76 FR 9028 - Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products; Availability

Science.gov (United States)

2011-02-16

...] Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products; Availability AGENCY: Food and... Therapy Products'' dated January 2011. The guidance document provides manufacturers of cellular and gene... for Industry: Potency Tests for Cellular and Gene Therapy Products'' dated January 2011. The guidance...

Regulatory structures for gene therapy medicinal products in the European Union.

Science.gov (United States)

Klug, Bettina; Celis, Patrick; Carr, Melanie; Reinhardt, Jens

2012-01-01

Taking into account the complexity and technical specificity of advanced therapy medicinal products: (gene and cell therapy medicinal products and tissue engineered products), a dedicated European regulatory framework was needed. Regulation (EC) No. 1394/2007, the "ATMP Regulation" provides tailored regulatory principles for the evaluation and authorization of these innovative medicines. The majority of gene or cell therapy product development is carried out by academia, hospitals, and small- and medium-sized enterprises (SMEs). Thus, acknowledging the particular needs of these types of sponsors, the legislation also provides incentives for product development tailored to them. The European Medicines Agency (EMA) and, in particular, its Committee for Advanced Therapies (CAT) provide a variety of opportunities for early interaction with developers of ATMPs to enable them to have early regulatory and scientific input. An important tool to promote innovation and the development of new medicinal products by micro-, small-, and medium-sized enterprises is the EMA's SME initiative launched in December 2005 to offer financial and administrative assistance to smaller companies. The European legislation also foresees the involvement of stakeholders, such as patient organizations, in the development of new medicines. Considering that gene therapy medicinal products are developed in many cases for treatment of rare diseases often of monogenic origin, the involvement of patient organizations, which focus on rare diseases and genetic and congenital disorders, is fruitful. Two such organizations are represented in the CAT. Research networks play another important role in the development of gene therapy medicinal products. The European Commission is funding such networks through the EU Sixth Framework Program. Copyright © 2012 Elsevier Inc. All rights reserved.

Candidate genes for drought tolerance and improved productivity in ...

Indian Academy of Sciences (India)

Madhu

tropics. Improving drought tolerance and productivity is one of the most difficult tasks for cereal breeders. The diffi- culty arises from the diverse strategies adopted by plants themselves to combat drought stress depending on the timing,. Candidate genes for drought tolerance and improved productivity in rice (Oryza sativa L.).

Escherichia coli yjjPB genes encode a succinate transporter important for succinate production.

Science.gov (United States)

Fukui, Keita; Nanatani, Kei; Hara, Yoshihiko; Yamakami, Suguru; Yahagi, Daiki; Chinen, Akito; Tokura, Mitsunori; Abe, Keietsu

2017-09-01

Under anaerobic conditions, Escherichia coli produces succinate from glucose via the reductive tricarboxylic acid cycle. To date, however, no genes encoding succinate exporters have been established in E. coli. Therefore, we attempted to identify genes encoding succinate exporters by screening an E. coli MG1655 genome library. We identified the yjjPB genes as candidates encoding a succinate transporter, which enhanced succinate production in Pantoea ananatis under aerobic conditions. A complementation assay conducted in Corynebacterium glutamicum strain AJ110655ΔsucE1 demonstrated that both YjjP and YjjB are required for the restoration of succinate production. Furthermore, deletion of yjjPB decreased succinate production in E. coli by 70% under anaerobic conditions. Taken together, these results suggest that YjjPB constitutes a succinate transporter in E. coli and that the products of both genes are required for succinate export.

Radiochemical identification of the kil gene product of bacteriophage lambda

International Nuclear Information System (INIS)

Greer, H.; Ausubel, F.M.

1979-01-01

The coliphage lambda kil gene product has been identified using a differential labeling technique . The kil gene polypeptide has a molecular weight of about 16,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration of the kil protein indicates that it may exist as a tetramer in native form

Detection of biosurfactants in Bacillus species: genes and products identification.

Science.gov (United States)

Płaza, G; Chojniak, J; Rudnicka, K; Paraszkiewicz, K; Bernat, P

2015-10-01

To screen environmental Bacillus strains for detection of genes encoding the enzymes involved in biosurfactant synthesis and to evaluate their products e.g. surfactin, iturin and fengycin. The taxonomic identification of isolated from the environment Bacillus strains was performed by Microgene ID Bacillus panel and GEN III Biolog system. The polymerase chain reaction (PCR) strategy for screening of genes in Bacillus strains was set up. Liquid chromatography-mass spectrometry (LC-MS/MS) method was used for the identification of lipopeptides (LPs). All studied strains exhibited the presence of srfAA gene and produced surfactin mostly as four homologues (C13 to C16). Moreover, in 2 strains (KP7, T'-1) simultaneous co-production of 3 biosurfactants: surfactin, iturin and fengycin was observed. Additionally, it was found out that isolate identified as Bacillus subtilis ssp. subtilis (KP7), beside LPs co-production, synthesizes surfactin with the efficiency much higher than other studied strains (40·2 mg l(-1) ) and with the yield ranging from 0·8 to 8·3 mg l(-1) . We showed that the combined methodology based on PCR and LC-MS/MS technique is an optimal tool for the detection of genes encoding enzymes involved in biosurfactant synthesis as well as their products, e.g. surfactin, iturin and fengycin. This approach improves the screening and the identification of environmental Bacillus co-producing biosurfactants-stimulating and facilitating the development of this area of science. The findings of this work will help to improve screening of biosurfactant producers. Discovery of novel biosurfactants and biosurfactants co-production ability has shed light on their new application fields and for the understanding of their interactions and properties. © 2015 The Society for Applied Microbiology.

Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

International Nuclear Information System (INIS)

Malpass, Gloria E.; Arimilli, Subhashini; Prasad, G.L.; Howlett, Allyn C.

2014-01-01

Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes

Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Malpass, Gloria E., E-mail: gloria.malpass@gmail.com [Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States); Arimilli, Subhashini, E-mail: sarimill@wakehealth.edu [Department of Microbiology and Immunology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States); Prasad, G.L., E-mail: prasadg@rjrt.com [R and D Department, R.J. Reynolds Tobacco Company, Winston-Salem, NC 27102 (United States); Howlett, Allyn C., E-mail: ahowlett@wakehealth.edu [Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States)

2014-09-01

Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes.

Improvement of oxytetracycline production mediated via cooperation of resistance genes in Streptomyces rimosus.

Science.gov (United States)

Yin, Shouliang; Wang, Xuefeng; Shi, Mingxin; Yuan, Fang; Wang, Huizhuan; Jia, Xiaole; Yuan, Fang; Sun, Jinliang; Liu, Tiejun; Yang, Keqian; Zhang, Yuxiu; Fan, Keqiang; Li, Zilong

2017-09-01

Increasing the self-resistance levels of Streptomyces is an effective strategy to improve the production of antibiotics. To increase the oxytetracycline (OTC) production in Streptomyces rimosus, we investigated the cooperative effect of three co-overexpressing OTC resistance genes: one gene encodes a ribosomal protection protein (otrA) and the other two express efflux proteins (otrB and otrC). Results indicated that combinational overexpression of otrA, otrB, and otrC (MKABC) exerted a synergetic effect. OTC production increased by 179% in the recombinant strain compared with that of the wild-type strain M4018. The resistance level to OTC was increased by approximately two-fold relative to the parental strain, thereby indicating that applying the cooperative effect of self-resistance genes is useful to improve OTC production. Furthermore, the previously identified cluster-situated activator OtcR was overexpressed in MKABC in constructing the recombinant strain MKRABC; such strain can produce OTC of approximately 7.49 g L -1 , which represents an increase of 19% in comparison with that of the OtcR-overexpressing strain alone. Our work showed that the cooperative overexpression of self-resistance genes is a promising strategy to enhance the antibiotics production in Streptomyces.

Candidate genes for drought tolerance and improved productivity in ...

Indian Academy of Sciences (India)

Madhu

Improving drought tolerance and productivity is one of the most difficult tasks for ... Keywords. Candidate gene; mapping population; polymerase chain reaction; single marker analysis. .... ple and the mean value computed. 2.4 Isolation of DNA.

Coregulation of terpenoid pathway genes and prediction of isoprene production in Bacillus subtilis using transcriptomics

Energy Technology Data Exchange (ETDEWEB)

Hess, Becky M.; Xue, Junfeng; Markillie, Lye Meng; Taylor, Ronald C.; Wiley, H. S.; Ahring, Birgitte K.; Linggi, Bryan E.

2013-06-19

The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding the system level regulation and control of the pathway. To address this limitation, we examined Bacillus subtilis grown under multiple conditions and then determined the relationship between altered isoprene production and the pattern of gene expression. We found that terpenoid genes appeared to fall into two distinct subsets with opposing correlations with respect to the amount of isoprene produced. The group whose expression levels positively correlated with isoprene production included dxs, the gene responsible for the commitment step in the pathway, as well as ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. This analysis showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model which accurately predicts production of this secondary metabolite across many simulated environmental conditions.

Vasopressin Gene-Related Products in the Management of Breast Cancer

National Research Council Canada - National Science Library

North, William

1999-01-01

...), and this information coupled with an absence of vasopressin gene-related products from fibrocystic disease potentially provides us with a new screening test for distinguishing both breast cancer...

Guidelines for the naming of genes, gene products, and mutants in the opportunistic protists.

Science.gov (United States)

Limper, Andrew H; Weiss, Louis M

2011-01-01

The opportunistic protists encompass a wide diversity of organisms including Pneumocystis, Toxoplasma, cryptosporidia, microsporidia, and related genera. Recent advances in the molecular biology and cellular biochemistry of these organisms have led to the identification of an ever growing numbers of key genes and their cognate proteins. Until now, these molecules have not been designated using any consistent nomenclature system, leading to considerable confusion. The participants of the 11th International Workshop on Opportunistic Protists met on August 3, 2010 to reach consensus of a nomenclature system for genes, gene products, and mutants in the opportunistic protists. The following summary reports the consensus agreement to move toward a unified nomenclature system for these organisms. The system is adapted from that used for Saccharomyces cerevisiae. © 2011 The Author(s). Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.

DNA repair synthesis dependent on the uvrA,B gene products

International Nuclear Information System (INIS)

Moses, R.E.; Moody, E.E.M.

1975-01-01

Ultraviolet irradiation of toluene-treated Escherichia coli causes an inhibition of replicative DNA synthesis. This is followed by the appearance of nonconservative DNA repair synthesis which does not require either the polymerase or 5' → 3' exonucleolytic activities of DNA polymerase I. The repair synthesis may be catalyzed by DNA polymerase III activity but does not require a functional DNA polymerase II. The ultraviolet-induced synthesis requires ATP and is dependent on a functional uvrA and uvrB gene product. However, other uvr gene products are not required for the synthesis. The recB function is also not required

Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect L-Lysine Production in Corynebacterium glutamicum.

Science.gov (United States)

Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

2016-03-09

Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in L-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport--NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885--were also expressed at significantly higher levels in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, L-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.

The Aspergillus flavus Homeobox Gene, hbx1, Is Required for Development and Aflatoxin Production

Directory of Open Access Journals (Sweden)

Jeffrey W. Cary

2017-10-01

Full Text Available Homeobox proteins, a class of well conserved transcription factors, regulate the expression of targeted genes, especially those involved in development. In filamentous fungi, homeobox genes are required for normal conidiogenesis and fruiting body formation. In the present study, we identified eight homeobox (hbx genes in the aflatoxin-producing ascomycete, Aspergillus flavus, and determined their respective role in growth, conidiation and sclerotial production. Disruption of seven of the eight genes had little to no effect on fungal growth and development. However, disruption of the homeobox gene AFLA_069100, designated as hbx1, in two morphologically different A. flavus strains, CA14 and AF70, resulted in complete loss of production of conidia and sclerotia as well as aflatoxins B1 and B2, cyclopiazonic acid and aflatrem. Microscopic examination showed that the Δhbx1 mutants did not produce conidiophores. The inability of Δhbx1 mutants to produce conidia was related to downregulation of brlA (bristle and abaA (abacus, regulatory genes for conidiophore development. These mutants also had significant downregulation of the aflatoxin pathway biosynthetic genes aflC, aflD, aflM and the cluster-specific regulatory gene, aflR. Our results demonstrate that hbx1 not only plays a significant role in controlling A. flavus development but is also critical for the production of secondary metabolites, such as aflatoxins.

New insights about antibiotic production by Pseudomonas aeruginosa: a gene expression analysis

Science.gov (United States)

Gionco, Bárbara; Tavares, Eliandro R.; de Oliveira, Admilton G.; Yamada-Ogatta, Sueli F.; do Carmo, Anderson O.; Pereira, Ulisses de Pádua; Chideroli, Roberta T.; Simionato, Ane S.; Navarro, Miguel O. P.; Chryssafidis, Andreas L.; Andrade, Galdino

2017-09-01

The bacterial resistance for antibiotics is one of the most important problems in public health and only a small number of new products are in development. Antagonistic microorganisms from soil are a promising source of new candidate molecules. Products of secondary metabolism confer adaptive advantages for their producer, in the competition for nutrients in the microbial community. The biosynthesis process of compounds with antibiotic activity is the key to optimize their production and the transcriptomic study of microorganisms is of great benefit for the discovery of these metabolic pathways. Pseudomonas aeruginosa LV strain growing in the presence of copper chloride produces a bioactive organometallic compound, which has a potent antimicrobial activity against various microorganisms. The objective of this study was to verify overexpressed genes and evaluate their relation to the organometallic biosynthesis in this microorganism. P. aeruginosa LV strain was cultured in presence and absence of copper chloride. Two methods were used for transcriptomic analysis, genome reference-guided assembly and de novo assembly. The genome referenced analysis identified nine upregulated genes when bacteria were exposed to copper chloride, while the De Novo Assembly identified twelve upregulated genes. Nineteen genes can be related to an increased microbial metabolism for the extrusion process of exceeding intracellular copper. Two important genes are related to the biosynthesis of phenazine and tetrapyrroles compounds, which can be involved in the bioremediation of intracellular copper and biosynthesis of the organometallic compound. Additional studies are being carried out to further prove the function of the described genes and relate them to the biosynthetic pathway of the organometallic compound.

A second pectin lyase gene (pel2) from Aspergillus oryzae KBN616: its sequence analysis and overexpression, and characterization of the gene products.

Science.gov (United States)

Kitamoto, N; Yoshino-Yasuda, S; Ohmiya, K; Tsukagoshi, N

2001-01-01

A second pectin lyase gene, designated pel2, was isolated from a shoyu koji mold Aspergillus oryzae KBN616 and characterized. The structural gene comprised 1306 bp with three introns. The ORF encoded 375 amino acids with a signal peptide of 19 amino acids. The deduced amino acid sequence showed high similarity to those of A. oryzae Pel1, Aspergillus niger pectin lyases and Glomerella cingulata Pn1A. The pel2 gene was overexpressed under the control of the promoter of the A. oryzae TEF1 gene for purification and enzymatic characterization of its gene product. The gene product exhibited two molecular masses of 48 and 44 kDa due to different degrees of glycosylation. Both proteins had the same pH optimum of 6.0 and temperature optimum of 50 degrees C.

Major genes and QTL influencing wool production and quality: a review

Directory of Open Access Journals (Sweden)

Purvis Ian

2005-12-01

Full Text Available Abstract The opportunity exists to utilise our knowledge of major genes that influence the economically important traits in wool sheep. Genes with Mendelian inheritance have been identified for many important traits in wool sheep. Of particular importance are genes influencing pigmentation, wool quality and the keratin proteins, the latter of which are important for the morphology of the wool fibre. Gene mapping studies have identified some chromosomal regions associated with variation in wool quality and production traits. The challenge now is to build on this knowledge base in a cost-effective way to deliver molecular tools that facilitate enhanced genetic improvement programs for wool sheep.

Regulatory Oversight of Cell and Gene Therapy Products in Canada.

Science.gov (United States)

Ridgway, Anthony; Agbanyo, Francisca; Wang, Jian; Rosu-Myles, Michael

2015-01-01

Health Canada regulates gene therapy products and many cell therapy products as biological drugs under the Canadian Food and Drugs Act and its attendant regulations. Cellular products that meet certain criteria, including minimal manipulation and homologous use, may be subjected to a standards-based approach under the Safety of Human Cells, Tissues and Organs for Transplantation Regulations. The manufacture and clinical testing of cell and gene therapy products (CGTPs) presents many challenges beyond those for protein biologics. Cells cannot be subjected to pathogen removal or inactivation procedures and must frequently be administered shortly after final formulation. Viral vector design and manufacturing control are critically important to overall product quality and linked to safety and efficacy in patients through concerns such as replication competence, vector integration, and vector shedding. In addition, for many CGTPs, the value of nonclinical studies is largely limited to providing proof of concept, and the first meaningful data relating to appropriate dosing, safety parameters, and validity of surrogate or true determinants of efficacy must come from carefully designed clinical trials in patients. Addressing these numerous challenges requires application of various risk mitigation strategies and meeting regulatory expectations specifically adapted to the product types. Regulatory cooperation and harmonisation at an international level are essential for progress in the development and commercialisation of these products. However, particularly in the area of cell therapy, new regulatory paradigms may be needed to harness the benefits of clinical progress in situations where the resources and motivation to pursue a typical drug product approval pathway may be lacking.

New Insights about Antibiotic Production by Pseudomonas aeruginosa: A Gene Expression Analysis

Directory of Open Access Journals (Sweden)

Bárbara Gionco

2017-09-01

Full Text Available The bacterial resistance for antibiotics is one of the most important problems in public health and only a small number of new products are in development. Antagonistic microorganisms from soil are a promising source of new candidate molecules. Products of secondary metabolism confer adaptive advantages for their producer, in the competition for nutrients in the microbial community. The biosynthesis process of compounds with antibiotic activity is the key to optimize their production and the transcriptomic study of microorganisms is of great benefit for the discovery of these metabolic pathways. Pseudomonas aeruginosa LV strain growing in the presence of copper chloride produces a bioactive organometallic compound, which has a potent antimicrobial activity against various microorganisms. The objective of this study was to verify overexpressed genes and evaluate their relation to the organometallic biosynthesis in this microorganism. P. aeruginosa LV strain was cultured in presence and absence of copper chloride. Two methods were used for transcriptomic analysis, genome reference-guided assembly and de novo assembly. The genome referenced analysis identified nine upregulated genes when bacteria were exposed to copper chloride, while the De Novo Assembly identified 12 upregulated genes. Nineteen genes can be related to an increased microbial metabolism for the extrusion process of exceeding intracellular copper. Two important genes are related to the biosynthesis of phenazine and tetrapyrroles compounds, which can be involved in the bioremediation of intracellular copper and we suggesting that may involve in the biosynthesis of the organometallic compound. Additional studies are being carried out to further prove the function of the described genes and relate them to the biosynthetic pathway of the organometallic compound.

Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins

International Nuclear Information System (INIS)

Strebel, K.; Beck, E.; Strohmaier, K.; Schaller, H.

1986-01-01

Defined segments of the cloned foot-and-mouth disease virus genome corresponding to all parts of the coding region were expressed in Escherichia coli as fusions to the N-terminal part of the MS2-polymerase gene under the control of the inducible λPL promoter. All constructs yielded large amounts of proteins, which were purified and used to raise sequence-specific antisera in rabbits. These antisera were used to identify the corresponding viral gene products in 35 S-labeled extracts from foot-and-mouth disease virus-infected BHK cells. This allowed us to locate unequivocally all mature foot-and-mouth disease virus gene products in the nucleotide sequence, to identify precursor-product relationships, and to detect several foot-and mouth disease virus gene products not previously identified in vivo or in vitro

Increasing Avermectin Production in Streptomyces avermitilis by Manipulating the Expression of a Novel TetR-Family Regulator and Its Target Gene Product.

Science.gov (United States)

Liu, Wenshuai; Zhang, Qinling; Guo, Jia; Chen, Zhi; Li, Jilun; Wen, Ying

2015-08-01

Avermectins produced by Streptomyces avermitilis are commercially important anthelmintic agents. The detailed regulatory mechanisms of avermectin biosynthesis remain unclear. Here, we identified SAV3619, a TetR-family transcriptional regulator designated AveT, to be an activator for both avermectin production and morphological differentiation in S. avermitilis. AveT was shown to indirectly stimulate avermectin production by affecting transcription of the cluster-situated activator gene aveR. AveT directly repressed transcription of its own gene (aveT), adjacent gene pepD2 (sav_3620), sav_7490 (designated aveM), and sav_7491 by binding to an 18-bp perfect palindromic sequence (CGAAACGKTKYCGTTTCG, where K is T or G and Y is T or C and where the underlining indicates inverted repeats) within their promoter regions. aveM (which encodes a putative transmembrane efflux protein belonging to the major facilitator superfamily [MFS]), the important target gene of AveT, had a striking negative effect on avermectin production and morphological differentiation. Overexpression of aveT and deletion of aveM in wild-type and industrial strains of S. avermitilis led to clear increases in the levels of avermectin production. In vitro gel-shift assays suggested that C-5-O-B1, the late pathway precursor of avermectin B1, acts as an AveT ligand. Taken together, our findings indicate positive-feedback regulation of aveT expression and avermectin production by a late pathway intermediate and provide the basis for an efficient strategy to increase avermectin production in S. avermitilis by manipulation of AveT and its target gene product, AveM. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Identification and localization of a gene that specifies production of Escherichia coli DNA topoisomerase I

International Nuclear Information System (INIS)

Trucksis, M.; Depew, R.E.

1981-01-01

A gene that specifies production of Escherichia coli DNA topoisomerase I (ω protein) was identified with the aid of a radioimmunoassay for this protein. E. coli DNA topoisomerase I was produced by Salmonella typhimurium merodiploids that harbored E. coli plasmid F' 123, but not by strains that lost this plasmid. Analysis of strains with spontaneous deletions of F' 123 showed that the gene, topA, required for production of the E. coli ω protein was between the trp operon and the cysB gene. Deletions that eliminated topA also eliminated the supX gene. We suggest that topA is the structural gene of E. coli DNA topoisomerase I and that topA is identical to supX

Efficient production of antibody Fab fragment by transient gene expression in insect cells.

Science.gov (United States)

Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

2017-08-01

Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins.

Science.gov (United States)

Yoon, Jaewoo; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2011-02-01

Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double (tppA, and pepE) and quintuple (tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes (alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae.

Single gene insertion drives bioalcohol production by a thermophilic archaeon

Energy Technology Data Exchange (ETDEWEB)

Basen, M; Schut, GJ; Nguyen, DM; Lipscomb, GL; Benn, RA; Prybol, CJ; Vaccaro, BJ; Poole, FL; Kelly, RM; Adams, MWW

2014-12-09

Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 degrees C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.

Diversity, distribution and quantification of antibiotic resistance genes in goat and lamb slaughterhouse surfaces and meat products.

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Leyre Lavilla Lerma

Full Text Available The distribution and quantification of tetracycline, sulfonamide and beta-lactam resistance genes were assessed in slaughterhouse zones throughout meat chain production and the meat products; this study represents the first to report quantitatively monitor antibiotic resistance genes (ARG in goat and lamb slaughterhouse using a culture independent approach, since most studies focused on individual bacterial species and their specific resistance types. Quantitative PCR (qPCR revealed a high prevalence of tetracycline resistance genes tetA and tetB in almost all slaughterhouse zones. Sulfonamide resistance genes were largely distributed, while beta-lactam resistance genes were less predominant. Statistical analysis revealed that resistant bacteria, in most cases, were spread by the same route in almost all slaughterhouse zones, except for tetB, blaCTX and blaTEM genes, which occurred in few zones as isolated 'hot spots.' The sum of all analyzed ARG indicated that slaughterhouse surfaces and end products act as reservoirs of ARG, mainly tet genes, which were more prevalent in slaughtering room (SR, cutting room (CR and commercial meat products (MP. Resistance gene patterns suggest they were disseminated throughout slaughterhouse zones being also detected in commercial meat products, with significant correlations between different sampling zones/end products and total resistance in SR, CR and white room (WR zones, and also refrigerator 4 (F4 and MP were observed. Strategically controlling key zones in slaughterhouse (SR, CR and WR by adequate disinfection methods could strategically reduce the risks of ARG transmission and minimize the issues of food safety and environment contamination.

Diversity, distribution and quantification of antibiotic resistance genes in goat and lamb slaughterhouse surfaces and meat products.

Science.gov (United States)

Lavilla Lerma, Leyre; Benomar, Nabil; Knapp, Charles W; Correa Galeote, David; Gálvez, Antonio; Abriouel, Hikmate

2014-01-01

The distribution and quantification of tetracycline, sulfonamide and beta-lactam resistance genes were assessed in slaughterhouse zones throughout meat chain production and the meat products; this study represents the first to report quantitatively monitor antibiotic resistance genes (ARG) in goat and lamb slaughterhouse using a culture independent approach, since most studies focused on individual bacterial species and their specific resistance types. Quantitative PCR (qPCR) revealed a high prevalence of tetracycline resistance genes tetA and tetB in almost all slaughterhouse zones. Sulfonamide resistance genes were largely distributed, while beta-lactam resistance genes were less predominant. Statistical analysis revealed that resistant bacteria, in most cases, were spread by the same route in almost all slaughterhouse zones, except for tetB, blaCTX and blaTEM genes, which occurred in few zones as isolated 'hot spots.' The sum of all analyzed ARG indicated that slaughterhouse surfaces and end products act as reservoirs of ARG, mainly tet genes, which were more prevalent in slaughtering room (SR), cutting room (CR) and commercial meat products (MP). Resistance gene patterns suggest they were disseminated throughout slaughterhouse zones being also detected in commercial meat products, with significant correlations between different sampling zones/end products and total resistance in SR, CR and white room (WR) zones, and also refrigerator 4 (F4) and MP were observed. Strategically controlling key zones in slaughterhouse (SR, CR and WR) by adequate disinfection methods could strategically reduce the risks of ARG transmission and minimize the issues of food safety and environment contamination.

A shortest-path graph kernel for estimating gene product semantic similarity

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Alvarez Marco A

2011-07-01

Full Text Available Abstract Background Existing methods for calculating semantic similarity between gene products using the Gene Ontology (GO often rely on external resources, which are not part of the ontology. Consequently, changes in these external resources like biased term distribution caused by shifting of hot research topics, will affect the calculation of semantic similarity. One way to avoid this problem is to use semantic methods that are "intrinsic" to the ontology, i.e. independent of external knowledge. Results We present a shortest-path graph kernel (spgk method that relies exclusively on the GO and its structure. In spgk, a gene product is represented by an induced subgraph of the GO, which consists of all the GO terms annotating it. Then a shortest-path graph kernel is used to compute the similarity between two graphs. In a comprehensive evaluation using a benchmark dataset, spgk compares favorably with other methods that depend on external resources. Compared with simUI, a method that is also intrinsic to GO, spgk achieves slightly better results on the benchmark dataset. Statistical tests show that the improvement is significant when the resolution and EC similarity correlation coefficient are used to measure the performance, but is insignificant when the Pfam similarity correlation coefficient is used. Conclusions Spgk uses a graph kernel method in polynomial time to exploit the structure of the GO to calculate semantic similarity between gene products. It provides an alternative to both methods that use external resources and "intrinsic" methods with comparable performance.

