its highest specificity for diadenosine triphosphate (ApppA). However, the trypanosome enzyme contains a unique amino-terminal extension, and hydrolysis of cap dinucleotides with monomethylated guanosine or dimethylated guanosine always yielded m(7)GMP (or m(2,7)GMP) as one of the reaction products. Interestingly, m(7)Gpppm(3)(N6, N6, 2'O)A was preferred among the methylated substrates. This hypermethylated dinucleotide is unique to trypanosomes and may be an intermediate in the decay of cap 4, i.e., m(7)Gpppm(3)(N6, N6, 2'O)Apm(2'O)Apm(2'O)Cpm(2)(N3, 2'O)U, that occurs in these organisms.

  7. Gene therapy enhances chemotherapy tolerance and efficacy in glioblastoma patients.

    PubMed

    Adair, Jennifer E; Johnston, Sandra K; Mrugala, Maciej M; Beard, Brian C; Guyman, Laura A; Baldock, Anne L; Bridge, Carly A; Hawkins-Daarud, Andrea; Gori, Jennifer L; Born, Donald E; Gonzalez-Cuyar, Luis F; Silbergeld, Daniel L; Rockne, Russell C; Storer, Barry E; Rockhill, Jason K; Swanson, Kristin R; Kiem, Hans-Peter

    2014-09-01

    Temozolomide (TMZ) is one of the most potent chemotherapy agents for the treatment of glioblastoma. Unfortunately, almost half of glioblastoma tumors are TMZ resistant due to overexpression of methylguanine methyltransferase (MGMT(hi)). Coadministration of O6-benzylguanine (O6BG) can restore TMZ sensitivity, but causes off-target myelosuppression. Here, we conducted a prospective clinical trial to test whether gene therapy to confer O6BG resistance in hematopoietic stem cells (HSCs) improves chemotherapy tolerance and outcome. We enrolled 7 newly diagnosed glioblastoma patients with MGMT(hi) tumors. Patients received autologous gene-modified HSCs following single-agent carmustine administration. After hematopoietic recovery, patients underwent O6BG/TMZ chemotherapy in 28-day cycles. Serial blood samples and tumor images were collected throughout the study. Chemotherapy tolerance was determined by the observed myelosuppression and recovery following each cycle. Patient-specific biomathematical modeling of tumor growth was performed. Progression-free survival (PFS) and overall survival (OS) were also evaluated. Gene therapy permitted a significant increase in the mean number of tolerated O6BG/TMZ cycles (4.4 cycles per patient, P < 0.05) compared with historical controls without gene therapy (n = 7 patients, 1.7 cycles per patient). One patient tolerated an unprecedented 9 cycles and demonstrated long-term PFS without additional therapy. Overall, we observed a median PFS of 9 (range 3.5-57+) months and OS of 20 (range 13-57+) months. Furthermore, biomathematical modeling revealed markedly delayed tumor growth at lower cumulative TMZ doses in study patients compared with patients that received standard TMZ regimens without O6BG. These data support further development of chemoprotective gene therapy in combination with O6BG and TMZ for the treatment of glioblastoma and potentially other tumors with overexpression of MGMT. Clinicaltrials.gov NCT00669669. R01CA114218, R

  8. 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.

  9. 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

  10. Novelty Indicator for Enhanced Prioritization of Predicted Gene Ontology Annotations.

    PubMed

    Chicco, Davide; Palluzzi, Fernando; Masseroli, Marco

    2017-04-18

    Biomolecular controlled annotations have become pivotal in computational biology, because they allow scientists to analyze large amounts of biological data to better understand test results, and to infer new knowledge. Yet, biomolecular annotation databases are incomplete by definition, like our knowledge of biology, and might contain errors and inconsistent information. In this context, machine-learning algorithms able to predict and prioritize new annotations are both effective and efficient, especially if compared with time-consuming trials of biological validation. To limit the possibility that these techniques predict obvious and trivial high-level features, and to help prioritizing their results, we introduce a new element that can improve accuracy and relevance of the results of an annotation prediction and prioritization pipeline. We propose a novelty indicator able to state the level of "originality" of the annotations predicted for a specific gene to Gene Ontology (GO) terms. This indicator, joint with our previously introduced prediction steps, helps prioritizing the most novel interesting annotations predicted. We performed an accurate biological functional analysis of the prioritized annotations predicted with high accuracy by our indicator and previously proposed methods. The relevance of our biological findings proves effectiveness and trustworthiness of our indicator and of its prioritization of predicted annotations.

  11. Consequences of Eukaryotic Enhancer Architecture for Gene Expression Dynamics, Development, and Fitness

    PubMed Central

    Kittler, Ralf; White, Kevin P.; Kreitman, Martin

    2011-01-01

    The regulatory logic of time- and tissue-specific gene expression has mostly been dissected in the context of the smallest DNA fragments that, when isolated, recapitulate native expression in reporter assays. It is not known if the genomic sequences surrounding such fragments, often evolutionarily conserved, have any biological function or not. Using an enhancer of the even-skipped gene of Drosophila as a model, we investigate the functional significance of the genomic sequences surrounding empirically identified enhancers. A 480 bp long “minimal stripe element” is able to drive even-skipped expression in the second of seven stripes but is embedded in a larger region of 800 bp containing evolutionarily conserved binding sites for required transcription factors. To assess the overall fitness contribution made by these binding sites in the native genomic context, we employed a gene-replacement strategy in which whole-locus transgenes, capable of rescuing even-skipped- lethality to adulthood, were substituted for the native gene. The molecular phenotypes were characterized by tagging Even-skipped with a fluorescent protein and monitoring gene expression dynamics in living embryos. We used recombineering to excise the sequences surrounding the minimal enhancer and site-specific transgenesis to create co-isogenic strains differing only in their stripe 2 sequences. Remarkably, the flanking sequences were dispensable for viability, proving the sufficiency of the minimal element for biological function under normal conditions. These sequences are required for robustness to genetic and environmental perturbation instead. The mutant enhancers had measurable sex- and dose-dependent effects on viability. At the molecular level, the mutants showed a destabilization of stripe placement and improper activation of downstream genes. Finally, we demonstrate through live measurements that the peripheral sequences are required for temperature compensation. These results imply that

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. Power enhancement via multivariate outlier testing with gene expression arrays.

    PubMed

    Asare, Adam L; Gao, Zhong; Carey, Vincent J; Wang, Richard; Seyfert-Margolis, Vicki

    2009-01-01

    As the use of microarrays in human studies continues to increase, stringent quality assurance is necessary to ensure accurate experimental interpretation. We present a formal approach for microarray quality assessment that is based on dimension reduction of established measures of signal and noise components of expression followed by parametric multivariate outlier testing. We applied our approach to several data resources. First, as a negative control, we found that the Affymetrix and Illumina contributions to MAQC data were free from outliers at a nominal outlier flagging rate of alpha=0.01. Second, we created a tunable framework for artificially corrupting intensity data from the Affymetrix Latin Square spike-in experiment to allow investigation of sensitivity and specificity of quality assurance (QA) criteria. Third, we applied the procedure to 507 Affymetrix microarray GeneChips processed with RNA from human peripheral blood samples. We show that exclusion of arrays by this approach substantially increases inferential power, or the ability to detect differential expression, in large clinical studies. http://bioconductor.org/packages/2.3/bioc/html/arrayMvout.html and http://bioconductor.org/packages/2.3/bioc/html/affyContam.html affyContam (credentials: readonly/readonly)

  2. 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.

  3. PCR-free Quantification of Multiple Splice Variants in Cancer Gene by Surface Enhanced Raman Spectroscopy

    PubMed Central

    Sun, Lan; Irudayaraj, Joseph

    2009-01-01

    We demonstrate a surface enhanced Raman spectroscopy (SERS) based array platform to monitor gene expression in cancer cells in a multiplex and quantitative format without amplification steps. A strategy comprising of DNA/RNA hybridization, S1 nuclease digestion, and alkaline hydrolysis was adopted to obtain DNA targets specific to two splice junction variants Δ(9, 10) and Δ(5) of the breast cancer susceptibility gene 1 (BRCA1) from MCF-7 and MDA-MB-231 breast cancer cell lines. These two targets were identified simultaneously and their absolute quantities were estimated by a SERS strategy utilizing the inherent plasmon-phonon Raman mode of gold nanoparticle probes as a self-referencing standard to correct for variability in surface enhancement. Results were then validated by reverse transcription PCR (RT-PCR). Our proposed methodology could be expanded to a higher level of multiplexing for quantitative gene expression analysis of any gene without any amplification steps. PMID:19780515

  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. Activation-dependent intrachromosomal interactions formed by the TNF gene promoter and two distal enhancers

    PubMed Central

    Tsytsykova, Alla V.; Rajsbaum, Ricardo; Falvo, James V.; Ligeiro, Filipa; Neely, Simon R.; Goldfeld, Anne E.

    2007-01-01

    Here we provide a mechanism for specific, efficient transcription of the TNF gene and, potentially, other genes residing within multigene loci. We identify and characterize highly conserved noncoding elements flanking the TNF gene, which undergo activation-dependent intrachromosomal interactions. These elements, hypersensitive site (HSS)−9 and HSS+3 (9 kb upstream and 3 kb downstream of the TNF gene, respectively), contain DNase I hypersensitive sites in naive, T helper 1, and T helper 2 primary T cells. Both HSS-9 and HSS+3 inducibly associate with acetylated histones, indicative of chromatin remodeling, bind the transcription factor nuclear factor of activated T cells (NFAT)p in vitro and in vivo, and function as enhancers of NFAT-dependent transactivation mediated by the TNF promoter. Using the chromosome conformation capture assay, we demonstrate that upon T cell activation intrachromosomal looping occurs in the TNF locus. HSS-9 and HSS+3 each associate with the TNF promoter and with each other, circularizing the TNF gene and bringing NFAT-containing nucleoprotein complexes into close proximity. TNF gene regulation thus reveals a mode of intrachromosomal interaction that combines a looped gene topology with interactions between enhancers and a gene promoter. PMID:17940009

  6. Hairpin formation within the enhancer region of the human enkephalin gene

    SciTech Connect

    McMurray, C.T.; Douglass, J.O. ); Wilson, W.D. )

    1991-01-15

    The 3{prime},5{prime}-cyclic adenosine monophosphate (cAMP)-inducible enhancer of the human enkephaline gene is located within an imperfect palindrom of 23 base pairs. The authors have found that a 23-base-pair oligonucleotide duplex containing the enhancer undergoes a reversible conformational transition from the duplex to two individual hairpin structures each formed from one strand of the duplex. Each individual hairpin forms with mismatched base pairs, one containing two GT pairs and the other containing two AC pairs. The conformational transition is stabilized by proton transfer to the hairpin containing AC mismatched pairs. The unique physical and thermodynamic properties of the enkephalin enhancer DNA suggest a model in which DNA secondary structure within the enhancer region plays and active role incAMP-inducible activation of the human enkephalin gene via formation of cruciform structures.

  7. Inverted Quasi-Spherical Droplets on Polydopamine-TiO2 Substrates for Enhancing Gene Delivery.

    PubMed

    Kim, Seung-Hyun; Lee, Mihyun; Cho, Mira; Kim, Il-Sun; Park, Kook In; Lee, Haeshin; Jang, Jae-Hyung

    2017-08-15

    Devising efficient gene delivery systems is crucial to enhancing the therapeutic efficacy of gene-cell therapy approaches. Herein, inverted quasi-spherical (iQS) droplet systems, which enhance gene delivery efficiencies by reducing the path lengths of gene vectors, mediating motions of vectors at early stages, and raising the contact frequencies of vectors with cells, are developed by adopting the principle of 3D hanging-drop cell culture. Micrometer-sized polydopamine (pDA) holes are created on superhydrophobic titanium isopropoxide (TiO2 )-coated substrates by physical scraping; droplets are loaded on the pDA holes, and inversion of the substrate generates iQS droplets with large contact angles. Both human neural stem cells (hNSCs) and adeno-associated viral vectors are simultaneously incorporated into the iQS droplets to assess gene delivery efficiencies. The steep angles of iQS droplets and enhanced cell/vector contact frequencies facilitate the viral association with hNSCs and enhancing cell-cell interactions, thereby significantly promoting gene delivery efficiencies. Even with reduced viral quantities/exposure times and cell numbers, the iQS droplet systems elicit sufficient gene expression (i.e., interleukin-10). The ability of the iQS droplet systems to maximize beneficial gene delivery effects with minimal materials (e.g., medium, cells, and vectors) should enable their extensive use as a platform for preparing genetically stimulated cellular therapeutics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Enhancement of the Gene Targeting Efficiency of Non-Conventional Yeasts by Increasing Genetic Redundancy

    PubMed Central

    Chen, Zao; Sun, Hongbing; Li, Pengfei; He, Ning; Zhu, Taicheng; Li, Yin

    2013-01-01

    In contrast to model yeasts, gene targeting efficiencies of non-conventional yeasts are usually low, which greatly limits the research and applications of these organisms. In this study, we aimed to enhance the gene targeting efficiency of non-conventional yeasts by improving the fitness of mutant strains, particularly by increasing the genetic redundancy of host cells. To demonstrate this process, OCH1 gene deletion in Pichia pastoris was performed. Extra copies of the OCH1 gene on a helper plasmid were provided for the P. pastoris GS115 strain before the native OCH1 gene in the genomic DNA was knocked out. The redundancy in OCH1 gene significantly eliminated the growth defects of the och1 mutant and increased the deletion efficiency of the OCH1 gene by two orders of magnitude with the same length of homologous flanks. The same strategy was used to delete the KU70 and SGS1 genes. The targeting efficiencies of KU70 and SGS1 were increased by 1- and 23-fold, respectively. Therefore, this study provided an efficient strategy for the deletion of “stubborn” genes in non-conventional yeasts. This study further showed that cellular fitness is potentially an important factor that can limit the efficiency of gene targeting. PMID:23505447

  9. 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

  10. 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

  11. Heterogeneity of sucrose synthase genes in bean (Phaseolus vulgaris L.): evidence for a nodule-enhanced sucrose synthase gene.

    PubMed

    Silvente, Sonia; Camas, Alberto; Lara, Miguel

    2003-02-01

    Sucrose synthase (SS), the key sucrose hydrolytic enzyme (EC 2.4.1.13), plays an important role in N(2)-fixing nodule metabolism. It has also been proposed that N(2) fixation in soybean nodules could be mediated by the potential to metabolize sucrose. The isolation and characterization of a nodule-enhanced SS full-length cDNA clone from the bean Phaseolus vulgaris is reported here. Southern blot analysis indicated that there are at least two SS genes in beans. Using a 3' specific probe from this SS cDNA clone, it was possible to identify a nodule-enhanced SS gene (PvSSn), which is expressed almost exclusively in nodules. A second gene (PvSS), which is expressed in all tissues tested, was detected using a coding region probe. Nodule-enhanced PvSSn transcript levels, but not the enzyme activity or protein amount, is reduced during nodule development. These data indicated that this reduction could be due to a limitation on the carbon availability in the nodule. PvSSn expression is reduced in the asparagine-treated nodules. By contrast, PvSSn transcript levels in nodules increased in the presence of glutamine, allantoin and allopurinol. This result suggests a relationship between ureide transport and SS regulation and could help in understanding why the ureide transport mechanism is activated during nitrogen fixation in bean.

  12. 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.

  13. 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.

  14. Simulations of Enhancer Evolution Provide Mechanistic Insights into Gene Regulation

    PubMed Central

    Duque, Thyago; Samee, Md. Abul Hassan; Kazemian, Majid; Pham, Hannah N.; Brodsky, Michael H.; Sinha, Saurabh

    2014-01-01

    There is growing interest in models of regulatory sequence evolution. However, existing models specifically designed for regulatory sequences consider the independent evolution of individual transcription factor (TF)–binding sites, ignoring that the function and evolution of a binding site depends on its context, typically the cis-regulatory module (CRM) in which the site is located. Moreover, existing models do not account for the gene-specific roles of TF-binding sites, primarily because their roles often are not well understood. We introduce two models of regulatory sequence evolution that address some of the shortcomings of existing models and implement simulation frameworks based on them. One model simulates the evolution of an individual binding site in the context of a CRM, while the other evolves an entire CRM. Both models use a state-of-the art sequence-to-expression model to predict the effects of mutations on the regulatory output of the CRM and determine the strength of selection. We use the new framework to simulate the evolution of TF-binding sites in 37 well-studied CRMs belonging to the anterior–posterior patterning system in Drosophila embryos. We show that these simulations provide accurate fits to evolutionary data from 12 Drosophila genomes, which includes statistics of binding site conservation on relatively short evolutionary scales and site loss across larger divergence times. The new framework allows us, for the first time, to test hypotheses regarding the underlying cis-regulatory code by directly comparing the evolutionary implications of the hypothesis with the observed evolutionary dynamics of binding sites. Using this capability, we find that explicitly modeling self-cooperative DNA binding by the TF Caudal (CAD) provides significantly better fits than an otherwise identical evolutionary simulation that lacks this mechanistic aspect. This hypothesis is further supported by a statistical analysis of the distribution of intersite

  15. 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.

  16. Enhanced artemisinin yield by expression of rol genes in Artemisia annua.

    PubMed

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

    2015-10-29

    Despite of many advances in the treatment of malaria, it is still the fifth most prevalent disease worldwide and is one of the major causes of death in the developing countries which accounted for 584,000 deaths in 2013, as estimated by World Health Organization. Artemisinin from Artemisia annua is still one of the most effective treatments for malaria. Increasing the artemisinin content of A. annua plants by genetic engineering would improve the availability of this much-needed drug. In this regard, a high artemisinin-yielding hybrid of A. annua produced by the centre for novel agricultural products of the University of York, UK, was selected (artemisinin maximally 1.4 %). As rol genes are potential candidates of biochemical engineering, genetic transformation of A. annua with Agrobacterium tumefaciens GV3101 harbouring vectors with rol B and rol C genes was carried out with the objective of enhancement of artemisinin content. Transgenic lines produced were analysed by the LC-MS for quantitative analysis of artemisinin and analogues. These high artemisinin yielding transgenics were also analysed by real time quantitative PCR to find the molecular dynamics of artemisinin enhancement. Genes of artemisinin biosynthetic pathway were studied including amorphadiene synthase (ADS), cytochrome P450, (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1). Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis. Thus, real time qPCR analysis of the TAFR1 gene was carried out, and trichome density was determined. Transgenics of rol B gene showed two- to ninefold (the decimal adds nothing in the abstract, please simplify to two- to ninefold) increase in artemisinin, 4-12-fold increase in artesunate and 1.2-3-fold increase in dihydroartemisinin. Whereas in the case of rol C gene transformants, a fourfold increase in artemisinin, four to

  17. 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

  18. Gef gene therapy enhances the therapeutic efficacy of cytotoxics in colon cancer cells.

    PubMed

    Ortiz, Raúl; Prados, Jose; Melguizo, Consolación; Rama, Ana R; Alvarez, Pablo J; Rodríguez-Serrano, Fernando; Caba, Octavio; Boulaiz, Houria; Aranega, Antonia

    2012-10-01

    The potential use of gene therapy to improve the response of patients with advanced cancer is being intensively analyzed. We evaluated the cytotoxic impact of the gef gene, a suicide gene, which has a demonstrated antiproliferative activity in tumor cells, in colon carcinoma cells in order to improve the antitumour effect of chemotherapeutic drugs used as first line treatment in the management of advanced colon cancer. We found that the gef gene induced a marked decrease in cell viability (50% in 24h) in T-84 cells through cell death by apoptosis. Interestingly, when gef gene expression was combined with drugs of choice in the clinical treatment of colon cancer (5-fluorouracil, oxaliplatin and irinotecan), a strong synergistic effect was observed with approximately a 15-20% enhancement of the antiproliferative effect. Our data demonstrate, for the first time, that gef gene expression induces significant growth arrest in colon cancer cells and that it is able to enhance the effect of some cytotoxic drugs compared with a single therapeutic approach. These results indicate the potential therapeutic value of the gef gene in colon cancer combination therapy. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. An enhancement of binary particle swarm optimization for gene selection in classifying cancer classes

    PubMed Central

    2013-01-01

    Background Gene expression data could likely be a momentous help in the progress of proficient cancer diagnoses and classification platforms. Lately, many researchers analyze gene expression data using diverse computational intelligence methods, for selecting a small subset of informative genes from the data for cancer classification. Many computational methods face difficulties in selecting small subsets due to the small number of samples compared to the huge number of genes (high-dimension), irrelevant genes, and noisy genes. Methods We propose an enhanced binary particle swarm optimization to perform the selection of small subsets of informative genes which is significant for cancer classification. Particle speed, rule, and modified sigmoid function are introduced in this proposed method to increase the probability of the bits in a particle’s position to be zero. The method was empirically applied to a suite of ten well-known benchmark gene expression data sets. Results The performance of the proposed method proved to be superior to other previous related works, including the conventional version of binary particle swarm optimization (BPSO) in terms of classification accuracy and the number of selected genes. The proposed method also requires lower computational time compared to BPSO. PMID:23617960

  4. 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.