Regulation of metabolic products and gene expression in Fusarium asiaticum by agmatine addition.

Science.gov (United States)

Suzuki, Tadahiro; Kim, Young-Kyung; Yoshioka, Hifumi; Iwahashi, Yumiko

2013-05-01

The metabolic products resulting from the cultivation of F. asiaticum in agmatine were identified using capillary electrophoresis-time of flight mass spectrometry. Glyoxylic acid was detected from fungal cultures grown in agmatine, while it was absent in control cells. The abundance of other metabolic products of the glycolytic pathway also increased because of agmatine; however, there was no increase in the amounts of pyruvic acid or metabolites from the tricarboxylic acid cycle. Moreover, gene expression levels within Fusarium asiaticum exposed to agmatine were analyzed by DNA microarray. Changes in gene expression levels directed the changes in metabolic products. Our results suggest that acetyl coenzyme A, which is a starting substrate for the biosynthesis of deoxynivalenol (DON), was simultaneously produced by activated β-oxidation. Furthermore, the content of 4-aminobutyrate (GABA) was increased in the agmatine addition culture medium. GABA can be synthesized from agmatine through putrescine and might influence the regulation of DON-related genes.

Metabolic engineering of Escherichia coli for ethanol production without foreign genes

Science.gov (United States)

Kim, Youngnyun

Worldwide dependence on finite petroleum-based energy necessitates alternative energy sources that can be produced from renewable resources. A successful example of an alternative transportation fuel is bioethanol, produced by microorganisms, from corn starch that is blended with gasoline. However, corn, currently the main feedstock for bioethanol production, also occupies a significant role in human food and animal feed chains. As more corn is diverted to bioethanol, the cost of corn is expected to increase with an increase in the price of food, feed and ethanol. Using lignocellulosic biomass for ethanol production is considered to resolve this problem. However, this requires a microbial biocatalyst that can ferment hexoses and pentoses to ethanol. Escherichia coli is an efficient biocatalyst that can use all the monomeric sugars in lignocellulose, and recombinant derivatives of E. coli have been engineered to produce ethanol as the major fermentation product. In my study, ethanologenic E. coli strains were isolated from a ldhA-, pflB- derivative without introduction of foreign genes. These isolates grew anaerobically and produced ethanol as the main fermentation product. The mutation responsible for anaerobic growth and ethanol production was mapped in the lpdA gene and the mutation was identified as E354K in three of the isolates tested. Another three isolates carried an lpdA mutation, H352Y. Enzyme kinetic studies revealed that the mutated form of the dihydrolipoamide dehydrogenase (LPD) encoded by the lpdA was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity to NADH of the pyruvate dehydrogenase complex in strain SE2378. The net yield of 4 moles of NADH and 2 moles of acetyl-CoA per mole of glucose produced by a combination of glycolysis and PDH provided a logical basis to explain the production of 2 moles of ethanol per glucose. The development of E

Use of galerina marginata genes and proteins for peptide production

Science.gov (United States)

Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.; Luo, Hong

2018-04-03

The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

Use of Galerina marginata genes and proteins for peptide production

Energy Technology Data Exchange (ETDEWEB)

Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.; Luo, Hong

2017-03-21

The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

Id-1 and Id-2 genes and products as markers of epithelial cancer

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Desprez, Pierre-Yves [El Cerrito, CA; Campisi, Judith [Berkeley, CA

2008-09-30

A method for detection and prognosis of breast cancer and other types of cancer. The method comprises detecting expression, if any, for both an Id-1 and an Id-2 genes, or the ratio thereof, of gene products in samples of breast tissue obtained from a patient. When expressed, Id-1 gene is a prognostic indicator that breast cancer cells are invasive and metastatic, whereas Id-2 gene is a prognostic indicator that breast cancer cells are localized and noninvasive in the breast tissue.

EpsA is an essential gene in exopolysaccharide production in Lactobacillus johnsonii FI9785.

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Dertli, Enes; Mayer, Melinda J; Colquhoun, Ian J; Narbad, Arjan

2016-07-01

Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)-1 and heteropolymeric EPS-2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS-1 and EPS-2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild-type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS-2 as the (1,4)-linked βGlcp unit, with the acetyl group located at O-6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Expression of nm23-H1 gene product in esophageal squamous cell carcinoma and its association with vessel invasion and survival

International Nuclear Information System (INIS)

Tomita, Masaki; Ayabe, Takanori; Matsuzaki, Yasunori; Edagawa, Masao; Maeda, Masayuki; Shimizu, Tetsuya; Hara, Masaki; Onitsuka, Toshio

2001-01-01

We assessed the nm23-H1 gene product expression and its relationship with lymphatic and blood vessel invasion in patients with esophageal squamous cell carcinoma. Formalin-fixed and paraffin-embedded tissue sections from 45 patients who were treated surgically were used in this study. Pathologists graded lymphatic and blood vessel invasion in each of the tissue samples. Expression of nm23-Hl gene product was determined using a specific monoclonal antibody. Expression of nm23-H1 gene product was present in 17 (37.8%) cases. We found an inverse correlation between nm23-H1 gene product expression and lymphatic vessel invasion, whereas no correlation between nm23-H1 gene product expression and blood vessel invasion. Overall survival rate was not different between nm23-H1 gene product positive and negative patients (p = 0.21). However, reduced expression of nm23-H1 gene product was associated with shorter overall survival in patients with involved lymph nodes (p < 0.05), but not in patients without involved lymph nodes (p = 0.87). In patients with esophageal squamous cell carcinoma, there appears to be an inverse relationship between nm23-H1 gene product expression and lymphatic vessel invasion. Furthermore, nm23-H1 gene product expression might be a prognostic marker in patients with involved lymph nodes. Our data does not demonstrate any correlation between nm23-H1 gene product expression and blood vessel invasion

Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger

DEFF Research Database (Denmark)

Meijer, Susan Lisette; Nielsen, Michael Lynge; Olsson, Lisbeth

2009-01-01

With the availability of the genome sequence of the filamentous fungus Aspergillus niger, the use of targeted genetic modifications has become feasible. This, together with the fact that A. niger is well established industrially, makes this fungus an attractive micro-organism for creating a cell...... factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which...... the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger....

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

Science.gov (United States)

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.

Improvement of heterologous protein production in Aspergillus oryzae by RNA interference with alpha-amylase genes.

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Nemoto, Takashi; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2009-11-01

Aspergillus oryzae RIB40 has three alpha-amylase genes (amyA, amyB, and amyC), and secretes alpha-amylase abundantly. However, large amounts of endogenous secretory proteins such as alpha-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of alpha-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three alpha-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of alpha-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in alpha-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of alpha-amylase is effective in heterologous protein production in A. oryzae.

Excision of foreign gene product with cathepsin D in chicken hepatoma cell line

International Nuclear Information System (INIS)

Sato, Masaharu; Kawashima, Tsuyoshi; Aosasa, Masayoshi; Horiuchi, Hiroyuki; Furusawa, Shuichi; Matsuda, Haruo

2005-01-01

To easily and rapidly recover exogenous gene products from chicken egg yolk, we constructed pVTG-catD (VTG, vitellogenin; catD, cathepsin D), a vector cassette carrying two catD-recognition signal peptides (catD-RSPs) in addition to the cloning site. An enhanced green fluorescence protein (EGFP)-encoding DNA fragment was ligated into the pVTG-catD. When the resultant construct pVTG-EGFP-catD containing histidine- and myc-tags was transfected into the chicken hepatoma cell line LMH, EGFP-expression at 24 h post-cultivation was confirmed by fluorescence microscopy. Because a signal peptide (NTVLAEF) encoded in pVTG-EGFP-catD is recognized by catD, the VTG-EGFP fusion protein digested with catD was detectable by Western blotting. Digested exogenous gene product was recovered with nickel resin. These results indicate that catD-recognition sites bearing pVTG-catD and His-tags are functional in chicken LMH cells. Therefore, the system described here may be of use in making excision exogenous gene products in the chicken and in creating homozygous knock-in chickens

Associations between variants of the HAL gene and milk production traits in Chinese Holstein cows.

Science.gov (United States)

Wang, Haifei; Jiang, Li; Wang, Wenwen; Zhang, Shengli; Yin, Zongjun; Zhang, Qin; Liu, Jian-Feng

2014-11-25

The histidine ammonia-lyse gene (HAL) encodes the histidine ammonia-lyase, which catalyzes the first reaction of histidine catabolism. In our previous genome-wide association study in Chinese Holstein cows to identify genetic variants affecting milk production traits, a SNP (rs41647754) located 357 bp upstream of HAL, was found to be significantly associated with milk yield and milk protein yield. In addition, the HAL gene resides within the reported QTLs for milk production traits. The aims of this study were to identify genetic variants in HAL and to test the association between these variants and milk production traits. Fifteen SNPs were identified within the regions under study of the HAL gene, including three coding mutations, seven intronic mutations, one promoter region mutation, and four 3'UTR mutations. Nine of these identified SNPs were chosen for subsequent genotyping and association analyses. Our results showed that five SNP markers (ss974768522, ss974768525, ss974768531, ss974768533 and ss974768534) were significantly associated with one or more milk production traits. Haplotype analysis showed that two haplotype blocks were significantly associated with milk yield and milk protein yield, providing additional support for the association between HAL variants and milk production traits in dairy cows (P HAL gene and milk production traits in Chinese Holstein cows, indicating the potential role of HAL variants in these traits. These identified SNPs may serve as genetic markers used in genomic selection schemes to accelerate the genetic gains of milk production traits in dairy cattle.

Characterization, expression profiles, intracellular distribution and association analysis of porcine PNAS-4 gene with production traits

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Wang Heng

2008-06-01

Full Text Available Abstract Background In a previous screen to identify differentially expressed genes associated with embryonic development, the porcine PNAS-4 gene had been found. Considering differentially expressed genes in early stages of muscle development are potential candidate genes to improve meat quality and production efficiency, we determined how porcine PNAS-4 gene regulates meat production. Therefore, this gene has been sequenced, expression analyzed and associated with meat production traits. Results We cloned the full-length cDNA of porcine PNAS-4 gene encoding a protein of 194 amino acids which was expressed in the Golgi complex. This gene was mapped to chromosome 10, q11–16, in a region of conserved synteny with human chromosome 1 where the human homologous gene was localized. Real-time PCR revealed that PNAS-4 mRNA was widely expressed with highest expression levels in skeletal muscle followed by lymph, liver and other tissues, and showed a down-regulated expression pattern during prenatal development while a up-regulated expression pattern after weaning. Association analysis revealed that allele C of SNP A1813C was prevalent in Chinese indigenous breeds whereas A was dominant allele in Landrace and Large White, and the pigs with homozygous CC had a higher fat content than those of the pigs with other genotypes (P Conclusion Porcine PNAS-4 protein tagged with green fluorescent protein accumulated in the Golgi complex, and its mRNA showed a widespread expression across many tissues and organs in pigs. It may be an important factor affecting the meat production efficiency, because its down-regulated expression pattern during early embryogenesis suggests involvement in increase of muscle fiber number. In addition, the SNP A1813C associated with fat traits might be a genetic marker for molecular-assisted selection in animal breeding.

Impact of nitrogen concentration on validamycin A production and related gene transcription in fermentation of Streptomyces hygroscopicus 5008.

Science.gov (United States)

Wei, Zhen-Hua; Bai, Linquan; Deng, Zixin; Zhong, Jian-Jiang

2012-09-01

Validamycin A (VAL-A) is an important and widely used agricultural antibiotic. In this study, statistical screening designs were applied to identify significant medium variables for VAL-A production and to find their optimal levels. The optimized medium caused 70% enhancement of VAL-A production. The difference between optimized medium and original medium suggested that low nitrogen source level might attribute to the enhancement of VAL-A production. The addition of different nitrogen sources to the optimized medium inhibited VAL-A production, which confirmed the importance of nitrogen concentration for VAL-A production. Furthermore, differences in structural gene transcription and enzyme activity between the two media were assayed. The results showed that lower nitrogen level in the optimized medium could regulate VAL-A production in gene transcriptional level. Our previous study indicated that the transcription of VAL-A structural genes could be enhanced at elevated temperature. In this work, the increased fermentation temperature from 37 to 42 °C with the optimized medium enhanced VAL-A production by 39%, which testified to the importance of structural gene transcription in VAL-A production. The information is useful for further VAL-A production enhancement.

Dairy Product Consumption Interacts with Glucokinase (GCK Gene Polymorphisms Associated with Insulin Resistance

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Marine S. Da Silva

2017-08-01

Full Text Available Dairy product intake and a person’s genetic background have been reported to be associated with the risk of type 2 diabetes (T2D. The objective of this study was to examine the interaction between dairy products and genes related to T2D on glucose-insulin homeostasis parameters. A validated food frequency questionnaire, fasting blood samples, and glucokinase (GCK genotypes were analyzed in 210 healthy participants. An interaction between rs1799884 in GCK and dairy intake on the homeostasis model assessment of insulin resistance was identified. Secondly, human hepatocellular carcinoma cells (HepG2 were grown in a high-glucose medium and incubated with either 1-dairy proteins: whey, caseins, and a mixture of whey and casein; and 2-four amino acids (AA or mixtures of AA. The expression of GCK-related genes insulin receptor substrate-1 (IRS-1 and fatty acid synthase (FASN was increased with whey protein isolate or hydrolysate. Individually, leucine increased IRS-1 expression, whereas isoleucine and valine decreased FASN expression. A branched-chain AA mixture decreased IRS-1 and FASN expression. In conclusion, carriers of the A allele for rs1799884 in the GCK gene may benefit from a higher intake of dairy products to maintain optimal insulin sensitivity. Moreover, the results show that whey proteins affect the expression of genes related to glucose metabolism.

Improving the Safety of Cell Therapy Products by Suicide Gene Transfer

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Antonio eDi Stasi

2014-11-01

Full Text Available Adoptive T-cell therapy can involve donor lymphocyte infusion (DLI after allogeneic hematopoietic stem cell transplantation, the administration of tumor infiltrating lymphocyte (TILs expanded ex-vivo, or more recently the use of T cell receptor (TCR or chimeric antigen receptor (CAR redirected T cells. However cellular therapies can pose significant risks, including graft-versus-host-disease and other on and off-target effects, and therefore strategies need to be implemented to permanently reverse any sign of toxicity. A suicide gene is a genetically encoded molecule that allows selective destruction of adoptively transferred cells. Suicide gene addition to cellular therapeutic products can lead to selective ablation of gene-modified cells, preventing collateral damage to contiguous cells and/or tissues. The ‘ideal’ suicide gene would ensure the safety of gene modified cellular applications by granting irreversible elimination of ‘all’ and ‘only’ the cells responsible for the unwanted toxicity. This review presents the suicide gene safety systems reported to date, with a focus on the state-of-the-art and potential applications regarding two of the most extensively validated suicide genes, including the clinical setting: herpes-simplex-thymidine-kinase (HSV-TK and inducible-caspase-9 (iCasp9.

The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products.

Science.gov (United States)

Rattay, Stephanie; Trilling, Mirko; Megger, Dominik A; Sitek, Barbara; Meyer, Helmut E; Hengel, Hartmut; Le-Trilling, Vu Thuy Khanh

2015-08-01

Transcription of mouse cytomegalovirus (MCMV) immediate early ie1 and ie3 is controlled by the major immediate early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids; pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-hemagglutinin (IE3-HA) virus by insertion of an in-frame HA epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts code for proteins. This prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as posttranscriptional regulation of MCMV gene products by pIE611. Cytomegaloviruses are pathogenic betaherpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immunosuppression, immunoimmaturity, or inflammation. The switch from latency to reactivation requires expression of immediate early genes. Therefore, understanding of immediate early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate early 3 protein (611 amino acids; pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted

Overexpression of D-Xylose Reductase (xyl1 Gene and Antisense Inhibition of D-Xylulokinase (xyiH Gene Increase Xylitol Production in Trichoderma reesei

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Yuanyuan Hong

2014-01-01

Full Text Available T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH, which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable. The copy number of the xylose reductase gene (xyl1 in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol.

Overexpression of D-Xylose Reductase (xyl1) Gene and Antisense Inhibition of D-Xylulokinase (xyiH) Gene Increase Xylitol Production in Trichoderma reesei

Science.gov (United States)

Hong, Yuanyuan; Dashtban, Mehdi; Kepka, Greg; Chen, Sanfeng; Qin, Wensheng

2014-01-01

T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol. PMID:25013760

Role of plnB gene in the regulation of bacteriocin production in Lactobacillus paraplantarum L-XM1.

Science.gov (United States)

Zhang, Xiangmei; Shang, Nan; Zhang, Xu; Gui, Meng; Li, Pinglan

2013-06-12

Homologues of plnB gene have been shown to participate in regulation of bacteriocin production through quorum sensing system in other organisms, to investigate the possible role of plnB gene in Lactobacillus paraplantarum L-XM1, we cloned and insertionally inactivated the plnB gene. The plnB knockout mutant ΔplnB21 showed loss of bacteriocin production, its Bac⁺ phenotype could not be restored even after the addition of PlnA. Furthermore, reverse transcription-PCR analysis from total RNA preparations showed that the bacteriocin structural genes of the plnEF and plnJK were not transcribed in the plnB knockout mutant compared with the wild-type strain. It was therefore concluded that plnB is invovled in a quorum sensing based bacteriocin production. This is the first demonstration of a role for plnB by gene knockout in L. paraplantarum. Copyright © 2012 Elsevier GmbH. All rights reserved.

Regulation of the E. coli SOS response by the lexA gene product

International Nuclear Information System (INIS)

Brent, R.

1983-01-01

In an Escherichia coli that is growing normally, transcription of many genes is repressed by the product of the lexA gene. If cellular DNA is damaged, proteolytically competent recA protein (recA protease) inactivates lexA protein and these genes are induced. Many of the cellular phenomena observed during the cellular response to DNA damage (the SOS response) are the consequence of the expression of these lexA-prepressed genes. Since the SOS response of E. coli has recently been the subject of a comprehensive review, in this paper I would like to concentrate on some modifications to the picture based on new data. 12 references, 2 figures

Bombyx mori nucleopolyhedrovirus BM5 protein regulates progeny virus production and viral gene expression

International Nuclear Information System (INIS)

Kokusho, Ryuhei; Koh, Yoshikazu; Fujimoto, Masaru; Shimada, Toru; Katsuma, Susumu

2016-01-01

Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulation of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. -- Highlights: •The role of BmNPV BM5 protein was examined in B. mori cultured cells and larvae. •BM5 contributes to efficient production of budded viruses and occlusion bodies. •BM5 regulates viral gene expression at the very late stage of infection. •BM5 dominantly localizes to the nuclear membrane. •Bm5 mutant showed v-cath down-regulation and resulting delay of larval death.

Bombyx mori nucleopolyhedrovirus BM5 protein regulates progeny virus production and viral gene expression

Energy Technology Data Exchange (ETDEWEB)

Kokusho, Ryuhei, E-mail: kokusho@ss.ab.a.u-tokyo.ac.jp; Koh, Yoshikazu; Fujimoto, Masaru; Shimada, Toru; Katsuma, Susumu, E-mail: katsuma@ss.ab.a.u-tokyo.ac.jp

2016-11-15

Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulation of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. -- Highlights: •The role of BmNPV BM5 protein was examined in B. mori cultured cells and larvae. •BM5 contributes to efficient production of budded viruses and occlusion bodies. •BM5 regulates viral gene expression at the very late stage of infection. •BM5 dominantly localizes to the nuclear membrane. •Bm5 mutant showed v-cath down-regulation and resulting delay of larval death.

Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production

Science.gov (United States)

Bailey, Andy M.; Alberti, Fabrizio; Kilaru, Sreedhar; Collins, Catherine M.; de Mattos-Shipley, Kate; Hartley, Amanda J.; Hayes, Patrick; Griffin, Alison; Lazarus, Colin M.; Cox, Russell J.; Willis, Christine L.; O'Dwyer, Karen; Spence, David W.; Foster, Gary D.

2016-05-01

Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus, no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi.

Identification of Enzyme Genes Using Chemical Structure Alignments of Substrate-Product Pairs.

Science.gov (United States)

Moriya, Yuki; Yamada, Takuji; Okuda, Shujiro; Nakagawa, Zenichi; Kotera, Masaaki; Tokimatsu, Toshiaki; Kanehisa, Minoru; Goto, Susumu

2016-03-28

Although there are several databases that contain data on many metabolites and reactions in biochemical pathways, there is still a big gap in the numbers between experimentally identified enzymes and metabolites. It is supposed that many catalytic enzyme genes are still unknown. Although there are previous studies that estimate the number of candidate enzyme genes, these studies required some additional information aside from the structures of metabolites such as gene expression and order in the genome. In this study, we developed a novel method to identify a candidate enzyme gene of a reaction using the chemical structures of the substrate-product pair (reactant pair). The proposed method is based on a search for similar reactant pairs in a reference database and offers ortholog groups that possibly mediate the given reaction. We applied the proposed method to two experimentally validated reactions. As a result, we confirmed that the histidine transaminase was correctly identified. Although our method could not directly identify the asparagine oxo-acid transaminase, we successfully found the paralog gene most similar to the correct enzyme gene. We also applied our method to infer candidate enzyme genes in the mesaconate pathway. The advantage of our method lies in the prediction of possible genes for orphan enzyme reactions where any associated gene sequences are not determined yet. We believe that this approach will facilitate experimental identification of genes for orphan enzymes.

Dynamics of plc gene transcription and α-toxin production during growth of Clostridium perfringens strains with contrasting α-toxin production

DEFF Research Database (Denmark)

Abildgaard, Lone; Schramm, Andreas; Rudi, Knut

2009-01-01

The aim of the present study was to investigate transcription dynamics of the α-toxin-encoding plc gene relative to two housekeeping genes (gyrA and rplL) in batch cultures of three Clostridium perfringens strains with low, intermediate, and high levels of α-toxin production, respectively. The plc...... transcript level was always low in the low α-toxin producing strain. For the two other strains, plc transcription showed an inducible pattern and reached a maximum level in the late exponential growth phase. The transcription levels were however inversely correlated to α-toxin production for the two strains....... We propose that this discrepancy is due to differences in plc translation rates between the strains and that strain-specific translational rates therefore must be determined before α-toxin production can be extrapolated from transcript levels in C. perfringens....

Pleiotropic regulatory genes bldA, adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin.

Science.gov (United States)

Makitrynskyy, Roman; Ostash, Bohdan; Tsypik, Olga; Rebets, Yuriy; Doud, Emma; Meredith, Timothy; Luzhetskyy, Andriy; Bechthold, Andreas; Walker, Suzanne; Fedorenko, Victor

2013-10-23

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production-bldA, adpA and absB-exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNA(Leu)UAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs-that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.

Gene Disruption Technologies Have the Potential to Transform Stored Product Insect Pest Control.

Science.gov (United States)

Perkin, Lindsey C; Adrianos, Sherry L; Oppert, Brenda

2016-09-19

Stored product insects feed on grains and processed commodities manufactured from grain post-harvest, reducing the nutritional value and contaminating food. Currently, the main defense against stored product insect pests is the pesticide fumigant phosphine. Phosphine is highly toxic to all animals, but is the most effective and economical control method, and thus is used extensively worldwide. However, many insect populations have become resistant to phosphine, in some cases to very high levels. New, environmentally benign and more effective control strategies are needed for stored product pests. RNA interference (RNAi) may overcome pesticide resistance by targeting the expression of genes that contribute to resistance in insects. Most data on RNAi in stored product insects is from the coleopteran genetic model, Tribolium castaneum, since it has a strong RNAi response via injection of double stranded RNA (dsRNA) in any life stage. Additionally, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been suggested as a potential resource for new pest control strategies. In this review we discuss background information on both gene disruption technologies and summarize the advances made in terms of molecular pest management in stored product insects, mainly T. castaneum, as well as complications and future needs.

Bayesian Computational Approaches for Gene Regulation Studies of Bioethanol and Biohydrogen Production. Final Scientific/Technical Report

Energy Technology Data Exchange (ETDEWEB)

Newberg, Lee; McCue, Lee Anne; Van Roey, Patrick

2014-04-17

The project developed mathematical models and first-version software tools for the understanding of gene regulation across multiple related species. The project lays the foundation for understanding how certain alpha-proteobacterial species control their own genes for bioethanol and biohydrogen production, and sets the stage for exploiting bacteria for the production of fuels. Enabling such alternative sources of fuel is a high priority for the Department of Energy and the public.

Glutamic acid promotes monacolin K production and monacolin K biosynthetic gene cluster expression in Monascus.

Science.gov (United States)

Zhang, Chan; Liang, Jian; Yang, Le; Chai, Shiyuan; Zhang, Chenxi; Sun, Baoguo; Wang, Chengtao

2017-12-01

This study investigated the effects of glutamic acid on production of monacolin K and expression of the monacolin K biosynthetic gene cluster. When Monascus M1 was grown in glutamic medium instead of in the original medium, monacolin K production increased from 48.4 to 215.4 mg l -1 , monacolin K production increased by 3.5 times. Glutamic acid enhanced monacolin K production by upregulating the expression of mokB-mokI; on day 8, the expression level of mokA tended to decrease by Reverse Transcription-polymerase Chain Reaction. Our findings demonstrated that mokA was not a key gene responsible for the quantity of monacolin K production in the presence of glutamic acid. Observation of Monascus mycelium morphology using Scanning Electron Microscope showed glutamic acid significantly increased the content of Monascus mycelium, altered the permeability of Monascus mycelium, enhanced secretion of monacolin K from the cell, and reduced the monacolin K content in Monascus mycelium, thereby enhancing monacolin K production.

Correlation of gene expression and protein production rate - a system wide study

Directory of Open Access Journals (Sweden)

Arvas Mikko

2011-12-01

Full Text Available Abstract Background Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype. Results We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR. We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response. Conclusions Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR.