  5. Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription

    PubMed Central

    Lam, Michael T.Y.; Cho, Han; Lesch, Hanna P.; Gosselin, David; Heinz, Sven; Tanaka-Oishi, Yumiko; Benner, Christopher; Kaikkonen, Minna U.; Kim, Aneeza S.; Kosaka, Mika; Lee, Cindy Y.; Watt, Andy; Grossman, Tamar R.; Rosenfeld, Michael G.; Evans, Ronald M.; Glass, Christopher K.

    2013-01-01

    Rev-Erbα and Rev-Erbβ are nuclear receptors that regulate the expression of genes involved in the control of circadian rhythm1,2, metabolism3,4, and inflammatory responses5. Rev-Erbs function as transcriptional repressors by recruiting NCoR/HDAC3 co-repressor complexes to Rev-Erb response elements in enhancers and promoters of target genes6-8, but the molecular basis for cell-specific programs of repression is not known. Here, we present evidence that in macrophages, Rev-Erbs regulate target gene expression by inhibiting the functions of distal enhancers that are selected by macrophage lineage-determining factors, thereby establishing a macrophage-specific program of repression. Remarkably, the repressive functions of Rev-Erbs are associated with their ability to inhibit the transcription of enhancer-derived RNAs (eRNAs). Furthermore, targeted degradation of eRNAs at two enhancers subject to negative regulation by Rev-Erbs resulted in reduced expression of nearby mRNAs, implying a direct role of these eRNAs in enhancer function. By precisely defining eRNA start sites using a method that quantifies nascent 5′ ends (5′-GRO-Seq), we show that transfer of full enhancer activity to a target promoter requires both the sequences mediating transcription factor binding and the specific sequences encoding the eRNA transcript. These studies provide evidence for direct roles of eRNAs in contributing to enhancer functions and suggest that Rev-Erbs act to suppress gene expression at a distance by repressing eRNA transcription. PMID:23728303

  6. Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes.

    PubMed

    Yamamoto, Keisuke; Hara, Kiyotaka Y; Morita, Toshihiko; Nishimura, Akira; Sasaki, Daisuke; Ishii, Jun; Ogino, Chiaki; Kizaki, Noriyuki; Kondo, Akihiko

    2016-09-13

    Red yeast, Xanthophyllomyces dendrorhous is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) widely used in the aquaculture, food, pharmaceutical and cosmetic industries. The potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Recently, we developed a multiple gene expression system in X. dendrorhous and enhanced the mevalonate synthetic pathway to increase astaxanthin production. In contrast, the mevalonate synthetic pathway is suppressed by ergosterol through feedback inhibition. Therefore, releasing the mevalonate synthetic pathway from this inhibition through the deletion of genes involved in ergosterol synthesis is a promising strategy to improve isoprenoid production. An efficient method for deleting diploid genes in X. dendrorhous, however, has not yet been developed. Xanthophyllomyces dendrorhous was cultivated under gradually increasing concentrations of antibiotics following the introduction of antibiotic resistant genes to be replaced with target genes. Using this method, double CYP61 genes encoding C-22 sterol desaturases relating to ergosterol biosynthesis were deleted sequentially. This double CYP61 deleted strain showed decreased ergosterol biosynthesis compared with the parental strain and single CYP61 disrupted strain. Additionally, this double deletion of CYP61 genes showed increased astaxanthin production compared with the parental strain and the single CYP61 knockout strain. Finally, astaxanthin production was enhanced by 1.4-fold compared with the parental strain, although astaxanthin production was not affected in the single CYP61 knockout strain. In this study, we developed a system to completely delete target diploid genes in X. dendrorhous. Using this method, we deleted diploid CYP61 genes involved in the synthesis of ergosterol that inhibits the pathway for mevalonate, which is a common

  7. Expression of methylation-modulated tumor-related genes in endoscopically resected early esophageal squamous neoplasia

    PubMed Central

    Hosoda, Kohei; Yashima, Kazuo; Tamoto, Akihiro; Yamamoto, Sohei; Kawata, Soichiro; Ikebuchi, Yuichiro; Matsumoto, Kazuya; Kawaguchi, Koichiro; Harada, Kenichi; Murawaki, Yoshikazu; Isomoto, Hajime

    2017-01-01

    Smoking and alcohol consumption are major risk factors for the development of esophageal squamous cell carcinoma (ESCC). Recent studies have demonstrated that smoking and alcohol consumption may be associated with altered DNA methylation in human cancer development. The aim of the present study was to evaluate methylation-modulated protein expression of tumor-related genes (TRGs) in the early stages of esophageal squamous neoplasia (ESN). ESN tissue samples (n=141) comprising 19 cases of low-grade intraepithelial neoplasia (LGIN), 70 of high-grade intraepithelial neoplasia/carcinoma in situ (HGIN/CIS) and 52 of invasive cancer, were endoscopically resected. The methylation-modulated protein expression of 5 TRGs [fragile histidine triad (FHIT), E-cadherin, MutL homolog 1 (MLH1) /MutS homolog 2 (MSH2) and cyclooxygenase-2 (COX-2)] as well as p53 was examined with immunohistochemistry, and their expression was compared with patient clinicopathological characteristics. Reduced or loss of FHIT, E-cadherin, MLH1/MSH2 and COX-2 expression was detected in 26.3 (5/19), 5.3 (1/19), 0 (0/19) and 63.2% (12/19) of LGIN cases, 61.4 (43/70), 18.6 (13/70), 7.1 (5/70) and 65.7% (46/70) of HGIN/CIS cases, and 78.8 (41/52), 50.0 (26/52), 11.5 (6/52) and 59.6% (31/52) of invasive cancer cases, respectively. Reduced or absent expression of FHIT and E-cadherin was significantly associated with neoplastic progression (FHIT, P=0.0007; E-cadherin, P=0.00014). The mean number of TRGs (FHIT, E-cadherin, MLH1/MSH2, and COX-2) that exhibited reduced or absent expression in LGIN, HGIN/CIS and invasive cancer specimens was 1.12±0.61, 1.66±0.93 and 2.09±0.96, respectively, demonstrating a significant stepwise increment from LGIN to HGIN/CIS and then to invasive cancer (P<0.05). p53 overexpression was frequently detected in ESN with head and neck carcinomas. However p53 overexpression was not significantly associated with ESN progression. An increase in the number of the 5 TRG proteins with

  8. 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

  9. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. 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.

  11. 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.

  12. 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

  13. Isolation of the gene encoding the Hin recombinational enhancer binding protein.

    PubMed Central

    Johnson, R C; Ball, C A; Pfeffer, D; Simon, M I

    1988-01-01

    In vitro DNA inversion mediated by the protein Hin requires the presence of a recombinational enhancer sequence located in cis relative to the recombination sites and a protein, Fis, which binds to the enhancer. We have cloned and determined the primary sequence of the gene encoding Fis. The deduced amino acid sequence of Fis indicates that the protein is 98 amino acids long and contains a potential helix-turn-helix DNA binding motif at its carboxyl terminus. The gene encoding Fis maps at 72 min on the Escherichia coli chromosome. The construction of mutant strains of E. coli that lack a functional fis gene demonstrates that Fis is not essential for cell growth under laboratory conditions but is required for high rates of Hin-mediated site-specific inversion in vivo. PMID:2835774

  14. DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes.

    PubMed

    Guha, Mithu; Saare, Mario; Maslovskaja, Julia; Kisand, Kai; Liiv, Ingrid; Haljasorg, Uku; Tasa, Tõnis; Metspalu, Andres; Milani, Lili; Peterson, Pärt

    2017-04-21

    The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Host-Induced Silencing of Pathogenicity Genes Enhances Resistance to Fusarium oxysporum Wilt in Tomato.

    PubMed

    Bharti, Poonam; Jyoti, Poonam; Kapoor, Priya; Sharma, Vandana; Shanmugam, V; Yadav, Sudesh Kumar

    2017-08-01

    This study presents a novel approach of controlling vascular wilt in tomato by RNAi expression directed to pathogenicity genes of Fusarium oxysporum f. sp. lycopersici. Vascular wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici leads to qualitative and quantitative loss of the crop. Limitation in the existing control measures necessitates the development of alternative strategies to increase resistance in the plants against pathogens. Recent findings paved way to RNAi, as a promising method for silencing of pathogenicity genes in fungus and provided effective resistance against fungal pathogens. Here, two important pathogenicity genes FOW2, a Zn(II)2Cys6 family putative transcription regulator, and chsV, a putative myosin motor and a chitin synthase domain, were used for host-induced gene silencing through hairpinRNA cassettes of these genes against Fusarium oxysporum f. sp. lycopersici. HairpinRNAs were assembled in appropriate binary vectors and transformed into tomato plant targeting FOW2 and chsV genes, for two highly pathogenic strains of Fusarium oxysporum viz. TOFOL-IHBT and TOFOL-IVRI. Transgenic tomatoes were analyzed for possible attainment of resistance in transgenic lines against fungal infection. Eight transgenic lines expressing hairpinRNA cassettes showed trivial disease symptoms after 6-8 weeks of infection. Hence, the host-induced posttranscriptional gene silencing of pathogenicity genes in transgenic tomato plants has enhanced their resistance to vascular wilt disease caused by Fusarium oxysporum.

  16. Scaffold-fused riboregulators for enhanced gene activation in Synechocystis sp. PCC 6803.

    PubMed

    Sakai, Yuta; Abe, Koichi; Nakashima, Saki; Ellinger, James J; Ferri, Stefano; Sode, Koji; Ikebukuro, Kazunori

    2015-08-01

    Cyanobacteria are an attractive host for biofuel production because they can produce valuable chemical compounds from CO2 fixed by photosynthesis. However, the available genetic tools that enable precise gene regulation for the applications of synthetic biology are insufficient. Previously, we engineered an RNA-based posttranscriptional regulator, termed riboregulator, for the control of target gene expression in cyanobacterium Synechocystis sp. PCC 6803. Moreover, we enhanced the gene regulation ability of the riboregulators in Escherichia coli by fusing and engineering a scaffold sequence derived from naturally occurring E. coli noncoding small RNAs. Here, we demonstrated that the scaffold sequence fused to the riboregulators improved their gene regulation ability in Synechocystis sp. PCC 6803. To further improve gene regulation, we expressed an exogenous RNA chaperone protein that is responsible for noncoding small RNA-mediated gene regulation, which resulted in higher target gene expression. The scaffold sequence derived from natural E. coli noncoding small RNAs is effective for designing RNA-based genetic tools and scaffold-fused riboregulators are a strong RNA-tool to regulate gene expression in cyanobacteria. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  17. 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

  18. Reduced expression of Paternally Expressed Gene-3 enhances somatic cell reprogramming through mitochondrial activity perturbation.

    PubMed

    Theka, Ilda; Sottile, Francesco; Aulicino, Francesco; Garcia, Alvaro Castells; Cosma, Maria Pia

    2017-08-29

    Imprinted genes control several cellular and metabolic processes in embryonic and adult tissues. In particular, paternally expressed gene-3 (Peg3) is active in the adult stem cell population and during muscle and neuronal lineage development. Here we have investigated the role of Peg3 in mouse embryonic stem cells (ESCs) and during the process of somatic cell reprogramming towards pluripotency. Our data show that Peg3 knockdown increases expression of pluripotency genes in ESCs and enhances reprogramming efficiency of both mouse embryonic fibroblasts and neural stem cells. Interestingly, we observed that altered activity of Peg3 correlates with major perturbations of mitochondrial gene expression and mitochondrial function, which drive metabolic changes during somatic cell reprogramming. Overall, our study shows that Peg3 is a regulator of pluripotent stem cells and somatic cell reprogramming.

  19. 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

  20. Propranolol enhances cell cycle-related gene expression in pressure overloaded hearts

    PubMed Central

    Musumeci, Marco; Maccari, Sonia; Sestili, Paola; Signore, Michele; Molinari, Paola; Ambrosio, Caterina; Stati, Tonino; Colledge, William H; Grace, Andrew A; Catalano, Liviana; Marano, Giuseppe

    2011-01-01

    BACKGROUND AND PURPOSE Cell cycle regulators are regarded as essential for cardiomyocyte hypertrophic growth. Given that the β-adrenoceptor antagonist propranolol blunts cardiomyocyte hypertrophic growth, we determined whether propranolol alters the expression of cell cycle-related genes in mouse hearts subjected to pressure overload. EXPERIMENTAL APPROACH Pressure overload was induced by transverse aortic constriction (TAC), whereas the expression levels of 84 cell cycle-related genes were assayed by real-time PCR. Propranolol (80 mg·kg−1·day−1) was administered in drinking water for 14 days. KEY RESULTS Two weeks after surgery, TAC caused a 46% increase in the left ventricular weight-to-body weight (LVW/BW) ratio but no significant changes in cell cycle gene expression. Propranolol, at plasma concentrations ranging from 10 to 140 ng·mL−1, blunted the LVW/BW ratio increase in TAC mice, while significantly increasing expression of 10 cell cycle genes including mitotic cyclins and proliferative markers such as Ki67. This increase in cell cycle gene expression was paralleled by a significant increase in the number of Ki67-positive non-cardiomyocyte cells as revealed by immunohistochemistry and confocal microscopy. β-Adrenoceptor signalling was critical for cell cycle gene expression changes, as genetic deletion of β-adrenoceptors also caused a significant increase in cyclins and Ki67 in pressure overloaded hearts. Finally, we found that metoprolol, a β1-adrenoceptor antagonist, failed to enhance cell cycle gene expression in TAC mice. CONCLUSIONS AND IMPLICATIONS Propranolol treatment enhances cell cycle-related gene expression in pressure overloaded hearts by increasing the number of cycling non-cardiomyocyte cells. These changes seem to occur via β2-adrenoceptor-mediated mechanisms. PMID:21615725

  1. 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.

  2. 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.

  3. 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.

  4. 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

  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. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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.

  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. Synergistic enhansons located within an acute phase responsive enhancer modulate glucocorticoid induction of angiotensinogen gene transcription.

    PubMed

    Brasier, A R; Ron, D; Tate, J E; Habener, J F

    1990-12-01

    The hepatic transcription of the angiotensinogen gene is regulated by both glucocorticoids and cytokines generated as products of the acute phase reaction. We have identified a multimodular enhancer in the 5'-flanking region of the rat angiotensinogen gene that mediates these responses and consists of an acute phase response element (APRE) flanked on both sides by adjacent glucocorticoid response element consensus motifs (GREs). Induction of transcription by the cytokine interleukin-1 (IL-1) is glucocorticoid dependent and mediated through the APRE. The APRE binds in a mutually exclusive manner a cytokine/phorbol ester-inducible protein (BPi), indistinguishable from nuclear factor kB, and a family of constitutive liver proteins (BPcs) related to the heat-stable transcription factor C/EBP. Using mutated 5'-flanking sequences of the angiotensinogen gene fused to a firefly luciferase reporter gene transfected into hepatoblastoma (HepG2) cells, we have mapped enhanson sequences required for the transcriptional response to glucocorticoids. Two functionally distinct GREs are identified by deletion and site-directed mutagenesis, both of which mediate glucocorticoid-stimulated transcription in vivo. Glucocorticoid-induced transcription mediated by the angiotensinogen gene enhancer is, furthermore, dependent on the occupancy of the APRE by either the BPi or a member of the BPc family because a mutant APRE that binds neither BPi nor BPc exhibits an attenuated glucocorticoid responsiveness. Mutant APREs that permit exclusive binding of either BPi or BPc synergistically transmit the glucocorticoid response mediated by one or the other of the adjacent GREs. Thus, the induction of angiotensinogen gene transcription involves interaction between the glucocorticoid receptor and either one of the APRE-binding proteins: either the cytokine-inducible NFkB or the constitutive family of C/EBP-like proteins, bound to adjacent enhansons in a mutually synergistic enhancer complex.

  14. Human coxsackie adenovirus receptor (CAR) expression in transgenic mouse prostate tumors enhances adenoviral delivery of genes.

    PubMed

    Bao, Yunhua; Peng, Weidan; Verbitsky, Amy; Chen, Jiping; Wu, Lily; Rauen, Katherine A; Sawicki, Janet A

    2005-09-01

    Transgenic mice that recapitulate the progression of human diseases are potentially useful models for testing the effectiveness of new therapeutic strategies. Their use in pre-clinical testing of adenovirally-delivered gene therapies, however, is limited because of restricted cell surface expression of Coxsackie adenovirus receptor (CAR) in mice. To develop a more suitable transgenic mouse model for testing adenoviral-based gene therapies for prostate cancer, we generated prostate specific antigen/human CAR (PSA/hCAR) transgenic mice in which a chimeric enhancer/promoter sequence of the human PSA gene drives expression of a functional hCAR coding sequence. Expression of an adenovirally-delivered luciferase reporter gene in prostate tumor cells in bigenic mice (PSA/hCAR + TRAMP) was enhanced compared to the level in tumor cells lacking the PSA/hCAR transgene. Breeding PSA/hCAR mice to existing transgenic mouse models for prostate cancer (e.g., TRAMP) results in improved mouse models for testing adenovirally-delivered therapeutic genes. Copyright 2005 Wiley-Liss, Inc.

  15. Combinatorial control of temporal gene expression in the Drosophila wing by enhancers and core promoters

    PubMed Central

    2012-01-01

    Background The transformation of a developing epithelium into an adult structure is a complex process, which often involves coordinated changes in cell proliferation, metabolism, adhesion, and shape. To identify genetic mechanisms that control epithelial differentiation, we analyzed the temporal patterns of gene expression during metamorphosis of the Drosophila wing. Results We found that a striking number of genes, approximately 50% of the Drosophila transcriptome, exhibited changes in expression during a time course of wing development. While cis-acting enhancer sequences clearly correlated with these changes, a stronger correlation was discovered between core-promoter types and the dynamic patterns of gene expression within this differentiating tissue. In support of the hypothesis that core-promoter type influences the dynamics of expression, expression levels of several TATA-box binding protein associated factors (TAFs) and other core promoter-associated components changed during this developmental time course, and a testes-specific TAF (tTAF) played a critical role in timing cellular differentiation within the wing. Conclusions Our results suggest that the combinatorial control of gene expression via cis-acting enhancer sequences and core-promoter types, determine the complex changes in gene expression that drive morphogenesis and terminal differentiation of the Drosophila wing epithelium. PMID:22992320

  16. Biosurfactant MEL-A enhances cellular association and gene transfection by cationic liposome.

    PubMed

    Igarashi, Saki; Hattori, Yoshiyuki; Maitani, Yoshie

    2006-05-30

    Mannnosylerythritol lipid A (MEL-A), a biosurfactant produced by microorganisms, has many biological activities. To enhance the gene transfection efficiency of a cationic liposome, we prepared a MEL-liposome (MEL-L) composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), dioleoyl phosphatidylethanolamine (DOPE) and MEL-A, and investigated its transfection efficiency in human cervix carcinoma Hela cells. MEL-L was about 40 nm in size, and the MEL-L/plasmid DNA complex (MEL-lipoplex) remained an injectable size (169 nm). MEL-A induced a significantly higher level of gene expression, compared to commercially available Tfx20 and the liposome without MEL-A (Cont-L). Analysis of flow cytometric profiles clearly indicated that the amount of DNA associated with the cells was rapidly increased and sustained by addition of MEL-A to the liposome. Confocal microscopic observation indicated that the MEL-lipoplex distributed widely in the cytoplasm, and the DNA was detected strongly in the cytoplasm and around the nucleus, compared with Cont-L. These results suggested that MEL-A increased gene expression by enhancing the association of the lipoplexes with the cells in serum. MEL-L might prove a remarkable non-viral vector for gene transfection and gene therapy.

  17. Proteasome Inhibitors Enhance Bacteriophage Lambda (λ) Mediated Gene Transfer in Mammalian Cells

    PubMed Central

    Volcy, Ketna; Dewhurst, Stephen

    2009-01-01

    Bacteriophage lambda vectors can transfer their genomes into mammalian cells, resulting in expression of phage-encoded genes. However, this process is inefficient. Experiments were therefore conducted to delineate the rate limiting step(s) involved, using a phage vector that contains a mammalian luciferase reporter gene cassette. The efficiency of phage-mediated gene transfer in mammalian cells was quantitated, in the presence or absence of pharmacologic inhibitors of cell uptake and degradation pathways. Inhibitors of lysosomal proteases and proteasome inhibitors strongly enhanced phage-mediated luciferase expression, suggesting that these pathways contribute to the destruction of intracellular phage particles. In contrast, inhibition of endosome acidification had no effect on phage-mediated gene transfer, presumably because phage lambda is tolerant to extended exposure to low pH. These findings provide insights into the pathways by which phage vectors enter and transduce mammalian cells, and suggest that it may be possible to pharmacologically enhance the efficiency of phage-mediated gene transfer in mammalian cells. Finally, the data also suggest that the proteasome complex may serve as an innate defense mechanism that restricts the infection of mammalian cells by diverse viral agents. PMID:19064273

  18. 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

  19. 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

  20. 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.

  1. 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

  2. Salicylic Acid-Regulated Antioxidant Mechanisms and Gene Expression Enhance Rosemary Performance under Saline Conditions.

    PubMed

    El-Esawi, Mohamed A; Elansary, Hosam O; El-Shanhorey, Nader A; Abdel-Hamid, Amal M E; Ali, Hayssam M; Elshikh, Mohamed S

    2017-01-01

    Salinity stress as a major agricultural limiting factor may influence the chemical composition and bioactivity of Rosmarinus officinallis L. essential oils and leaf extracts. The application of salicylic acid (SA) hormone may alleviate salinity stress by modifying the chemical composition, gene expression and bioactivity of plant secondary metabolites. In this study, SA was applied to enhance salinity tolerance in R. officinallis. R. officinallis plants were subjected to saline water every 2 days (640, 2,000, and 4,000 ppm NaCl) and 4 biweekly sprays of SA at 0, 100, 200, and 300 ppm for 8 weeks. Simulated salinity reduced all vegetative growth parameters such as plant height, plant branches and fresh and dry weights. However, SA treatments significantly enhanced these plant growth and morphological traits under salinity stress. Salinity affected specific major essential oils components causing reductions in α-pinene, β-pinene, and cineole along with sharp increases in linalool, camphor, borneol, and verbenone. SA applications at 100-300 ppm largely reversed the effects of salinity. Interestingly, SA treatments mitigated salinity stress effects by increasing the total phenolic, chlorophyll, carbohydrates, and proline contents of leaves along with decline in sodium and chloride. Importantly, this study also proved that SA may stimulate the antioxidant enzymatic mechanism pathway including catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX) as well as increasing the non-enzymatic antioxidants such as free and total ascorbate in plants subjected to salinity. Quantitative real-time PCR analysis revealed that APX and 3 SOD genes showed higher levels in SA-treated rosemary under salinity stress, when compared to non-sprayed plants. Moreover, the expression level of selected genes conferring tolerance to salinity (bZIP62, DREB2, ERF3, and OLPb) were enhanced in SA-treated rosemary under salt stress, indicating that SA treatment resulted in the

  3. 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.

  4. 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. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

  5. 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.

  6. Addiction-related gene regulation: risks of exposure to cognitive enhancers vs. other psychostimulants.

    PubMed

    Steiner, Heinz; Van Waes, Vincent

    2013-01-01

    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 two 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. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. 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

  8. Enhancer Variants Synergistically Drive Dysfunction of a Gene Regulatory Network In Hirschsprung Disease

    SciTech Connect

    Chatterjee, Sumantra; Kapoor, Ashish; Akiyama, Jennifer A.; Auer, Dallas R.; Lee, Dongwon; Gabriel, Stacey; Berrios, Courtney; Pennacchio, Len A.; Chakravarti, Aravinda

    2016-10-01

    Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidence that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders.

  9. Circadian Enhancers Coordinate Multiple Phases of Rhythmic Gene Transcription In Vivo

    PubMed Central

    Fang, Bin; Everett, Logan J.; Jager, Jennifer; Briggs, Erika; Armour, Sean M.; Feng, Dan; Roy, Ankur; Gerhart-Hines, Zachary; Sun, Zheng; Lazar, Mitchell A.

    2014-01-01

    SUMMARY Mammalian transcriptomes display complex circadian rhythms with multiple phases of gene expression that cannot be accounted for by current models of the molecular clock. We have determined the underlying mechanisms by measuring nascent RNA transcription around the clock in mouse liver. Unbiased examination of eRNAs that cluster in specific circadian phases identified functional enhancers driven by distinct transcription factors (TFs). We further identify on a global scale the components of the TF cistromes that function to orchestrate circadian gene expression. Integrated genomic analyses also revealed novel mechanisms by which a single circadian factor controls opposing transcriptional phases. These findings shed new light on the diversity and specificity of TF function in the generation of multiple phases of circadian gene transcription in a mammalian organ. PMID:25416951

  10. 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.

  11. [Investigation of transcriptional regulation of Oct4 (POU5F1) gene with distal enhancer].

    PubMed

    Nazarov, I B; Krasnoborova, V A; Mittenberg, A G; Chikhirzhina, E V; Davydov-Sinitsyn, A P; Liskovykh, M A; Tomilin, A N

    2013-01-01

    Investigations of transcriptional regulation of Oct4 gene in mouse embryonic stem cells have revealed an important cis-element--the distal enhancer (DE). DE consists of two functionally significant elements--DEa and DEb. Both elements are necessary to complete the DE-mediated expression of Oct4 gene in pluripotent cells. The most likely candidates for the binding site DEb are Oct4 itself in complex with Sox2 protein. It remains unclear which transcriptional proteins bind to the DEa site and what is the mechanism of the co-operation between the DEa and the DEb. Through the use of using the EMSA and chromatographic fractionation of proteins from extracts of mouse embryonic stem cells and mouse tissues, were isolated proteins specifically interacting with the sequence DEa Oct4 gene.

  12. 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.

  13. 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.

  14. 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

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

    PubMed

    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.

  16. Knockdown of Broad-Complex Gene Expression of Bombyx mori by Oligopyrrole Carboxamides Enhances Silk Production.

    PubMed

    Ali, Asfa; Bovilla, Venugopal Reddy; Mysarla, Danti Kumari; Siripurapu, Prasanthi; Pathak, Rashmi U; Basu, Bhakti; Mamillapalli, Anitha; Bhattacharya, Santanu

    2017-04-11

    Bombyx mori (B. mori) is important due to its major role in the silk production. Though DNA binding ligands often influence gene expression, no attempt has been made to exploit their use in sericulture. The telomeric heterochromatin of B. mori is enriched with 5'-TTAGG-3' sequences. These sequences were also found to be present in several genes in the euchromatic regions. We examined three synthetic oligopyrrole carboxamides that target 5'-TTAGG-3' sequences in controlling the gene expression in B. mori. The ligands did not show any defect or feeding difference in the larval stage, crucial for silk production. The ligands caused silencing of various isoforms of the broad-complex transcription factor and cuticle proteins which resulted in late pupal developmental defects. Furthermore, treatment with such drugs resulted in statistically enhanced cocoon weight, shell weight, and silk yield. This study shows for the first time use of oligopyrrole carboxamide drugs in controlling gene expression in B. mori and their long term use in enhancing silk production.

  17. Overexpression of Muscadinia rotundifolia CBF2 gene enhances biotic and abiotic stress tolerance in Arabidopsis.

    PubMed

    Wu, Jiao; Folta, Kevin M; Xie, Yifan; Jiang, Wenming; Lu, Jiang; Zhang, Yali

    2017-01-01

    C-repeat-binding factor dehydration-responsive element-binding factor 1C (CBF2/DREB1C) gene encodes a small family of transcriptional activator that has been described as playing an important role in freezing tolerance and cold acclimation of plants. We here report that CBF2 gene also plays an important role in the early response to the pathogen infection of grapevine downy mildew disease. The expression level of CBF2 increased dramatically and reached a peak at 7 h after infection in immune grapevine Muscadinia rotundifolia 'Noble', which was much faster than moderate resistant Vitis amurensis 'PI1288' and susceptible Vitis vinifera 'Cabernet Sauvignon'. Muscadinia rotundifolia MrCBF2 exhibited amino acid domains characteristic of Vitis CBF2 proteins with unique features including rich serine repeats and slight differences in NLS, DSAWRL, and AP2 domains. The MrCBF2 gene was introduced to Arabidopsis 'COL0' which are susceptible to downy mildew pathogen. The transgenic lines showed an increased resistance to downy mildew disease and more accumulation of SA as well as higher expression of pathogenesis-related (PR) genes (AtPR1, AtPR4, and AtPR5) as a consequence of MrCBF2 overexpression. Besides, constitutive expression of MrCBF2 enhanced phytohormone abscisic acid (ABA)-independent drought tolerance of transgenic plants. Freezing tolerance of transgenic lines was also enhanced accompanied with an increase in the expression of the cold-regulated genes AtCOR, AtCOR15A, AtKIN1, AtRD29A, and AtSuSy. In addition, the development of MrCBF2-overexpressing plants was seen to be altered and resulted in growth retardation, dwarfism, late flowering, and prone rosette leaves, which may be because of an increase in the gene expression of partial DELLA proteins and DDF1.

  18. Soybean GmbZIP123 gene enhances lipid content in the seeds of transgenic Arabidopsis plants.

    PubMed

    Song, Qing-Xin; Li, Qing-Tian; Liu, Yun-Feng; Zhang, Feng-Xia; Ma, Biao; Zhang, Wan-Ke; Man, Wei-Qun; Du, Wei-Guang; Wang, Guo-Dong; Chen, Shou-Yi; Zhang, Jin-Song

    2013-11-01

    Soybean is one of most important oil crops and a significant increase in lipid content in soybean seeds would facilitate vegetable oil production in the world. Although the pathways for lipid biosynthesis in higher plants have been uncovered, our understanding of regulatory mechanism controlling lipid accumulation is still limited. In this study, we identified 87 transcription factor genes with a higher abundance at the stage of lipid accumulation in soybean seeds. One of these genes, GmbZIP123, was selected to further study its function in regulation of lipid accumulation. Overexpression of GmbZIP123 enhanced lipid content in the seeds of transgenic Arabidopsis thaliana plants. The GmbZIP123 transgene promoted expression of two sucrose transporter genes (SUC1 and SUC5) and three cell-wall invertase genes (cwINV1, cwINV3, and cwINV6) by binding directly to the promoters of these genes. Consistently, the cell-wall invertase activity and sugar translocation were all enhanced in siliques of GmbZIP123 transgenic plants. Higher levels of glucose, fructose, and sucrose were also found in seeds of GmbZIP123 transgenic plants. These results suggest that GmbZIP123 may participate in regulation of lipid accumulation in soybean seeds by controlling sugar transport into seeds from photoautotrophic tissues. This study provides novel insights into the regulatory mechanism for lipid accumulation in seeds and may facilitate improvements in oil production in soybean and other oil crops through genetic manipulation of the GmbZIP123 gene.

  19. 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

  20. MGMT DNA repair gene promoter/enhancer haplotypes alter transcription factor binding and gene expression.

    PubMed

    Xu, Meixiang; Cross, Courtney E; Speidel, Jordan T; Abdel-Rahman, Sherif Z

    2016-10-01

    The O(6)-methylguanine-DNA methyltransferase (MGMT) protein removes O(6)-alkyl-guanine adducts from DNA. MGMT expression can thus alter the sensitivity of cells and tissues to environmental and chemotherapeutic alkylating agents. Previously, we defined the haplotype structure encompassing single nucleotide polymorphisms (SNPs) in the MGMT promoter/enhancer (P/E) region and found that haplotypes, rather than individual SNPs, alter MGMT promoter activity. The exact mechanism(s) by which these haplotypes exert their effect on MGMT promoter activity is currently unknown, but we noted that many of the SNPs comprising the MGMT P/E haplotypes are located within or in close proximity to putative transcription factor binding sites. Thus, these haplotypes could potentially affect transcription factor binding and, subsequently, alter MGMT promoter activity. In this study, we test the hypothesis that MGMT P/E haplotypes affect MGMT promoter activity by altering transcription factor (TF) binding to the P/E region. We used a promoter binding TF profiling array and a reporter assay to evaluate the effect of different P/E haplotypes on TF binding and MGMT expression, respectively. Our data revealed a significant difference in TF binding profiles between the different haplotypes evaluated. We identified TFs that consistently showed significant haplotype-dependent binding alterations (p ≤ 0.01) and revealed their role in regulating MGMT expression using siRNAs and a dual-luciferase reporter assay system. The data generated support our hypothesis that promoter haplotypes alter the binding of TFs to the MGMT P/E and, subsequently, affect their regulatory function on MGMT promoter activity and expression level.

  1. 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

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

    PubMed

    Dilshad, Erum; Cusido, Rosa Maria; Ramirez Estrada, Karla; 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.

  3. A Single Enhancer Regulating the Differential Expression of Duplicated Red-Sensitive Opsin Genes in Zebrafish

    PubMed Central

    Tsujimura, Taro; Hosoya, Tomohiro; Kawamura, Shoji

    2010-01-01

    A fundamental step in the evolution of the visual system is the gene duplication of visual opsins and differentiation between the duplicates in absorption spectra and expression pattern in the retina. However, our understanding of the mechanism of expression differentiation is far behind that of spectral tuning of opsins. Zebrafish (Danio rerio) have two red-sensitive cone opsin genes, LWS-1 and LWS-2. These genes are arrayed in a tail-to-head manner, in this order, and are both expressed in the long member of double cones (LDCs) in the retina. Expression of the longer-wave sensitive LWS-1 occurs later in development and is thus confined to the peripheral, especially ventral-nasal region of the adult retina, whereas expression of LWS-2 occurs earlier and is confined to the central region of the adult retina, shifted slightly to the dorsal-temporal region. In this study, we employed a transgenic reporter assay using fluorescent proteins and P1-artificial chromosome (PAC) clones encompassing the two genes and identified a 0.6-kb “LWS-activating region” (LAR) upstream of LWS-1, which regulates expression of both genes. Under the 2.6-kb flanking upstream region containing the LAR, the expression pattern of LWS-1 was recapitulated by the fluorescent reporter. On the other hand, when LAR was directly conjugated to the LWS-2 upstream region, the reporter was expressed in the LDCs but also across the entire outer nuclear layer. Deletion of LAR from the PAC clones drastically lowered the reporter expression of the two genes. These results suggest that LAR regulates both LWS-1 and LWS-2 by enhancing their expression and that interaction of LAR with the promoters is competitive between the two genes in a developmentally restricted manner. Sharing a regulatory region between duplicated genes could be a general way to facilitate the expression differentiation in duplicated visual opsins. PMID:21187910

  4. 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

  5. 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.

  6. 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.

  7. Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island.

    PubMed

    Ling, Jun; Wang, Hui; Wu, Ping; Li, Tao; Tang, Yu; Naseer, Nawar; Zheng, Huiming; Masson-Boivin, Catherine; Zhong, Zengtao; Zhu, Jun

    2016-11-29

    Horizontal gene transfer (HGT) of genomic islands is a driving force of bacterial evolution. Many pathogens and symbionts use this mechanism to spread mobile genetic elements that carry genes important for interaction with their eukaryotic hosts. However, the role of the host in this process remains unclear. Here, we show that plant compounds inducing the nodulation process in the rhizobium-legume mutualistic symbiosis also enhance the transfer of symbiosis islands. We demonstrate that the symbiosis island of the Sesbania rostrata symbiont, Azorhizobium caulinodans, is an 87.6-kb integrative and conjugative element (ICE(Ac)) that is able to excise, form a circular DNA, and conjugatively transfer to a specific site of gly-tRNA gene of other rhizobial genera, expanding their host range. The HGT frequency was significantly increased in the rhizosphere. An ICE(Ac)-located LysR-family transcriptional regulatory protein AhaR triggered the HGT process in response to plant flavonoids that induce the expression of nodulation genes through another LysR-type protein, NodD. Our study suggests that rhizobia may sense rhizosphere environments and transfer their symbiosis gene contents to other genera of rhizobia, thereby broadening rhizobial host-range specificity.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26720500

  9. 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

  10. Enhanced gene delivery of low molecular weight PEI by flower-like ZnO microparticles.

    PubMed

    Chen, Ming; Tang, Yaqin; Wang, Tingting; Long, Qipeng; Zeng, Ziying; Chen, Houwen; Feng, Xuli

    2016-12-01

    Low molecular weight (1.8 kDa) branched polyethylenimine (PEI) has been used as non-viral vector for gene delivery because of its low toxicity, however, its further application in biomedical field has been restricted due to its low gene transfection efficiency. Herein, ZnO microflowers were prepared to increase the gene expression level mediated by PEI. Four methods have been applied to tune the shape of ZnO microstructures. Scanning electron microscopy (SEM) demonstrated the successful preparation of four kinds of flower like ZnO microparticles. By loading PEI/pDNA into ZnO microparticles, the formed new complexes showed enhanced gene transfection compared to PEI/pDNA alone. Cell uptaking experiments explained a possible mechanism that the tips of ZnO microflowers penetrated into the surface of cells, thus facilitating the entry of gene cargo into cells. These findings highlight the potential of needle like microstructure as adjuvant for efficient biomacromolecular delivery. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. RNA stem-loop enhanced expression of previously non-expressible genes.