A positive feedback-based gene circuit to increase the production of a membrane protein

Directory of Open Access Journals (Sweden)

Gennis Robert B

2010-05-01

Full Text Available Abstract Background Membrane proteins are an important class of proteins, playing a key role in many biological processes, and are a promising target in pharmaceutical development. However, membrane proteins are often difficult to produce in large quantities for the purpose of crystallographic or biochemical analyses. Results In this paper, we demonstrate that synthetic gene circuits designed specifically to overexpress certain genes can be applied to manipulate the expression kinetics of a model membrane protein, cytochrome bd quinol oxidase in E. coli, resulting in increased expression rates. The synthetic circuit involved is an engineered, autoinducer-independent variant of the lux operon activator LuxR from V. fischeri in an autoregulatory, positive feedback configuration. Conclusions Our proof-of-concept experiments indicate a statistically significant increase in the rate of production of the bd oxidase membrane protein. Synthetic gene networks provide a feasible solution for the problem of membrane protein production.

Effects of thiamine on growth, aflatoxin production, and aflr gene expression in A. parasiticus

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Ladan Nazemi

2015-01-01

Results: The minimum inhibitory concentration was yielded as > 500 mg/ml. However, HPLC analysis results showed that aflatoxin production reduced in samples treated with 500 mg/ml of thiamine. In addition, the level of afIR gene expression was significantly reduced after treating with 500 and 250 mg/ml of vitamin B1. Conclusion: Based on the obtained results, thiamine could not inhibit the fungal growth completely. However, the rate of afIR gene expression and aflatoxin production was significantly reduced after fungal treating with thiamine. Consequently, using natural compounds such as vitamins may be regarded as potential antitoxic agent in food industry and the industries related to agriculture.

Increasing cocoa butter-like lipid production of Saccharomyces cerevisiae by expression of selected cocoa genes

DEFF Research Database (Denmark)

Wei, Yongjun; Gossing, Michael; Bergenholm, David

2017-01-01

for CB biosynthesis from the cocoa genome using a phylogenetic analysis approach. By expressing the selected cocoa genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some S. cerevisiae strains. The relative CBL content in three yeast...... higher level of CBL compared with the control strain. In summary, CBL production by S. cerevisiae were increased through expressing selected cocoa genes potentially involved in CB biosynthesis.......Cocoa butter (CB) extracted from cocoa beans mainly consists of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0-C18:1-C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol(POS,C16:0C18:1-C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18...

Gene Disruption Technologies Have the Potential to Transform Stored Product Insect Pest Control

Directory of Open Access Journals (Sweden)

Lindsey C. Perkin

2016-09-01

Full Text Available Stored product insects feed on grains and processed commodities manufactured from grain post-harvest, reducing the nutritional value and contaminating food. Currently, the main defense against stored product insect pests is the pesticide fumigant phosphine. Phosphine is highly toxic to all animals, but is the most effective and economical control method, and thus is used extensively worldwide. However, many insect populations have become resistant to phosphine, in some cases to very high levels. New, environmentally benign and more effective control strategies are needed for stored product pests. RNA interference (RNAi may overcome pesticide resistance by targeting the expression of genes that contribute to resistance in insects. Most data on RNAi in stored product insects is from the coleopteran genetic model, Tribolium castaneum, since it has a strong RNAi response via injection of double stranded RNA (dsRNA in any life stage. Additionally, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR technology has been suggested as a potential resource for new pest control strategies. In this review we discuss background information on both gene disruption technologies and summarize the advances made in terms of molecular pest management in stored product insects, mainly T. castaneum, as well as complications and future needs.

Synthesis of bacteriophage-coded gene products during infection of Escherichia coli with amber mutants of T3 and T7 defective in gene 1

DEFF Research Database (Denmark)

Issinger, O G; Hausmann, R

1973-01-01

During nonpermissive infection by a T7 amber mutant in gene 1 (phage RNA polymerase-deficient), synthesis of the products of the phage genes 3 (endonuclease), 3, 5 (lysozyme), 5 (DNA polymerase), and 17 (serum blocking power) was shown to occur at about half the rate as during wild-type infection...

Recombinant hepatitis B surface antigen production in Aspergillus niger: evaluating the strategy of gene fusion to native glucoamylase

CSIR Research Space (South Africa)

James, ER

2012-10-01

Full Text Available Microbiology and Biotechnology October 2012/ Vol. 96, No.2 Recombinant hepatitis B surface antigen production in Aspergillus niger: evaluating the strategy of gene fusion to native glucoamylase ER James a,c & WH van Zyl b & PJ van Zyl c & JF Görgens..., Pretoria 0001, South Africa Abstract This study demonstrates the potential of Aspergillus niger as a candidate expression system for virus- like particle production using gene fusion. Hepatitis B surface antigen (HBsAg) production, targeted...

Improvements in algal lipid production: a systems biology and gene editing approach.

Science.gov (United States)

Banerjee, Avik; Banerjee, Chiranjib; Negi, Sangeeta; Chang, Jo-Shu; Shukla, Pratyoosh

2018-05-01

In the wake of rising energy demands, microalgae have emerged as potential sources of sustainable and renewable carbon-neutral fuels, such as bio-hydrogen and bio-oil. For rational metabolic engineering, the elucidation of metabolic pathways in fine detail and their manipulation according to requirements is the key to exploiting the use of microalgae. Emergence of site-specific nucleases have revolutionized applied research leading to biotechnological gains. Genome engineering as well as modulation of the endogenous genome with high precision using CRISPR systems is being gradually employed in microalgal research. Further, to optimize and produce better algal platforms, use of systems biology network analysis and integration of omics data is required. This review discusses two important approaches: systems biology and gene editing strategies used on microalgal systems with a focus on biofuel production and sustainable solutions. It also emphasizes that the integration of such systems would contribute and compliment applied research on microalgae. Recent advances in microalgae are discussed, including systems biology, gene editing approaches in lipid bio-synthesis, and antenna engineering. Lastly, it has been attempted here to showcase how CRISPR/Cas systems are a better editing tool than existing techniques that can be utilized for gene modulation and engineering during biofuel production.

Immunohistochemical Mapping of TRK-Fused Gene Products in the Rat Brainstem

International Nuclear Information System (INIS)

Takeuchi, Shigeko; Masuda, Chiaki; Maebayashi, Hisae; Tooyama, Ikuo

2012-01-01

The TRK-fused gene (TFG in human, Tfg in rat) was originally identified in human papillary thyroid cancer as a chimeric form of the NTRK1 gene. It was since reported that the gene product (TFG) plays a role in regulating phosphotyrosine-specific phosphatase-1 activity. As shown in the accompanying paper, we produced an antibody to rat TFG and used it to localize TFG to selected neurons in specific regions. In the present study, we mapped the TFG-positive neurons in the brainstem, cerebellum, and spinal cord of rats. In the brainstem, neurons intensely positive for TFG were distributed in the raphe nuclei, the gigantocellular reticular nucleus, the reticulotegmental nucleus of the pons, and some cranial nerve nuclei such as the trigeminal nuclei, the vestibulocochlear nuclei, and the dorsal motor nucleus of the vagus. Purkinje cells in the cerebellum and motor neurons in the spinal anterior horn were also positive for TFG. These results provide fundamental data for studying the functions of TFG in the brain

Essential Oils Modulate Gene Expression and Ochratoxin A Production in Aspergillus carbonarius

International Nuclear Information System (INIS)

Atoui, A.; El Khoury, R.; Verheecke, C; Mathieu, F.; Maroun, R.; El Khoury, A.

2016-01-01

Ochratoxin A (OTA) is a mycotoxin, mainly produced on grapes by Aspergillus carbonarius, that causes massive health problems for humans. This study aims to reduce the occurrence of OTA by using the ten following essential oils (E.Os): fennel, cardamom, anise, chamomile, celery, cinnamon, thyme, taramira, oregano and rosemary at μL/mL and 5 μL/mL for each E.O. As a matter of fact, their effects on the OTA production and the growth of A. carbonarius S402 cultures were evaluated, after four days at 28 °C on a Synthetic Grape Medium (SGM). Results showed that A. carbonarius growth was reduced up to 100%, when cultured with the E.Os of cinnamon, taramira, and oregano at both concentrations and the thyme at 5 μL/mL. As for the other six E.Os, their effect on A. carbonarius growth was insignificant, but highly important on the OTA production. Interestingly, the fennel E.O at 5 _L/mL reduced the OTA production up to 88.9% compared to the control, with only 13.8% of fungal growth reduction. We further investigated the effect of these E.Os on the expression levels of the genes responsible for the OTA biosynthesis (acOTApks and acOTAnrps along with the acpks gene) as well as the two regulatory genes laeA and vea, using the quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) method. The results revealed that these six E.Os reduced the expression of the five studied genes, where the ackps was downregulated by 99.2% (the highest downregulation in this study) with 5 μL/mL of fennel E.O. As for the acOTApks, acOTAnrps, veA and laeA, their reduction levels ranged between 10% and 96% depending on the nature of the E.O and its concentration in the medium. (author)

Lambda bacteriophage gene products and x-ray sensitivity of Escherichia coli: comparison of red-dependent and gam-dependent radioresistance

International Nuclear Information System (INIS)

Trgovcevic, Z.; Rupp, W.D.

1975-01-01

When gene products of lambda bacteriophage are introduced into a cell by transient induction of a lysogen, increased resistance of the cells to x rays results. This phenomenon has been called phage-induced radioresistance. Genetic studies show at least two classes of induced radioresistance. The first type depends on the products of the lambda red genes and is observed in bacteria that are mutated in the recB gene. It is thought that the lambda red products compensate for the missing RecBC nuclease in the repair of x-ray damage. An optimal effect is obtained even when the lambda red products are supplied 1 h after irradiation. The lesions that are affected by the red-dependent process are probably not deoxyribonucleic acid strand breaks because the extent of deoxyribonucleic acid strand rejoining is not altered by the red products. The second type of phage-induced radioresistance requires the gam product of lambda and is observed in wild-type and polA strains. The lambda gam + gene product must be present immediately after irradiation to exert its full effect. In its presence, DNA breakdown is decreased, and a greater fraction of DNA is converted back to high molecular weight. Strains carrying lex, recA, or certain other combinations of mutations do not show any detectable phage-induced radioresistance. (U.S.)

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

Directory of Open Access Journals (Sweden)

Mohammad H Dezfulian

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

Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities

Science.gov (United States)

Mackie, R.I.; Koike, S.; Krapac, I.; Chee-Sanford, J.; Maxwell, Susan; Aminov, R.I.

2006-01-01

Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment.To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators-including inorganic ions, antibiotics, and antibiotic resistance genes-were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 ??g/L.Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and

Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes.

Science.gov (United States)

Ziemons, Sandra; Koutsantas, Katerina; Becker, Kordula; Dahlmann, Tim; Kück, Ulrich

2017-02-16

Multi-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster. We performed pulsed-field gel electrophoresis (PFGE), quantitative qRT-PCR, and penicillin bioassays to investigate production, deletion and overexpression strains generated in the P. chrysogenum P2niaD18 background, in order to determine the copy number of the penicillin biosynthesis gene cluster, and study the expression of one penicillin biosynthesis gene, and the penicillin titer. Analysis of production and recombinant strain showed that the enhanced penicillin titer did not depend on the copy number of the penicillin gene cluster. Our assumption was strengthened by results with a penicillin null strain lacking pcbC encoding isopenicillin N synthase. Reintroduction of one or two copies of the cluster into the pcbC deletion strain restored transcriptional high expression of the pcbC gene, but recombinant strains showed no significantly different penicillin titer compared to parental strains. Here we present a molecular genetic analysis of production and recombinant strains in the P2niaD18 background carrying different copy numbers of the penicillin biosynthesis gene cluster. Our analysis shows that the enhanced penicillin titer does not strictly depend on the copy number of the cluster. Based on these overall findings, we hypothesize that instead, complex regulatory mechanisms are prominently implicated in increased penicillin biosynthesis in production strains.

Applications of gene-based technologies for improving animal production and health in developing countries

International Nuclear Information System (INIS)

Makkar, H.P.S.; Viljoen, G.J.

2005-01-01

This book provides a compilation of peer-reviewed scientific contributions from authoritative researchers attending an international symposium convened by the Animal Production and Health Sub-programme of the Animal Production and Health (APH), Joint FAO/IAEA Programme in cooperation with the Animal Production and Health Division of the FAO. These Proceedings contain invaluable information on the role and future potential of gene-based technologies for improving animal production and health, possible applications and constraints in the use of this technology in developing countries and their specific research needs

The presence of c-erbB-2 gene product-related protein in culture medium conditioned by breast cancer cell line SK-BR-3

International Nuclear Information System (INIS)

Alper, O.; Yamaguchi, K.; Hitomi, J.; Honda, S.; Matsushima, T.; Abe, K.

1990-01-01

The Mr 185,000 glycoprotein encoded by human c-erbB-2/neu/HER2 gene, termed c-erbB-2 gene product, shows a close structural similarity with epidermal growth factor receptor and is now regarded to be a growth factor receptor for an as yet unidentified ligand. Abundant c-erbB-2 mRNA was demonstrated by Northern blot studies in the human breast cancer cell line SK-BR-3. Cellular radiolabeling experiments followed by immunoprecipitation with three different anti-c-erbB-2 gene product antibodies, recognizing extracellular domain, kinase domain, and carboxyl-terminal portion, respectively, demonstrated the production of a large amount of c-erbB-2 gene product which had the capacity to be phosphorylated. Immunization of mice with concentrated culture medium conditioned by SK-BR-3 cells always generated antibodies against c-erbB-2 gene product, demonstrating that this culture medium contained substance(s) immunologically indistinguishable from c-erbB-2 gene product. This observation was supported by the successful development of a monoclonal antibody against c-erbB-2 gene product, GFD-OA-p185-1, by immunizing mice with this culture medium. The biochemical nature of the substance(s) present in the culture medium was further characterized. When the culture medium conditioned by [35S]cysteine-labeled SK-BR-3 cells was immunoprecipitated by three different anti-c-erbB-2 gene product antibodies, only the antibody recognizing extracellular domain precipitated the [35S]-labeled protein with a molecular weight of 110,000, namely p110. The newly developed monoclonal antibody also immunoprecipitated this protein

A mutation in the aroE gene affects pigment production, virulence, and chemotaxis in Xanthomonas oryzae pv. oryzae.

Science.gov (United States)

Kim, Hong-Il; Noh, Tae-Hwan; Lee, Chang-Soo; Park, Young-Jin

2015-01-01

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB) in rice. To study its function, a random insertion mutation library of Xoo was constructed using the Tn5 transposon. A mutant strain with decreased virulence against the susceptible rice cultivar IR24 was isolated from the library (aroE mutant), which also had extremely low pigment production. Thermal asymmetric interlaced-polymerase chain reaction (TAIL-PCR) and sequence analysis of the mutant revealed that the transposon was inserted into the aroE gene (encoding shikimate dehydrogenase). To investigate gene expression changes in the pigment- and virulence-deficient mutant, DNA microarray analysis was performed, which showed downregulation of 20 genes involved in the chemotaxis of Xoo. Our findings reveal that mutation of the aroE gene affects virulence and pigment production, as well as expression of genes involved in Xoo chemotaxis. Copyright © 2014 Elsevier GmbH. All rights reserved.

Intrinsic incompatibilities evolving as a by-product of divergent ecological selection: Considering them in empirical studies on divergence with gene flow.

Science.gov (United States)

Kulmuni, J; Westram, A M

2017-06-01

The possibility of intrinsic barriers to gene flow is often neglected in empirical research on local adaptation and speciation with gene flow, for example when interpreting patterns observed in genome scans. However, we draw attention to the fact that, even with gene flow, divergent ecological selection may generate intrinsic barriers involving both ecologically selected and other interacting loci. Mechanistically, the link between the two types of barriers may be generated by genes that have multiple functions (i.e., pleiotropy), and/or by gene interaction networks. Because most genes function in complex networks, and their evolution is not independent of other genes, changes evolving in response to ecological selection can generate intrinsic barriers as a by-product. A crucial question is to what extent such by-product barriers contribute to divergence and speciation-that is whether they stably reduce gene flow. We discuss under which conditions by-product barriers may increase isolation. However, we also highlight that, depending on the conditions (e.g., the amount of gene flow and the strength of selection acting on the intrinsic vs. the ecological barrier component), the intrinsic incompatibility may actually destabilize barriers to gene flow. In practice, intrinsic barriers generated as a by-product of divergent ecological selection may generate peaks in genome scans that cannot easily be interpreted. We argue that empirical studies on divergence with gene flow should consider the possibility of both ecological and intrinsic barriers. Future progress will likely come from work combining population genomic studies, experiments quantifying fitness and molecular studies on protein function and interactions. © 2017 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

Identification of an ovine atadenovirus gene whose product activates the viral E2 promoter: possible involvement of E2F-1

International Nuclear Information System (INIS)

Kuemin, Daniel; Hofmann, Christian; Uckert, Wolfgang; Both, Gerald W.; Loeser, Peter

2004-01-01

Activation of the adenoviral E2 promoter is an early step in adenovirus gene expression. For members of the mast- and aviadenoviruses, this requires induction of the cellular transcription factor E2F by virally encoded gene products such as E1A, E4orf6/7 and orf22/GAM-1. The newly recognized genus atadenovirus, of which the ovine isolate OAdV is the prototype, lacks any sequence homology to those genes. To find a possible link between E2 promoter activation and OAdV gene expression, we utilized a screening method to search for genes within the OAdV genome that were capable of stimulating the viral E2 promoter. One such gene, E43, was identified within the proposed E4 region toward the right-hand end of the OAdV genome. The E43 gene product was also found to be capable of stimulating E2F-1-dependent gene expression. A closer inspection of the E2 promoter revealed the presence of a non-palindromic E2F binding site within the OAdV E2 promoter. Mutation of this site markedly reduced both E2F-1- and E43-dependent promoter activation. Moreover, a direct protein-protein interaction of the E43 gene product with E2F, but not with the retinoblastoma protein pRb, suggested a possible cooperation between these two proteins in activating the E2 promoter. The importance of the E43 gene product for virus replication is also underlined by the finding that an OAdV recombinant with a functionally inactivated E43 gene showed severely inhibited virus growth

Functional Reconstitution of a Fungal Natural Product Gene Cluster by Advanced Genome Editing.

Science.gov (United States)

Weber, Jakob; Valiante, Vito; Nødvig, Christina S; Mattern, Derek J; Slotkowski, Rebecca A; Mortensen, Uffe H; Brakhage, Axel A

2017-01-20

Filamentous fungi produce varieties of natural products even in a strain dependent manner. However, the genetic basis of chemical speciation between strains is still widely unknown. One example is trypacidin, a natural product of the opportunistic human pathogen Aspergillus fumigatus, which is not produced among different isolates. Combining computational analysis with targeted gene editing, we could link a single nucleotide insertion in the polyketide synthase of the trypacidin biosynthetic pathway and reconstitute its production in a nonproducing strain. Thus, we present a CRISPR/Cas9-based tool for advanced molecular genetic studies in filamentous fungi, exploiting selectable markers separated from the edited locus.

Cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the E.coli bacteria

Directory of Open Access Journals (Sweden)

Masome Zeinali

2016-09-01

Full Text Available Introduction: Ethanol made by a biomass is one of the useful strategies in terms of economic and environmental and as a clean and safe energy to replace fossil fuels considered and examined. Materials and methods: In this study, key enzyme in the production of ethanol (Pyruvate decarboxylase from Zymomonas mobilis bacteria was isolated and cloned at E. coli bacteria by freeze and thaw method. For gene cloning, we used specific primers of pdc and PCR reaction and then pdc gene isolated and pET 28a plasmid double digested with (Sal I and Xho I enzymes. Digestion Products were ligated by T4 DNA ligase in 16 °C for 16 hours. Results: Results of bacteria culture showed that a few colonies containing pET 28a plasmid could grow. Result of colony pcr of pdc gene with specific primers revealed 1700 bp bands in 1% agarose gel electrophoresis. The results of PCR with T7 promotor forward primer and pdc revers primer have proved the accurate direction of integration of pdc gene into plasmid and revealed 1885 bp band. Double digestion of recombinant plasmid with SalI and XhoI enzymes revealed same bands. Finally, RT showed the expected band of 1700 bp that implies the desired gene expression in the samples. Discussion and conclusion: Due to the increased production of ethanol via pyruvate decarboxylase gene cloning in expression plasmids with a strong promoter upstream of the cloning site can conclude that, pyruvate decarboxylase cloning as a key gene would be useful and according to beneficial properties of E. coli bacteria, transfering the gene to bacteria appears to be reasonable.

Effects of excretory/secretory products from Anisakis simplex (Nematoda) on immune gene expression in rainbow trout (Oncorhynchus mykiss)

DEFF Research Database (Denmark)

Bahlool, Qusay Zuhair Mohammad; Skovgaard, Alf; Kania, Per Walter

2013-01-01

Excretory/secretory (ES) products are molecules produced by parasitic nematodes, including larval Anisakis simplex, a parasite occurring in numerous marine fish hosts. The effects of these substances on host physiology have not been fully described. The present work elucidates the influence of ES...... substances on the fish immune system by measuring immune gene expression in spleen and liver of rainbow trout (Oncorhynchus mykiss) injected intraperitoneally with ES products isolated from A. simplex third stage larvae. The overall gene expression profile of exposed fish showed a generalized down....... This type of hydrolytic enzyme activity may play a role in nematode penetration of host tissue. In addition, based on the notion that A. simplex ES products may have an immune-depressive effect (by minimizing immune gene expression) it could also be suggested that worm enzymes directly target host immune...

Expression and Localization of TRK-Fused Gene Products in the Rat Brain and Retina

International Nuclear Information System (INIS)

Maebayashi, Hisae; Takeuchi, Shigako; Masuda, Chiaki; Makino, Satoshi; Fukui, Kenji; Kimura, Hiroshi; Tooyama, Ikuo

2012-01-01

The TRK-fused gene (TFG in human, Tfg in rat) was originally identified in human papillary thyroid cancer as a chimeric form of the NTRK1 gene. It has been reported that the gene product (TFG) plays a role in regulating phosphotyrosine-specific phosphatase-1 activity. However, no information regarding the localization of Tfg in rat tissues is available. In this study, we investigated the expression of Tfg mRNA in normal rat tissues using reverse transcription-polymerase chain reaction (RT-PCR). We also produced an antibody against Tfg gene products and examined the localization of TFG in the rat brain and retina. The RT-PCR experiments demonstrated that two types of Tfg mRNA were expressed in rat tissues: the conventional form of Tfg (cTfg) and a novel variant form, retinal Tfg (rTfg). RT-PCR analyses demonstrated that cTfg was ubiquitously expressed in rat tissues, while rTfg was predominantly expressed in the brain and retina. Western blot analysis demonstrated two bands with molecular weights of about 30 kDa and 50 kDa in the rat brain. Immunohistochemistry indicated that TFG proteins were predominantly expressed by neurons in the brain. In the rat retina, intense TFG-immunoreactivity was detected in the layer of rods and cones and the outer plexiform layer

Genome Wide Association Analysis Reveals New Production Trait Genes in a Male Duroc Population.

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Kejun Wang

Full Text Available In this study, 796 male Duroc pigs were used to identify genomic regions controlling growth traits. Three production traits were studied: food conversion ratio, days to 100 KG, and average daily gain, using a panel of 39,436 single nucleotide polymorphisms. In total, we detected 11 genome-wide and 162 chromosome-wide single nucleotide polymorphism trait associations. The Gene ontology analysis identified 14 candidate genes close to significant single nucleotide polymorphisms, with growth-related functions: six for days to 100 KG (WT1, FBXO3, DOCK7, PPP3CA, AGPAT9, and NKX6-1, seven for food conversion ratio (MAP2, TBX15, IVL, ARL15, CPS1, VWC2L, and VAV3, and one for average daily gain (COL27A1. Gene ontology analysis indicated that most of the candidate genes are involved in muscle, fat, bone or nervous system development, nutrient absorption, and metabolism, which are all either directly or indirectly related to growth traits in pigs. Additionally, we found four haplotype blocks composed of suggestive single nucleotide polymorphisms located in the growth trait-related quantitative trait loci and further narrowed down the ranges, the largest of which decreased by ~60 Mb. Hence, our results could be used to improve pig production traits by increasing the frequency of favorable alleles via artificial selection.

Production of β-ionone by combined expression of carotenogenic and plant CCD1 genes in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Lopez, Javiera; Essus, Karen; Kim, Il-Kwon

2015-01-01

cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of beta-ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy......, the carotenogenic crtYB, crtI genes and the plant PhCCD1 gene-the highest β-ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods.......Background: Apocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plants-like raspberries and roses-by the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour...

Isoepoxydon dehydrogenase (idh) gene expression in relation to patulin production by Penicillium expansum under different temperature and atmosphere.

Science.gov (United States)

De Clercq, N; Vlaemynck, G; Van Pamel, E; Van Weyenberg, S; Herman, L; Devlieghere, F; De Meulenaer, B; Van Coillie, E

2016-03-02

Penicillium expansum growth and patulin production occur mainly at post-harvest stage during the long-term storage of apples. Low temperature in combination with reduced oxygen concentrations is commonly applied as a control strategy to extend apple shelf life and supply the market throughout the year. Our in vitro study investigated the effect of temperature and atmosphere on expression of the idh gene in relation to the patulin production by P. expansum. The idh gene encodes the isoepoxydon dehydrogenase enzyme, a key enzyme in the patulin biosynthesis pathway. First, a reverse transcription real-time PCR (RT-qPCR) method was optimized to measure accurately the P. expansum idh mRNA levels relative to the mRNA levels of three reference genes (18S, β-tubulin, calmodulin), taking into account important parameters such as PCR inhibition and multiple reference gene stability. Subsequently, two P. expansum field isolates and one reference strain were grown on apple puree agar medium (APAM) under three conditions of temperature and atmosphere: 20 °C - air, 4 °C - air and 4 °C - controlled atmosphere (CA; 3% O2). When P. expansum strains reached a 0.5 and 2.0 cm colony diameter, idh expression and patulin concentrations were determined by means of the developed RT-qPCR and an HPLC-UV method, respectively. The in vitro study showed a clear reduction in patulin production and down-regulation of the idh gene expression when P. expansum was grown under 4 °C - CA. The results suggest that stress (low temperature and oxygen level) caused a delay of the fungal metabolism rather than a complete inhibition of toxin biosynthesis. A good correlation was found between the idh expression and patulin production, corroborating that temperature and atmosphere affected patulin production by acting at the transcriptional level of the idh gene. Finally, a reliable RT-qPCR can be considered as an alternative tool to investigate the effect of control strategies on the toxin formation in

The YJR127C/ZMS1 gene product is involved in glycerol-based respiratory growth of the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Lu, Lin; Roberts, George G; Oszust, Cynthia; Hudson, Alan P

2005-10-01

A putative yeast mitochondrial upstream activating sequence (UAS) was used in a one-hybrid screening procedure that identified the YJR127C ORF on chromosome X. This gene was previously designated ZMS1 and is listed as a transcription factor on the SGD website. Real time RT-PCR assays showed that expression of YJR127C/ZMS1 was glucose-repressible, and a deletion mutant for the gene showed a growth defect on glycerol-based but not on glucose- or ethanol-based medium. Real time RT-PCR analyses identified severely attenuated transcript levels from GUT1 and GUT2 to be the source of that growth defect, the products of GUT1 and GUT2 are required for glycerol utilization. mRNA levels from a large group of mitochondria- and respiration-related nuclear genes also were shown to be attenuated in the deletion mutant. Importantly, transcript levels from the mitochondrial OLI1 gene, which has an associated organellar UAS, were attenuated in the DeltaYJR127C mutant during glycerol-based growth, but those from COX3 (OXI2), which lacks an associated mitochondrial UAS, were not. Transcriptome analysis of the glycerol-grown deletion mutant showed that genes in several metabolic and other categories are affected by loss of this gene product, including protein transport, signal transduction, and others. Thus, the product of YJR127C/ZMS1 is involved in transcriptional control for genes in both cellular genetic compartments, many of which specify products required for glycerol-based growth, respiration, and other functions.