    PubMed

    Paulus, Michael; Haslbeck, Martin; Watzele, Manfred

    2004-05-26

    The key step in bacterial translation is formation of the pre-initiation complex. This requires initial contacts between mRNA, fMet-tRNA and the 30S subunit of the ribosome, steps that limit the initiation of translation. Here we report a method for improving translational initiation, which allows expression of several previously non-expressible genes. This method has potential applications in heterologous protein synthesis and high-throughput expression systems. We introduced a synthetic RNA stem-loop (stem length, 7 bp; DeltaG(0) = -9.9 kcal/mol) in front of various gene sequences. In each case, the stem-loop was inserted 15 nt downstream from the start codon. Insertion of the stem-loop allowed in vitro expression of five previously non-expressible genes and enhanced the expression of all other genes investigated. Analysis of the RNA structure proved that the stem-loop was formed in vitro, and demonstrated that stabilization of the ribosome binding site is due to stem-loop introduction. By theoretical RNA structure analysis we showed that the inserted RNA stem-loop suppresses long-range interactions between the translation initiation domain and gene-specific mRNA sequences. Thus the inserted RNA stem-loop supports the formation of a separate translational initiation domain, which is more accessible to ribosome binding.

  12. 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.

  13. Transcriptional Enhancers Induce Insertional Gene Deregulation Independently From the Vector Type and Design

    PubMed Central

    Maruggi, Giulietta; Porcellini, Simona; Facchini, Giulia; Perna, Serena K; Cattoglio, Claudia; Sartori, Daniela; Ambrosi, Alessandro; Schambach, Axel; Baum, Christopher; Bonini, Chiara; Bovolenta, Chiara; Mavilio, Fulvio; Recchia, Alessandra

    2009-01-01

    The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) γ-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase—PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a γ-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design. PMID:19293778

  14. Transcriptional enhancers induce insertional gene deregulation independently from the vector type and design.

    PubMed

    Maruggi, Giulietta; Porcellini, Simona; Facchini, Giulia; Perna, Serena K; Cattoglio, Claudia; Sartori, Daniela; Ambrosi, Alessandro; Schambach, Axel; Baum, Christopher; Bonini, Chiara; Bovolenta, Chiara; Mavilio, Fulvio; Recchia, Alessandra

    2009-05-01

    The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) gamma-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase--PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a gamma-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design.

  15. 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.

  16. HUA ENHANCER2, a putative DExH-box RNA helicase, maintains homeotic B and C gene expression in Arabidopsis

    PubMed Central

    Western, Tamara L.; Cheng, Yulan; Liu, Jun; Chen, Xuemei

    2016-01-01

    SUMMARY Reproductive organ identity in Arabidopsis is controlled by the B, C and SEPALLATA classes of floral homeotic genes. We have identified a recessive mutation in a novel gene, HUA ENHANCER2, which, when combined with mutations in two weak class C genes, HUA1 and HUA2, leads to the production of third whorl sepal-petal-stamens and fourth whorl sepal-carpels. Quadruple mutant analysis and in situ localization of A, B, C and SEPALLATA floral homeotic RNAs suggest that HUA ENHANCER2 is required for the maintenance of B and C gene expression in the reproductive whorls. In addition to its role in floral homeotic gene expression, HUA ENHANCER2 is required for normal spacing and number of perianth organ primordia. We show that HUA ENHANCER2 encodes a putative DExH-box RNA helicase that is expressed in specific patterns in the inflorescence meristem and developing flowers. As a possible ortholog of the yeast exosome-associated protein, Dob1p (Mtr4p), HUA ENHANCER2 may affect floral organ spacing and identity through the regulation of protein synthesis or mRNA degradation. Therefore, our studies on HUA ENHANCER2 not only demonstrate that B and C gene expression is established and maintained separately, but also implicate the existence of post-transcriptional mechanisms in the maintenance of B and C gene expression. PMID:11923195

  17. 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

  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. Hepatic-Specific Accessibility of Igf1 Gene Enhancers Is Independent of Growth Hormone Signaling

    PubMed Central

    Santhanam, Mahalakshmi

    2013-01-01

    The diverse roles of IGF-1 in physiology include acting as the endocrine intermediate to elicit the anabolic actions of GH. The majority of serum IGF-1 is synthesized in liver, where GH stimulates Igf1 gene transcription via the transcription factor, signal transducer and activator of transcription (Stat)5b. We and others have identified multiple Stat5-binding domains at the Igf1 locus that function in gene regulation, but it remains unclear whether the roles of these domains are tissue specific. Survey of the chromatin landscape of regulatory domains can provide insight about mechanisms of gene regulation, with chromatin accessibility regarded as a hallmark feature of regulatory domains. We prepared chromatin from liver, kidney, and spleen of C57BL/6 mice, and used formaldehyde-associated isolation of regulatory elements to assess chromatin accessibility at the major Igf1 promoter and 7 -binding enhancers. Whereas the promoters of other prototypical tissue-specific genes are open in a tissue-specific way, the major Igf1 promoter is open in all 3 tissues, albeit moderately more so in liver. In contrast, chromatin accessibility at Igf1 Stat5-binding domains is essentially restricted to liver, indicating that the enhancers are driving extensive differences in tissue expression. Furthermore, studies with Ghrhrlit/lit mice reveal that prior GH exposure is not necessary to establish open chromatin at these domains. Lastly, formaldehyde-associated isolation of regulatory elements of human liver samples confirms open chromatin at IGF1 Promoter 1, but unexpectedly, homologous Stat5-binding motifs are not accessible. We conclude that robust GH-stimulated hepatic Igf1 gene transcription utilizes tissue-specific mechanisms of epigenetic regulation that are established independent of GH signaling. PMID:24109593

  20. 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.

  1. 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.

  2. 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

  3. Enhancement of Aerosol Cisplatin Chemotherapy with Gene Therapy Expressing ABC10 protein in Respiratory System

    PubMed Central

    Hohenforst-Schmidt, Wolfgang; Zarogoulidis, Paul; Linsmeier, Bernd; Kioumis, Ioannis; Li, Qiang; Huang, Haidong; Sachpatzidou, Despoina; Lampaki, Sofia; Organtzis, John; Domvri, Kalliopi; Sakkas, Leonidas; Zachariadis, George A.; Archontas, Konstantinos N.; Kallianos, Anastasios; Rapti, Aggeliki; Yarmus, Lonny; Zarogoulidis, Konstantinos; Brachmann, Johannes

    2014-01-01

    Inhaled therapy for lung cancer is a local form of treatment. Currently inhaled non-specific cytotoxic agents have been evaluated as a future treatment for local disease control and distant metastasis control. There are few information regarding the influence of local transporters and gene expression of the respiratory epithelium to the absorption of administered drugs. In the current work we used adenoviral-type 5(dE1/E3) (Cytomegalovirus promoter) with human ABCA10 transgene (Ad-h-ABCA10) purchased from Vector Labs® in order to investigate whether gene therapy can be used as a pre-treatment to enhance the efficiency of inhaled cisplatin. We included the following groups to our work: a) control, b) aerosol vector, c) aerosol vector plus cisplatin, d) aerosol cisplatin, e) intratumoral cisplatin administration, f) intratumoral vector plus cisplatin administration. The results indicate that the aerosol cisplatin group had a long term survival with the intratumoral cisplatin group following. The enhancement of the ABCA family locally to the respiratory system prior to the aerosol cisplatin administration can be used safely and efficiently. Future treatment design of local therapies should include the investigation of local transporters and genes. PMID:24723977

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation.

    PubMed

    Xiang, D; Liu, C-C; Wang, M-J; Li, J-X; Chen, F; Yao, H; Yu, B; Lu, L; Borjigin, U; Chen, Y-X; Zhong, L; Wangensteen, K J; He, Z-Y; Wang, X; Hu, Y-P

    2014-05-22

    Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)(-/-) mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation.

  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. 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.

  13. 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.

  14. 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

  15. Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy.

    PubMed

    Powell, Sara Kathleen; Rivera-Soto, Ricardo; Gray, Steven James

    2015-01-01

    Over the last five years, the number of clinical trials involving AAV (adeno-associated virus) and lentiviral vectors continue to increase by about 150 trials each year. For continued success, AAV and lentiviral expression cassettes need to be designed to meet each disease's specific needs. This review discusses how viral vector expression cassettes can be engineered with elements to enhance target specificity and increase transgene expression. The key differences relating to target specificity between ubiquitous and tissue-specific promoters are discussed, as well as how endogenous miRNAs and their target sequences have been used to restrict transgene expression. Specifically, relevant studies indicating how cis-acting elements such as introns, WPRE, polyadenylation signals, and the CMV enhancer are highlighted to show their utility for enhancing transgene expression in gene therapy applications. All discussion bears in mind that expression cassettes have space constraints. In conclusion, this review can serve as a menu of vector genome design elements and their cost in terms of space to thoughtfully engineer viral vectors for gene therapy.

  16. GENE THERAPY APPROACHES TO ENHANCING PLASTICITY AND REGENERATION AFTER SPINAL CORD INJURY

    PubMed Central

    Franz, Steffen; Weidner, Norbert; Blesch, Armin

    2011-01-01

    During the past decades, new insights into mechanisms that limit plasticity and functional recovery after spinal cord injury have spurred the development of novel approaches to enhance axonal regeneration and rearrangement of spared circuitry. Gene therapy may provide one means to address mechanisms that underlie the insufficient regenerative response of injured neurons and can also be used to identify factors important for axonal growth. Several genetic approaches aimed to modulate the environment of injured axons, for example by localized expression of growth factors, to enhance axonal sprouting and regeneration and to guide regenerating axons towards their target have been described. In addition, genetic modification of injured neurons via intraparenchymal injection, or via retrograde transport of viral vectors has been used to manipulate the intrinsic growth capacity of injured neurons. In this review we will summarize some of the progress and limitations of cell transplantation and gene therapy to enhance axonal bridging and regeneration across a lesion site, and to maximize the function, collateral sprouting and connectivity of spared axonal systems. PMID:21281633

  17. Evolution of a genomic regulatory domain: The role of gene co-option and gene duplication in the Enhancer of split complex

    PubMed Central

    Duncan, Elizabeth J.; Dearden, Peter K.

    2010-01-01

    The Drosophila Enhancer of split complex [E(spl)-C] is a remarkable complex of genes many of which are effectors or modulators of Notch signaling. The complex contains different classes of genes including four bearded genes and seven basic helix-loop-helix (bHLH) genes. We examined the evolution of this unusual complex by identifying bearded and bHLH genes in the genome sequences of Arthropods. We find that a four-gene E(spl)-C, containing three bHLH genes and one bearded gene, is an ancient component of the genomes of Crustacea and Insects. The complex is well conserved in insects but is highly modified in Drosophila, where two of the ancestral genes of the complex are missing, and the remaining two have been duplicated multiple times. Through examining the expression of E(spl)-C genes in honeybees, aphids, and Drosophila, we determined that the complex ancestrally had a role in Notch signaling. The expression patterns of genes found inserted into the complex in some insects, or that of ancestral E(spl)-C genes that have moved out of the complex, imply that the E(spl)-C is a genomic domain regulated as a whole by Notch signaling. We hypothesize that the E(spl)-C is a Notch-regulated genomic domain conserved in Arthropod genomes for around 420 million years. We discuss the consequence of this conserved domain for the recruitment of novel genes into the Notch signaling cascade. PMID:20458100

  18. 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.

  19. The Enhancer Binding Protein Nla6 Regulates Developmental Genes That Are Important for Myxococcus xanthus Sporulation

    PubMed Central

    Giglio, Krista M.; Zhu, Chengjun; Klunder, Courtney; Kummer, Shelley

    2015-01-01

    ABSTRACT In the bacterium Myxococcus xanthus, starvation triggers the formation of multicellular fruiting bodies containing thousands of stress-resistant spores. Recent work showed that fruiting body development is regulated by a cascade of transcriptional activators called enhancer binding proteins (EBPs). The EBP Nla6 is a key component of this cascade; it regulates the promoters of other EBP genes, including a downstream-functioning EBP gene that is crucial for sporulation. In recent expression studies, hundreds of Nla6-dependent genes were identified, suggesting that the EBP gene targets of Nla6 may be part of a much larger regulon. The goal of this study was to identify and characterize genes that belong to the Nla6 regulon. Accordingly, a direct repeat [consensus, C(C/A)ACGNNGNC] binding site for Nla6 was identified using in vitro and in vivo mutational analyses, and the sequence was subsequently used to find 40 potential developmental promoter (88 gene) targets. We showed that Nla6 binds to the promoter region of four new targets (asgE, exo, MXAN2688, and MXAN3259) in vitro and that Nla6 is important for their normal expression in vivo. Phenotypic studies indicate that all of the experimentally confirmed targets of Nla6 are primarily involved in sporulation. These targets include genes involved in transcriptional regulation, cell-cell signal production, and spore differentiation and maturation. Although sporulation occurs late in development, all of the developmental loci analyzed here show an Nla6-dependent burst in expression soon after starvation is induced. This finding suggests that Nla6 starts preparing cells for sporulation very early in the developmental process. IMPORTANCE Bacterial development yields a remarkable array of complex multicellular forms. One such form, which is commonly found in nature, is a surface-associated aggregate of cells known as a biofilm. Mature biofilms are structurally complex and contain cells that are highly resistant to

  20. The enhancer binding protein Nla6 regulates developmental genes that are important for Myxococcus xanthus sporulation.

    PubMed

    Giglio, Krista M; Zhu, Chengjun; Klunder, Courtney; Kummer, Shelley; Garza, Anthony G

    2015-04-01

    In the bacterium Myxococcus xanthus, starvation triggers the formation of multicellular fruiting bodies containing thousands of stress-resistant spores. Recent work showed that fruiting body development is regulated by a cascade of transcriptional activators called enhancer binding proteins (EBPs). The EBP Nla6 is a key component of this cascade; it regulates the promoters of other EBP genes, including a downstream-functioning EBP gene that is crucial for sporulation. In recent expression studies, hundreds of Nla6-dependent genes were identified, suggesting that the EBP gene targets of Nla6 may be part of a much larger regulon. The goal of this study was to identify and characterize genes that belong to the Nla6 regulon. Accordingly, a direct repeat [consensus, C(C/A)ACGNNGNC] binding site for Nla6 was identified using in vitro and in vivo mutational analyses, and the sequence was subsequently used to find 40 potential developmental promoter (88 gene) targets. We showed that Nla6 binds to the promoter region of four new targets (asgE, exo, MXAN2688, and MXAN3259) in vitro and that Nla6 is important for their normal expression in vivo. Phenotypic studies indicate that all of the experimentally confirmed targets of Nla6 are primarily involved in sporulation. These targets include genes involved in transcriptional regulation, cell-cell signal production, and spore differentiation and maturation. Although sporulation occurs late in development, all of the developmental loci analyzed here show an Nla6-dependent burst in expression soon after starvation is induced. This finding suggests that Nla6 starts preparing cells for sporulation very early in the developmental process. Bacterial development yields a remarkable array of complex multicellular forms. One such form, which is commonly found in nature, is a surface-associated aggregate of cells known as a biofilm. Mature biofilms are structurally complex and contain cells that are highly resistant to antibacterial agents

  1. 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.

  2. 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.

  3. 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.

  4. Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight

    PubMed Central

    Mackintosh, Caroline A.; Lewis, Janet; Radmer, Lorien E.; Shin, Sanghyun; Heinen, Shane J.; Smith, Lisa A.; Wyckoff, Meagen N.; Dill-Macky, Ruth; Evans, Conrad K.; Kravchenko, Sasha; Baldridge, Gerald D.; Zeyen, Richard J.

    2006-01-01

    Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions. PMID:17103001

  5. 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

  6. 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.

  7. 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.

  8. Mutation analysis of NR2E3 and NRL genes in Enhanced S Cone Syndrome.

    PubMed

    Wright, Alan F; Reddick, Adam C; Schwartz, Sharon B; Ferguson, Julie S; Aleman, Tomas S; Kellner, Ulrich; Jurklies, Bernhard; Schuster, Andreas; Zrenner, Eberhart; Wissinger, Bernd; Lennon, Alan; Shu, Xinhua; Cideciyan, Artur V; Stone, Edwin M; Jacobson, Samuel G; Swaroop, Anand

    2004-11-01

    Ten new and seventeen previously reported Enhanced S Cone Syndrome (ESCS) subjects were used to search for genetic heterogeneity. All subjects were diagnosed with ESCS on the basis of clinical, psychophysical and/or electroretinography testing using published criteria. Mutation analysis was performed on the NR2E3 nuclear receptor gene by single strand conformation analysis and direct sequencing, which revealed either homozygous (N=13) or compound heterozygous (N=11) mutations in 24 subjects (89%), heterozygous mutations in 2 subjects (7%) and no mutations in 1 subject (4%). Fifteen different mutations were identified, including six not previously reported. The subject (Patient A) with no detected NR2E3 mutation had features not usually associated with ESCS, in particular moderate rod photoreceptor function in peripheral retina and an abnormally thick retinal nerve fibre layer. Mutation analysis of the NRL, CRX, NR1D1 and THRB genes in this individual revealed a heterozygous one base-pair insertion in exon 2 of the NRL gene, which results in a predicted truncation of the NRL protein. Loss-of-function NRL alleles have not been described previously in humans, but since the same mutation was present in unaffected family members, it raises the possibility that the abnormal ESCS phenotype in Patient A may result from a digenic mechanism, with a heterozygous NRL mutation and a mutation in another unknown gene. Copyright 2004 Wiley-Liss, Inc.

  9. 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.

  10. PEGylated polyplex with optimized PEG shielding enhances gene introduction in lungs by minimizing inflammatory responses.