Increased production of free fatty acids in Aspergillus oryzae by disruption of a predicted acyl-CoA synthetase gene.

Science.gov (United States)

Tamano, Koichi; Bruno, Kenneth S; Koike, Hideaki; Ishii, Tomoko; Miura, Ai; Umemura, Myco; Culley, David E; Baker, Scott E; Machida, Masayuki

2015-04-01

Fatty acids are attractive molecules as source materials for the production of biodiesel fuel. Previously, we attained a 2.4-fold increase in fatty acid production by increasing the expression of fatty acid synthesis-related genes in Aspergillus oryzae. In this study, we achieved an additional increase in the production of fatty acids by disrupting a predicted acyl-CoA synthetase gene in A. oryzae. The A. oryzae genome is predicted to encode six acyl-CoA synthetase genes and disruption of AO090011000642, one of the six genes, resulted in a 9.2-fold higher accumulation (corresponding to an increased production of 0.23 mmol/g dry cell weight) of intracellular fatty acid in comparison to the wild-type strain. Furthermore, by introducing a niaD marker from Aspergillus nidulans to the disruptant, as well as changing the concentration of nitrogen in the culture medium from 10 to 350 mM, fatty acid productivity reached 0.54 mmol/g dry cell weight. Analysis of the relative composition of the major intracellular free fatty acids caused by disruption of AO090011000642 in comparison to the wild-type strain showed an increase in stearic acid (7 to 26 %), decrease in linoleic acid (50 to 27 %), and no significant changes in palmitic or oleic acid (each around 20-25 %).

Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

KAUST Repository

Li, Yongxin

2015-03-24

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning plug-and-playa approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

Science.gov (United States)

Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S.; Qian, Pei-Yuan

2015-03-01

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning ``plug-and-play'' approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

Effects of impurities in biodiesel-derived glycerol on growth and expression of heavy metal ion homeostasis genes and gene products in Pseudomonas putida LS46.

Science.gov (United States)

Fu, Jilagamazhi; Sharma, Parveen; Spicer, Vic; Krokhin, Oleg V; Zhang, Xiangli; Fristensky, Brian; Wilkins, John A; Cicek, Nazim; Sparling, Richard; Levin, David B

2015-07-01

Biodiesel production-derived waste glycerol (WG) was previously investigated as potential carbon source for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46. In this study, we evaluated the effect of impurities in the WG on P. putida LS46 physiology during exponential growth and corresponding changes in transcription and protein expression profiles compared with cells grown on pure, reagent grade glycerol. High concentration of metal ions, such as Na(+), and numbers of heavy metals ion, such as copper, ion, zinc, were detected in biodiesel-derived WG. Omics analysis from the corresponding cultures suggested altered expression of genes involved in transport and metabolism of ammonia and heavy metal ions. Expression of three groups of heavy metal homeostasis genes was significantly changed (mostly upregulated) in WG cultures and included the following: copper-responded cluster 1 and 2 genes, primarily containing cusABC; two copies of copAB and heavy metal translocating P-type ATPase; Fur-regulated, TonB-dependent siderophore receptor; and several cobalt/zinc/cadmium transporters. Expression of these genes suggests regulation of intracellular concentrations of heavy metals during growth on biodiesel-derived glycerol. Finally, a number of genes involved in adapting to, or metabolizing free fatty acids and other nonheavy metal contaminants, such as Na(+), were also upregulated in P. putida LS46 grown on biodiesel-derived glycerol.

Identification of genes related to high royal jelly production in the honey bee (Apis mellifera) using microarray analysis.

Science.gov (United States)

Nie, Hongyi; Liu, Xiaoyan; Pan, Jiao; Li, Wenfeng; Li, Zhiguo; Zhang, Shaowu; Chen, Shenglu; Miao, Xiaoqing; Zheng, Nenggan; Su, Songkun

2017-01-01

China is the largest royal jelly producer and exporter in the world, and high royal jelly-yielding strains have been bred in the country for approximately three decades. However, information on the molecular mechanism underlying high royal jelly production is scarce. Here, a cDNA microarray was used to screen and identify differentially expressed genes (DEGs) to obtain an overview on the changes in gene expression levels between high and low royal jelly producing bees. We developed a honey bee gene chip that covered 11,689 genes, and this chip was hybridised with cDNA generated from RNA isolated from heads of nursing bees. A total of 369 DEGs were identified between high and low royal jelly producing bees. Amongst these DEGs, 201 (54.47%) genes were up-regulated, whereas 168 (45.53%) were down-regulated in high royal jelly-yielding bees. Gene ontology (GO) analyses showed that they are mainly involved in four key biological processes, and pathway analyses revealed that they belong to a total of 46 biological pathways. These results provide a genetic basis for further studies on the molecular mechanisms involved in high royal jelly production.

Identification of genes related to high royal jelly production in the honey bee (Apis mellifera using microarray analysis

Directory of Open Access Journals (Sweden)

Hongyi Nie

2017-10-01

Full Text Available Abstract China is the largest royal jelly producer and exporter in the world, and high royal jelly-yielding strains have been bred in the country for approximately three decades. However, information on the molecular mechanism underlying high royal jelly production is scarce. Here, a cDNA microarray was used to screen and identify differentially expressed genes (DEGs to obtain an overview on the changes in gene expression levels between high and low royal jelly producing bees. We developed a honey bee gene chip that covered 11,689 genes, and this chip was hybridised with cDNA generated from RNA isolated from heads of nursing bees. A total of 369 DEGs were identified between high and low royal jelly producing bees. Amongst these DEGs, 201 (54.47% genes were up-regulated, whereas 168 (45.53% were down-regulated in high royal jelly-yielding bees. Gene ontology (GO analyses showed that they are mainly involved in four key biological processes, and pathway analyses revealed that they belong to a total of 46 biological pathways. These results provide a genetic basis for further studies on the molecular mechanisms involved in high royal jelly production.

Abundance and distribution of Macrolide-Lincosamide-Streptogramin resistance genes in an anaerobic-aerobic system treating spiramycin production wastewater.

Science.gov (United States)

Liu, Miaomiao; Ding, Ran; Zhang, Yu; Gao, Yingxin; Tian, Zhe; Zhang, Tong; Yang, Min

2014-10-15

The behaviors of the Macrolide-Lincosamide-Streptogramin (MLS) resistance genes were investigated in an anaerobic-aerobic pilot-scale system treating spiramycin (SPM) production wastewater. After screening fifteen typical MLS resistance genes with different mechanisms using conventional PCR, eight detected genes were determined by quantitative PCR, together with three mobile elements. Aerobic sludge in the pilot system exhibited a total relative abundance of MLS resistance genes (per 16S rRNA gene) 2.5 logs higher than those in control samples collected from sewage and inosine wastewater treatment systems (P resistance genes. However, the total relative gene abundance in anaerobic sludge (4.3 × 10(-1)) was lower than that in aerobic sludge (3.7 × 10(0)) despite of the higher SPM level in anaerobic reactor, showing the advantage of anaerobic treatment in reducing the production of MLS resistance genes. The rRNA methylase genes (erm(B), erm(F), erm(X)) were the most abundant in the aerobic sludge (5.3 × 10(-1)-1.7 × 10(0)), followed by esterase gene ere(A) (1.3 × 10(-1)) and phosphorylase gene mph(B) (5.7 × 10(-2)). In anaerobic sludge, erm(B), erm(F), ere(A), and msr(D) were the major ones (1.2 × 10(-2)-3.2 × 10(-1)). These MLS resistance genes (except for msr(D)) were positively correlated with Class 1 integron (r(2) = 0.74-0.93, P < 0.05), implying the significance of horizontal transfer in their proliferation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Whole-Genome Microarray and Gene Deletion Studies Reveal Regulation of the Polyhydroxyalkanoate Production Cycle by the Stringent Response in Ralstonia eutropha H16

Energy Technology Data Exchange (ETDEWEB)

Brigham, CJ; Speth, DR; Rha, C; Sinskey, AJ

2012-10-22

Poly(3-hydroxybutyrate) (PHB) production and mobilization in Ralstonia eutropha are well studied, but in only a few instances has PHB production been explored in relation to other cellular processes. We examined the global gene expression of wild-type R. eutropha throughout the PHB cycle: growth on fructose, PHB production using fructose following ammonium depletion, and PHB utilization in the absence of exogenous carbon after ammonium was resupplied. Our results confirm or lend support to previously reported results regarding the expression of PHB-related genes and enzymes. Additionally, genes for many different cellular processes, such as DNA replication, cell division, and translation, are selectively repressed during PHB production. In contrast, the expression levels of genes under the control of the alternative sigma factor sigma(54) increase sharply during PHB production and are repressed again during PHB utilization. Global gene regulation during PHB production is strongly reminiscent of the gene expression pattern observed during the stringent response in other species. Furthermore, a ppGpp synthase deletion mutant did not show an accumulation of PHB, and the chemical induction of the stringent response with DL-norvaline caused an increased accumulation of PHB in the presence of ammonium. These results indicate that the stringent response is required for PHB accumulation in R. eutropha, helping to elucidate a thus-far-unknown physiological basis for this process.

The Natural Product Osthole Attenuates Yeast Growth by Extensively Suppressing the Gene Expressions of Mitochondrial Respiration Chain.

Science.gov (United States)

Wang, Zhe; Shen, Yan

2017-03-01

The fast growing evidences have indicated that the natural product osthole is a promising drug candidate for fighting several serious human diseases, for example, cancer and inflammation. However, the mode-of-action (MoA) of osthole remains largely incomplete. In this study, we investigated the growth inhibition activity of osthole using fission yeast as a model, with the goal of understanding the osthole's mechanism of action, especially from the molecular level. Microarray analysis indicated that osthole has significant impacts on gene transcription levels (In total, 214 genes are up-regulated, and 97 genes are down-regulated). Gene set enrichment analysis (GSEA) indicated that 11 genes belong to the "Respiration module" category, especially including the components of complex III and V of mitochondrial respiration chain. Based on GSEA and network analysis, we also found that 54 up-regulated genes belong to the "Core Environmental Stress Responses" category, particularly including many transporter genes, which suggests that the rapidly activated nutrient exchange between cell and environment is part of the MoA of osthole. In summary, osthole can greatly impact on fission yeast transcriptome, and it primarily represses the expression levels of the genes in respiration chain, which next causes the inefficiency of ATP production and thus largely explains osthole's growth inhibition activity in Schizosaccharomyces pombe (S. pombe). The complexity of the osthole's MoA shown in previous studies and our current research demonstrates that the omics approach and bioinformatics tools should be applied together to acquire the complete landscape of osthole's growth inhibition activity.

Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae.

Science.gov (United States)

Zhu, Lin; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2013-07-01

The filamentous fungus Aspergillus oryzae is used as one of the most favored hosts for heterologous protein production due to its ability to secrete large amounts of proteins into the culture medium. We previously generated a hyper-producing mutant strain of A. oryzae, AUT1, which produced 3.2- and 2.6-fold higher levels of bovine chymosin (CHY) and human lysozyme (HLY), respectively, compared with the wild-type strain. However, further enhancement of heterologous protein production by multiple gene disruption is difficult because of the low gene-targeting efficiency in strain AUT1. Here, we disrupted the ligD gene, which is involved in nonhomologous recombination, and the pyrG gene to create uridine/uracil auxotrophy in strain AUT1, to generate a hyper-producing mutant applicable to pyrG marker recycling with highly efficient gene targeting. We generated single and double disruptants of the tripeptidyl peptidase gene AosedD and vacuolar sorting receptor gene Aovps10 in the hyper-producing mutant background, and found that all disruptants showed significant increases in heterologous protein production. Particularly, double disruption of the Aovps10 and AosedD genes increased the production levels of CHY and HLY by 1.6- and 2.1-fold, respectively, compared with the parental strain. Thus, we successfully generated a fungal host for further enhancing the heterologous protein production ability by combining mutational and molecular breeding techniques.

Overexpression of the p53 tumor suppressor gene product in primary lung adenocarcinomas is associated with cigarette smoking

NARCIS (Netherlands)

Westra, W. H.; Offerhaus, G. J.; Goodman, S. N.; Slebos, R. J.; Polak, M.; Baas, I. O.; Rodenhuis, S.; Hruban, R. H.

1993-01-01

Mutations in the p53 tumor suppressor gene are frequently observed in primary lung adenocarcinomas, suggesting that these mutations are critical events in the malignant transformation of airway cells. These mutations are often associated with stabilization of the p53 gene product, resulting in the

Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3.

Science.gov (United States)

Zhang, Wei; He, Yulian; Gao, Weixia; Feng, Jun; Cao, Mingfeng; Yang, Chao; Song, Cunjiang; Wang, Shufang

2015-02-01

Here, we attempted to elevate poly-gamma-glutamic acid (γ-PGA) production by modifying genes involved in glutamate metabolism in Bacillus amyloliquefaciens LL3. Products of rocR, rocG and gudB facilitate the conversion from glutamate to 2-oxoglutarate in Bacillus subtillis. The gene odhA is responsible for the synthesis of a component of the 2-oxoglutarate dehydrogenase complex that catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl coenzyme A. In-frame deletions of these four genes were performed. In shake flask experiments the gudB/rocG double mutant presented enhanced production of γ-PGA, a 38 % increase compared with wild type. When fermented in a 5-L fermenter with pH control, the γ-PGA yield of the rocR mutant was increased to 5.83 g/L from 4.55 g/L for shake flask experiments. The gudB/rocG double mutant produced 5.68 g/L γ-PGA compared with that of 4.03 g/L for the wild type, a 40 % increase. Those results indicated the possibility of improving γ-PGA production by modifying glutamate metabolism, and identified potential genetic targets to improve γ-PGA production.

Determination of transcriptional units and gene products from the ftsA region of Escherichia coli.

Science.gov (United States)

Lutkenhaus, J F; Wu, H C

1980-01-01

Lambda transducing phage gamma 16-2 carries the genes envA, ftsZ, ftsA, ddl, and murC and directs the synthesis of six unique proteins in ultraviolet-irradiated cells. Various derivatives of gamma 16-2 carrying smaller segments of the bacterial deoxyribonucleic acid have also been analyzed for their capacity to direct protein synthesis in ultraviolet-irradiated cells. These results, in combination with genetic results, have allowed the gene product of each of these genes to be assigned. In addition, an unidentified gene was located counterclockwise to murC between murC and murF. Analysis of the direction of transcription indicates that murC, ddl, ftsA, and ftsZ are transcribed clockwise on the Escherichia coli genetic map, and envA is transcribed counterclockwise. In addition, it is shown that each of the genes envA, ftsZ, and ftsA can be expressed independently. Images PMID:6447690

Characterization and Heterologous Expression of the Genes Encoding Enterocin A Production, Immunity, and Regulation in Enterococcus faecium DPC1146

Science.gov (United States)

O’Keeffe, Triona; Hill, Colin; Ross, R. Paul

1999-01-01

Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by Enterococcus faecium DPC1146. The sequence of a 10,879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes entA, entI, and entF encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. Immediately downstream of the entT and entD genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the yteI gene of Bacillus subtilis and to E. coli protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the entA gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the entF gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in Enterococcus faecalis, while a four-gene construct (entAITD) under the

An allele of an ancestral transcription factor dependent on a horizontally acquired gene product.

Science.gov (United States)

Chen, H Deborah; Jewett, Mollie W; Groisman, Eduardo A

2012-01-01

Changes in gene regulatory circuits often give rise to phenotypic differences among closely related organisms. In bacteria, these changes can result from alterations in the ancestral genome and/or be brought about by genes acquired by horizontal transfer. Here, we identify an allele of the ancestral transcription factor PmrA that requires the horizontally acquired pmrD gene product to promote gene expression. We determined that a single amino acid difference between the PmrA proteins from the human adapted Salmonella enterica serovar Paratyphi B and the broad host range S. enterica serovar Typhimurium rendered transcription of PmrA-activated genes dependent on the PmrD protein in the former but not the latter serovar. Bacteria harboring the serovar Typhimurium allele exhibited polymyxin B resistance under PmrA- or under PmrA- and PmrD-inducing conditions. By contrast, isogenic strains with the serovar Paratyphi B allele displayed PmrA-regulated polymyxin B resistance only when experiencing activating conditions for both PmrA and PmrD. We establish that the two PmrA orthologs display quantitative differences in several biochemical properties. Strains harboring the serovar Paratyphi B allele showed enhanced biofilm formation, a property that might promote serovar Paratyphi B's chronic infection of the gallbladder. Our findings illustrate how subtle differences in ancestral genes can impact the ability of horizontally acquired genes to confer new properties.

Restriction fragment length polymorphism (RFLP) analysis of PCR products amplified from 18S ribosomal RNA gene of Trypanosoma congolense

International Nuclear Information System (INIS)

Osanyo, A.; Majiwa, P.W.

2006-01-01

Oligonucleotide primers were designed from the conserved nucleotide sequences of 18S ribosomal RNA (18S rRNA) gene of protozoans: Trypanosoma brucei, Leishmania donovani, Triponema aequale and Lagenidium gigantum. The primers were used in polymerace chain reaction (PCR) to generate PCR products of approximately 1 Kb using genomic DNA from T. brucei and the four genotypic groups of T. congolense as template. The five PCR products so produced were digested with several restriction enzymes and hybridized to a DNA probe made from T. brucei PCR product of the same 18S rRNA gene region. Most restriction enzyme digests revealed polymorphism with respect to the location of their recognition sites on the five PCR products. The restriction fragment length polymorphism (RFLP) pattern observed indicate that the 18S rRNA gene sequences of trypanosomes: T. brucei and the four genotypes of T.congolence group are heterogeneous. The results further demonstrate that the region that was amplified can be used in specific identification of trypanosomes species and subspecies.(author)

A Two-Layer Gene Circuit for Decoupling Cell Growth from Metabolite Production.

Science.gov (United States)

Lo, Tat-Ming; Chng, Si Hui; Teo, Wei Suong; Cho, Han-Saem; Chang, Matthew Wook

2016-08-01

We present a synthetic gene circuit for decoupling cell growth from metabolite production through autonomous regulation of enzymatic pathways by integrated modules that sense nutrient and substrate. The two-layer circuit allows Escherichia coli to selectively utilize target substrates in a mixed pool; channel metabolic resources to growth by delaying enzymatic conversion until nutrient depletion; and activate, terminate, and re-activate conversion upon substrate availability. We developed two versions of controller, both of which have glucose nutrient sensors but differ in their substrate-sensing modules. One controller is specific for hydroxycinnamic acid and the other for oleic acid. Our hydroxycinnamic acid controller lowered metabolic stress 2-fold and increased the growth rate 2-fold and productivity 5-fold, whereas our oleic acid controller lowered metabolic stress 2-fold and increased the growth rate 1.3-fold and productivity 2.4-fold. These results demonstrate the potential for engineering strategies that decouple growth and production to make bio-based production more economical and sustainable. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Characterization of the product of a nonribosomal peptide synthetase-like (NRPS-like) gene using the doxycycline dependent Tet-on system in Aspergillus terreus.

Science.gov (United States)

Sun, Wei-Wen; Guo, Chun-Jun; Wang, Clay C C

2016-04-01

Genome sequencing of the fungus Aspergillus terreus uncovered a number of silent core structural biosynthetic genes encoding enzymes presumed to be involved in the production of cryptic secondary metabolites. There are five nonribosomal peptide synthetase (NRPS)-like genes with the predicted A-T-TE domain architecture within the A. terreus genome. Among the five genes, only the product of pgnA remains unknown. The Tet-on system is an inducible, tunable and metabolism-independent expression system originally developed for Aspergillus niger. Here we report the adoption of the Tet-on system as an effective gene activation tool in A. terreus. Application of this system in A. terreus allowed us to uncover the product of the cryptic NRPS-like gene, pgnA. Furthermore expression of pgnA in the heterologous Aspergillus nidulans host suggested that the pgnA gene alone is necessary for phenguignardic acid (1) biosynthesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Targeted capture and heterologous expression of the Pseudoalteromonas alterochromide gene cluster in Escherichia coli represents a promising natural product exploratory platform.

Science.gov (United States)

Ross, Avena C; Gulland, Lauren E S; Dorrestein, Pieter C; Moore, Bradley S

2015-04-17

Marine pseudoalteromonads represent a very promising source of biologically important natural product molecules. To access and exploit the full chemical capacity of these cosmopolitan Gram-(-) bacteria, we sought to apply universal synthetic biology tools to capture, refactor, and express biosynthetic gene clusters for the production of complex organic compounds in reliable host organisms. Here, we report a platform for the capture of proteobacterial gene clusters using a transformation-associated recombination (TAR) strategy coupled with direct pathway manipulation and expression in Escherichia coli. The ~34 kb pathway for production of alterochromide lipopeptides by Pseudoalteromonas piscicida JCM 20779 was captured and heterologously expressed in E. coli utilizing native and E. coli-based T7 promoter sequences. Our approach enabled both facile production of the alterochromides and in vivo interrogation of gene function associated with alterochromide's unusual brominated lipid side chain. This platform represents a simple but effective strategy for the discovery and biosynthetic characterization of natural products from marine proteobacteria.

Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production.

Science.gov (United States)

Brueggeman, Andrew J; Kuehler, Daniel; Weeks, Donald P

2014-09-01

Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial-scale algal production facilities against contamination by organisms sensitive to these broad-spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×-fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO-inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene-based gene sequences found in various norflurazon-resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large-scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 μm and ~0.1 μm, respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large-scale algal production facilities in suppressing growth of organisms sensitive to these herbicides. © 2014 Society for Experimental Biology, Association of Applied Biologists and

Gene doping: gene delivery for olympic victory

OpenAIRE

Gould, David

2012-01-01

With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted...

FUM gene expression profile and fumonisin production by Fusarium verticillioides inoculated in Bt and non-Bt maize

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Liliana Oliveira Rocha

2016-01-01

Full Text Available This study aimed to determine the levels of fumonisins produced by F. verticillioides and FUM gene expression on Bt (Bacillus thuringiensis and non-Bt maize, post harvest, during different periods of incubation. Transgenic hybrids 30F35 YG, 2B710 Hx and their isogenic (30F35 and 2B710 were collected from the field and a subset of 30 samples selected for the experiments. Maize samples were sterilized by gamma radiation at a dose of 20 kGy. Samples were then inoculated with Fusarium verticillioides and analysed under controlled conditions of temperature and relative humidity for fumonisin B1 and B2 (FB¬1 and FB2 production and FUM1, FUM3, FUM6, FUM7, FUM8, FUM13, FUM14, FUM15 and FUM19 expression. 2B710 Hx and 30F35 YG kernel samples were virtually intact when compared to the non-Bt hybrids that came from the field. Statistical analysis showed that FB¬1 production was significantly lower in 30F35 YG and 2B710 Hx than in the 30F35 and 2B710 hybrids (P 0.05. The kernel injuries observed in the non-Bt samples have possibly facilitated F. verticillioides penetration and promoted FB1 production under controlled conditions. FUM genes were expressed by F. verticillioides in all of the samples. However, there was indication of lower expression of a few FUM genes in the Bt hybrids; and a weak association between FB1 production and the relative expression of some of the FUM genes were observed in the 30F35 YG hybrid.

Expression regulation of design process gene in product design

DEFF Research Database (Denmark)

Li, Bo; Fang, Lusheng; Li, Bo

2011-01-01

To improve the design process efficiency, this paper proposes the principle and methodology that design process gene controls the characteristics of design process under the framework of design process reuse and optimization based on design process gene. First, the concept of design process gene...... is proposed and analyzed, as well as its three categories i.e., the operator gene, the structural gene and the regulator gene. Second, the trigger mechanism that design objectives and constraints trigger the operator gene is constructed. Third, the expression principle of structural gene is analyzed...... with the example of design management gene. Last, the regulation mode that the regulator gene regulates the expression of the structural gene is established and it is illustrated by taking the design process management gene as an example. © (2011) Trans Tech Publications....

Lactobacillus reuteri-specific immunoregulatory gene rsiR modulates histamine production and immunomodulation by Lactobacillus reuteri.

Science.gov (United States)

Hemarajata, P; Gao, C; Pflughoeft, K J; Thomas, C M; Saulnier, D M; Spinler, J K; Versalovic, J

2013-12-01

Human microbiome-derived strains of Lactobacillus reuteri potently suppress proinflammatory cytokines like human tumor necrosis factor (TNF) by converting the amino acid l-histidine to the biogenic amine histamine. Histamine suppresses mitogen-activated protein (MAP) kinase activation and cytokine production by signaling via histamine receptor type 2 (H2) on myeloid cells. Investigations of the gene expression profiles of immunomodulatory L. reuteri ATCC PTA 6475 highlighted numerous genes that were highly expressed during the stationary phase of growth, when TNF suppression is most potent. One such gene was found to be a regulator of genes involved in histidine-histamine metabolism by this probiotic species. During the course of these studies, this gene was renamed the Lactobacillus reuteri-specific immunoregulatory (rsiR) gene. The rsiR gene is essential for human TNF suppression by L. reuteri and expression of the histidine decarboxylase (hdc) gene cluster on the L. reuteri chromosome. Inactivation of rsiR resulted in diminished TNF suppression in vitro and reduced anti-inflammatory effects in vivo in a trinitrobenzene sulfonic acid (TNBS)-induced mouse model of acute colitis. A L. reuteri strain lacking an intact rsiR gene was unable to suppress colitis and resulted in greater concentrations of serum amyloid A (SAA) in the bloodstream of affected animals. The PhdcAB promoter region targeted by rsiR was defined by reporter gene experiments. These studies support the presence of a regulatory gene, rsiR, which modulates the expression of a gene cluster known to mediate immunoregulation by probiotics at the transcriptional level. These findings may point the way toward new strategies for controlling gene expression in probiotics by dietary interventions or microbiome manipulation.

Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells

International Nuclear Information System (INIS)

Tsujimoto, Yoshihide

1989-01-01

The biological activity of the human BCL-2 gene product was analyzed in an Epstein-Barr virus (EBV)-infected human lymphoblastoid B-cell line transfected with BCL-2 sequences driven by the simian virus 40 promoter and enhancer. Overproduction of the BCL-2 protein conferred a selective growth advantage to the EBV-infected B cells as compared with control transfectants in low-serum medium and also after seeding at limiting dilution but did not render the cells tumorigenic in athymic nude mice. This growth enhancement was also seen in cells transfected with the BCL-2 gene with its own promoter juxtaposed to the immunoglobulin heavy chain gene enhancer, which represents the translocated form of the BCL-2 gene observed in follicular lymphomas with the t(14;18) translocation. The growth advantage of EBV-infected B cells overproducing the BCL-2 protein is neither due to the enhanced growth factor production nor due to an enhanced sensitivity of the BCL-2 transfectants to interleukins 1 or 6, although both lymphokines are known to stimulate proliferation of EBV-infected B-cell lines. The growth advantage of EBV-infected B-cell lines. The growth advantage of EBV-infected B cells by overproduction of the BCL-2 protein suggests the direct involvement of the BCL-2 gene product in the pathogenesis of follicular lymphoma

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

Directory of Open Access Journals (Sweden)

Pereira Francisco B

2011-12-01

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

Functional Reconstitution of a Fungal Natural Product Gene Cluster by Advanced Genome Editing

DEFF Research Database (Denmark)

Weber, Jakob; Valiante, Vito; Nødvig, Christina Spuur

2017-01-01

is not produced among different isolates. Combining computational analysis with targeted gene editing, we could link a single nucleotide insertion in the polyketide synthase of the trypacidin biosynthetic pathway and reconstitute its production in a nonproducing strain. Thus, we present a CRISPR/Cas9-based tool...... for advanced molecular genetic studies in filamentous fungi, exploiting selectable markers separated from the edited locus....

Study on isolation, molecular detection of virulence gene and antibiotic sensitivity pattern of Escherichia coli isolated from milk and milk products

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M. N. Brahmbhatt

2013-06-01

Full Text Available Aim: The study was undertaken to isolate pathogenic E. coli from milk and various milk products, detection of virulence gene using Polymerase chain reaction (PCR and investigate their antibiotic sensitivity pattern. Materials and Methods: Altogether 250 milk and various milk products samples consisting of raw milk (50, cheese (50, ice-cream (50, mawa (50 and dahi (50 were collected from milk vendors, retail shops located in Anand city, under aseptic precautions. For the enrichment of the organism from the collected samples, MacConkey broth was used and inoculation was carried out on MacConkey agar and EMB agar. Later on, to confirm the isolates, various biochemical tests such as IMViC test, Urease test were performed. Evaluation of antibiotic sensitivity pattern of E. coli was assessed by disk diffusion method. Finally the E. coli isolates were screened for the presence of virulence associated genes by PCR . Results: The prevalence of E. coli was observed 32 % in the samples comprising of milk (52.00%, cheese (28.00%, icecream (20.00%, mawa (44.00%, and dahi (16.00%. Antibiotic sensitivity was recorded high for Co-trimoxazole (100% followed by Gentamicin (96.73%, Trimithoprime (93.47% and Doxycycline hydochloride (92.39%. Least sensitivity was recorded for Ampicillin (8.69%. In this study, out of 80 E. coli isolates, 25 isolates (31.25% were positive for stx genes, of which 7 (8.75% isolates were positive for stx1 gene only, while 12 (15.00% isolates were positive for stx2 gene only and 5 (6.25% isolates were positive for both stx1 and stx2, 7 isolates (8.75% were positive for eaeA gene and all the isolate were negetive for rfb O157 gene. Conclusions: Current study supports the finding that raw milk and various milk products can be regarded as critical source of pathogenic E. coli This explains the need of strict monitoring and surveillance for effective measures of hygiene and sanitary practice during production of milk and various milk

Sulfur source-mediated transcriptional regulation of the rhlABC genes involved in biosurfactants production by Pseudomonas sp. strain AK6U.

Science.gov (United States)

Ismail, Wael; El Nayal, Ashraf M; Ramadan, Ahmed R; Abotalib, Nasser

2014-01-01

Despite the nutritional significance of sulfur, its influence on biosurfactants production has not been sufficiently studied. We investigated the expression of key biosurfactants production genes, rhlABC, in cultures of Pseudomonas sp. AK6U grown with inorganic or organic sulfur sources. AK6U grew with either inorganic sulfate (MgSO4), dibenzothiophene (DBT), or DBT-sulfone as a sole sulfur source in the presence of glucose as a carbon source. The AK6U cultures produced variable amounts of biosurfactants depending on the utilized sulfur source. Biosurfactants production profile of the DBT cultures was significantly different from that of the DBT-sulfone and inorganic sulfate cultures. The last two cultures were very similar in terms of biosurfactants productivity. Biosurfactants yield in the DBT cultures (1.3 g/L) was higher than that produced by the DBT-sulfone (0.5 g/L) and the inorganic sulfate (0.44 g/L) cultures. Moreover, the surface tension reduction in the DBT cultures (33 mN/m) was much stronger than that measured in the DBT-sulfone (58 mN/m) or inorganic sulfate (54 mN/m) cultures. RT-qPCR revealed variations in the expression levels of the rhlABC genes depending on the sulfur source. The DBT cultures had higher expression levels for the three genes as compared to the DBT-sulfone and inorganic sulfate cultures. There was no significant difference in the expression profiles between the DBT-sulfone and the MgSO4 cultures. The increased expression of rhlC in the DBT cultures is indicative for production of higher amounts of dirhamnolipids compared to the DBT-sulfone and inorganic sulfate cultures. The gene expression results were in good agreement with the biosurfactants production yields and surface tension measurements. The sulfur source mediates a fine-tuned mechanism of transcriptional regulation of biosurfactants production genes. Our findings can have an impact on industrial production of biosurfactants and other biotechnological processes like

DnaB gene product-independence of DNA polymerase III-directed repair synthesis in Escherichia coli K-12

International Nuclear Information System (INIS)

Billen, D.; Hellermann, G.R.

1977-01-01

An investigation has been carried out into the role of dnaB gene product in X-ray-induced repair synthesis carried out by DNA polymerase III in toluene-treated Escherichia coli K-12. A polAl polBlOO dnaB mutant deficient in both DNA polymerase I and II activities was used, and it was shown that the level of X-ray-induced, ATP-dependent, non-conservative DNA synthesis was, unlike semi-conservative DNA synthesis, unaffected by a temperature shift from 30 0 to 42 0 C. The dnaB gene product was not therefore necessary for DNA polymerase III-directed repair synthesis, which occurred in the absence of replicative synthesis. (U.K.)

Relationship between fumonisin production and FUM gene expression in Fusarium verticillioides under different environmental conditions.

Science.gov (United States)

Fanelli, Francesca; Iversen, Anita; Logrieco, Antonio F; Mulè, Giuseppina

2013-01-01

Fusarium verticillioides is the main source of fumonisins, a group of mycotoxins that can contaminate maize-based food and feed and cause diseases in humans and animals. The study of the effect of different environmental conditions on toxin production should provide information that can be used to develop strategies to minimize the risk. This study analysed the effect of temperature (15°C-35°C), water activity (a(w): 0.999-0.93), salinity (0-125 g l(-1) NaCl) and pH (5-8) on the growth and production of fumonisins B(1) (FB1), B(2) (FB2) and B(3) (FB3) and the expression of FUM1 and FUM21 in F. verticillioides. The highest growth rate was measured at 25°C, a(w) of 0.998-0.99 and 0-25 g l(-1) of NaCl. Optimal conditions for fumonisin production were 30°C, a(w) of 0.99, 25 g l(-1) of NaCl and pH 5; nevertheless, the strain showed a good adaptability and was able to produce moderate levels of fumonisins under a wide range of conditions. Gene expression mirrored fumonisin production profile under all conditions with the exception of temperature: FUM1 and FUM21 expression was highest at 15°C, while maximum fumonisin production was at 30°C. These data indicate that a post-transcriptional regulation mechanism could account for the different optimal temperatures for FUM gene expression and fumonisin production.

Polymorphisms of POU1F1 and STAT5A genes and their associate on with milk production traits in cattle

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Sonia Zakizadeh

2015-04-01

Full Text Available Specific trait candidate genes are sequenced genes with known biological activity. The effects of POU1F1 and STAT5A on milk production traits have been studied in several studies. POU1F1 affects on transcription of prolactin and growth hormone gene, as well as, STAT5A is known as a main mediator of growth hormone action on target genes and intracellular mediator of prolactin signaling. Since these genes are essential for development of mammary system, the aim of this study was to determine association of their polymorphism with milk production breeding values in Brown Swiss cattle. Blood of ninety milking cow were randomly obtained. DNA was extracted from whole blood using modified salting out method, then the desired fragments were PCR amplified and digested by specific restriction endonuclease enzymes. Gene and genotype frequencies, heterozygosity indexes, the real and effective allele number were calculated by PopGene software; and the breeding values of production traits were estimated by DFREML. SAS software was used to analyze association between genotypes and breeding values. The frequency of 'A' and 'C' alleles of POU1F1 and STAT5A were 0.455 and 0.489, respectively. This population was in hardy-weinburg equilibrium for both loci. There was no significant association between genotypes and breeding values, although POU1F1*B tended to produce higher milk and POU1F1*A showed higher fat and protein percent.

A systems biology approach to investigate the effect of pH-induced gene regulation on solvent production by Clostridium acetobutylicum in continuous culture

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Bahl Hubert

2011-01-01

Full Text Available Abstract Background Clostridium acetobutylicum is an anaerobic bacterium which is known for its solvent-producing capabilities, namely regarding the bulk chemicals acetone and butanol, the latter being a highly efficient biofuel. For butanol production by C. acetobutylicum to be optimized and exploited on an industrial scale, the effect of pH-induced gene regulation on solvent production by C. acetobutylicum in continuous culture must be understood as fully as possible. Results We present an ordinary differential equation model combining the metabolic network governing solvent production with regulation at the genetic level of the enzymes required for this process. Parameterizing the model with experimental data from continuous culture, we demonstrate the influence of pH upon fermentation products: at high pH (pH 5.7 acids are the dominant product while at low pH (pH 4.5 this switches to solvents. Through steady-state analyses of the model we focus our investigations on how alteration in gene expression of C. acetobutylicum could be exploited to increase butanol yield in a continuous culture fermentation. Conclusions Incorporating gene regulation into the model of solvent production by C. acetobutylicum enables an accurate representation of the pH-induced switch to solvent production to be obtained and theoretical investigations of possible synthetic-biology approaches to be pursued. Steady-state analyses suggest that, to increase butanol yield, alterations in the expression of single solvent-associated genes are insufficient; a more complex approach targeting two or more genes is required.

[Reconstruction of Leptospira interrogans lipL21 gene and characteristics of its expression product].

Science.gov (United States)

Luo, Dong-jiao; Hu, Ye; Dennin, R H; Yan, Jie

2007-09-01

To reconstruct the nucleotide sequence of Leptospira interrogans lipL21 gene for increasing the output of prokaryotic expression and to understand the changes on immunogenicity of the expression products before and after reconstruction, and to determine the position of envelope lipoprotein LipL21 on the surface of leptospiral body. According to the preferred codons of E.coli, the nucleotide sequence of lipL21 gene was designed and synthesized, and then its prokaryotic expression system was constructed. By using SDS-PAGE plus BioRad agarose image analysor, the expression level changes of lipL21 genes before and after reconstruction were measured. A Western blot assay using rabbit anti-TR/Patoc I serum as the first antibody was performed to identify the immunoreactivity of the two target recombinant proteins rLipL21s before and after reconstruction. The changes of cross agglutination titers of antisera against two rLipL21s before and after reconstruction to the different leptospiral serogroups were demonstrated using microscope agglutination test (MAT). Immuno-electronmicroscopy was applied to confirm the location of LipL21s. The expression outputs of original and reconstructed lipL21 genes were 8.5 % and 46.5 % of the total bacterial proteins, respectively. Both the two rLipL21s could take place immune conjugation reaction with TR/Patoc I antiserum. After immunization with each of the two rLipL21s in rabbits, the animals could produce specific antibody. Similar MAT titers with 1:80 - 1:320 of the two antisera against rLipL21s were present. LipL21 was confirmed to locate on the surface of leptospiral envelope. LipL21 is a superficial antigen of Leptospira interrogans. The expression output of the reconstructed lipL21 gene is remarkably increased. The expression rLipL21 maintains fine antigenicity and immunoreactivity and its antibody still shows an extensive cross immunoagglutination activity. The high expression of the reconstructed lipL21 gene will offer a

Transcriptional regulation of genes involved in terpenoid índole alkaloid production in Catharanthus roseus seedlings

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Pedro J. Rocha

2002-07-01

Full Text Available Catharanthus roseus (L. G Don is a medicinal plant that produces a variety of terpenoid indole alkaloids (TIAs, some of which display pharmacological activity. C. roseus plants and cell cultures have been used to elucidate the TIAs biosynthetic pathway. A considerable number or enzymes have also been characterised, and their respective genes cloned. TIAs production in C. roseus plant and cell cultures is highly regulated at transcriptional-, develop-mental-, and environmental-level. Studies into TIAs biosynthetic gene regulation have been carried out using cell cultures. However, regulation in plants is almost unknown. Here, biosynthetic genes idc, strl, d4h and dat expres-sion levels are qualitatively examined in a developmental series of C. roseus seedlings. The effect of water- and light-stress and methyl jasmonate (MeJa and acetyl salicylic acid (ASA elicitation is also examined. Comparison between seedlings and cell cultures strongly suggests that TIAs biosynthetic gene transcriptional regulation is different in C.roseus plants and cell cultures.

Direct capture and heterologous expression of Salinispora natural product genes for the biosynthesis of enterocin.

Science.gov (United States)

Bonet, Bailey; Teufel, Robin; Crüsemann, Max; Ziemert, Nadine; Moore, Bradley S

2015-03-27

Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora's promising secondary metabolome.

Effects of Copper on Hemocyte Apoptosis, ROS Production, and Gene Expression in White Shrimp Litopenaeus vannamei.

Science.gov (United States)

Guo, Hui; Li, Kexu; Wang, Wei; Wang, Chenggui; Shen, Yuchun

2017-10-01

Copper, a common chemical contaminant in aquatic environment, is known to be toxic to aquatic life at high concentrations. In the present study, we evaluated the apoptotic cell ratio and ROS production in hemocytes of the white shrimp Litopenaeus vannamei exposed to 1 or 5 mg L -1 Cu for 0, 3, 6, 12, 24, and 48 h. The expression changes of antioxidant biomarker genes, i.e., copper-zinc superoxide dismutase (Cu-Zn SOD) and catalase (CAT), apoptosis-related genes, i.e., caspase-3 and inhibitor of apoptosis protein (IAP), and a specific biomarker gene of heavy metal pollution, i.e., metallothionein (MT), were also determined in hemocytes. Significant increases in ROS production were observed in both treatment groups at each time points. The apoptotic cell ratios were significantly increased at 6-48 h among shrimp exposed to 1 mg L -1 Cu and at each time points in 5 mg L -1 Cu group. These results indicated that Cu would induce oxidative stress and apoptosis in the hemocyte of L. vannamei. Quantitative real-time PCR analysis revealed that the relative expression levels of Cu-Zn SOD, CAT, caspase-3, IAP, and MT were upregulated in a dose-dependent and time-dependent manner, suggesting the involvement of these genes in stress response against Cu exposure.

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

DEFF Research Database (Denmark)

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

2015-01-01

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

Detection of coding genes for enterotoxins in Bacillus cereus by PCR and their products by BCET-RPLA and ELISA Assay

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Marcela Vyletělová

2010-01-01

Full Text Available Determination of enterotoxin production, diarrhoeal and emetic gene identification was studied in 41 Bacillus cereus strains isolated from raw cows’ and raw goats’ milk, pasteurized milk, dairy products during technological processing and from dairy plant equipment. Presence of enterotoxins was detected by BCET-RPLA (HBL and ELISA immunoassay (NHE. Gene identification (nheA, nheB, nheC, hblA, hblC, hblD, bceT, cytK-1, cytK-2, entFM and ces was achieved by means of PCR. Enterotoxin HBL was detected in 32 strains, enterotoxin NHE in all 41 strains. Presence of all three genes nheA, nheB and nheC was confirmed in 40 strains and genes hblA, hblC and hblD in 29 strains. Comparison of used methods was as follow: 1 BCET-RPLA (which detects L2 component and PCR (positive or negative all three hblA, hblC and hblD gene detection were identical in 30 (73%; 2 ELISA (NheA and PCR (all three nheC, nheB and nheA gene expression were identical in 40 (98% cases isolated strains.

Light quality affects flavonoid production and related gene expression in Cyclocarya paliurus.

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Liu, Yang; Fang, Shengzuo; Yang, Wanxia; Shang, Xulan; Fu, Xiangxiang

2018-02-01

Understanding the responses of plant growth and secondary metabolites to differential light conditions is very important to optimize cultivation conditions of medicinal woody plants. As a highly valued and multiple function tree species, Cyclocarya paliurus is planted and managed for timber production and medical use. In this study, LED-based light including white light (WL), blue light (BL), red light (RL), and green light (GL) were used to affect leaf biomass production, flavonoid accumulation and related gene expression of one-year C. paliurus seedlings in controlled environments. After the treatments of 60 days, the highest leaf biomass appeared in the treatment of WL, while the lowest leaf biomass was found under GL. Compared to WL, the total flavonoid contents of C. paliurus leaves were significantly higher in BL, RL, and GL, but the highest values of selected flavonoids (kaempferol, isoquercitrin and quercetin) were observed under BL. Furthermore, the greatest yields of total and selected flavonoids in C. paliurus leaves per seedling were also achieved under BL, indicating that blue light was effective for inducing the production of flavonoids in C. paliurus leaves. Pearson's correlation analysis showed that there were significantly positive correlations between leaf flavonoid content and relative gene expression of key enzymes (phenylalanine ammonia lyase, PAL; 4-coumaroyl CoA-ligase, 4CL; and chalcone synthase, CHS) in the upstream, which converting phenylalanine into the flavonoid skeleton of tetrahydroxy chalcone. It is concluded that manipulating light quality may be potential mean to achieve the highest yields of flavonoids in C. paliurus cultivation, however this needs to be further verified by more field trials. Copyright © 2018 Elsevier B.V. All rights reserved.

Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant.

Science.gov (United States)

Tamano, Koichi; Miura, Ai

2016-09-01

Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression.

Genes encoding novel lipid transporters and their use to increase oil production in vegetative tissues of plants

Science.gov (United States)

Xu, Changcheng; Fan, Jilian; Yan, Chengshi; Shanklin, John

2017-12-26

The present invention discloses a novel gene encoding a transporter protein trigalactosyldiacylglycerol-5 (TGD5), mutations thereof and their use to enhance TAG production and retention in plant vegetative tissue.

Impacts of Macronutrients on Gene Expression: Recent Evidence to Understand Productive and Reproductive Performance of Livestock

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Md. Mahmodul Hasan Sohel

2018-03-01

Full Text Available In order to identify the effects of nutrients on gene expression and to assess the interactions between genes and nutrition by means of various cutting-edge technologies, the interdisciplinary branch ‘Nutrigenomics’ was created. Therefore, nutrigenomics corresponds to the use of knowledge and techniques of nutrition, genomics, transcriptomics, proteomics, epigenomics, and metabolomics to seek and explain the cross-talk between nutrition and genes in molecular level. Macronutrients are important dietary signals that control metabolic programming of cells and have important roles in maintaining cellular homeostasis by influencing specific gene expression. Recent advancements in molecular genetics studies, for instance, use of next-generation sequencing, microarray and qPCR array to investigate the expression of transcripts, genes, and miRNAs, has a crucial impact on understanding and quantitative measurement of the impact of dietary macronutrients on gene function. This review will shade a light on the interactions and mechanisms how the dietary source of macronutrients changes the expression of specific mRNA and miRNA. Furthermore, it will highlight the exciting recent findings in relation to animal performance characteristics which eventually help us to identify a dietary target to improve animal production.

The product of the ABC half-transporter gene ABCG2 (BCRP/MXR/ABCP) is expressed in the plasma membrane

DEFF Research Database (Denmark)

Rocchi, E; Khodjakov, A; Volk, E L

2000-01-01

by Western blot and immunohistochemistry. This protein is highly overexpressed in several drug-resistant cell lines and localizes predominantly to the plasma membrane, instead of to intracellular membranes as seen with all other known half-transporters. Therefore, BCRP/MXR is unique among the ABC half......The products of the ABC gene family can be generally classified as either full-transporters of half-transporters. Full-transporters are expressed in the plasma membrane, whereas half-transporters are usually found in intracellular membranes. Recently, an ABC half-transporter, the ABCG2 gene product......-transporters by being localized to the plasma membrane....

Isolation of Penicillium nalgiovense strains impaired in penicillin production by disruption of the pcbAB gene and application as starters on cured meat products.

Science.gov (United States)

Laich, Federico; Fierro, Francisco; Martin, Juan F

2003-06-01

The presence of some fungi on a variety of food products, like cheeses or cured meat products, is beneficial for the ripening of the product and for the development of specific flavour features. The utilization of these fungi as starters, which are inoculated normally as asexual spores on the food products at the beginning of the ripening process, is becoming a usual procedure in the food industry. The starter culture also prevents undesirable fungi or bacteria from growing on the product. Penicillium nalgiovense is the most frequently used starter for cured and fermented meat products, but the fact that this fungus can secrete penicillin to the meat product makes it important to get strains unable to synthesize this antibiotic. In this work we report that P. nalgiovense strains impaired in penicillin production can be obtained by disruption of the pcbAB gene (the first gene of the penicillin biosynthetic pathway). When applied as starter on cecina (a salted, smoke-cured beef meat product from the region of León, Spain), the pcbAB-disrupted strain showed no differences with respect to the parental penicillin-producing strain in its ability to colonize the meat pieces and to control their normal mycoflora. Both strains exerted a similar control on the presence of bacteria in cecina. A similar proportion of penicillin-sensitive and penicillin-resistant bacteria were isolated from pieces inoculated with the penicillin-producing or the non-producing P. nalgiovense strains. The decrease of the bacterial population on the surface of cecina seems to be due to the higher competition for nutrients as a consequence of the inoculation and development of the P. nalgiovense mycelium and not due to the production of penicillin by this fungus. Penicillin production was less affected than growth in a solid medium with high NaCl concentrations; this suggests that the high salt concentration present in cecina is not a limiting factor for penicillin production by P. nalgiovense.

Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

Energy Technology Data Exchange (ETDEWEB)

Tennyson, C.N.; Worton, R.G. [Univ. of Toronto and the Hospital for Sick Children, Ontario (Canada)

1994-09-01

The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

Gene Delivery into Plant Cells for Recombinant Protein Production

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Qiang Chen

2015-01-01

Full Text Available Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

Advanced Glycation End-Products affect transcription factors regulating insulin gene expression

International Nuclear Information System (INIS)

Puddu, A.; Storace, D.; Odetti, P.; Viviani, G.L.

2010-01-01

Advanced Glycation End-Products (AGEs) are generated by the covalent interaction of reducing sugars with proteins, lipids or nucleic acids. AGEs are implicated in diabetic complications and pancreatic β-cell dysfunction. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs leads to a significant decrease of insulin secretion and content. Insulin gene transcription is positively regulated by the beta cell specific transcription factor PDX-1 (Pancreatic and Duodenal Homeobox-1). On the contrary, the forkhead transcription factor FoxO1 inhibits PDX-1 gene transcription. Activity of FoxO1 is regulated by post-translational modifications: phosphorylation deactivates FoxO1, and acetylation prevents FoxO1 ubiquitination. In this work we investigated whether AGEs affect expression and subcellular localization of PDX-1 and FoxO1. HIT-T15 cells were cultured for 5 days in presence of AGEs. Cells were then lysed and processed for subcellular fractionation. We determined intracellular insulin content, then we assessed the expression and subcellular localization of PDX-1, FoxO1, phosphoFoxO1 and acetylFoxO1. As expected intracellular insulin content was lower in HIT-T15 cells cultured with AGEs. The results showed that AGEs decreased expression and nuclear localization of PDX-1, reduced phosphorylation of FoxO1, and increased expression and acetylation of FoxO1. These results suggest that AGEs decrease insulin content unbalancing transcription factors regulating insulin gene expression.

Noise minimization in eukaryotic gene expression.

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Hunter B Fraser

2004-06-01

Full Text Available All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or "noise." Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

Noise minimization in eukaryotic gene expression

Energy Technology Data Exchange (ETDEWEB)

Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.

2004-01-15

All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

Noise minimization in eukaryotic gene expression

International Nuclear Information System (INIS)

Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.

2004-01-01

All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection

Methanogenic Paraffin Biodegradation: Alkylsuccinate Synthase Gene Quantification and Dicarboxylic Acid Production.

Science.gov (United States)

Oberding, Lisa K; Gieg, Lisa M

2018-01-01

Paraffinic n -alkanes (>C 17 ) that are solid at ambient temperature comprise a large fraction of many crude oils. The comparatively low water solubility and reactivity of these long-chain alkanes can lead to their persistence in the environment following fuel spills and pose serious problems for crude oil recovery operations by clogging oil production wells. However, the degradation of waxy paraffins under the anoxic conditions characterizing contaminated groundwater environments and deep subsurface energy reservoirs is poorly understood. Here, we assessed the ability of a methanogenic culture enriched from freshwater fuel-contaminated aquifer sediments to biodegrade the model paraffin n -octacosane (C 28 H 58 ). Compared with that in controls, the consumption of n -octacosane was coupled to methane production, demonstrating its biodegradation under these conditions. Smithella was postulated to be an important C 28 H 58 degrader in the culture on the basis of its high relative abundance as determined by 16S rRNA gene sequencing. An identified assA gene (known to encode the α subunit of alkylsuccinate synthase) aligned most closely with those from other Smithella organisms. Quantitative PCR (qPCR) and reverse transcription qPCR assays for assA demonstrated significant increases in the abundance and expression of this gene in C 28 H 58 -degrading cultures compared with that in controls, suggesting n -octacosane activation by fumarate addition. A metabolite analysis revealed the presence of several long-chain α,ω-dicarboxylic acids only in the C 28 H 58 -degrading cultures, a novel observation providing clues as to how methanogenic consortia access waxy hydrocarbons. The results of this study broaden our understanding of how waxy paraffins can be biodegraded in anoxic environments with an application toward bioremediation and improved oil recovery. IMPORTANCE Understanding the methanogenic biodegradation of different classes of hydrocarbons has important

Role of nitric oxide and flavohemoglobin homolog genes in Aspergillus nidulans sexual development and mycotoxin production

Science.gov (United States)

Flavohemoglobins are widely distributed proteins in both prokaryotic and eukaryotic organisms, conferring resistance against nitrosative stress. In the present study we investigated the role of two flavohemoglobin homologous genes, fhbA and fhbB, in morphogenesis and in the production of the mycotox...