    PubMed

    Uchida, Satoshi; Itaka, Keiji; Chen, Qixian; Osada, Kensuke; Ishii, Takehiko; Shibata, Masa-Aki; Harada-Shiba, Mariko; Kataoka, Kazunori

    2012-06-01

    Safety is a critical issue in clinical applications of nonviral gene delivery systems. Safe and effective gene introduction into the lungs was previously achieved using polyplexes from poly(ethyleneglycol) (PEG)-block-polycation [PEG-block-PAsp(DET)] and plasmid DNA (pDNA). Although PEGylated polyplexes appeared to be safe, an excess ratio of polycation to pDNA was needed to obtain sufficient transgene expression, which may cause toxicities shortly after gene introduction. In the present study, we investigated the combined use of two polymers, PEG-block-PAsp(DET) (B) and homo PAsp(DET) (H) across a range of mixing ratios to construct polyplexes. Although transgene expressions following in vitro transfections increased in parallel with increased proportions of H, polyplexes with B/H = 50/50 formulation produced the highest expression level following in vivo intratracheal administration. Higher proportions of H elicited high levels of cytokine induction with significant inflammation as assessed by histopathological examinations. Based on the aggregation behavior of polyplexes in bronchoalveolar lavage fluids (BALFs), we suggested that rapid aggregation of polyplexes in the lung induced acute inflammatory responses, resulting in reduced transgene expression. B/H formulation of polyplex can help to improve gene therapy for the respiratory system because it achieves both effective PEG shielding of polyplexes and functioning of PAsp(DET) polycations to enhance endosomal escape.

  11. Rigid aromatic linking moiety in cationic lipids for enhanced gene transfection efficiency.

    PubMed

    Wang, Bing; Zhao, Rui-Mo; Zhang, Ji; Liu, Yan-Hong; Huang, Zheng; Yu, Qing-Ying; Yu, Xiao-Qi

    2017-08-18

    Although numerous cationic lipids have been developed as non-viral gene vectors, the structure-activity relationship (SAR) of these materials remains unclear and needs further investigation. In this work, a series of lysine-derived cationic lipids containing linkages with different rigidity were designed and synthesized. SAR studies showed that lipids with rigid aromatic linkage could promote the formation of tight liposomes and enhance DNA condensation, which is essential for the gene delivery process. These lipids could give much higher transfection efficiency than those containing more flexible aliphatic linkage in various cell lines. Moreover, the rigid aromatic linkage also affords the material higher serum tolerance ability. Flow cytometry assay revealed that the target lipids have good cellular uptake, while confocal microscopy observation showed weaker endosome escape than Lipofectamine 2000. To solve such problem and further increase the transfection efficiency, some lysosomotropic reagents were used to improve the endosome escape of lipoplex. As expected, higher transfection efficiency than Lipofectamine 2000 could be obtained via this strategy. Cytotoxicity assay showed that these lipids have lower toxicity in various cell lines than Lipofectamine 2000, suggesting their potential for further application. This work demonstrates that a rigid aromatic linkage might distinctly improve the gene transfection abilities of cationic lipids and affords information to construct safe and efficient gene vector towards practical application. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  12. Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility.

    PubMed

    LePage, Daniel P; Metcalf, Jason A; Bordenstein, Sarah R; On, Jungmin; Perlmutter, Jessamyn I; Shropshire, J Dylan; Layton, Emily M; Funkhouser-Jones, Lisa J; Beckmann, John F; Bordenstein, Seth R

    2017-03-09

    The genus Wolbachia is an archetype of maternally inherited intracellular bacteria that infect the germline of numerous invertebrate species worldwide. They can selfishly alter arthropod sex ratios and reproductive strategies to increase the proportion of the infected matriline in the population. The most common reproductive manipulation is cytoplasmic incompatibility, which results in embryonic lethality in crosses between infected males and uninfected females. Females infected with the same Wolbachia strain rescue this lethality. Despite more than 40 years of research and relevance to symbiont-induced speciation, as well as control of arbovirus vectors and agricultural pests, the bacterial genes underlying cytoplasmic incompatibility remain unknown. Here we use comparative and transgenic approaches to demonstrate that two differentially transcribed, co-diverging genes in the eukaryotic association module of prophage WO from Wolbachia strain wMel recapitulate and enhance cytoplasmic incompatibility. Dual expression in transgenic, uninfected males of Drosophila melanogaster crossed to uninfected females causes embryonic lethality. Each gene additively augments embryonic lethality in crosses between infected males and uninfected females. Lethality associates with embryonic defects that parallel those of wild-type cytoplasmic incompatibility and is notably rescued by wMel-infected embryos in all cases. The discovery of cytoplasmic incompatibility factor genes cifA and cifB pioneers genetic studies of prophage WO-induced reproductive manipulations and informs the continuing use of Wolbachia to control dengue and Zika virus transmission to humans.

  13. 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.

  14. 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.

  15. Degradable terpolymers with alkyl side chains demonstrate enhanced gene delivery potency and nanoparticle stability.

    PubMed

    Eltoukhy, Ahmed A; Chen, Delai; Alabi, Christopher A; Langer, Robert; Anderson, Daniel G

    2013-03-13

    Degradable, cationic poly(β-amino ester)s (PBAEs) with alkyl side chains are developed for non-viral gene delivery. Nanoparticles formed from these PBAE terpolymers exhibit significantly enhanced DNA transfection potency and resistance to aggregation. These hydrophobic PBAE terpolymers, but not PBAEs lacking alkyl side chains, support interaction with PEG-lipid conjugates, facilitating their functionalization with shielding and targeting moieties and accelerating the in vivo translation of these materials. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. Neural precursor-specific expression of multiple Drosophila genes is driven by dual enhancer modules with overlapping function

    PubMed Central

    Miller, Steven W.; Rebeiz, Mark; Atanasov, Jenny E.

    2014-01-01

    Transcriptional cis-regulatory modules (CRMs), or enhancers, are responsible for directing gene expression in specific territories and cell types during development. In some instances, the same gene may be served by two or more enhancers with similar specificities. Here we show that the utilization of dual, or “shadow”, enhancers is a common feature of genes that are active specifically in neural precursor (NP) cells in Drosophila. By genome-wide computational discovery of statistically significant clusters of binding motifs for both proneural activator (P) proteins and basic helix–loop–helix (bHLH) repressor (R) factors (a “P+R” regulatory code), we have identified NP-specific enhancer modules associated with multiple genes expressed in this cell type. These CRMs are distinct from those previously identified for the corresponding gene, establishing the existence of a dual-enhancer arrangement in which both modules reside close to the gene they serve. Using wild-type and mutant reporter gene constructs in vivo, we show that P sites in these modules mediate activation by proneural factors in “proneural cluster” territories, whereas R sites mediate repression by bHLH repressors, which serves to restrict expression specifically to NP cells. To our knowledge, our results identify the first direct targets of these bHLH repressors. Finally, using genomic rescue constructs for neuralized (neur), we demonstrate that each of the gene's two NP-specific enhancers is sufficient to rescue neur function in the lateral inhibition process by which adult sensory organ precursor (SOP) cells are specified, but that deletion of both enhancers results in failure of this event. PMID:25404315

  1. 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

  2. A novel pathway-based distance score enhances assessment of disease heterogeneity in gene expression.

    PubMed

    Yan, Xiting; Liang, Anqi; Gomez, Jose; Cohn, Lauren; Zhao, Hongyu; Chupp, Geoffrey L

    2017-06-20

    Distance based unsupervised clustering of gene expression data is commonly used to identify heterogeneity in biologic samples. However, high noise levels in gene expression data and relatively high correlation between genes are often encountered, so traditional distances such as Euclidean distance may not be effective at discriminating the biological differences between samples. An alternative method to examine disease phenotypes is to use pre-defined biological pathways. These pathways have been shown to be perturbed in different ways in different subjects who have similar clinical features. We hypothesize that differences in the expressions of genes in a given pathway are more predictive of differences in biological differences compared to standard approaches and if integrated into clustering analysis will enhance the robustness and accuracy of the clustering method. To examine this hypothesis, we developed a novel computational method to assess the biological differences between samples using gene expression data by assuming that ontologically defined biological pathways in biologically similar samples have similar behavior. Pre-defined biological pathways were downloaded and genes in each pathway were used to cluster samples using the Gaussian mixture model. The clustering results across different pathways were then summarized to calculate the pathway-based distance score between samples. This method was applied to both simulated and real data sets and compared to the traditional Euclidean distance and another pathway-based clustering method, Pathifier. The results show that the pathway-based distance score performs significantly better than the Euclidean distance, especially when the heterogeneity is low and genes in the same pathways are correlated. Compared to Pathifier, we demonstrated that our approach achieves higher accuracy and robustness for small pathways. When the pathway size is large, by downsampling the pathways into smaller pathways, our

  3. 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.

  4. 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

  5. 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.

  6. In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea

    PubMed Central

    2013-01-01

    Background The freshwater planarian Schmidtea mediterranea has emerged as a powerful model for studies of regenerative, stem cell, and germ cell biology. Whole-mount in situ hybridization (WISH) and whole-mount fluorescent in situ hybridization (FISH) are critical methods for determining gene expression patterns in planarians. While expression patterns for a number of genes have been elucidated using established protocols, determining the expression patterns for particularly low-abundance transcripts remains a challenge. Results We show here that a short bleaching step in formamide dramatically enhances signal intensity of WISH and FISH. To further improve signal sensitivity we optimized blocking conditions for multiple anti-hapten antibodies, developed a copper sulfate quenching step that virtually eliminates autofluorescence, and enhanced signal intensity through iterative rounds of tyramide signal amplification. For FISH on regenerating planarians, we employed a heat-induced antigen retrieval step that provides a better balance between permeabilization of mature tissues and preservation of regenerating tissues. We also show that azide most effectively quenches peroxidase activity between rounds of development for multicolor FISH experiments. Finally, we apply these modifications to elucidate the expression patterns of a few low-abundance transcripts. Conclusion The modifications we present here provide significant improvements in signal intensity and signal sensitivity for WISH and FISH in planarians. Additionally, these modifications might be of widespread utility for whole-mount FISH in other model organisms. PMID:23497040

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

    PubMed Central

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

    2012-01-01

    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. PMID:22499169

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

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

    PubMed Central

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

    2008-01-01

    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 FcγRI, but not its associated γ chain, and was not supported by other FcγR family members (FcγRIIA, FcγRIIB and FcγRIII). 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, FcγRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer. PMID:18191979

  14. 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.

  15. A novel double-enhanced suicide gene therapy in a colon cancer cell line mediated by gef and apoptin.

    PubMed

    Boulaiz, Houria; Aránega, Antonia; Cáceres, Blanca; Blanca, Cáceres; Alvarez, Pablo; Pablo, Alvarez; Serrano-Rodríguez, Fernando; Fernando, Rodríguez-Serrano; Carrillo, Esmeralda; Esmeralda, Carrillo; Melguizo, Consolación; Consolación, Melguizo; Prados, Jose; Jose, Prados

    2014-02-01

    Double-suicide gene therapy is a promising strategy for the treatment of advanced cancer. It has become an important research line in the development of gene therapy to overcome the drawbacks of single-gene therapy. The aim of this study was to investigate the usefulness of double-suicide gene therapy with the two suicide genes, gef and apoptin, in colon carcinoma. gef and apoptin genes were cloned into a doxycycline-regulated retrovirus-mediated gene expression system. Expression of both genes in the DLD-1 cell line was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Cell viability was determined with the sulforhodamine B colorimetric assay, and the cell cycle was studied by propidium iodide (PI) staining. Annexin V-FITC and PI assays were used to evaluate apoptosis, and the results were confirmed by electron microscopy. The mitochondrial membrane potential was measured by JC-1 assay. Our results showed that the combined expression of gef and apoptin genes was strikingly more effective than the expression of either gene alone. Co-expression of gef and apoptin synergistically enhanced the decrease in cell viability, increasing necrosis and inducing apoptosis in colon cancer cells via the mitochondrial pathway, which can be deficient in advanced or metastatic colon cancer. Double-suicide gene therapy based on gef and apoptin genes may be a candidate for the development of new colon cancer strategies, and further studies are warranted to establish the usefulness of double-suicide gene therapy in vivo.

  16. The pioneer transcription factor FoxA maintains an accessible nucleosome configuration at enhancers for tissue-specific gene activation

    PubMed Central

    Iwafuchi-Doi, Makiko; Donahue, Greg; Kakumanu, Akshay; Watts, Jason A.; Mahony, Shaun; Pugh, B. Franklin; Lee, Dolim; Kaestner, Klaus H.; Zaret, Kenneth S.

    2016-01-01

    SUMMARY Nuclear DNA wraps around core histones to form nucleosomes, which restricts the binding of transcription factors to gene regulatory sequences. Pioneer transcription factors can bind DNA sites on nucleosomes and initiate gene regulatory events, often leading to the local opening of chromatin. However, the nucleosomal configuration of open chromatin and the basis for its regulation is unclear. We combined low and high levels of MNase digestion along with core histone mapping to assess the nucleosomal configuration at enhancers and promoters in mouse liver. We find that MNase-accessible nucleosomes, bound by transcription factors, are retained more at liver-specific enhancers than at promoters and ubiquitous enhancers. The pioneer factor FoxA displaces linker histone H1, thereby keeping enhancer nucleosomes accessible in chromatin and allowing other liver-specific transcription factors to bind and stimulate transcription. Thus, nucleosomes are not exclusively repressive to gene regulation when they are retained with, and exposed by, pioneer factors. PMID:27058788

  17. 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.

  18. 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

  19. 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.

  20. A deficiency in the autophagy gene Atg16L1 enhances resistance to enteric bacterial infection

    PubMed Central

    Marchiando, Amanda M.; Ramanan, Deepshika; Ding, Yi; Gomez, Luis E.; Hubbard-Lucey, Vanessa M.; Maurer, Katie; Wang, Caihong; Ziel, Joshua W.; van Rooijen, Nico; Nuñez, Gabriel; Finlay, B. Brett; Mysorekar, Indira U.; Cadwell, Ken

    2013-01-01

    SUMMARY Polymorphisms in the essential autophagy gene Atg16L1 have been linked with susceptibility to Crohn’s disease, a major type of inflammatory bowel disease (IBD). Although the inability to control intestinal bacteria is thought to underlie IBD, the role of Atg16L1 during extracellular intestinal bacterial infections has not been sufficiently examined and compared to the function of other IBD susceptibility genes such as Nod2, which encodes a cytosolic bacterial sensor. We find that Atg16L1 mutant mice are resistant to intestinal disease induced by the model bacterial pathogen Citrobacter rodentium. An Atg16L1 deficiency alters the intestinal environment to mediate an enhanced immune response that is dependent on monocytic cells, but this hyper-immune phenotype and protective effects are lost in Atg16L1/Nod2 double mutant mice. These results reveal an immuno-suppressive function of Atg16L1, and suggest that gene variants affecting the autophagy pathway may have been evolutionarily maintained to protect against certain life-threatening infections. PMID:23954160

  1. A deficiency in the autophagy gene Atg16L1 enhances resistance to enteric bacterial infection.

    PubMed

    Marchiando, Amanda M; Ramanan, Deepshika; Ding, Yi; Gomez, Luis E; Hubbard-Lucey, Vanessa M; Maurer, Katie; Wang, Caihong; Ziel, Joshua W; van Rooijen, Nico; Nuñez, Gabriel; Finlay, B Brett; Mysorekar, Indira U; Cadwell, Ken

    2013-08-14

    Polymorphisms in the essential autophagy gene Atg16L1 have been linked with susceptibility to Crohn's disease, a major type of inflammatory bowel disease (IBD). Although the inability to control intestinal bacteria is thought to underlie IBD, the role of Atg16L1 during extracellular intestinal bacterial infections has not been sufficiently examined and compared to the function of other IBD susceptibility genes, such as Nod2, which encodes a cytosolic bacterial sensor. We find that Atg16L1 mutant mice are resistant to intestinal disease induced by the model bacterial pathogen Citrobacter rodentium. An Atg16L1 deficiency alters the intestinal environment to mediate an enhanced immune response that is dependent on monocytic cells, but this hyperimmune phenotype and its protective effects are lost in Atg16L1/Nod2 double-mutant mice. These results reveal an immunosuppressive function of Atg16L1 and suggest that gene variants affecting the autophagy pathway may have been evolutionarily maintained to protect against certain life-threatening infections. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Enhancer Variants Synergistically Drive Dysfunction of a Gene Regulatory Network In Hirschsprung Disease

    DOE PAGES

    Chatterjee, Sumantra; Kapoor, Ashish; Akiyama, Jennifer A.; ...

    2016-09-29

    Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidencemore » that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders.« less

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

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

    PubMed Central

    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

    2011-01-01

    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--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. PMID:21829177

  16. 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.

  17. Cell-Specific Actions of a Human LHX3 Gene Enhancer During Pituitary and Spinal Cord Development

    PubMed Central

    Park, Soyoung; Mullen, Rachel D.

    2013-01-01

    The LIM class of homeodomain protein 3 (LHX3) transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie complex pediatric syndromes featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. Here we characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in α-glycoprotein subunit -expressing cells secreting the TSHβ, LHβ, or FSHβ hormones and expressing the GATA2 and steroidogenic factor 1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module active in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression. Furthermore, these studies revealed significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice also provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. PMID:24100213

  18. Enhanced Horizontal Transfer of Antibiotic Resistance Genes in Freshwater Microcosms Induced by an Ionic Liquid

    PubMed Central

    Wang, Qing; Mao, Daqing; Mu, Quanhua; Luo, Yi

    2015-01-01

    The spread and propagation of antibiotic resistance genes (ARGs) is a worldwide public health concern. Ionic liquids (ILs), considered as “environmentally friendly” replacements for industrial organic solvents, have been widely applied in modern industry. However, few data have been collected regarding the potential ecological and environmental risks of ILs, which are important for preparing for their potential discharge into the environment. In this paper, the IL 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6]) (0.001-5.0 g/L) was tested for its effects on facilitating ARGs horizontal transfer mediated by plasmid RP4 in freshwater microcosms. In the horizontal transfer microcosms, the transfer frequency of plasmid RP4 was significantly enhanced (60-fold higher than untreated groups) by the IL [BMIm][PF6] (1.0 g/L). Meanwhile, two strains of opportunistic pathogen Acinetobacter spp. and Salmonella spp. were isolated among the transconjugants, illustrating plasmid RP4 mediated horizontal transfer of ARGs occurred in pathogen. This could increase the risk of ARGs dissemination to human pathogens and pose great threat to public health. The cause that [BMIm[PF6] enhanced the transfer frequency of plasmid RP4 was proposed by suppressed cell membrane barrier and enhanced cell membrane permeability, which was evidenced by flow cytometry (FCM). This is the first report that some ILs facilitate horizontal transfer of plasmid RP4 which is widely distributed in the environment and thus add the adverse effects of the environmental risk of ILs. PMID:25951456

  19. Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum

    PubMed Central

    Shin, Sanghyun; Mackintosh, Caroline A.; Lewis, Janet; Heinen, Shane J.; Radmer, Lorien; Dill-Macky, Ruth; Baldridge, Gerald D.; Zeyen, Richard J.; Muehlbauer, Gary J.

    2008-01-01

    Fusarium head blight (FHB; scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing wheat germplasm pools for FHB resistance is low and may not provide sufficient resistance to develop cultivars through traditional breeding approaches. Thus, genetic engineering provides an additional approach to enhance FHB resistance. The objectives of this study were to develop transgenic wheat expressing a barley class II chitinase and to test the transgenic lines against F. graminearum infection under greenhouse and field conditions. A barley class II chitinase gene was introduced into the spring wheat cultivar, Bobwhite, by biolistic bombardment. Seven transgenic lines were identified that expressed the chitinase transgene and exhibited enhanced Type II resistance in the greenhouse evaluations. These seven transgenic lines were tested under field conditions for percentage FHB severity, percentage visually scabby kernels (VSK), and DON accumulation. Two lines (C8 and C17) that exhibited high chitinase protein levels also showed reduced FHB severity and VSK compared to Bobwhite. One of the lines (C8) also exhibited reduced DON concentration compared with Bobwhite. These results showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions. PMID:18467324

  20. 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.

  1. 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.

  2. Butyrate Enhances Disease Resistance of Chickens by Inducing Antimicrobial Host Defense Peptide Gene Expression

    PubMed Central

    Sunkara, Lakshmi T.; Achanta, Mallika; Schreiber, Nicole B.; Bommineni, Yugendar R.; Dai, Gan; Jiang, Weiyu; Lamont, Susan; Lillehoj, Hyun S.; Beker, Ali; Teeter, Robert G.; Zhang, Guolong

    2011-01-01

    Host defense peptides (HDPs) constitute a large group of natural broad-spectrum antimicrobials and an important first line of immunity in virtually all forms of life. Specific augmentation of synthesis of endogenous HDPs may represent a promising antibiotic-alternative approach to disease control. In this study, we tested the hypothesis that exogenous administration of butyrate, a major type of short-chain fatty acids derived from bacterial fermentation of undigested dietary fiber, is capable of inducing HDPs and enhancing disease resistance in chickens. We have found that butyrate is a potent inducer of several, but not all, chicken HDPs in HD11 macrophages as well as in primary monocytes, bone marrow cells, and jejuna and cecal explants. In addition, butyrate treatment enhanced the antibacterial activity of chicken monocytes against Salmonella enteritidis, with a minimum impact on inflammatory cytokine production, phagocytosis, and oxidative burst capacities of the cells. Furthermore, feed supplementation with 0.1% butyrate led to a significant increase in HDP gene expression in the intestinal tract of chickens. More importantly, such a feeding strategy resulted in a nearly 10-fold reduction in the bacterial titer in the cecum following experimental infections with S. enteritidis. Collectively, the results indicated that butyrate-induced synthesis of endogenous HDPs is a phylogenetically conserved mechanism of innate host defense shared by mammals and aves, and that dietary supplementation of butyrate has potential for further development as a convenient antibiotic-alternative strategy to enhance host innate immunity and disease resistance. PMID:22073293

  3. A Novel Method for Gene-Specific Enhancement of Protein Translation by Targeting 5'UTRs of Selected Tumor Suppressors.

    PubMed

    Master, Adam; Wójcicka, Anna; Giżewska, Kamilla; Popławski, Piotr; Williams, Graham R; Nauman, Alicja

    2016-01-01

    Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators. We demonstrate that oligonucleotide-based trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5'-untranslated region (5'UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5'UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRβ1 5'UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5'UTR has higher translational regulatory potential when compared to the weakly folded TRβ1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5'UTRs of complex structure. This report represents the first method for gene-specific translation enhancement using selective trans-acting factors designed to target specific 5'UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene and may have future application in gene therapy

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. Engineering an enhanced, thermostable, monomeric bacterial luciferase gene as a reporter in plant protoplasts.

    PubMed

    Cui, Boyu; Zhang, Lifeng; 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.

  9. Expression of sweet pepper Hrap gene in banana enhances resistance to Xanthomonas campestris pv. musacearum.

    PubMed

    Tripathi, Leena; Mwaka, Henry; Tripathi, Jaindra Nath; Tushemereirwe, Wilberforce Kateera

    2010-11-01

    Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum, is the most devastating disease of banana in the Great Lakes region of Africa. The pathogen's rapid spread has threatened the livelihood of millions of Africans who rely on banana fruit for food security and income. The disease is very destructive, infecting all banana varieties, including both East African Highland bananas and exotic types of banana. In the absence of natural host plant resistance among banana cultivars, the constitutive expression of the hypersensitivity response-assisting protein (Hrap) gene from sweet pepper (Capsicum annuum) was evaluated for its ability to confer resistance to BXW. Transgenic lines expressing the Hrap gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of two banana cultivars: 'Sukali Ndiizi' and 'Mpologoma'. These lines were characterized by molecular analysis, and were challenged with Xanthomonas campestris pv. musacearum to analyse the efficacy of the Hrap gene against BXW. The majority of transgenic lines (six of eight) expressing Hrap did not show any symptoms of infection after artificial inoculation of potted plants in the screenhouse, whereas control nontransgenic plants showed severe symptoms resulting in complete wilting. This study demonstrates that the constitutive expression of the sweet pepper Hrap gene in banana results in enhanced resistance to BXW. We describe the development of transgenic banana varieties resistant to BXW, which will boost the arsenal available to fight this epidemic disease and save livelihoods in the Great Lakes region of East and Central Africa. © 2010 IITA/NARO. Molecular Plant Pathology © 2010 BSPP and Blackwell Publishing Ltd.

  10. Expression of Terminal Effector Genes in Mammalian Neurons Is Maintained by a Dynamic Relay of Transient Enhancers.

    PubMed

    Rhee, Ho Sung; Closser, Michael; Guo, Yuchun; Bashkirova, Elizaveta V; Tan, G Christopher; Gifford, David K; Wichterle, Hynek

    2016-12-21

    Generic spinal motor neuron identity is established by cooperative binding of programming transcription factors (TFs), Isl1 and Lhx3, to motor-neuron-specific enhancers. How expression of effector genes is maintained following downregulation of programming TFs in maturing neurons remains unknown. High-resolution exonuclease (ChIP-exo) mapping revealed that the majority of enhancers established by programming TFs are rapidly deactivated following Lhx3 downregulation in stem-cell-derived hypaxial motor neurons. Isl1 is released from nascent motor neuron enhancers and recruited to new enhancers bound by clusters of Onecut1 in maturing neurons. Synthetic enhancer reporter assays revealed that Isl1 operates as an integrator factor, translating the density of Lhx3 or Onecut1 binding sites into transient enhancer activity. Importantly, independent Isl1/Lhx3- and Isl1/Onecut1-bound enhancers contribute to sustained expression of motor neuron effector genes, demonstrating that outwardly stable expression of terminal effector genes in postmitotic neurons is controlled by a dynamic relay of stage-specific enhancers. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. 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. © 2016 Wiley Periodicals, Inc.

  12. 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.

  13. Transforming function of proto-ras genes depends on heterologous promoters and is enhanced by specific point mutations.

    PubMed Central

    Chakraborty, A K; Cichutek, K; Duesberg, P H

    1991-01-01

    Based on transfection into cells in culture or natural transduction into retroviruses, proto-ras genes seem to derive transforming function either from heterologous promoters or from point mutations. Here we ask how such different events could achieve the same results. To identify homologous regulatory elements, about 3 kilobases of rat DNA upstream of the first untranslated proto-Ha-ras exon was sequenced. Surprisingly, the sequence shares at -1858 a homology of 148 nucleotides with Harvey (Ha) sarcoma virus, 5' of viral ras, signaling possibly a second untranslated proto-Ha-ras exon. In addition the sequence contains a perfect repeat of 25 CA dinucleotides at -2655. A retroviral promoter, even from upstream of the poly(CA), conferred transforming function on proto-Ha-ras and increased transcription greater than 100-fold compared with that of unrearranged proto-ras. Point mutations were not necessary for transforming function of rat and human proto-Ha-ras genes with retroviral promoters but did enhance it greater than 10-fold. A unifying hypothesis proposes that proto-ras genes depend on high expression from heterologous promoters or enhancers for transforming function, which is modulated by ras point mutations. The hypothesis makes two testable predictions. (i) Unrearranged proto-ras genes with point mutations, which occur in some cancers, have no transforming function. Indeed, tumors with mutated proto-ras genes, even those that also lack hypothetical tumor-suppressor genes, are indistinguishable from counterparts with normal proto-ras genes. (ii) Proto-ras genes in transfected cells derive transforming function from heterologous promoters or enhancers acquired via illegitimate recombination from vector DNAs and particularly from viral helper genes that must be cotransfected for transformation of primary cells. Indeed, expression of exogenous proto-ras genes in cells transformed by transfection is as high as for viral ras genes and is much higher than in the

  14. 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

  15. Immunoglobulin heavy-chain enhancer is required to maintain transfected gamma 2A gene expression in a pre-B-cell line.

    PubMed Central

    Porton, B; Zaller, D M; Lieberson, R; Eckhardt, L A

    1990-01-01

    The immunoglobulin heavy-chain (IgH) enhancer serves to activate efficient and accurate transcription of cloned IgH genes when introduced into B lymphomas or myelomas. The role of this enhancer after gene activation, however, is unclear. The endogenous IgH genes in several cell lines, for example, have lost the IgH enhancer by deletion and yet continue to be expressed. This might be explained if the role of the enhancer were to establish high-level gene transcription but not to maintain it. Alternatively, other enhancers might lie adjacent to endogenous IgH genes, substituting their activity for that of the lost IgH enhancer. To address both of these alternatives, we searched for enhancer activity within the flanking regions of one of these IgH enhancer-independent genes and designed an experiment that allowed us to consider separately the establishment and maintenance of expression of a transfected gene. For the latter experiment we generated numerous pre-B cell lines stably transformed with a gamma 2a gene. In this gene, the IgH enhancer lay at a site outside the heavy-chain transcription unit, between DH and JH gene segments. After expression of the transfected gene was established, selective conditions were chosen for the outgrowth of subclones that had undergone D-J joining and thus IgH enhancer deletion. Measurements of gamma 2a expression before and after enhancer deletion revealed that the enhancer was required for maintenance of expression of the transfected gene. The implication of this finding for models of enhancer function in endogenous genes is discussed. Images PMID:2106067

  16. Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene.

    PubMed

    Geckil, Hikmet; Barak, Ze'ev; Chipman, David M; Erenler, Sebnem O; Webster, Dale A; Stark, Benjamin C

    2004-10-01

    Microbial production of butanediol and acetoin has received increasing interest because of their diverse potential practical uses. Although both products are fermentative in nature, their optimal production requires a low level of oxygen. In this study, the use of a recombinant oxygen uptake system on production of these metabolites was investigated. Enterobacter aerogenes was transformed with a pUC8-based plasmid carrying the gene (vgb) encoding Vitreoscilla (bacterial)hemoglobin (VHb). The presence of vgb and production of VHb by this strain resulted in an increase in viability from 72 to 96 h in culture, but no overall increase in cell mass. Accumulation of the fermentation products acetoin and butanediol were enhanced (up to 83%) by the presence of vgb/VHb. This vgb/VHb related effect appears to be due to an increase of flux through the acetoin/butanediol pathway, but not at the expense of acid production.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. Rational design of a super core promoter that enhances gene expression.

    PubMed

    Juven-Gershon, Tamar; Cheng, Susan; Kadonaga, James T

    2006-11-01

    Transcription is a critical component in the expression of genes. Here we describe the design and analysis of a potent core promoter, termed super core promoter 1 (SCP1), which directs high amounts of transcription by RNA polymerase II in metazoans. SCP1 contains four core promoter motifs-the TATA box, initiator (Inr), motif ten element (MTE) and downstream promoter element (DPE)-in a single promoter, and is distinctly stronger than the cytomegalovirus (CMV) IE1 and adenovirus major late (AdML) core promoters both in vitro and in vivo. Each of the four core promoter motifs is needed for full SCP1 activity. SCP1 is bound efficiently by TFIID and exhibits a high propensity to form productive transcription complexes. SCP1 and related super core promoters (SCPs) with multiple core promoter motifs will be useful for the biophysical analysis of TFIID binding to DNA, the biochemical investigation of the transcription process and the enhancement of gene expression in cells.

  4. 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

  5. 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

  6. 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).

  7. Transgenic banana expressing Pflp gene confers enhanced resistance to Xanthomonas wilt disease.

    PubMed

    Namukwaya, B; Tripathi, L; Tripathi, J N; Arinaitwe, G; Mukasa, S B; Tushemereirwe, W K

    2012-08-01

    Banana Xanthomonas wilt (BXW), caused by Xanthomonas campestris pv. musacearum, is one of the most important diseases of banana (Musa sp.) and currently considered as the biggest threat to banana production in Great Lakes region of East and Central Africa. The pathogen is highly contagious and its spread has endangered the livelihood of millions of farmers who rely on banana for food and income. The development of disease resistant banana cultivars remains a high priority since farmers are reluctant to employ labor-intensive disease control measures and there is no host plant resistance among banana cultivars. In this study, we demonstrate that BXW can be efficiently controlled using transgenic technology. Transgenic bananas expressing the plant ferredoxin-like protein (Pflp) gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of banana. These transgenic lines were characterized by molecular analysis. After challenge with X. campestris pv. musacearum transgenic lines showed high resistance. About 67% of transgenic lines evaluated were completely resistant to BXW. These transgenic lines did not show any disease symptoms after artificial inoculation of in vitro plants under laboratory conditions as well as potted plants in the screen-house, whereas non-transgenic control plants showed severe symptoms resulting in complete wilting. This study confirms that expression of the Pflp gene in banana results in enhanced resistance to BXW. This transgenic technology can provide a timely solution to the BXW pandemic.

  8. 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

  9. Enhanced water stress tolerance of transgenic maize plants over-expressing LEA Rab28 gene.

    PubMed

    Amara, Imen; Capellades, Montserrat; Ludevid, M Dolors; Pagès, Montserrat; Goday, Adela

    2013-06-15

    Late Embryogenesis Abundant (LEA) proteins participate in plant stress responses and contribute to the acquisition of desiccation tolerance. In this report Rab28 LEA gene has been over-expressed in maize plants under a constitutive maize promoter. The expression of Rab28 transcripts led to the accumulation and stability of Rab28 protein in the transgenic plants. Native Rab28 protein is localized to nucleoli in wild type maize embryo cells; here we find by whole-mount immunocytochemistry that in root cells of Rab28 transgenic and wild-type plants the protein is also associated to nucleolar structures. Transgenic plants were tested for stress tolerance and resulted in sustained growth under polyethyleneglycol (PEG)-mediated dehydration compared to wild-type controls. Under osmotic stress transgenic seedlings showed increased leaf and root areas, higher relative water content (RWC), reduced chlorophyll loss and lower Malondialdehyde (MDA) production in relation to wild-type plants. Moreover, transgenic seeds exhibited higher germination rates than wild-type seeds under water deficit. Overall, our results highlight the presence of transgenic Rab28 protein in nucleolar structures and point to the potential of group 5 LEA Rab28 gene as candidate to enhance stress tolerance in maize plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

  10. 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.

  11. DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus

    PubMed Central

    2013-01-01

    Background DNA methylation is of pivotal importance during development. Previous genome-wide studies identified numerous differentially methylated regions upon differentiation of stem cells, many of them associated with transcriptional start sites. Results We present the first genome-wide, single-base-resolution view into DNA methylation dynamics during differentiation of a mammalian epithelial stem cell: the mouse small intestinal Lgr5+ stem cell. Very little change was observed at transcriptional start sites and our data suggest that differentiation-related genes are already primed for expression in the stem cell. Genome-wide, only 50 differentially methylated regions were identified. Almost all of these loci represent enhancers driving gene expression in the differentiated part of the small intestine. Finally, we show that binding of the transcription factor Tcf4 correlates with hypo-methylation and demonstrate that Tcf4 is one of the factors contributing to formation of differentially methylated regions. Conclusions Our results reveal limited DNA methylation dynamics during small intestine stem cell differentiation and an impact of transcription factor binding on shaping the DNA methylation landscape during differentiation of stem cells in vivo. PMID:23714178

  12. Overexpression of tomato SpMPK3 gene in Arabidopsis enhances the osmotic tolerance.

    PubMed

    Li, Cui; Chang, Pei-Pei; Ghebremariam, Kibreab Mehretead; Qin, Lei; Liang, Yan

    2014-01-10

    Mitogen-activated protein kinase (MAPK) class plays diverse roles in plant response to abiotic stresses. SpMPK3, a serine/threonine protein kinase containing a Thr-Glu-Tyr (TEY) activation domain, has been reported to function against bacterial, fungus and insect attack in tomatoes. But, its roles in abiotic stress response are poorly experienced. Herein, the experiment we have conducted demonstrates that there is a substantial increase of SpMPK3 expression in tomato leaves by drought, salt and cold stress. Transgenic Arabidopsis plants overexpressing SpMPK3 showed improved seed germination and antioxidant capacity under osmotic stress, but decreased seed germination with ABA treatment compared to wile-type. In addition, SpMPK3 overexpression enhanced the transcription levels of ABA inducible genes-RD29A, RAB18 and RD22, and transcription factor genes-ZAT6, ZAT10 and MYB44, in transgenic Arabidopsis compared with wild-type in response to ABA or salt stress. These results suggest that SpMPK3 is a positive regulator in response to osmotic stress in Arabidopsis at germination and development stage. Copyright © 2014. Published by Elsevier Inc.

  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. Expression of the novel wheat gene TM20 confers enhanced cadmium tolerance to bakers' yeast.

    PubMed

    Kim, Yu-Young; Kim, Do-Young; Shim, Donghwan; Song, Won-Yong; Lee, Joohyun; Schroeder, Julian I; Kim, Sanguk; Moran, Nava; Lee, Youngsook

    2008-06-06

    Cadmium causes the generation of reactive oxygen species, which in turn causes cell damage. We isolated a novel gene from a wheat root cDNA library, which conferred Cd(II)-specific tolerance when expressed in yeast (Saccharomyces cerevisiae). The gene, which we called TaTM20, for Triticum aestivum transmembrane 20, encodes a putative hydrophobic polypeptide of 889 amino acids, containing 20 transmembrane domains arranged as a 5-fold internal repeating unit of 4 transmembrane domains each. Expression of TaTM20 in yeast cells stimulated Cd(II) efflux resulting in a decrease in the content of yeast intracellular cadmium. TaTM20-induced Cd(II) tolerance was maintained in yeast even under conditions of reduced GSH. These results demonstrate that TaTM20 enhances Cd(II) tolerance in yeast through the stimulation of Cd(II) efflux from the cell, partially independent of GSH. Treatment of wheat seedlings with Cd(II) induced their expression of TaTM20, decreasing subsequent root Cd(II) accumulation and suggesting a possible role for TaTM20 in Cd(II) tolerance in wheat.

  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. 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.

  17. 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.

  18. 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.