Improvement of iturin A production in Bacillus subtilis ZK0 by overexpression of the comA and sigA genes.

Science.gov (United States)

Zhang, Z; Ding, Z T; Zhong, J; Zhou, J Y; Shu, D; Luo, D; Yang, J; Tan, H

2017-06-01

Bacillus subtilis ZK0, which was isolated from cotton, produces a type of lipopeptide antibiotic iturin A that inhibits the growth of pathogenic fungi on agricultural crops. However, the low level of iturin A production by B. subtilis ZK0 does not support its large-scale application. In this study, B. subtilis ZK0 was subjected to genetic manipulation to improve iturin A production. By the independent or simultaneous overexpression of two regulatory genes (comA and sigA), iturin A production by B. subtilis ZK0 was significantly increased. When both genes were simultaneously overexpressed under optimal conditions, iturin A production increased up to 215 mg l -1 (an approximate 43-fold increase compared with B. subtilis ZK0). Moreover, overexpression of both genes was unexpectedly found to inhibit biofilm formation by B. subtilis ZK0, indicating that the phenomenon of 'stuck fermentation' would be avoided during B. subtilis ZK0 fermentation. In conclusion, a genetic manipulation method that improves iturin A production and inhibits biofilm formation in B. subtilis ZK0 is reported for the first time and this method has the potential to be widely applied in B. subtilis ZK0 commercial fermentation. This study provides new perspectives on improving iturin A production by Bacillus subtilis. Our newly engineered strains could be applied to commercial fermentation by enhancing yields of iturin A and reducing the rate of 'stuck fermentation'. Increased production would facilitate more widespread application of this powerful antibiotic. © 2017 The Society for Applied Microbiology.

Global gene expression in muscle from fasted/refed trout reveals up-regulation of genes promoting myofibre hypertrophy but not myofibre production.

Science.gov (United States)

Rescan, Pierre-Yves; Le Cam, Aurelie; Rallière, Cécile; Montfort, Jérôme

2017-06-07

Compensatory growth is a phase of rapid growth, greater than the growth rate of control animals, that occurs after a period of growth-stunting conditions. Fish show a capacity for compensatory growth after alleviation of dietary restriction, but the underlying cellular mechanisms are unknown. To learn more about the contribution of genes regulating hypertrophy (an increase in muscle fibre size) and hyperplasia (the generation of new muscle fibres) in the compensatory muscle growth response in fish, we used high-density microarray analysis to investigate the global gene expression in muscle of trout during a fasting-refeeding schedule and in muscle of control-fed trout displaying normal growth. The compensatory muscle growth signature, as defined by genes up-regulated in muscles of refed trout compared with control-fed trout, showed enrichment in functional categories related to protein biosynthesis and maturation, such as RNA processing, ribonucleoprotein complex biogenesis, ribosome biogenesis, translation and protein folding. This signature was also enriched in chromatin-remodelling factors of the protein arginine N-methyl transferase family. Unexpectedly, functional categories related to cell division and DNA replication were not inferred from the molecular signature of compensatory muscle growth, and this signature contained virtually none of the genes previously reported to be up-regulated in hyperplastic growth zones of the late trout embryo myotome and to potentially be involved in production of new myofibres, notably genes encoding myogenic regulatory factors, transmembrane receptors essential for myoblast fusion or myofibrillar proteins predominant in nascent myofibres. Genes promoting myofibre growth, but not myofibre formation, were up-regulated in muscles of refed trout compared with continually fed trout. This suggests that a compensatory muscle growth response, resulting from the stimulation of hypertrophy but not the stimulation of hyperplasia

Genes contributing to prion pathogenesis

DEFF Research Database (Denmark)

Tamgüney, Gültekin; Giles, Kurt; Glidden, David V

2008-01-01

incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes...... show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1...

Overexpression of the Squalene Epoxidase Gene Alone and in Combination with the 3-Hydroxy-3-methylglutaryl Coenzyme A Gene Increases Ganoderic Acid Production in Ganoderma lingzhi.

Science.gov (United States)

Zhang, De-Huai; Jiang, Lu-Xi; Li, Na; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

2017-06-14

The squalene epoxidase (SE) gene from the biosynthetic pathway of ganoderic acid (GA) was cloned and overexpressed in Ganoderma lingzhi. The strain that overexpressed the SE produced approximately 2 times more GA molecules than the wild-type (WT) strain. Moreover, SE overexpression upregulated lanosterol synthase gene expression in the biosynthetic pathway. These results indicated that SE stimulates GA accumulation. Then, the SE and 3-hydroxy-3-methylglutaryl coenzyme A (HMGR) genes were simultaneously overexpressed in G. lingzhi. Compared with the individual overexpression of SE or HMGR, the combined overexpression of the two genes further enhanced individual GA production. The overexpressing strain produced maximum GA-T, GA-S, GA-Mk, and GA-Me contents of 90.4 ± 7.5, 35.9 ± 5.4, 6.2 ± 0.5, and 61.8 ± 5.8 μg/100 mg dry weight, respectively. These values were 5.9, 4.5, 2.4, and 5.8 times higher than those produced by the WT strain. This is the first example of the successful manipulation of multiple biosynthetic genes to improve GA content in G. lingzhi.

Scarless and sequential gene modification in Pseudomonas using PCR product flanked by short homology regions

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Liang Rubing

2010-08-01

Full Text Available Abstract Background The lambda Red recombination system has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR fragment containing antibiotic cassette flanked by homology regions to the target locus into a strain that can express the lambda Red proteins (Gam, Bet, Exo. Results Here a scarless gene modification strategy based on the Red recombination system has been developed to modify Pseudomonas genome DNA via sequential deletion of multiple targets. This process was mediated by plasmid pRKaraRed encoding the Red proteins regulated by PBAD promoter, which was functional in P. aeruginosa as well as in other bacteria. First the target gene was substituted for the sacB-bla cassette flanked by short homology regions (50 bp, and then this marker gene cassette could be replaced by the PCR fragment flanking itself, generating target-deleted genome without any remnants and no change happened to the surrounding region. Twenty genes involved in the synthesis and regulation pathways of the phenazine derivate, pyocyanin, were modified, including one single-point mutation and deletion of two large operons. The recombination efficiencies ranged from 88% to 98%. Multiple-gene modification was also achieved, generating a triple-gene deletion strain PCA (PAO1, ΔphzHΔphzMΔphzS, which could produce another phenazine derivate, phenazine-1-carboxylic acid (PCA, efficiently and exclusively. Conclusions This lambda Red-based technique can be used to generate scarless and sequential gene modification mutants of P. aeruginosa efficiently, using one-step PCR product flanked by short homology regions. Single-point mutation, scarless deletion of genes can be achieved easily in less than three days. This method may give a new way to construct genetically modified P. aeruginosa strains more efficiently and advance the regulatory network study of this organism.

Associations of the variation in the porcine myogenin gene with muscle fibre characteristics, lean meat production and meat quality traits.

Science.gov (United States)

Kim, J M; Choi, B D; Kim, B C; Park, S S; Hong, K C

2009-04-01

Pig breeding is aimed at improving lean meat production ability as well as meat quality, and muscle fibre characteristics may be important for enhancing these traits. Therefore, new molecular markers have been demanded for selecting lean meat production ability and meat quality in live animals. Myogenin belongs to the MyoD gene family, and is a candidate gene responsible for muscle fibre characteristics. We identified a new single nucleotide polymorphism (SNP) site in the 5' upstream region of the myogenin gene (nucleotides C and T). A total of 252 pigs of three breeds were genotyped by polymerase chain reaction-restriction fragment length polymorphism using BspCNI. Additionally, they were genotyped for the previously detected MspI site in the 3'-flanking region (alleles A and B). The CCBB diplotype had the highest frequency over breeds, followed by TCBB and CCAB. The other diplotypes were not found in studied pigs. Association analysis performed for the markers found that the TCBB diplotype has desirable effects on the total number of fibres (p lean meat production ability with good meat quality.

Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism

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Karhumaa Kaisa

2011-07-01

Full Text Available Abstract Background Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric n-butanol. Saccharomyces cerevisiae was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials. Results The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in anaerobic fermentation of glucose in mineral medium in S. cerevisiae. Isobutanol yield was further improved by twofold by the additional overexpression of BAT2, encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of ILV6, encoding the regulatory subunit of Ilv2, in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the ILV2 ILV3 ILV5 overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD complex medium under aerobic conditions, respectively. Conclusions Overexpression of genes ILV2, ILV3, ILV5, and BAT2 in valine metabolism led to an increase in isobutanol production in S. cerevisiae. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of ILV2, ILV3, and ILV5 on isobutanol production. Aerobic cultivations of the ILV2 ILV3 ILV5 overexpression strain and the reference strain showed that supplying amino acids in cultivation media

Production of transgenic brassica juncea with the synthetic chitinase gene (nic) conferring resistance to alternaria brassicicola

International Nuclear Information System (INIS)

Munir, I.; Hussan, W.; Kazi, M.; Mian, A.

2016-01-01

Brassica juncea is an important oil seed crop throughout the world. The demand and cultivation of oil seed crops has gained importance due to rapid increase in world population and industrialization. Fungal diseases pose a great threat to Brassica productivity worldwide. Absence of resistance genes against fungal infection within crossable germplasms of this crop necessitates deployment of genetic engineering approaches to produce transgenic plants with resistance against fungal infections. In the current study, hypocotyls and cotyledons of Brassica juncea, used as explants, were transformed with Agrobacterium tumefacien strain EHA101 harboring binary vector pEKB/NIC containing synthetic chitinase gene (NIC), an antifungal gene under the control of cauliflower mosaic virus promoter (CaMV35S). Bar genes and nptII gene were used as selectable markers. Presence of chitinase gene in trangenic lines was confirmed by PCR and southern blotting analysis. Effect of the extracted proteins from non-transgenic and transgenic lines was observed on the growth of Alternaria brassicicola, a common disease causing pathogen in brassica crop. In comparison to non-transgenic control lines, the leaf tissue extracts of the transgenic lines showed considerable resistance and antifungal activity against A. brassicicola. The antifungal activity in transgenic lines was observed as corresponding to the transgene copy number. (author)

Subtle variation within conserved effector operon gene products contributes to T6SS-mediated killing and immunity.

Science.gov (United States)

Alteri, Christopher J; Himpsl, Stephanie D; Zhu, Kevin; Hershey, Haley L; Musili, Ninette; Miller, Jessa E; Mobley, Harry L T

2017-11-01

Type VI secretion systems (T6SS) function to deliver lethal payloads into target cells. Many studies have shown that protection against a single, lethal T6SS effector protein requires a cognate antidote immunity protein, both of which are often encoded together in a two-gene operon. The T6SS and an effector-immunity pair is sufficient for both killing and immunity. HereIn this paper we describe a T6SS effector operon that differs from conventional effector-immunity pairs in that eight genes are necessary for lethal effector function, yet can be countered by a single immunity protein. In this study, we investigated the role that the PefE T6SS immunity protein plays in recognition between two strains harboring nearly identical effector operons. Interestingly, despite containing seven of eight identical effector proteins, the less conserved immunity proteins only provided protection against their native effectors, suggesting that specificity and recognition could be dependent on variation within an immunity protein and one effector gene product. The variable effector gene product, PefD, is encoded upstream from pefE, and displays toxic activity that can be countered by PefE independent of T6SS-activity. Interestingly, while the entire pef operon was necessary to exert toxic activity via the T6SS in P. mirabilis, production of PefD and PefE alone was unable to exert this effector activity. Chimeric PefE proteins constructed from two P. mirabilis strains were used to localize immunity function to three amino acids. A promiscuous immunity protein was created using site-directed mutagenesis to change these residues from one variant to another. These findings support the notion that subtle differences between conserved effectors are sufficient for T6SS-mediated kin discrimination and that PefD requires additional factors to function as a T6SS-dependent effector.

The transport of antibiotic resistance genes and residues in groundwater near swine production facilities

Science.gov (United States)

Lin, Y. F.; Yannarell, A. C.; Mackie, R. I.; Krapac, I. G.; Chee-Sanford, J. S.; Koike, S.

2008-12-01

The use of antibiotics at concentrated animal feeding operations (CAFOs) for disease prevention, disease treatment, and growth promotion can contribute to the spread of antibiotic compounds, their breakdown products, and antibiotic resistant bacteria and/or the genes that confer resistance. In addition, constitutive use of antibiotics at sub-therapeutic levels can select for antibiotic resistance among the bacteria that inhabit animal intestinal tracts, onsite manure treatment facilities, and any environments receiving significant inputs of manure (e.g. through waste lagoon leakage or fertilizer amendments to farm soils). If the antibiotic resistant organisms persist in these new environments, or if they participate in genetic exchanges with the native microflora, then CAFOs may constitute a significant reservoir for the spread of antibiotic resistance to the environment at large. Our results have demonstrated that leakage from waste treatment lagoons can influence the presence and persistence of tetracycline resistance genes in the shallow aquifer adjacent to swine CAFOs, and molecular phylogeny allowed us to distinguish "native" tetracycline resistance genes in control groundwater wells from manure-associated genes introduced from the lagoon. We have also been able to detect the presence of erythromycin resistance genes in CAFO surface and groundwater even though erythromycin is strictly reserved for use in humans and thus is not utilized at any of these sites. Ongoing research, including modeling of particle transport in groundwater, will help to determine the potential spatial and temporal extent of CAFO-derived antibiotic resistance.

Identification of immediate early gene products of bovine herpes virus 1 (BHV-1) as dominant antigens recognized by CD8 T cells in immune cattle

DEFF Research Database (Denmark)

Hart, Jane; MacHugh, Niall D.; Sheldrake, Tara

2017-01-01

candidate viral gene products with CD8 T-cell lines from 3 BHV-1-immune cattle of defined MHC genotypes identified 4 antigens, including 3 immediate early (IE) gene products (ICP4, ICP22 and Circ) and a tegument protein (UL49). Identification of the MHC restriction specificities revealed that the antigens...... cases refined, the identity of the epitopes. Analyses of the epitope specificity of the CD8 T-cell lines showed that a large component of the response is directed against these IE epitopes. The results indicate that these IE gene products are dominant targets of the CD8 T-cell response in BHV...

The Cyclic Di-GMP Phosphodiesterase Gene Rv1357c/BCG1419c Affects BCG Pellicle Production and In Vivo Maintenance.

Science.gov (United States)

Flores-Valdez, Mario Alberto; Aceves-Sánchez, Michel de Jesús; Pedroza-Roldán, César; Vega-Domínguez, Perla Jazmín; Prado-Montes de Oca, Ernesto; Bravo-Madrigal, Jorge; Laval, Françoise; Daffé, Mamadou; Koestler, Ben; Waters, Christopher M

2015-02-01

Bacteria living in a surface-attached community that contains a heterogeneous population, coated with an extracellular matrix, and showing drug tolerance (biofilms) are often linked to chronic infections. In mycobacteria, the pellicle mode of growth has been equated to an in vitro biofilm and meets several of the criteria mentioned above, while tuberculosis infection presents a chronic (latent) phase of infection. As mycobacteria lack most genes required to control biofilm production by other microorganisms, we deleted or expressed from the hsp60 strong promoter the only known c-di-GMP phosphodiesterase (PDE) gene in Mycobacterium bovis BCG. We found changes in pellicle production, cellular protein profiles, lipid production, resistance to nitrosative stress and maintenance in lungs and spleens of immunocompetent BALB/mice. Our results show that pellicle production and capacity to remain within the host are linked in BCG. © 2015 International Union of Biochemistry and Molecular Biology.

Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco.

Science.gov (United States)

Ji, Chang Yoon; Kim, Yun-Hee; Kim, Ho Soo; Ke, Qingbo; Kim, Gun-Woo; Park, Sung-Chul; Lee, Haeng-Soon; Jeong, Jae Cheol; Kwak, Sang-Soo

2016-09-01

Tocopherol (vitamin E) is a chloroplast lipid that is presumed to be involved in the plant response to oxidative stress. In this study, we isolated and characterized five tocopherol biosynthetic genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including genes encoding 4-hydroxyphenylpyruvate dioxygenase (IbHPPD), homogentisate phytyltransferase (IbHPT), 2-methyl-6-phytylbenzoquinol methyltransferase (IbMPBQ MT), tocopherol cyclase (IbTC) and γ-tocopherol methyltransferase (IbTMT). Fluorescence microscope analysis indicated that four proteins localized into the chloroplast, whereas IbHPPD observed in the nuclear. Quantitative RT-PCR analysis revealed that the expression patterns of the five tocopherol biosynthetic genes varied in different plant tissues and under different stress conditions. All five genes were highly expressed in leaf tissues, whereas IbHPPD and IbHPT were highly expressed in the thick roots. The expression patterns of these five genes significantly differed in response to PEG, NaCl and H2O2-mediated oxidative stress. IbHPPD was strongly induced following PEG and H2O2 treatment and IbHPT was strongly induced following PEG treatment, whereas IbMPBQ MT and IbTC were highly expressed following NaCl treatment. Upon infection of the bacterial pathogen Pectobacterium chrysanthemi, the expression of IbHPPD increased sharply in sweetpotato leaves, whereas the expression of the other genes was reduced or unchanged. Additionally, transient expression of the five tocopherol biosynthetic genes in tobacco (Nicotiana bentamiana) leaves resulted in increased transcript levels of the transgenes expressions and tocopherol production. Therefore, our results suggested that the five tocopherol biosynthetic genes of sweetpotato play roles in the stress defense response as transcriptional regulators of the tocopherol production. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Enhancement of malate-production and increase in sensitivity to dimethyl succinate by mutation of the VID24 gene in Saccharomyces cerevisiae.

Science.gov (United States)

Negoro, Hiroaki; Kotaka, Atsushi; Matsumura, Kengo; Tsutsumi, Hiroko; Hata, Yoji

2016-06-01

Malate in sake (a Japanese alcoholic beverage) is an important component for taste that is produced by yeasts during alcoholic fermentation. To date, many researchers have developed methods for breeding high-malate-producing yeasts; however, genes responsible for the high-acidity phenotype are not known. We determined the mutated gene involved in high malate production in yeast, isolated as a sensitive mutant to dimethyl succinate. In the comparative whole genome analysis between high-malate-producing strain and its parent strain, one of the non-synonymous substitutions was identified in the VID24 gene. The mutation of VID24 resulted in enhancement of malate-productivity and sensitivity to dimethyl succinate. The mutation appeared to lead to a deficiency in Vid24p function. Furthermore, disruption of cytoplasmic malate dehydrogenase (Mdh2p) gene in the VID24 mutant inhibited the high-malate-producing phenotype. Vid24p is known as a component of the multisubunit ubiquitin ligase and participates in the degradation of gluconeogenic enzymes such as Mdh2p. We suggest that the enhancement of malate-productivity results from an accumulation of Mdh2p due to the loss of Vid24p function. These findings propose a novel mechanism for the regulation of organic acid production in yeast cells by the component of ubiquitin ligase, Vid24p. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Occurrence of Extended-Spectrum β-Lactamases, Plasmid-Mediated Quinolone Resistance, and Disinfectant Resistance Genes in Escherichia coli Isolated from Ready-To-Eat Meat Products

DEFF Research Database (Denmark)

Li, Lili; Ye, Lei; Kromann, Sofie

2017-01-01

There are growing concerns about the coselection of resistance against antibiotics and disinfectants in bacterial pathogens. The aim of this study was to characterize the antimicrobial susceptibility profiles, the prevalence of extended-spectrum β-lactamases (ESBLs), plasmid-mediated quinolone...... resistance genes (PMQRs), and quaternary ammonium compound resistance genes (QACs) in Escherichia coli isolated from ready-to-eat (RTE) meat products obtained in Guangzhou, China, and to determine whether these genes were colocalized in the isolates. A total of 64 E. coli isolates were obtained from 720 RTE...... isolates from RTE meat products. The E. coli isolates with multiple antimicrobial resistance genes may transmit to humans through food chain and thus require further investigation and increased awareness....

MitoRes: a resource of nuclear-encoded mitochondrial genes and their products in Metazoa.

Science.gov (United States)

Catalano, Domenico; Licciulli, Flavio; Turi, Antonio; Grillo, Giorgio; Saccone, Cecilia; D'Elia, Domenica

2006-01-24

Mitochondria are sub-cellular organelles that have a central role in energy production and in other metabolic pathways of all eukaryotic respiring cells. In the last few years, with more and more genomes being sequenced, a huge amount of data has been generated providing an unprecedented opportunity to use the comparative analysis approach in studies of evolution and functional genomics with the aim of shedding light on molecular mechanisms regulating mitochondrial biogenesis and metabolism. In this context, the problem of the optimal extraction of representative datasets of genomic and proteomic data assumes a crucial importance. Specialised resources for nuclear-encoded mitochondria-related proteins already exist; however, no mitochondrial database is currently available with the same features of MitoRes, which is an update of the MitoNuc database extensively modified in its structure, data sources and graphical interface. It contains data on nuclear-encoded mitochondria-related products for any metazoan species for which this type of data is available and also provides comprehensive sequence datasets (gene, transcript and protein) as well as useful tools for their extraction and export. MitoRes http://www2.ba.itb.cnr.it/MitoRes/ consolidates information from publicly external sources and automatically annotates them into a relational database. Additionally, it also clusters proteins on the basis of their sequence similarity and interconnects them with genomic data. The search engine and sequence management tools allow the query/retrieval of the database content and the extraction and export of sequences (gene, transcript, protein) and related sub-sequences (intron, exon, UTR, CDS, signal peptide and gene flanking regions) ready to be used for in silico analysis. The tool we describe here has been developed to support lab scientists and bioinformaticians alike in the characterization of molecular features and evolution of mitochondrial targeting sequences. The

Identification of genes for melatonin synthetic enzymes in 'Red Fuji' apple (Malus domestica Borkh.cv.Red) and their expression and melatonin production during fruit development.

Science.gov (United States)

Lei, Qiong; Wang, Lin; Tan, Dun-Xian; Zhao, Yu; Zheng, Xiao-Dong; Chen, Hao; Li, Qing-Tian; Zuo, Bi-Xiao; Kong, Jin

2013-11-01

Melatonin is present in many edible fruits; however, the presence of melatonin in apple has not previously been reported. In this study, the genes for melatonin synthetic enzymes including tryptophan decarboxylase, tryptamine 5-hydroxylase (T5H), arylalkylamine N-acetyltransferase, and N-acetylserotonin methyltransferase were identified in 'Red Fuji' apple. Each gene has several homologous genes. Sequence analysis shows that these genes have little homology with those of animals and they only have limited homology with known genes of rice melatonin synthetic enzymes. Multiple origins of melatonin synthetic genes during the evolution are expected. The expression of these genes is fully coordinated with melatonin production in apple development. Melatonin levels in apple exhibit an inverse relationship with the content of malondialdehyde, a product of lipid peroxidation. Two major melatonin synthetic peaks appeared on July 17 and on October 8 in both unbagged and bagged apple samples. At the periods mentioned above, apples experienced rapid expansion and increased respiration. These episodes significantly elevate reactive oxygen species production in the apple. Current data further confirmed that melatonin produced in apple was used to neutralize the toxic oxidants and protect the developing apple against oxidative stress. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Integrating toxin gene expression, growth and fumonisin B1 and B2 production by a strain of Fusarium verticillioides under different environmental factors

Science.gov (United States)

Medina, Angel; Schmidt-Heydt, Markus; Cárdenas-Chávez, Diana L.; Parra, Roberto; Geisen, Rolf; Magan, Naresh

2013-01-01

The objective of this study was to integrate data on the effect of water activity (aw; 0.995–0.93) and temperature (20–35°C) on activation of the biosynthetic FUM genes, growth and the mycotoxins fumonisin (FB1, FB2) by Fusarium verticillioides in vitro. The relative expression of nine biosynthetic cluster genes (FUM1, FUM7, FUM10, FUM11, FUM12, FUM13, FUM14, FUM16 and FUM19) in relation to the environmental factors was determined using a microarray analysis. The expression was related to growth and phenotypic FB1 and FB2 production. These data were used to develop a mixed-growth-associated product formation model and link this to a linear combination of the expression data for the nine genes. The model was then validated by examining datasets outside the model fitting conditions used (35°C). The relationship between the key gene (FUM1) and other genes in the cluster (FUM11, FUM13, FUM9, FUM14) were examined in relation to aw, temperature, FB1 and FB2 production by developing ternary diagrams of relative expression. This model is important in developing an integrated systems approach to develop prevention strategies to control fumonisin biosynthesis in staple food commodities and could also be used to predict the potential impact that climate change factors may have on toxin production. PMID:23697716

Gene doping: gene delivery for olympic victory.

Science.gov (United States)

Gould, David

2013-08-01

With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called 'gene doping'. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. © 2012 The Author. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.

Amplification of the uvrA gene product of Escherichia coli to 7% of cellular protein by linkage to the p/sub L/ promoter of pKC30

International Nuclear Information System (INIS)

Yoakum, G.H.; Yeung, A.T.; Mattes, W.B.; Grossman, L.