  19. Enhancement of sciatic nerve regeneration after vascular endothelial growth factor (VEGF) gene therapy.

    PubMed

    Pereira Lopes, F R; Lisboa, B C G; Frattini, F; Almeida, F M; Tomaz, M A; Matsumoto, P K; Langone, F; Lora, S; Melo, P A; Borojevic, R; Han, S W; Martinez, A M B

    2011-10-01

    Recent studies have emphasized the beneficial effects of the vascular endothelial growth factor (VEGF) on neurone survival and Schwann cell proliferation. VEGF is a potent angiogenic factor, and angiogenesis has long been recognized as an important and necessary step during tissue repair. Here, we investigated the effects of VEGF on sciatic nerve regeneration. Using light and electron microscopy, we evaluated sciatic nerve regeneration after transection and VEGF gene therapy. We examined the survival of the neurones in the dorsal root ganglia and in lumbar 4 segment of spinal cord. We also evaluated the functional recovery using the sciatic functional index and gastrocnemius muscle weight. In addition, we evaluated the VEGF expression by immunohistochemistry. Fluorescein isothiocyanate-dextran (FITC-dextran) fluorescence of nerves and muscles revealed intense staining in the VEGF-treated group. Quantitative analysis showed that the numbers of myelinated fibres and blood vessels were significantly higher in VEGF-treated animals. VEGF also increased the survival of neurone cell bodies in dorsal root ganglia and in spinal cord. The sciatic functional index and gastrocnemius muscle weight reached significantly higher values in VEGF-treated animals. We demonstrate a positive relationship between increased vascularization and enhanced nerve regeneration, indicating that VEGF administration can support and enhance the growth of regenerating nerve fibres, probably through a combination of angiogenic, neurotrophic and neuroprotective effects. © 2011 The Authors. Neuropathology and Applied Neurobiology © 2011 British Neuropathological Society.

  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. 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.

  2. 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.

  3. Enhancement in Motor Learning through Genetic Manipulation of the Lynx1 Gene

    PubMed Central

    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. PMID:23139735

  4. Urea uptake enhances barrier function and antimicrobial defense in humans by regulating epidermal gene expression

    PubMed Central

    Grether-Beck, Susanne; Felsner, Ingo; Brenden, Heidi; Kohne, Zippora; Majora, Marc; Marini, Alessandra; Jaenicke, Thomas; Rodriguez-Martin, Marina; Trullas, Carles; Hupe, Melanie; Elias, Peter M.; Krutmann, Jean

    2012-01-01

    Urea is an endogenous metabolite, known to enhance stratum corneum hydration. Yet, topical urea anecdotally also improves permeability barrier function, and it appears to exhibit antimicrobial activity. Hence, we hypothesized that urea is not merely a passive metabolite, but a small-molecule regulator of epidermal structure and function. In 21 human volunteers, topical urea improved barrier function in parallel with enhanced antimicrobial peptide (LL-37 and β-defensin-2) expression. Urea both stimulates expression of, and is transported into keratinocytes by two urea transporters, UT-A1 and UT-A2, and by aquaporin 3, 7 and 9. Inhibitors of these urea transporters block the downstream biological effects of urea, which include increased mRNA and protein levels for: (i) transglutaminase-1, involucrin, loricrin and filaggrin; (ii) epidermal lipid synthetic enzymes, and (iii) cathelicidin/LL-37 and β-defensin-2. Finally, we explored the potential clinical utility of urea, showing that topical urea applications normalized both barrier function and antimicrobial peptide expression in a murine model of atopic dermatitis (AD). Together, these results show that urea is a small-molecule regulator of epidermal permeability barrier function and antimicrobial peptide expression after transporter uptake, followed by gene regulatory activity in normal epidermis, with potential therapeutic applications in diseased skin. PMID:22418868

  5. 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.

  6. 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

  7. A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression.

    PubMed

    Rodríguez-Mejía, José-Luis; Roldán-Salgado, Abigail; Osuna, Joel; Merino, Enrique; Gaytán, Paul

    2017-01-01

    Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions. © 2016 S. Karger AG, Basel.

  8. Functional Enhancers at the Gene-Poor 8q24 Cancer-Linked Locus

    PubMed Central

    Pomerantz, Mark; Jaschek, Rami; Herman, Paula; Reich, David; Yan, Chunli; Khalid, Omar; Kantoff, Phil; Oh, William; Manak, J. Robert; Berman, Benjamin P.; Henderson, Brian E.; Frenkel, Baruch; Haiman, Christopher A.; Freedman, Matthew; Tanay, Amos; Coetzee, Gerhard A.

    2009-01-01

    Multiple discrete regions at 8q24 were recently shown to contain alleles that predispose to many cancers including prostate, breast, and colon. These regions are far from any annotated gene and their biological activities have been unknown. Here we profiled a 5-megabase chromatin segment encompassing all the risk regions for RNA expression, histone modifications, and locations occupied by RNA polymerase II and androgen receptor (AR). This led to the identification of several transcriptional enhancers, which were verified using reporter assays. Two enhancers in one risk region were occupied by AR and responded to androgen treatment; one contained a single nucleotide polymorphism (rs11986220) that resides within a FoxA1 binding site, with the prostate cancer risk allele facilitating both stronger FoxA1 binding and stronger androgen responsiveness. The study reported here exemplifies an approach that may be applied to any risk-associated allele in non-protein coding regions as it emerges from genome-wide association studies to better understand the genetic predisposition of complex diseases. PMID:19680443

  9. An integrated holo-enhancer unit defines tissue and gene specificity of the Fgf8 regulatory landscape.

    PubMed

    Marinić, Mirna; Aktas, Tugce; Ruf, Sandra; Spitz, François

    2013-03-11

    Fgf8 encodes a key signaling factor, and its precise regulation is essential for embryo patterning. Here, we identified the regulatory modules that control Fgf8 expression during mammalian embryogenesis. These enhancers are interspersed with unrelated genes along a large region of 220 kb; yet they act on Fgf8 only. Intriguingly, this region also contains additional genuine enhancer activities that are not transformed into gene expression. Using genomic engineering strategies, we showed that these multiple and distinct regulatory modules act as a coherent unit and influence genes depending on their position rather than on their promoter sequence. These findings highlight how the structure of a locus regulates the autonomous intrinsic activities of the regulatory elements it contains and contributes to their tissue and target specificities. We discuss the implications of such regulatory systems regarding the evolution of gene expression and the impact of human genomic structural variations. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Icodextrin enhances survival in an intraperitoneal ovarian cancer murine model utilizing gene therapy.

    PubMed

    Rocconi, Rodney P; Numnum, Michael T; Zhu, Zeng B; Lu, Baogen; Wang, Minghui; Rivera, Angel A; Stoff-Khalili, Mariam; Alvarez, Ronald D; Curiel, David T; Makhija, Sharmila

    2006-12-01

    Icodextrin, a novel glucose polymer solution utilized for peritoneal dialysis, has been demonstrated to have prolonged intraperitoneal (IP) instillation volumes in comparison to standard PBS solutions. In an animal model of ovarian cancer, we explored whether a survival advantage exists utilizing icodextrin rather than PBS as a delivery solution for an infectivity enhanced virotherapy approach. Initial experiments evaluated whether icodextrin would adversely affect replication of a clinical grade infectivity enhanced conditionally replicative adenovirus (Delta24-RGD). Virus was added to prepared blinded solutions of PBS or icodextrin (20%) and then evaluated in vitro in various human ovarian cancer cell lines (SKOV3.ip1, PA-1, and Hey) and in vivo in a SKOV3.ip1 human ovarian cancer IP murine model. Viral replication was measured by detecting adenovirus E4 gene levels utilizing QRT-PCR. Survival was subsequently evaluated in a separate SKOV3.ip1 ovarian cancer IP murine model. Cohorts of mice were treated in blinded fashion with PBS alone, icodextrin alone, PBS+Delta24-RGD, or icodextrin+Delta24-RGD. Survival data were plotted on Kaplan-Meier curve and statistical calculations performed using the log-rank test. There was no adverse affect of icodextrin on vector replication in the ovarian cancer cell lines nor murine model tumor samples evaluated. Median survival in the IP treated animal cohorts was 23 days for the PBS group, 40 days for the icodextrin group, 65 days for the PBS+Delta24-RGD group, and 105 days for icodextrin+Delta24-RGD (p=0.023). Of note, 5 of the 10 mice in the icodextrin+Delta24-RGD group were alive at the end of the study period, all without evidence of tumor (120 days). These experiments suggest that the use of dialysates such as icodextrin may further enhance the therapeutic effects of novel IP virotherapy and other gene therapy strategies for ovarian cancer. Phase I studies utilizing icodextrin-based virotherapy for ovarian cancer are

  17. Deep developmental transcriptome sequencing uncovers numerous new genes and enhances gene annotation in the sponge Amphimedon queenslandica.

    PubMed

    Fernandez-Valverde, Selene L; Calcino, Andrew D; Degnan, Bernard M

    2015-05-15

    The demosponge Amphimedon queenslandica is amongst the few early-branching metazoans with an assembled and annotated draft genome, making it an important species in the study of the origin and early evolution of animals. Current gene models in this species are largely based on in silico predictions and low coverage expressed sequence tag (EST) evidence. Amphimedon queenslandica protein-coding gene models are improved using deep RNA-Seq data from four developmental stages and CEL-Seq data from 82 developmental samples. Over 86% of previously predicted genes are retained in the new gene models, although 24% have additional exons; there is also a marked increase in the total number of annotated 3' and 5' untranslated regions (UTRs). Importantly, these new developmental transcriptome data reveal numerous previously unannotated protein-coding genes in the Amphimedon genome, increasing the total gene number by 25%, from 30,060 to 40,122. In general, Amphimedon genes have introns that are markedly smaller than those in other animals and most of the alternatively spliced genes in Amphimedon undergo intron-retention; exon-skipping is the least common mode of alternative splicing. Finally, in addition to canonical polyadenylation signal sequences, Amphimedon genes are enriched in a number of unique AT-rich motifs in their 3' UTRs. The inclusion of developmental transcriptome data has substantially improved the structure and composition of protein-coding gene models in Amphimedon queenslandica, providing a more accurate and comprehensive set of genes for functional and comparative studies. These improvements reveal the Amphimedon genome is comprised of a remarkably high number of tightly packed genes. These genes have small introns and there is pervasive intron retention amongst alternatively spliced transcripts. These aspects of the sponge genome are more similar unicellular opisthokont genomes than to other animal genomes.

  18. Gef gene therapy enhances the therapeutic efficacy of doxorubicin to combat growth of MCF-7 breast cancer cells.

    PubMed

    Prados, Jose; Melguizo, Consolación; Rama, Ana Rosa; Ortiz, Rául; Segura, Ana; Boulaiz, Houria; Vélez, Celia; Caba, Octavio; Ramos, Juan Luís; Aránega, Antonia

    2010-05-01

    The potential use of combined therapy is under intensive study including the association between classical cytotoxic and genes encoding toxic proteins which enhanced the antitumour activity. The main aim of this work was to evaluate whether the gef gene, a suicide gene which has a demonstrated antiproliferative activity in tumour cells, improved the antitumour effect of chemotherapeutic drugs used as first-line treatment in the management of advanced breast cancer. MCF-7 human breast cancer cells were transfected with gef gene using pcDNA3.1-TOPO expression vector. To determine the effect of the combined therapy, MCF-7 transfected and non-transfected cells were exposed to paclitaxel, docetaxel and doxorubicin at different concentrations. The growth-inhibitory effect of gef gene and/or drugs was assessed by MTT assay. Apoptosis modulation was determined by flow cytometric analysis, DNA fragmentation and morphological analysis. Multicellular tumour spheroids (MTS) from MCF-7 cells were used to confirm effectiveness of combined therapy (gef gene and drug). Our results demonstrate that combined therapy gef gene/drugs (paclitaxel, docetaxel or doxurubicin) caused a decrease in cell viability. However, only the gef-doxorubicin (10 microM) combination induced a greater enhancement in the antitumour activity in MCF-7 cells. Most importantly, this combined strategy resulted in a significant synergistic effect, thus allowing lower doses of the drug to be used to achieve the same therapeutic effect. These results were confirmed using MTS in which volume decrease with combined therapy was greater than obtained using the gene therapy or chemotherapy alone, or the sum of both therapies. The cytotoxic effect of gef gene in breast cancer cells enhances the chemotherapeutic effect of doxorubicin. This therapeutic approach has the potential to overcome some of the major limitations of conventional chemotherapy, and may therefore constitute a promising strategy for future

  19. 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.

  20. 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

  1. 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.

  2. Disruption of the MIG1 gene enhances lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica ACA-DC 50109.

    PubMed

    Wang, Zhi-Peng; Xu, Hong-Mei; Wang, Guang-Yuan; Chi, Zhe; Chi, Zhen-Ming

    2013-04-01

    In this study, the MIG1 gene in the oleaginous yeast Yarrowia lipolytica ACA-DC 50109 (the parent yeast) was disrupted and the disruptant M25 obtained could grow in yeast nitrogen base-N5000 medium without uracil or the medium with 2-deoxy-D-glucose. It was found that the cells of the disruptant M25 had more lipid bodies than those of its parent yeast. The disruptant M25 contained 48.7% (w/w) of oil based on its cell weight while the parent yeast only contained 36.0% (w/w) of oil. Transcript levels of many genes relevant to lipid biosynthesis in the disruptant M25 were enhanced compared to those of the same genes in the parent yeast. However, transcript level of the MFE1 gene, one of the genes relevant to fatty acid degradation was reduced in the disruptant M25 compared to that of the same gene in the parent yeast. Such changes in gene expression profile may cause the increased lipid biosynthesis in the disruptant M25. Biosynthesis of C18:1 fatty acid in the disruptant M25 was greatly enhanced compared to that in the parent yeast. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. 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.

  4. StAR Enhances Transcription of Genes Encoding the Mitochondrial Proteases Involved in Its Own Degradation

    PubMed Central

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

    2014-01-01

    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. PMID:24422629

  5. Enhanced biosurfactant production through cloning of three genes and role of esterase in biosurfactant release

    PubMed Central

    2011-01-01

    Background Biosurfactants have been reported to utilize a number of immiscible substrates and thereby facilitate the biodegradation of panoply of polyaromatic hydrocarbons. Olive oil is one such carbon source which has been explored by many researchers. However, studying the concomitant production of biosurfactant and esterase enzyme in the presence of olive oil in the Bacillus species and its recombinants is a relatively novel approach. Results Bacillus species isolated from endosulfan sprayed cashew plantation soil was cultivated on a number of hydrophobic substrates. Olive oil was found to be the best inducer of biosurfactant activity. The protein associated with the release of the biosurfactant was found to be an esterase. There was a twofold increase in the biosurfactant and esterase activities after the successful cloning of the biosurfactant genes from Bacillus subtilis SK320 into E.coli. Multiple sequence alignment showed regions of similarity and conserved sequences between biosurfactant and esterase genes, further confirming the symbiotic correlation between the two. Biosurfactants produced by Bacillus subtilis SK320 and recombinant strains BioS a, BioS b, BioS c were found to be effective emulsifiers, reducing the surface tension of water from 72 dynes/cm to as low as 30.7 dynes/cm. Conclusion The attributes of enhanced biosurfactant and esterase production by hyper-producing recombinant strains have many utilities from industrial viewpoint. This study for the first time has shown a possible association between biosurfactant production and esterase activity in any Bacillus species. Biosurfactant-esterase complex has been found to have powerful emulsification properties, which shows promising bioremediation, hydrocarbon biodegradation and pharmaceutical applications. PMID:21707984

  6. 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.

  7. Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells

    PubMed Central

    Nie, Fang; Xu, Hui-Xiong; Tang, Qing; Lu, Ming-De

    2006-01-01

    AIM: To explore the effects of ultrasound exposure combined with microbubble contrast agent (SonoVue) on the permeability of the cellular membrane and on the expression of plasmid DNA encoding enhanced green fluorescent protein (pEGFP) transfer into human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs with fluorescein isothiocyanate-dextran (FD500) and HUVECs with pEGFP were exposed to continuous wave (1.9 MHz, 80.0 mW/cm2) for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytometry, respectively. RESULTS: The percentage of FD500-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone (24.0% ± 5.5% vs 66.6% ± 4.1%, P < 0.001). Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue (16.1% ± 1.9% vs 1.5% ± 0.2%, P < 0.001). No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue (94.1% ± 2.3% vs 91.1% ± 4.1%). CONCLUSION: The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non-invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors. PMID:17167842

  8. Activation of the glutaredoxin-1 gene by Nuclear Factor kappa B enhances signaling

    PubMed Central

    Aesif, Scott W.; Kuipers, Ine; van der Velden, Jos; Tully, Jane E.; Guala, Amy S.; Anathy, Vikas; Sheely, Juliana I.; Reynaert, Niki L.; Wouters, Emiel F. M.; van der Vliet, Albert; Janssen-Heininger, Yvonne M. W.

    2011-01-01

    The transcription factor, Nuclear Factor kappa B (NF-κB) is a critical regulator of inflammation and immunity, and is negatively regulated via S-glutathionylation. The inhibitory effect of S-glutathionylation is overcome by glutaredoxin-1 (Grx1), which under physiological conditions catalyses deglutathionylation and enhances NF-κB activation. The mechanisms whereby expression of the Glrx1 gene is regulated remain unknown. Here we examined the role of NF-κB in regulating activation of Glrx1. Transgenic mice which express a doxycyclin-inducible constitutively active version of inhibitory kappa B kinase-beta (CA-IKKβ) demonstrate elevated expression of Grx1. Transient transfection of CA-IKKβ also resulted in significant induction of Grx1. A 2kb region Glrx1 promoter that contains two putative NF-κB binding sites was activated by CA-IKKβ, RelA/p50, and lipopolysaccharide (LPS). Chromatin immunoprecipitation experiments confirmed binding of RelA to the promoter of Glrx1 in response to LPS. Stimulation of C10 lung epithelial cells with LPS caused transient increases in Grx1 mRNA expression, and time-dependent increases in S-glutathionylation of IKKβ. Overexpression of Grx1 decreased S-glutathionylation of IKKβ, prolonged NF-κB activation, and increased levels of pro-inflammatory mediators. Collectively, this study demonstrates that the Glrx1 gene is positively regulated by NF-κB, and suggests a feed forward mechanism to promote NF-κB signaling by decreasing S-glutathionylation. PMID:21762778

  9. Roles of Plasmalemma Aquaporin Gene StPIP1 in Enhancing Drought Tolerance in Potato

    PubMed Central

    Wang, Li; Liu, Yuhui; Feng, Shoujiang; Yang, Jiangwei; Li, Dan; Zhang, Junlian

    2017-01-01

    Survival and mortality of plants in response to severe drought may be related to carbon starvation, but little is known about how plasma membrane intrinsic proteins may help alleviate the drought-induced damage. Here, we determined the roles of plasmalemma aquaporin gene in improving plant water status, maintaining carbon accumulation, and thereby enhancing drought tolerance. Seven StPIP1 transformed potato (Solanum tuberosum L.) lines (namely T1, T2…T7) were compared with non-transgenic control plant at molecule and whole-plant levels. The relative expression of StPIP1 gene was found in leaves, stems and roots, with the most abundant expression being in the roots. The transgenic lines T6 and T7 had the highest StPIP1 expression, averaging 7.2 times that of the control and the greatest differences occurred 48 h after mannitol osmotic stress treatment. Using an evaluation index to quantifying the degree of drought tolerance, we found that the StPIP1 transgenic lines T6 and T7 had the highest drought tolerance, averaging 8.5 times that of the control. Measured at 30 days in drought stress treatment, the control plant decreased net photosynthetic rate by 33 and 56%, respectively, under moderate and severe stresses; also decreased stomatal conductance by 39 and 65%; and lowered transpiration rate by 49 and 69%, compared to the no-stress treatment, whereas the transgenic lines T6 and T7 maintained a relatively stable level with slight decreases in these properties. The constitutive overexpression of StPIP1 in potato improved plant water use efficiency and increased nonstructural carbohydrate concentration, which helped alleviate carbon starvation and minimized the loss of biomass and tuber yield due to drought stress. We conclude that the expression of StPIPs improves overall water relations in the plant and helps maintain photosynthesis and stomatal conductance; these help minimize carbon starvation and enhance the whole plant tolerance to drought stress. PMID

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

    USDA-ARS?s Scientific Manuscript database

    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...

  11. Disruption of the CAR1 gene encoding arginase enhances freeze tolerance of the commercial baker's yeast Saccharomyces cerevisiae.

    PubMed

    Shima, Jun; Sakata-Tsuda, Yuko; Suzuki, Yasuo; Nakajima, Ryouichi; Watanabe, Hajime; Kawamoto, Shinichi; Takano, Hiroyuki

    2003-01-01

    The effect of intracellular charged amino acids on freeze tolerance in dough was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.

  12. Learning about mammalian gene regulation from functional enhancer assays in the mouse.

    PubMed

    Nord, Alex S

    2015-09-01

    Enhancer biology is emerging as a critical area of research that informs studies of evolution, development, and disease. From early experiments that defined and mapped the first enhancers to recent enhancer models of human disease, functional experiments in the mouse have played a central role in revealing enhancer biology. Three decades of in vivo enhancer studies in mouse have laid the groundwork for the current understanding of mammalian enhancers, demonstrating the developmental and tissue-specific activity of enhancers and illuminating general features of enhancer evolution and function. Recent studies offer an emerging perspective on the importance of chromosomal context, combinatorial enhancer activity, and the functional consequences of enhancer sequence variation. This review describes the basic principles of functional testing in mouse, summarizes the contributions these studies have made to our understanding of enhancer biology, and describes limitations and future outlook of in vivo mouse enhancer studies. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. 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.

  14. 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

  15. 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.

  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. 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.

  1. 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.

  2. 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.

  3. 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

  4. Genetic variants in the enhancer region of the thymidylate synthase gene in the Chilean population

    PubMed Central

    Acuña, M; Eaton, L; Cifuentes, L; Massardo, D

    2006-01-01

    Aims Thymidylate synthase (TYMS) is an important target enzyme for the fluoropyrimidines. The TYMS gene enhancer region possesses tandemly repeated (TSER) sequences that are polymorphic in humans and different among ethnic groups. The aims of this study were to estimate the frequencies of the TSER variants in two hospital samples located in the northern (HSJ) and eastern (CLC) parts of Santiago, Chile, and compare them with the frequencies in other populations of different ethnic origin. Methods Genotyping of TSER variants in 368 Chilean subjects (HSJ = 178 and CLC = 190) by polymerase chain reaction; products of amplification were electrophoresed, obtaining fragments of 250 bp for allele TSER*3 and 220 bp for allele TSER*2. Results The two hospital samples had different degrees of Amerindian admixture (HSJ 34.5%; CLC 15.9%), which was not reflected in the observed frequencies of the CLC TSER*3: 56.8% and HSJ TSER*3: 53.4%. Conclusions Our results are unexpected, considering that genetic markers in the Chilean population generally show allele frequencies between those observed in European Caucasians and Amerindians and that the percentage of Amerindian admixture in CLC is lower than in HSJ. Both hospitals should have had greater frequencies of TSER*3 than were found and the frequency should have been greater in HSJ than in CLC; the only logical explanation of our results is that the frequency of this allele in aboriginal Chilean people is much lower than the 80% estimated for Mongoloid populations. PMID:16722845

  5. Programming a nonvolatile memory-like sensor for KRAS gene sensing and signal enhancement.

    PubMed

    Lin, Yi-Ting; Purwidyantri, Agnes; Luo, Ji-Dung; Chiou, Chiuan-Chian; Yang, Chia-Ming; Lo, Chih-Hong; Hwang, Tsann-Long; Yen, Tzung-Hai; Lai, Chao-Sung

    2016-05-15

    A programmable field effect-based electrolyte-insulator-semiconductor (EIS) sensor constructed with a nonvolatile memory-like structure is proposed for KRAS gene DNA hybridization detection. This programmable EIS structure was fabricated with silicon oxide (SiO2)/silicon nitride (Si3N4)/silicon oxide on a p-type silicon wafer, namely electrolyte-oxide-nitride-oxide-Si (EONOS). In this research, voltage stress programming from 4 to 20V was applied to trigger holes confinement in the nitride-trapping layer that, consequently, enhances the DNA attachment onto the sensing surface due to additional electrostatic interaction. Not solely resulting from the higher DNA load, the programming may affect the orientation of the DNA that finally contributes to the change in capacitance. Findings have shown that a higher voltage program is able to increase the total capacitance and results in ~3.5- and ~5.5-times higher sensitivities for a series of concentrations for complementary DNA and wild type versus mutant DNA hybridization detection, respectively. Overall, it has been proven that the voltage program on the nonvolatile memory-like structure of EONOS is a notable candidate for genosensor development, scoping the diagnosis of a single nucleotide polymorphism (SNP)-related disease.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. Gene gun transferring-bone morphogenetic protein 2 (BMP-2) gene enhanced bone fracture healing in rabbits

    PubMed Central

    Li, Wenju; Wei, Haifeng; Xia, Chunmei; Zhu, Xiaomeng; Hou, Guozhu; Xu, Feng; Song, Xinghua; Zhan, Yulin

    2015-01-01

    Purpose: Transferring the bone morphogenetic protein 2 (BMP-2) genes into the tissues or cells can improve the bone healing of the fracture has been widely accepted. We evaluated the efficiency of using gene gun to transfer the BMP-2 gene thereby affected the healing of a fractured bone. Methods: The vector coding for BMP-2 was constructed by a non-replicating encephalo-myocarditis virus (ECMV)-based vector. The segmental bone defect (1.5 cm) model was created by a wire-saw at the middle part of the radius bone of the New Zealand white rabbits. Then either BMP-2 gene or control vector without BMP-2 gene was injected into the tissues around the fracture site. Healing of the defects was monitored radiographically for 9 weeks, bone consolidation was determined by the Lane-Sandhu score pre- and post-operatively, which can evaluated bone formation, bone connect and bone plasticity. Results: The radiographic score and bone consolidation rates were significantly higher in animals injected with BMP-2 gene group as compared with control vector-injected animals (P<0.05). The control group still showed no radiological signs of stable healing. Western-blot and RT-PCR showed BMP-2 expression was significant increase in the tissues around the site of osseous lesions in comparison with the control vector-injected animals (P<0.05). Conclusions: Our results suggested that BMP-2 gene transferred by gene gun could increase the expression of BMP-2 protein and improved the bone callus formation therefore shortened the time of bone defect healing. PMID:26884910

  12. Automated Discovery of Tissue-Targeting Enhancers and Transcription Factors from Binding Motif and Gene Function Data

    PubMed Central

    Tuteja, Geetu; Moreira, Karen Betancourt; Chung, Tisha; Chen, Jenny; Wenger, Aaron M.; Bejerano, Gill

    2014-01-01

    Identifying enhancers regulating gene expression remains an important and challenging task. While recent sequencing-based methods provide epigenomic characteristics that correlate well with enhancer activity, it remains onerous to comprehensively identify all enhancers across development. Here we introduce a computational framework to identify tissue-specific enhancers evolving under purifying selection. First, we incorporate high-confidence binding site predictions with target gene functional enrichment analysis to identify transcription factors (TFs) likely functioning in a particular context. We then search the genome for clusters of binding sites for these TFs, overcoming previous constraints associated with biased manual curation of TFs or enhancers. Applying our method to the placenta, we find 33 known and implicate 17 novel TFs in placental function, and discover 2,216 putative placenta enhancers. Using luciferase reporter assays, 31/36 (86%) tested candidates drive activity in placental cells. Our predictions agree well with recent epigenomic data in human and mouse, yet over half our loci, including 7/8 (87%) tested regions, are novel. Finally, we establish that our method is generalizable by applying it to 5 additional tissues: heart, pancreas, blood vessel, bone marrow, and liver. PMID:24499934

  13. Fluoxetine potentiates methylphenidate-induced gene regulation in addiction-related brain regions: Concerns for use of cognitive enhancers?

    PubMed Central

    Steiner, Heinz; Van Waes, Vincent; Marinelli, Michela

    2009-01-01

    Background There is growing use of psychostimulant cognitive enhancers such as methylphenidate (Ritalin). Methylphenidate differs from the psychostimulant cocaine because it does not enhance brain levels of serotonin. We investigated whether exposure to methylphenidate combined with a serotonin-enhancing medication, the prototypical selective serotonin reuptake inhibitor (SSRI) fluoxetine (Prozac), would produce more “cocaine-like” molecular and behavioral changes. Methods We measured the effects of fluoxetine on gene expression induced by the cognitive enhancer methylphenidate in the striatum and nucleus accumbens of rats, by in situ hybridization histochemistry. We also determined whether fluoxetine modified behavioral effects of methylphenidate. Results Fluoxetine robustly potentiated methylphenidate-induced expression of the transcription factors c-fos and zif 268 throughout the striatum and to some degree in the nucleus accumbens. Fluoxetine also enhanced methylphenidate-induced stereotypical behavior. Conclusions Both potentiated gene regulation in the striatum and the behavioral effects indicate that combining the SSRI fluoxetine with the cognitive enhancer methylphenidate mimics cocaine effects, consistent with an increased risk for substance use disorder. PMID:19931852

  14. Enhancement of reporter gene detection sensitivity by insertion of specific mini-peptide-coding sequences.

    PubMed

    Cutrera, J; Dibra, D; Xia, X; Li, S

    2010-02-01

    Two important aspects of gene therapy are to increase the level of gene expression and track the gene delivery site and expression, and a sensitive reporter gene may be one of the options for preclinical studies and possibly for human clinical trials. We report the novel concept of increasing the activity of the gene products. With the insertion of the mini-peptide-coding sequence CWDDWLC into the plasmid DNA of a SEAP reporter gene, we observed vast increases in the enzyme activity in vitro in all murine and human cell lines used. In addition, in vivo injection of this CWDDWLC-SEAP-encoding gene resulted in the same increases in reporter gene activity, but these increases did not correspond to alterations in the level of the gene products in the serum. Minor sequence changes in this mini-peptide negate the activity increase of the reporter gene. We report the novel concept of increasing the activity of gene products as another method to improve the reporting sensitivity of reporter genes. This improved reporter gene could complement any improved vector for maximizing the reporter sensitivity. Moreover, this strategy has the potential to be used to discover peptides that improve the activity of therapeutic genes.

  15. Enhancement of Reporter Gene Detection Sensitivity by Insertion of Specific Mini-Peptide-Coding Sequences

    PubMed Central

    Cutrera, Jeffry; Dibra, Denada; Xia, Xueqing; Li, Shulin

    2009-01-01

    Two important aspects for gene therapy are to increase the level of gene expression and track the gene delivery site and expression, and a sensitive reporter gene may be one of the options for preclinical studies and possibly for human clinical trials. We report the novel concept of increasing the activity of the gene products. With the insertion of the mini-peptide-coding sequence CWDDWLC into the plasmid DNA of a SEAP reporter gene, we observed vast increases in the enzyme activity in vitro in all murine and human cell lines used. Also, in vivo injection of this CWDDWLC-SEAP encoding gene resulted in the same increases in reporter gene activity, but these increases did not correspond to alterations in the level of the gene products in the serum. Minor sequence changes in this mini-peptide negate the activity increase of the reporter gene. We report the novel concept of increasing the activity of gene products as another method to improve the reporting sensitivity of reporter genes. This improved reporter gene could complement any improved vector for maximizing the reporter sensitivity. Also, this strategy has the potential to be used to discover peptides that improve the activity of therapeutic genes. PMID:19713998

  16. 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.

  17. 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.

  18. A system biology approach highlights a hormonal enhancer effect on regulation of genes in a nitrate responsive "biomodule"

    PubMed Central

    Nero, Damion; Krouk, Gabriel; Tranchina, Daniel; Coruzzi, Gloria M

    2009-01-01

    Background Nitrate-induced reprogramming of the transcriptome has recently been shown to be highly context dependent. Herein, a systems biology approach was developed to identify the components and role of cross-talk between nitrate and hormone signals, likely to be involved in the conditional response of NO3- signaling. Results Biclustering was used to identify a set of genes that are N-responsive across a range of Nitrogen (N)-treatment backgrounds (i.e. nitrogen treatments under different growth conditions) using a meta-dataset of 76 Affymetrix ATH1 chips from 5 different laboratories. Twenty-one biclusters were found to be N-responsive across subsets of this meta-dataset. N-bicluster 9 (126 genes) was selected for further analysis, as it was shown to be reproducibly responsive to NO3- as a signal, across a wide-variety of background conditions and datasets. N-bicluster 9 genes were then used as "seed" to identify putative cross-talk mechanisms between nitrate and hormone signaling. For this, the 126 nitrate-regulated genes in N-bicluster 9 were biclustered over a meta-dataset of 278 ATH1 chips spanning a variety of hormone treatments. This analysis divided the bicluster 9 genes into two classes: i) genes controlled by NO3- only vs. ii) genes controlled by both NO3- and hormones. The genes in the latter group showed a NO3- response that is significantly enhanced, compared to the former. In silico analysis identified two Cis-Regulatory Elements candidates (CRE) (E2F, HSE) potentially involved the interplay between NO3- and hormonal signals. Conclusion This systems analysis enabled us to derive a hypothesis in which hormone signals are proposed to enhance the nitrate response, providing a potential mechanistic explanation for the link between nitrate signaling and the control of plant development. PMID:19500399

  19. 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

  20. 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.

  1. 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

  2. Genome-wide profiling of 24 hr diel rhythmicity in the water flea, Daphnia pulex: network analysis reveals rhythmic gene expression and enhances functional gene annotation.

    PubMed

    Rund, Samuel S C; Yoo, Boyoung; Alam, Camille; Green, Taryn; Stephens, Melissa T; Zeng, Erliang; George, Gary F; Sheppard, Aaron D; Duffield, Giles E; Milenković, Tijana; Pfrender, Michael E

    2016-08-18

    Marine and freshwater zooplankton exhibit daily rhythmic patterns of behavior and physiology which may be regulated directly by the light:dark (LD) cycle and/or a molecular circadian clock. One of the best-studied zooplankton taxa, the freshwater crustacean Daphnia, has a 24 h diel vertical migration (DVM) behavior whereby the organism travels up and down through the water column daily. DVM plays a critical role in resource tracking and the behavioral avoidance of predators and damaging ultraviolet radiation. However, there is little information at the transcriptional level linking the expression patterns of genes to the rhythmic physiology/behavior of Daphnia. Here we analyzed genome-wide temporal transcriptional patterns from Daphnia pulex collected over a 44 h time period under a 12:12 LD cycle (diel) conditions using a cosine-fitting algorithm. We used a comprehensive network modeling and analysis approach to identify novel co-regulated rhythmic genes that have similar network topological properties and functional annotations as rhythmic genes identified by the cosine-fitting analyses. Furthermore, we used the network approach to predict with high accuracy novel gene-function associations, thus enhancing current functional annotations available for genes in this ecologically relevant model species. Our results reveal that genes in many functional groupings exhibit 24 h rhythms in their expression patterns under diel conditions. We highlight the rhythmic expression of immunity, oxidative detoxification, and sensory process genes. We discuss differences in the chronobiology of D. pulex from other well-characterized terrestrial arthropods. This research adds to a growing body of literature suggesting the genetic mechanisms governing rhythmicity in crustaceans may be divergent from other arthropod lineages including insects. Lastly, these results highlight the power of using a network analysis approach to identify differential gene expression and provide novel

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

    PubMed Central

    Geng, Fan‐Suo; Manning, Elizabeth; Suster, Maximiliano; Kawakami, Koichi; Becker, Thomas S.

    2015-01-01

    Summary 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. genesis 53:640–651, 2015. © 2015 The Authors. genesis Published by Wiley Periodicals, Inc. PMID:26271004

  4. 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. © 2015 The Authors. genesis Published by Wiley Periodicals, Inc.

  5. 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

  6. 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

  7. Adenoviral Mediated Gene Transfer of IGF-1 Enhances Wound Healing and Induces Angiogenesis

    PubMed Central

    Balaji, S.; LeSaint, M.; Bhattacharya, S. S.; Moles, C.; Dhamija, Y.; Kidd, M.; Le, L.D.; King, A.; Shaaban, A.; Crombleholme, T. M.; Bollyky, P.; Keswani, S. G.

    2014-01-01

    to control treatments. Conclusions In two different models, our data demonstrates that adenoviral mediated gene transfer of IGF-1 results in enhanced wound healing and induces angiogenesis via a VEGF-independent pathway. Understanding the underlying mechanisms of IGF-1 effects on angiogenesis may help produce novel therapeutics for chronic wounds or diseases characterized by a deficit in neovascularization. PMID:24725678

  8. 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

  9. AAV-mediated gene targeting is significantly enhanced by transient inhibition of nonhomologous end joining or the proteasome in vivo.

    PubMed

    Paulk, Nicole K; Loza, Laura Marquez; Finegold, Milton J; Grompe, Markus

    2012-06-01

    Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah(-/-)Ku70(-/-) double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. Mutations in chicory FEH genes are statistically associated with enhanced resistance to post-harvest inulin depolymerization.

    PubMed

    Dauchot, Nicolas; Raulier, Pierre; Maudoux, Olivier; Notté, Christine; Bertin, Pierre; Draye, Xavier; Van Cutsem, Pierre

    2014-01-01

    Nucleotidic polymorphisms were identified in fructan exohydrolases genes which are statistically associated with enhanced susceptibility to post-harvest inulin depolymerization. Industrial chicory (Cichorium intybus L.) root is the main commercial source of inulin, a linear fructose polymer used as dietary fiber. Post-harvest, inulin is depolymerized into fructose which drastically increases processing cost. To identify genetic variations associated with enhanced susceptibility to post-harvest inulin depolymerization and related free sugars content increase, we used a candidate-gene approach focused on inulin and sucrose synthesis and degradation genes, all members of the family 32 of glycoside hydrolases (GH32). Polymorphism in these genes was first investigated by carrying out EcoTILLING on two groups of chicory breeding lines exhibiting contrasted response to post-harvest inulin depolymerization. This allowed the identification of polymorphisms significantly associated with depolymerization in three fructan exohydrolase genes (FEH). This association was confirmed on a wider panel of 116 unrelated families in which the FEH polymorphism explained 35 % of the post-harvest variance for inulin content, 36 % of variance for sucrose content, 18 % for inulin degree of polymerization, 23 % for free fructose content and 22 % for free glucose content. These polymorphisms were associated with significant post-harvest changes of inulin content, inulin chain length and free sugars content.

  17. 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 enhanci