1982-01-01

Researchers have constructed a hybrid pKC30-uvrA plasmid (pGHY5003) in which transcription of the uvrA gene can be induced under p/sub L/ control to amplify the uvrA gene product to 7% of cellular protein. To construct pGHY5003, researchers developed a genetic selection using the basal level of expression (30 0 C) from p/sub L/ in thermosensitive cI857 lysogens to isolate appropriately tailored repair genes inserted at the Hpa I site of pKC30 from recombinant DNA mixtures with a variety of products. In addition, a post-uv-irradiation radiolabeling method was adapted to screen inserts for temperature-inducible polypeptide synthesis directed by transcription under p/sub L/ control rapidly. This should prove generally useful for isolating genes inserted at the Hpa I site of plasmid pKC30 with the following characteristics: (1) genetically functional hybrid plasmids selected from a large population of exonucleolytically tailored fragments ligated into Hpa I of pKC30 and (2) production of high-level amplification for the gene product of interest by screening for post-uv-irradiation temperature inducibility of polypeptides synthesized from hybrid plasmids. The level of amplification obtained for the uvrA gene product from pGHY5003 is approximately 10,000-fold higher than estimates of the level of uvrA protein in logarithmic phase Escherichia coli

Identifying genes involved in the interaction of Aggregatibacter actinomycetemcomitans with Maillard reaction products (MRP)

Science.gov (United States)

Jaha, Raniah Abdulmohsen

Aggregatibacter (Actinobacillus) actinomycelemcomitcrns is a gram-negative bacterium that is a facultative anaerobe which can grow in either aerobic or anaerobic conditions. The bacteria cause localized aggressive periodontitis that can result in the loss of teeth and endocarditis, which is an infection of the heart valves. A rich medium is an essential requirement for its growth. There arc some difficulties associated with growing the bacteria as they easily switch from the rough to smooth phenotype under no specific conditions. The bacteria start to lose viability after about 19 hours of growth in broth or about three days on plates. Colonies in the dense part of the streak on plates die earlier. It was shown that acid secreted by the colonies is responsible for the loss of viability as the bacteria are extremely sensitive to low pH. Autoclaving the growth medium for A. actinomycetemcomitans causes the bacteria to grow slowly because of the formation of Maillard reaction products (MRPs). A method has been developed to make the A. actinomycetemcomitans growth medium using the microwave instead of the autoclave. This method produces much less of the inhibitory product since the heating time is only six minutes, compared to more than an hour when using the autoclave. Two approaches were sought in this research. The first approach was the identification of genes responsible for the interaction between the MRP and A. actinomycetemcomitans. The gene responsible for this interaction was found to be a Lys M protein which is found in many genes responsible for the cell wall integrity. The second approach was to develop a new drug made of glucose and lysine with a minimum inhibitory concentration as 75mM.

Characterization of CG6178 gene product with high sequence similarity to firefly luciferase in Drosophila melanogaster.

Science.gov (United States)

Oba, Yuichi; Ojika, Makoto; Inouye, Satoshi

2004-03-31

This is the first identification of a long-chain fatty acyl-CoA synthetase in Drosophila by enzymatic characterization. The gene product of CG6178 (CG6178) in Drosophila melanogaster genome, which has a high sequence similarity to firefly luciferase, has been expressed and characterized. CG6178 showed long-chain fatty acyl-CoA synthetic activity in the presence of ATP, CoA and Mg(2+), suggesting a fatty acyl adenylate is an intermediate. Recently, it was revealed that firefly luciferase has two catalytic functions, monooxygenase (luciferase) and AMP-mediated CoA ligase (fatty acyl-CoA synthetase). However, unlike firefly luciferase, CG6178 did not show luminescence activity in the presence of firefly luciferin, ATP, CoA and Mg(2+). The enzymatic properties of CG6178 including substrate specificity, pH dependency and optimal temperature were close to those of firefly luciferase and rat fatty acyl-CoA synthetase. Further, phylogenic analyses strongly suggest that the firefly luciferase gene may have evolved from a fatty acyl-CoA synthetase gene as a common ancestral gene.

Tissue-specific production of limonene in Camelina sativa with the Arabidopsis promoters of genes BANYULS and FRUITFULL

NARCIS (Netherlands)

Borghi, Monica; Xie, De Yu

2016-01-01

Main conclusion: Arabidopsis promoters of genesBANYULSandFRUITFULLare transcribed in Camelina. They triggered the transcription oflimonene synthaseand induced higher limonene production in seeds and fruits thanCaMV 35Spromoter.Camelina sativa (Camelina) is an oilseed crop of relevance for the

Why commercialization of gene therapy stalled; examining the life cycles of gene therapy technologies.

Science.gov (United States)

Ledley, F D; McNamee, L M; Uzdil, V; Morgan, I W

2014-02-01

This report examines the commercialization of gene therapy in the context of innovation theories that posit a relationship between the maturation of a technology through its life cycle and prospects for successful product development. We show that the field of gene therapy has matured steadily since the 1980s, with the congruent accumulation of >35 000 papers, >16 000 US patents, >1800 clinical trials and >$4.3 billion in capital investment in gene therapy companies. Gene therapy technologies comprise a series of dissimilar approaches for gene delivery, each of which has introduced a distinct product architecture. Using bibliometric methods, we quantify the maturation of each technology through a characteristic life cycle S-curve, from a Nascent stage, through a Growing stage of exponential advance, toward an Established stage and projected limit. Capital investment in gene therapy is shown to have occurred predominantly in Nascent stage technologies and to be negatively correlated with maturity. Gene therapy technologies are now achieving the level of maturity that innovation research and biotechnology experience suggest may be requisite for efficient product development. Asynchrony between the maturation of gene therapy technologies and capital investment in development-focused business models may have stalled the commercialization of gene therapy.

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

Science.gov (United States)

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.

Functional Genomics Reveals That a Compact Terpene Synthase Gene Family Can Account for Terpene Volatile Production in Apple1[W

Science.gov (United States)

Nieuwenhuizen, Niels J.; Green, Sol A.; Chen, Xiuyin; Bailleul, Estelle J.D.; Matich, Adam J.; Wang, Mindy Y.; Atkinson, Ross G.

2013-01-01

Terpenes are specialized plant metabolites that act as attractants to pollinators and as defensive compounds against pathogens and herbivores, but they also play an important role in determining the quality of horticultural food products. We show that the genome of cultivated apple (Malus domestica) contains 55 putative terpene synthase (TPS) genes, of which only 10 are predicted to be functional. This low number of predicted functional TPS genes compared with other plant species was supported by the identification of only eight potentially functional TPS enzymes in apple ‘Royal Gala’ expressed sequence tag databases, including the previously characterized apple (E,E)-α-farnesene synthase. In planta functional characterization of these TPS enzymes showed that they could account for the majority of terpene volatiles produced in cv Royal Gala, including the sesquiterpenes germacrene-D and (E)-β-caryophyllene, the monoterpenes linalool and α-pinene, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene. Relative expression analysis of the TPS genes indicated that floral and vegetative tissues were the primary sites of terpene production in cv Royal Gala. However, production of cv Royal Gala floral-specific terpenes and TPS genes was observed in the fruit of some heritage apple cultivars. Our results suggest that the apple TPS gene family has been shaped by a combination of ancestral and more recent genome-wide duplication events. The relatively small number of functional enzymes suggests that the remaining terpenes produced in floral and vegetative and fruit tissues are maintained under a positive selective pressure, while the small number of terpenes found in the fruit of modern cultivars may be related to commercial breeding strategies. PMID:23256150

Extended region of nodulation genes in Rhizobium meliloti 1021. II. Nucleotide sequence, transcription start sites and protein products

International Nuclear Information System (INIS)

Fisher, R.F.; Swanson, J.A.; Mulligan, J.T.; Long, S.R.

1987-01-01

The authors have established the DNA sequence and analyzed the transcription and translation products of a series of putative nodulation (nod) genes in Rhizobium meliloti strain 1021. Four loci have been designated nodF, nodE, nodG and nodH. The correlation of transposon insertion positions with phenotypes and open reading frames was confirmed by sequencing the insertion junctions of the transposons. The protein products of these nod genes were visualized by in vitro expression of cloned DNA segments in a R. meliloti transcription-translation system. In addition, the sequence for nodG was substantiated by creating translational fusions in all three reading frames at several points in the sequence; the resulting fusions were expressed in vitro in both E. coli and R. meliloti transcription-translation systems. A DNA segment bearing several open reading frames downstream of nodG corresponds to the putative nod gene mutated in strain nod-216. The transcription start sites of nodF and nodH were mapped by primer extension of RNA from cells induced with the plant flavone, luteolin. Initiation of transcription occurs approximately 25 bp downstream from the conserved sequence designated the nod box, suggesting that this conserved sequence acts as an upstream regulator of inducible nod gene expression. Its distance from the transcription start site is more suggestive of an activator binding site rather than an RNA polymerase binding site

Detection of biosynthetic gene and phytohormone production by endophytic actinobacteria associated with Solanum lycopersicum and their plant-growth-promoting effect.

Science.gov (United States)

Passari, Ajit Kumar; Chandra, Preeti; Zothanpuia; Mishra, Vineet Kumar; Leo, Vincent Vineeth; Gupta, Vijai Kumar; Kumar, Brijesh; Singh, Bhim Pratap

2016-10-01

In the present study, fifteen endophytic actinobacterial isolates recovered from Solanum lycopersicum were studied for their antagonistic potential and plant-growth-promoting (PGP) traits. Among them, eight isolates showed significant antagonistic and PGP traits, identified by amplification of the 16S rRNA gene. Isolate number DBT204, identified as Streptomyces sp., showed multiple PGP traits tested in planta and improved a range of growth parameters in seedlings of chili (Capsicum annuum L.) and tomato (S. lycopersicum L.). Further, genes of indole acetic acid (iaaM) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS) were successively amplified from five strains. Six antibiotics (trimethoprim, fluconazole, chloramphenicol, nalidixic acid, rifampicin and streptomycin) and two phytohormones [indole acetic acid (IAA) and kinetin (KI)] were detected and quantified in Streptomyces sp. strain DBT204 using UPLC-ESI-MS/MS. The study indicates the potential of these PGP strains for production of phytohormones and shows the presence of biosynthetic genes responsible for production of secondary metabolites. It is the first report showing production of phytohormones (IAA and KI) by endophytic actinobacteria having PGP and biosynthetic potential. We propose Streptomyces sp. strain DBT204 for inoculums production and development of biofertilizers for enhancing growth of chili and tomato seedlings. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch.

Science.gov (United States)

Poulain, Adeline; Perret, Sylvie; Malenfant, Félix; Mullick, Alaka; Massie, Bernard; Durocher, Yves

2017-08-10

To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHO BRI/rcTA ) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHO BRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHO BRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Volatile Gas Production by Methyl Halide Transferase: An In Situ Reporter Of Microbial Gene Expression In Soil.

Science.gov (United States)

Cheng, Hsiao-Ying; Masiello, Caroline A; Bennett, George N; Silberg, Jonathan J

2016-08-16

Traditional visual reporters of gene expression have only very limited use in soils because their outputs are challenging to detect through the soil matrix. This severely restricts our ability to study time-dependent microbial gene expression in one of the Earth's largest, most complex habitats. Here we describe an approach to report on dynamic gene expression within a microbial population in a soil under natural water levels (at and below water holding capacity) via production of methyl halides using a methyl halide transferase. As a proof-of-concept application, we couple the expression of this gas reporter to the conjugative transfer of a bacterial plasmid in a soil matrix and show that gas released from the matrix displays a strong correlation with the number of transconjugant bacteria that formed. Gas reporting of gene expression will make possible dynamic studies of natural and engineered microbes within many hard-to-image environmental matrices (soils, sediments, sludge, and biomass) at sample scales exceeding those used for traditional visual reporting.

Experimental Model to Study the Role of Retinoblastoma Gene Product (pRb) for Determination of Adipocyte Differentiation.

Science.gov (United States)

Popov, B V; Shilo, P S; Zhidkova, O V; Zaichik, A M; Petrov, N S

2015-06-01

Using stable constitutive expression of retinoblastoma gene product (pRb) in polypotent mesenchymal 10T1/2 cells we obtained stable cell lines hyperexpressing functionally active or inactive mutant pRb. The cells producing active exogenous pRb demonstrated high sensitivity to adipocyte differentiation inductors, whereas production of inactive form of the exogenous protein suppressed adipocyte differentiation. The obtained lines can serve as the experimental model for studying the role of pRb in determination of adipocyte differentiation.

Mutation in the peroxin-coding gene PEX22 contributing to high malate production in Saccharomyces cerevisiae.

Science.gov (United States)

Negoro, Hiroaki; Sakamoto, Mitsuru; Kotaka, Atsushi; Matsumura, Kengo; Hata, Yoji

2018-02-01

Saccharomyces cerevisiae produces organic acids such as succinate, acetate, and malate during alcoholic fermentation. Since malate contributes to the pleasant taste of sake (a Japanese alcoholic beverage), various methods for breeding high-malate-producing yeast strains have been developed. Here, a high-malate-producing yeast strain F-701H was isolated. This mutant was sensitive to dimethyl succinate (DMS) and harbored a nonsense mutation in the peroxin gene PEX22, which was identified as the cause of high malate production by comparative genome analysis. This mutation, which appeared to cause Pex22p dysfunction, was sufficient to confer increased malate productivity and DMS sensitivity to yeast cells. Next, we investigated the mechanism by which this mutation led to high malate production in yeast cells. Peroxins, such as Pex22p, maintain peroxisomal biogenesis. Analysis of 29 PEX disruptants revealed an increased malate production by deletion of the genes encoding peroxins responsible for importing proteins (containing peroxisomal targeting signal 1, PTS1) into the peroxisomal matrix, and those responsible for the assembly of peroxins themselves in the peroxisomal membrane. A defect in peroxisomal malate dehydrogenase (Mdh3p), harboring endogenous PTS1, inhibited the high malate-producing phenotype in the PEX22 mutant. Moreover, Mdh3p, which was normally sorted to the peroxisomal matrix, was potentially mislocalized to the cytosol in the PEX22 mutant. This suggested that an increase in malate production resulted from the mislocalization of Mdh3p from the peroxisome to the cytoplasm due to the loss of peroxin-mediated transportation. Thus, the present study revealed a novel mechanism for organic acid productions in yeast during sake brewing. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

GABA production and structure of gadB/gadC genes in Lactobacillus and Bifidobacterium strains from human microbiota.

Science.gov (United States)

Yunes, R A; Poluektova, E U; Dyachkova, M S; Klimina, K M; Kovtun, A S; Averina, O V; Orlova, V S; Danilenko, V N

2016-12-01

Gamma-amino butyric acid (GABA) is an active biogenic substance synthesized in plants, fungi, vertebrate animals and bacteria. Lactic acid bacteria are considered the main producers of GABA among bacteria. GABA-producing lactobacilli are isolated from food products such as cheese, yogurt, sourdough, etc. and are the source of bioactive properties assigned to those foods. The ability of human-derived lactobacilli and bifidobacteria to synthesize GABA remains poorly characterized. In this paper, we screened our collection of 135 human-derived Lactobacillus and Bifidobacterium strains for their ability to produce GABA from its precursor monosodium glutamate. Fifty eight strains were able to produce GABA. The most efficient GABA-producers were Bifidobacterium strains (up to 6 g/L). Time profiles of cell growth and GABA production as well as the influence of pyridoxal phosphate on GABA production were studied for L. plantarum 90sk, L. brevis 15f, B. adolescentis 150 and B. angulatum GT102. DNA of these strains was sequenced; the gadB and gadC genes were identified. The presence of these genes was analyzed in 14 metagenomes of healthy individuals. The genes were found in the following genera of bacteria: Bacteroidetes (Bacteroides, Parabacteroides, Alistipes, Odoribacter, Prevotella), Proteobacterium (Esherichia), Firmicutes (Enterococcus), Actinobacteria (Bifidobacterium). These data indicate that gad genes as well as the ability to produce GABA are widely distributed among lactobacilli and bifidobacteria (mainly in L. plantarum, L. brevis, B. adolescentis, B. angulatum, B. dentium) and other gut-derived bacterial species. Perhaps, GABA is involved in the interaction of gut microbiota with the macroorganism and the ability to synthesize GABA may be an important feature in the selection of bacterial strains - psychobiotics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Trans-activation function of a 3' truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues

International Nuclear Information System (INIS)

Takada, Shinako; Koike, Katsuro

1990-01-01

To investigate the expression and transactivation function of the X gene in integrated hepatitis B virus (HBV) DNA from chronic hepatitis tissues, a series of transfectants containing cloned integrated HBV DNAs was made and analyzed for X mRNA expression and trans-activation activity by using a chloramphenicol acetyltransferase assay. Most of the integrated HBV DNAs expressed X mRNA and encoded a product with trans-activation activity in spite of the loss of the 3' end region of the X gene due to integration. From cDNA cloning and sequence analysis of X mRNA transcribed from native or integrated HBV DNA, the X protein was found to be translated from the X open reading frame without splicing. For integrated HBV DNA, transcription was extended to a cellular flanking DNA and an X gene-cell fusion transcript was terminated by using a cellular poly(A) signal. The amino acid sequence deduced from an X-cell fusion transcript indicated truncation of the carboxyl-terminal five amino acids, but the upstream region of seven amino acids conserved among hepadnaviruses was retained in the integrated HBV DNA, suggesting that this conserved region is essential for the transactivation function of the X protein. These findings support the following explanation for hepatocarcinogenesis by HBV DNA integration: the expression of a cellular oncogene(s) is transactivated at the time of chronic infection by the increasing amounts of the integrated HBV gene product(s), such as the X-cell fusion product

The chicken c-erbA alpha-product induces expression of thyroid hormone-responsive genes in 3,5,3'-triiodothyronine receptor-deficient rat hepatoma cells

DEFF Research Database (Denmark)

Muñoz, A; Höppner, W; Sap, J

1990-01-01

To determine the capacity of the chicken c-erbA (cTR-alpha) gene product in regulating expression of known thyroid hormone-responsive genes, both the cTR-alpha and the viral v-erbA genes were expressed in FAO cells, a rat hepatoma cell line defective for functional thyroid hormone receptors. Upon...

Suppression of Tla1 gene expression for improved solar conversion efficiency and photosynthetic productivity in plants and algae

Science.gov (United States)

Melis, Anastasios; Mitra, Mautusi

2010-06-29

The invention provides method and compositions to minimize the chlorophyll antenna size of photosynthesis by decreasing TLA1 gene expression, thereby improving solar conversion efficiencies and photosynthetic productivity in plants, e.g., green microalgae, under bright sunlight conditions.

Abundance and distribution of antibiotic resistance genes in a full-scale anaerobic-aerobic system alternately treating ribostamycin, spiramycin and paromomycin production wastewater.

Science.gov (United States)

Tang, Mei; Dou, Xiaomin; Wang, Chunyan; Tian, Zhe; Yang, Min; Zhang, Yu

2017-12-01

The occurrence of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs) has been intensively investigated for wastewater treatment systems treating single class of antibiotic in recent years. However, the impacts of alternately occurring antibiotics in antibiotic production wastewater on the behavior of ARGs in biological treatment systems were not well understood yet. Herein, techniques including high-capacity quantitative PCR and quantitative PCR (qPCR) were used to investigate the behavior of ARGs in an anaerobic-aerobic full-scale system. The system alternately treated three kinds of antibiotic production wastewater including ribostamycin, spiramycin and paromomycin, which referred to stages 1, 2 and 3. The aminoglycoside ARGs (52.1-79.3%) determined using high-capacity quantitative PCR were the most abundant species in all sludge samples of the three stages. The total relative abundances of macrolide-lincosamide-streptogramin (MLS) resistance genes and aminoglycoside resistance genes measured using qPCR were significantly higher (P  0.05) in both aerobic and anaerobic sludge samples. In aerobic sludge, one acetyltransferase gene (aacA4) and the other three nucleotidyltransferase genes (aadB, aadA and aadE) exhibited positive correlations with intI1 (r 2  = 0.83-0.94; P < 0.05), implying the significance of horizontal transfer in their proliferation. These results and facts will be helpful to understand the abundance and distribution of ARGs from antibiotic production wastewater treatment systems.

[Prokaryotic expression of Leptospira interrogans groEL gene and immunoprotection of its products in hamsters].

Science.gov (United States)

Li, Xiaoyu; Wang, Yinhuan; Yan, Jie; Cheng, Dongqing

2013-03-01

To construct a prokaryotic expression system of groEL gene of Leptospira interrogans serogroup Icterohaemorrhagia serovar Lai strain Lai, and to determine the immunoprotective effect of recombinant GroEL protein (rGroEL) in LVG hamsters. The groEL gene was amplified by high fidelity PCR and the amplification products were then sequenced. A prokaryotic expression system of groEL gene was constructed using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Gel Image Analyzer was applied to examine the expression and dissolubility of rGroEL protein while Ni-NTA affinity chromatography was used to extract the expressed rGroEL. The immunoprotective rate in rGroEL-immunized LVG hamsters was determined after challenge with L.interrogans strain Lai. The cross agglutination titers of sera from immunized hamsters with different L.interrogans serogroups were detected using MAT. The nucleotide and amino acid sequences of the cloned groEL gene were the same as those reported in GenBank. The constructed prokaryotic expression system of groEL gene expressed soluble rGroEL. The immunoprotective rates of 100 and 200 μg rGroEL in LVG hamsters were 50.0 % and 75.0%, respectively. The sera from the rGroEL-immunized LVG hamsters agglutinated all the L.interrogans serogroups tested with different levels. The GroEL protein is a genus-specific immunoprotective antigen of L.interrogans and can be used to develop an universal genetically engineering vaccine of Leptospira.

In vitro production of bovine embryos: cumulus/granulosa cell gene expression patterns point to early atresia as beneficial for oocyte competence

DEFF Research Database (Denmark)

Mazzoni, Gianluca; Razza, Eduardo; Pedersen, Hanne S.

2017-01-01

In vitro production (IW) of bovine embryos has become widespread technology implemented in cattle breeding and production. Here, we review novel data on cumulus/granulosa cell gene expression, as determined by RNAseq on cellular material from pooled follicular fluids at the single animal level...

CFH Y402H polymorphism and the complement activation product C5a: effects on NF-κB activation and inflammasome gene regulation.

Science.gov (United States)

Cao, Sijia; Wang, Jay Ching Chieh; Gao, Jiangyuan; Wong, Matthew; To, Elliott; White, Valerie A; Cui, Jing Z; Matsubara, Joanne A

2016-05-01

The Y402H polymorphism in the complement factor H (CFH) gene is an important risk factor for age-related macular degeneration (AMD). Complement activation products and proinflammatory cytokines are associated with this polymorphism at the systemic level, but less is known of the associations in the outer retina of the genotyped eye. Here we investigate complement activation products and their role in nuclear factor (NF)-κB activation and gene expression of the NLRP3 inflammasome pathway. Postmortem donor eyes were genotyped for the CFH Y402H polymorphism and assessed for complement C3a, C5a, interleukin (IL)-18 and tumour necrosis factor (TNF)-α. ARPE19 cells were stimulated basolaterally with C5a or TNF-α in polarised cultures. NF-κB activation was assessed with a reporter cell line. Gene expression of inflammasome-related (NLRP3, caspase-1, IL-1β and IL-18) and classic inflammatory (IL-6 and IL-8) genes was studied. The distribution of inflammasome products, IL-1β and IL-18, was studied in postmortem donor eyes with AMD pathologies. Eyes with the homozygous at-risk variant demonstrated higher levels of C5a, IL-18 and TNF-α in Bruch's membrane and choroid. C5a promoted NF-κB activation and upregulation of IL-18 in polarised ARPE19. TNF-α promoted NF-κB activation and gene expression of caspase-1, IL-1β, IL-18, IL-6 and IL-8, but downregulated NLRP3. In eyes with geographic atrophy, strong immunoreactivity was observed for inflammasome products IL-1β and IL-18 compared with age-matched controls. The at-risk polymorphism of the CFH Y402H may contribute to AMD disease process through increased complement and NF-κB activation, and the upregulation of IL-18, a product of inflammasome activation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Characterization of representative rpoB gene mutations leading to a significant change in toyocamycin production of Streptomyces diastatochromogenes 1628.

Science.gov (United States)

Ma, Zheng; Luo, Shuai; Xu, Xianhao; Bechthold, Andreas; Yu, Xiaoping

2016-04-01

Modification of enzymes involved in transcription- or translation-processes is an interesting way to increase secondary metabolite production in Streptomycetes. However, application of such methods has not been widely described for strains which produce nucleoside antibiotics. The nucleoside antibiotic toyocamycin (TM) is produced by Streptomyces diastatochromogenes 1628. For improving TM production in S. diastatochromogenes 1628, the strain was spread on rifamycin-resistant (Rif(r)) medium. Several spontaneous mutants were obtained with mutations in the rpoB gene which encodes a RNA polymerase β-subunit. The mutants which showed increased TM production were detected at a frequency of 7.5 % among the total Rif(r) mutants. Mutant 1628-T15 harboring amino acid substitution His437Arg was the best TM producer with a 4.5-fold increase in comparison to that of the wild-type strain. The worst producer was mutant 1628-T62 which also showed a poor sporulation behavior. RT-PCR was performed to study the transcription levels of the TM biosynthetic gene toyG in the parental strain as well as in mutants 1628-T15 and 1628-T62. The transcriptional level of toyG was higher in mutant 1628-T15 than that in parental strain 1628, while much lower in mutant 1628-T62. In mutant strain 1628-T62 the expression of adpA sd gene, which is required for morphological differentiation, was also much lower. Our studies also indicate that the introduction of mutations into rpoB is an effective strategy to improve the production of TM which is an important nucleoside antibiotic.

Gene function prediction based on Gene Ontology Hierarchy Preserving Hashing.

Science.gov (United States)

Zhao, Yingwen; Fu, Guangyuan; Wang, Jun; Guo, Maozu; Yu, Guoxian

2018-02-23

Gene Ontology (GO) uses structured vocabularies (or terms) to describe the molecular functions, biological roles, and cellular locations of gene products in a hierarchical ontology. GO annotations associate genes with GO terms and indicate the given gene products carrying out the biological functions described by the relevant terms. However, predicting correct GO annotations for genes from a massive set of GO terms as defined by GO is a difficult challenge. To combat with this challenge, we introduce a Gene Ontology Hierarchy Preserving Hashing (HPHash) based semantic method for gene function prediction. HPHash firstly measures the taxonomic similarity between GO terms. It then uses a hierarchy preserving hashing technique to keep the hierarchical order between GO terms, and to optimize a series of hashing functions to encode massive GO terms via compact binary codes. After that, HPHash utilizes these hashing functions to project the gene-term association matrix into a low-dimensional one and performs semantic similarity based gene function prediction in the low-dimensional space. Experimental results on three model species (Homo sapiens, Mus musculus and Rattus norvegicus) for interspecies gene function prediction show that HPHash performs better than other related approaches and it is robust to the number of hash functions. In addition, we also take HPHash as a plugin for BLAST based gene function prediction. From the experimental results, HPHash again significantly improves the prediction performance. The codes of HPHash are available at: http://mlda.swu.edu.cn/codes.php?name=HPHash. Copyright © 2018 Elsevier Inc. All rights reserved.

Targeted gene panels and microbiota analysis provide insight into the effects of effects of alternative production diet formulations on channel catfish nutritional physiology

Science.gov (United States)

The present research evaluated targeted gene panels and microbiota analysis to provide greater insight into the effects of alternatively-sourced dietary ingredients on production indices, gut health, changes in the gut microbiota and genes involved in the regulation of appetite, growth, metabolism, ...

A global RNA-seq-driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation.

Science.gov (United States)

Könitzer, Jennifer D; Müller, Markus M; Leparc, Germán; Pauers, Martin; Bechmann, Jan; Schulz, Patrick; Schaub, Jochen; Enenkel, Barbara; Hildebrandt, Tobias; Hampel, Martin; Tolstrup, Anne B

2015-09-01

Boehringer Ingelheim uses two CHO-DG44 lines for manufacturing biotherapeutics, BI-HEX-1 and BI-HEX-2, which produce distinct cell type-specific antibody glycosylation patterns. A recently established CHO-K1 descended host, BI-HEX-K1, generates antibodies with glycosylation profiles differing from CHO-DG44. Manufacturing process development is significantly influenced by these unique profiles. To investigate the underlying glycosylation related gene expression, we leveraged our CHO host and production cell RNA-seqtranscriptomics and product quality database together with the CHO-K1 genome. We observed that each BI-HEX host and antibody producing cell line has a unique gene expression fingerprint. CHO-DG44 cells only transcribe Fut10, Gfpt2 and ST8Sia6 when expressing antibodies. BI-HEX-K1 cells express ST8Sia6 at host cell level. We detected a link between BI-HEX-1/BI-HEX-2 antibody galactosylation and mannosylation and the gene expression of the B4galt gene family and genes controlling mannose processing. Furthermore, we found major differences between the CHO-DG44 and CHO-K1 lineages in the expression of sialyl transferases and enzymes synthesizing sialic acid precursors, providing a rationale for the lack of immunogenic NeuGc/NGNA synthesis in CHO. Our study highlights the value of systems biotechnology to understand glycoprotein synthesis and product glycoprofiles. Such data improve future production clone selection and process development strategies for better steering of biotherapeutic product quality. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional analysis of three type-2 DGAT homologue genes for triacylglycerol production in the green microalga Chlamydomonas reinhardtii.

Science.gov (United States)

La Russa, M; Bogen, C; Uhmeyer, A; Doebbe, A; Filippone, E; Kruse, O; Mussgnug, J H

2012-11-30

Photosynthetic organisms like plants and algae can use sunlight to produce lipids as important metabolic compounds. Plant-derived triacylglycerols (TAGs) are valuable for human and animal nutrition because of their high energy content and are becoming increasingly important for the production of renewable biofuels. Acyl-CoA:diacylglycerol acyltransferases (DGATs) have been demonstrated to play an important role in the accumulation of TAG compounds in higher plants. DGAT homologue genes have been identified in the genome of the green alga Chlamydomonas reinhardtii, however their function in vivo is still unknown. In this work, the three most promising type-2 DGAT candidate genes potentially involved in TAG lipid accumulation (CrDGAT2a, b and c) were investigated by constructing overexpression strains. For each of the genes, three strains were identified which showed enhanced mRNA levels of between 1.7 and 29.1 times that of the wild type (wt). Total lipid contents, neutral lipids and fatty acid profiles were determined and showed that an enhanced mRNA expression level of the investigated DGAT genes did not boost the intracellular TAG accumulation or resulted in alterations of the fatty acid profiles compared to wild type during standard growth condition or during nitrogen or sulfur stress conditions. We conclude that biotechnological efforts to enhance cellular TAG amount in microalgae need further insights into the complex network of lipid biosynthesis to identify potential bottlenecks of neutral lipid production. Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization, expression and complex formation of the murine Fanconi anaemia gene product Fancg.

Science.gov (United States)

van de Vrugt, Henri J; Koomen, Mireille; Berns, Mariska A D; de Vries, Yne; Rooimans, Martin A; van der Weel, Laura; Blom, Eric; de Groot, Jan; Schepers, Rik J; Stone, Stacie; Hoatlin, Maureen E; Cheng, Ngan Ching; Joenje, Hans; Arwert, Fré

2002-03-01

Fanconi anaemia (FA) is an autosomal recessive chromosomal instability disorder. Six distinct FA disease genes have been identified, the products of which function in an integrated pathway that is thought to support a nuclear caretaker function. Comparison of FA gene characteristics in different species may help to unravel the molecular function of the FA pathway. We have cloned the murine homologue of the Fanconi anaemia complementation group G gene, FANCG/XRCC9. The murine Fancg protein shows an 83% similarity to the human protein sequence, and has a predicted molecular weight of 68.5 kDa. Expression of mouse Fancg in human FA-G lymphoblasts fully corrects their cross-linker hypersensitivity. At mRNA and protein levels we detected the co-expression of Fancg and Fanca in murine tissues. In addition, mouse Fancg and Fanca proteins co-purify by immunoprecipitation. Upon transfection into Fanca-deficient mouse embryonic fibroblasts EGFP-Fancg chimeric protein was detectable in the nucleus. We identified a murine cDNA, Fancg, which cross-complements the cellular defect of human FA-G cells and thus represents a true homologue of human FANCG. Spleen, thymus and testis showed the highest Fancg expression levels. Although Fancg and Fanca are able to form a complex, this interaction is not required for Fancg to accumulate in the nuclear compartment.

Structure models of G72, the product of a susceptibility gene to schizophrenia.

Science.gov (United States)

Kato, Yusuke; Fukui, Kiyoshi

2017-02-01

The G72 gene is one of the most susceptible genes to schizophrenia and is contained exclusively in the genomes of primates. The product of the G72 gene modulates the activity of D-amino acid oxidase (DAO) and is a small protein prone to aggregate, which hampers its structural studies. In addition, lack of a known structure of a homologue makes it difficult to use the homology modelling method for the prediction of the structure. Thus, we first developed a hybrid ab initio approach for small proteins prior to the prediction of the structure of G72. The approach uses three known ab initio algorithms. To evaluate the hybrid approach, we tested our prediction of the structure of the amino acid sequences whose structures were already solved and compared the predicted structures with the experimentally solved structures. Based on these comparisons, the average accuracy of our approach was calculated to be ∼5 Å. We then applied the approach to the sequence of G72 and successfully predicted the structures of the N- and C-terminal domains (ND and CD, respectively) of G72. The predicted structures of ND and CD were similar to membrane-bound proteins and adaptor proteins, respectively. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Regulatory Considerations for Gene Therapy Products in the US, EU, and Japan.

Science.gov (United States)

Halioua-Haubold, Celine-Lea; Peyer, James G; Smith, James A; Arshad, Zeeshaan; Scholz, Matthew; Brindley, David A; MacLaren, Robert E

2017-12-01

Developers of gene therapy products (GTPs) must adhere to additional regulation beyond that of traditional small-molecule therapeutics, due to the unique mechanism-of-action of GTPs and the subsequent novel risks arisen. We have provided herein a summary of the regulatory structure under which GTPs fall in the United States, the European Union, and Japan, and a comprehensive overview of the regulatory guidance applicable to the developer of GTP. Understanding the regulatory requirements for seeking GTP market approval in these major jurisdictions is crucial for an effective and expedient path to market. The novel challenges facing GTP developers is highlighted by a case study of alipogene tiparvovec (Glybera).

Intellectual property rights and gene-based technologies for animal production and health. Issues for developing countries

International Nuclear Information System (INIS)

Dutfield, G.

2005-01-01

Intellectual property rights (IPR) are legal and institutional devices to protect creations of the mind. With respect to gene-based innovation, the most significant IPR is patents. Appropriate patent regimes have the potential to foster innovation in animal biotechnology and the transfer of gene-based technologies. Inappropriate patent systems may be counter-productive. Indeed, many critics are doubtful that the current international patent standards, based as they are on a combination of the United States of America' and European regimes, can help countries that lack the capacity to do much life science and biotechnology research to become more innovative o r contribute to the acquisition, absorption and, where desirable, the adaptation of new gene-based technologies from outside. Present legislation in Europe, North America and internationally is considered, together with the controversies and important policy questions for developing countries, and the choices facing countries seeking to enhance their scientific and technological capacities in these areas. (author)

Fermentative hydrogen production from Jerusalem artichoke by Clostridium tyrobutyricum expressing exo-inulinase gene.

Science.gov (United States)

Jiang, Ling; Wu, Qian; Xu, Qing; Zhu, Liying; Huang, He

2017-08-11

Clostridium tyrobutyricum ATCC25755 has been reported as being able to produce significant quantities of hydrogen. In this study, the exo-inulinase encoding gene cloned from Paenibacillus polymyxa SC-2 was into the expression plasmid pSY6 and expressed in the cells of C. tyrobutyricum. The engineered C. tyrobutyricum strain efficiently fermented the inulin-type carbohydrates from Jerusalem artichoke, without any pretreatment being necessary for the production of hydrogen. A comparatively high hydrogen yield (3.7 mol/mol inulin-type sugar) was achieved after 96 h in a batch process with simultaneous saccharification and fermentation (SSF), with an overall volumetric productivity rate of 620 ± 60 mL/h/L when the initial total sugar concentration of the inulin extract was increased to 100 g/L. Synthesis of inulinase in the batch SSF culture was closely associated with strain growth until the end of the exponential phase, reaching a maximum activity of 28.4 ± 0.26 U/mL. The overall results show that the highly productive and abundant biomass crop Jerusalem artichoke can be a good substrate for hydrogen production, and that the application of batch SSF for its conversion has the potential to become a cost-effective process in the near future.

Association of Single Nucleotide Polymorphisms in the IL-18 Gene with Production of IL-18 Protein by Mononuclear Cells from Healthy Donors

Directory of Open Access Journals (Sweden)

Khripko Olga Pavlovna

2008-01-01

Full Text Available IL-18 has proinflammatory effects and participates in both innate and adaptive cellular and humoral immunity. A number of SNPs that influence IL-18 production are found in the gene promoter region. We investigated the association of SNPs in the IL-18 promoter at −607 and −137 with the level of IL-18 protein production by PBMC from healthy donors from Southwestern Siberia. The genetic distribution of these SNPs in the promoter site was established by PCR. IL-18 protein production was determined by ELISA. Our results showed that PBMC from donors carrying allele 137C have lower levels of both spontaneous and LPS-stimulated IL-18 production. In contrast, PBMC from donors carrying allele 607A showed significant increases in spontaneous and stimulated IL-18 production compared to wild type. Our study suggests that the SNPs −607 and −137 in the promoter region of the IL-18 gene influence the level of IL-18 protein production by PBMC from healthy donors in Southwestern Siberia.

Production process reproducibility and product quality consistency of transient gene expression in HEK293 cells with anti-PD1 antibody as the model protein.

Science.gov (United States)

Ding, Kai; Han, Lei; Zong, Huifang; Chen, Junsheng; Zhang, Baohong; Zhu, Jianwei

2017-03-01

Demonstration of reproducibility and consistency of process and product quality is one of the most crucial issues in using transient gene expression (TGE) technology for biopharmaceutical development. In this study, we challenged the production consistency of TGE by expressing nine batches of recombinant IgG antibody in human embryonic kidney 293 cells to evaluate reproducibility including viable cell density, viability, apoptotic status, and antibody yield in cell culture supernatant. Product quality including isoelectric point, binding affinity, secondary structure, and thermal stability was assessed as well. In addition, major glycan forms of antibody from different batches of production were compared to demonstrate glycosylation consistency. Glycan compositions of the antibody harvested at different time periods were also measured to illustrate N-glycan distribution over the culture time. From the results, it has been demonstrated that different TGE batches are reproducible from lot to lot in overall cell growth, product yield, and product qualities including isoelectric point, binding affinity, secondary structure, and thermal stability. Furthermore, major N-glycan compositions are consistent among different TGE batches and conserved during cell culture time.

Physiological characterisation of Penicillium chrysogenum strains expressing the expandase gene from Streptomyces clavuligerus during batch cultivations. Growth and adipoyl-7- aminodeacetoxycephalosporanic acid production

DEFF Research Database (Denmark)

Robin, Jarno Jacky Christian; Jakobsen, M.; Beyer, M.

2001-01-01

The production of adipoyl-7-aminodeacetoxy-cephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase; and this pr......The production of adipoyl-7-aminodeacetoxy-cephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase...

Production of Recombinant Adenovirus Containing Human Interlukin-4 Gene

OpenAIRE

Mojarrad, Majid; Abdolazimi, Yassan; Hajati, Jamshid; Modarressi, Mohammad Hossein

2011-01-01

Objective(s) Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics ...

Isolation and characterization of Agouti: a diabetes/obesity related gene

Energy Technology Data Exchange (ETDEWEB)

Woychik, Richard P. (Knoxville, TN)

2000-06-27

The present invention relates to the cloning and expression of the Agouti gene and analogous genes in transformed, transfected and transgenic mice. The present invention provides an animal model for the study of diabetes, obesity and tumors for the testing of potential therapeutic agents. The present invention provides oligonucleotide probes for the detection of the Agouti gene and mutations in the gene. The present invention also relates to the isolation and recombinant production of the Agouti gene product, production of antibodies to the Agouti gene product and their use as diagnostic and therapeutic agents.

Isolation and characterization of Agouti: a diabetes/obesity related gene

Energy Technology Data Exchange (ETDEWEB)

Woychik, Richard P. (Knoxville, TN)

1998-01-01

The present invention relates to the cloning and expression of the Agouti gene and analogous genes in transformed, transfected and transgenic mice. The present invention provides an animal model for the study of diabetes, obesity and tumors for the testing of potential therapeutic agents. The present invention provides oligonucleotide probes for the detection of the Agouti gene and mutations in the gene. The present invention also relates to the isolation and recombinant production of the Agouti gene product, production of antibodies to the Agouti gene product and their use as diagnostic and therapeutic agents.

Effect of deletion of 2,3-butanediol dehydrogenase gene (bdhA) on acetoin production of Bacillus subtilis.

Science.gov (United States)

Zhang, Junjiao; Zhao, Xiangying; Zhang, Jiaxiang; Zhao, Chen; Liu, Jianjun; Tian, Yanjun; Yang, Liping

2017-09-14

The present work aims to block 2,3-butanediol synthesis in acetoin fermentation of Bacillus subtilis. First, we constructed a recombinant strain BS168D by deleting the 2,3-butanediol dehydrogenase gene bdhA of the B. subtilis168, and there was almost no 2,3-butanediol production in 20 g/L of glucose media. The acetoin yield of BS168D reached 6.61 g/L, which was about 1.5 times higher than that of the control B. subtilis168 (4.47 g/L). Then, when the glucose concentration was increased to 100 g/L, the acetoin yield reached 24.6 g/L, but 2.4 g/L of 2,3-butanediol was detected at the end of fermentation. The analysis of 2,3-butanediol chiral structure indicated that the main 2,3-butanediol production of BS168D was meso-2,3-butanediol, and the bdhA gene was only responsible for (2R,3R)-2,3-butanediol synthesis. Therefore, we speculated that there may exit another pathway relating to the meso-2,3-butanediol synthesis in the B. subtilis. In addition, the results of low oxygen condition fermentation showed that deletion of bdhA gene successfully blocked the reversible transformation between acetoin and 2,3-butanediol and eliminated the effect of dissolved oxygen on the transformation.

Gene therapy of cancer and development of therapeutic target gene

Energy Technology Data Exchange (ETDEWEB)

Kim, Chang Min; Kwon, Hee Chung

1998-04-01

We applied HSV-tk/GCV strategy to orthotopic rat hepatoma model and showed anticancer effects of hepatoma. The increased expression of Lac Z gene after adenovirus-mediated gene delivery throughout hepatic artery was thought that is increased the possibility of gene therapy for curing hepatoma. With the construction of kGLP-laboratory, it is possible to produce a good quantity and quality of adenovirus in lage-scale production and purification of adenovirus vector. Also, the analysis of hepatoma related genes by PCR-LOH could be used for the diagnosis of patients and the development of therapeutic gene.

Gene therapy of cancer and development of therapeutic target gene

International Nuclear Information System (INIS)

Kim, Chang Min; Kwon, Hee Chung

1998-04-01

We applied HSV-tk/GCV strategy to orthotopic rat hepatoma model and showed anticancer effects of hepatoma. The increased expression of Lac Z gene after adenovirus-mediated gene delivery throughout hepatic artery was thought that is increased the possibility of gene therapy for curing hepatoma. With the construction of kGLP-laboratory, it is possible to produce a good quantity and quality of adenovirus in lage-scale production and purification of adenovirus vector. Also, the analysis of hepatoma related genes by PCR-LOH could be used for the diagnosis of patients and the development of therapeutic gene

Effects of carbon, nitrogen and ambient pH on patulin production and related gene expression in Penicillium expansum.

Science.gov (United States)

Zong, Yuanyuan; Li, Boqiang; Tian, Shiping

2015-08-03

Patulin, a potent mycotoxin which can cause serious health concerns, is mainly produced in foods by Penicillium expansum. Environmental factors play important roles in regulating biosynthesis of mycotoxins; however, information about the effects of environmental factors on patulin production and the involved mechanisms in P. expansum is limited. Here, we investigated the effects of different carbon (C) and nitrogen (N) sources, and ambient pH on patulin production in three P. expansum strains T01, M1 and Pe21, and the expression profile of 15 genes involved in patulin biosynthetic pathway. It was found that C and N sources and pH had great influence on patulin production in P. expansum. In general, patulin production of all three P. expansum strains showed similar trends under different C and N sources and pH conditions, though there were some differences in the optimal conditions among these strains. Glucose-containing sugars, complex N sources, and acidic conditions were favorable conditions for patulin production. The results of RT-qPCR showed that the relative expressions of most of the patulin genes were up-regulated under patulin-permissive conditions, indicating that patulin biosynthesis was mainly regulated at transcriptional level by these environmental factors. These findings will provide useful information to better understand the regulation mechanisms of patulin biosynthesis, and be helpful in developing effective means for controlling patulin contamination. Copyright © 2015 Elsevier B.V. All rights reserved.

High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes.

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Mariela V Catone

Full Text Available Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB, a short chain length polyhydroxyalkanoate (sclPHA infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA. All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC in comparison with the mclPHA core genome genes (phaC1 and phaC2 indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases.

Lack of a functional VHL gene product sensitizes renal cell carcinoma cells to the apoptotic effects of the protein synthesis inhibitor verrucarin A.

Science.gov (United States)

Woldemichael, Girma M; Turbyville, Thomas J; Vasselli, James R; Linehan, W Marston; McMahon, James B

2012-08-01

Verrucarin A (VA) is a small molecule derived from the fungal plant pathogen Myrothecium verrucaria and was identified as a selective inhibitor of clear cell renal cell carcinoma (CCRCC) cell proliferation in a high-throughput screen of a library of naturally occurring small molecules. CCRCC arises as a result of loss-of-function mutations in the von Hippel-Lindau (VHL) gene. Here we show that VA inhibits protein translation initiation culminating in apoptosis through the extrinsic signaling pathway. Reintroduction of the VHL gene in CCRCC cells afforded resistance to VA's apoptotic effects. This resistance is mediated in part by the formation of stress granules that entrap signaling molecules that initiate the apoptotic signaling cascade. The VHL gene product was found to be a component of stress granules that develop as result of VA treatment. These findings reveal an important role for the VHL gene product in cytotoxic stress response and have important implications for the rational development of VA-related compounds in chemotherapeutic targeting of CCRCC.

Nucleotide sequences of the Erwinia chrysanthemi ogl and pelE genes negatively regulated by the kdgR gene product.

Science.gov (United States)

Reverchon, S; Huang, Y; Bourson, C; Robert-Baudouy, J

1989-12-21

The nucleotide sequences of the coding and regulatory regions of the genes encoding oligoglacturonate lyase (OGL) and pectate lyase e isoenzyme (PLe) from Erwinia chrysanthemi 3937 were determined. The ogl sequence contains an open reading frame (ORF) of 1164 bp coding for a 388-amino acid (aa) polypeptide with a predicted Mr of 44,124. A possible transcriptional start signal showing homology with the Escherichia coli promoter consensus sequence was detected. In addition, a sequence 3' to the coding region was found to be able to form a secondary structure which may function as an Rho-independent transcriptional termination signal. For the pelE sequence, a long ORF of 1212 bp coding for a 404-aa polypeptide was detected. PLe is secreted into the external medium by E. chrysanthemi, and a potential signal peptide sequence was identified in the pelE gene. In the 5' upstream pelE coding region, a putative promoter resembling E. coli promoter consensus sequences was detected. Furthermore, the region immediately 3' to the pelE translational stop codon may function as an Rho-independent translational termination signal. In strain 3937, the synthesis of OGL and PLe, as well as the other enzymes involved in the pectin-degradative pathway (particularly the kdgT product), are known to be regulated by the KdgR repressor, which mediates galacturonate and polygalacturonate induction. Synthesis of these enzymes is also regulated by the CRP-cAMP complex which mediates catabolite repression. Analysis of the regulatory regions of ogl and pelE allowed us to identify possible CRP-binding sites for these two genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Twenty Years of European Union Support to Gene Therapy and Gene Transfer.

Science.gov (United States)

Gancberg, David

2017-11-01

For 20 years and throughout its research programmes, the European Union has supported the entire innovation chain for gene transfer and gene therapy. The fruits of this investment are ripening as gene therapy products are reaching the European market and as clinical trials are demonstrating the safety of this approach to treat previously untreatable diseases.

The Effect of IL-4 Gene Polymorphisms on Cytokine Production in Patients with Chronic Periodontitis and in Healthy Controls

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Jirina Bartova

2014-01-01

Full Text Available Chronic periodontitis (CP is an inflammatory disease of the teeth-supporting tissues in which genetic predisposition, dental plaque bacteria, and immune mechanisms all play important roles. The aim of this study was to evaluate the occurrence of IL-4 gene polymorphisms in chronic periodontitis and to investigate the association between polymorphisms and cytokines production after bacterial stimulation. Sixty-two subjects (47 CP patients and 15 healthy controls with detected two polymorphisms in the IL-4 gene (-590C/T and intron 3 VNTR were examined. Production of cytokines (IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-17, TNFα, INFγ, and VEGF was studied after in vitro stimulation of isolated peripheral blood by mitogens (Pokeweed mitogen, Concanavalin A, dental plaque bacteria (Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, and Prevotella intermedia, and Heat Shock Protein (HSP 70 by the Luminex multiplex cytokine analysis system. The results were correlated with IL-4 genotypes in patients with CP and healthy controls. The mononuclear cells isolated from peripheral blood of CP patients with selected IL-4 polymorphisms significantly altered the production of IFNγ, IL-10, IL-1β, IL-1α, TNFα, and IL-6 after stimulation by HSP 70 or selected bacteria (from P<0.001 to P<0.05. IL-4 gene polymorphisms may influence the function of mononuclear cells to produce not only interleukin-4 but also other cytokines, especially in patients with CP.

FAO/IAEA international symposium on applications of gene-based technologies for improving animal production and health in developing countries. Book of extended synopses

Energy Technology Data Exchange (ETDEWEB)

NONE

2003-07-01

Genetic engineering is at the forefront of much biological research - basic, adaptive and applied or near market. Manipulation of genes to bring about the expression of a specific product, or to produce a characteristic or trait, offers exciting possibilities within both the plant and the animal kingdom. The opportunities, in terms of improving livestock productivity or reducing losses from disease, lie in a number of areas. In almost all areas of this research, isotopic markers are extensively used and are in most cases essential for achieving the levels of sensitivity required for genetic characterization and manipulation. Genetic engineering has the potential to solve many problems relating to animal productivity and health. At present the focus is on the problems that face livestock producers in the developed world. If the full benefit of this technology is to be realized globally, the problems confronting livestock farmers in developing countries will have to be considered. The characterization and application of methods in these regions has to be managed and exploited. It is hoped that this Symposium will stimulate the international exchange of information and ideas that contribute to greater accessibility and enhanced use of gene based technologies in animal agriculture in developing countries. OBJECTIVES: To create an interactive environment to discuss the role and future potential of gene based technologies for improving animal production and health; To identify constraints in the use of gene based technologies in developing countries and to determine how to use these technologies in a simple, practical way; To identify and prioritize specific research needs; To explore the possibility of international co-ordination in the area of gene based technologies in animal agriculture; To examine ethical, technological, policy and environmental issues and the role of nuclear techniques in the further development and application of gene based technologies with

FAO/IAEA international symposium on applications of gene-based technologies for improving animal production and health in developing countries. Book of extended synopses

International Nuclear Information System (INIS)

2003-01-01

Genetic engineering is at the forefront of much biological research - basic, adaptive and applied or near market. Manipulation of genes to bring about the expression of a specific product, or to produce a characteristic or trait, offers exciting possibilities within both the plant and the animal kingdom. The opportunities, in terms of improving livestock productivity or reducing losses from disease, lie in a number of areas. In almost all areas of this research, isotopic markers are extensively used and are in most cases essential for achieving the levels of sensitivity required for genetic characterization and manipulation. Genetic engineering has the potential to solve many problems relating to animal productivity and health. At present the focus is on the problems that face livestock producers in the developed world. If the full benefit of this technology is to be realized globally, the problems confronting livestock farmers in developing countries will have to be considered. The characterization and application of methods in these regions has to be managed and exploited. It is hoped that this Symposium will stimulate the international exchange of information and ideas that contribute to greater accessibility and enhanced use of gene based technologies in animal agriculture in developing countries. OBJECTIVES: To create an interactive environment to discuss the role and future potential of gene based technologies for improving animal production and health; To identify constraints in the use of gene based technologies in developing countries and to determine how to use these technologies in a simple, practical way; To identify and prioritize specific research needs; To explore the possibility of international co-ordination in the area of gene based technologies in animal agriculture; To examine ethical, technological, policy and environmental issues and the role of nuclear techniques in the further development and application of gene based technologies with

Lovastatin in Aspergillus terreus: fermented rice straw extracts interferes with methane production and gene expression in Methanobrevibacter smithii.

Science.gov (United States)

Faseleh Jahromi, Mohammad; Liang, Juan Boo; Ho, Yin Wan; Mohamad, Rosfarizan; Goh, Yong Meng; Shokryazdan, Parisa; Chin, James

2013-01-01

Lovastatin, a natural byproduct of some fungi, is able to inhibit HMG-CoA (3-hydroxy-3 methyl glutaryl CoA) reductase. This is a key enzyme involved in isoprenoid synthesis and essential for cell membrane formation in methanogenic Archaea. In this paper, experiments were designed to test the hypothesis that lovastatin secreted by Aspergillus terreus in fermented rice straw extracts (FRSE) can inhibit growth and CH4 production in Methanobrevibacter smithii (a test methanogen). By HPLC analysis, 75% of the total lovastatin in FRSE was in the active hydroxyacid form, and in vitro studies confirmed that this had a stronger effect in reducing both growth and CH4 production in M. smithii compared to commercial lovastatin. Transmission electron micrographs revealed distorted morphological divisions of lovastatin- and FRSE-treated M. smithii cells, supporting its role in blocking normal cell membrane synthesis. Real-time PCR confirmed that both commercial lovastatin and FRSE increased (P < 0.01) the expression of HMG-CoA reductase gene (hmg). In addition, expressions of other gene transcripts in M. smithii. with a key involvement in methanogenesis were also affected. Experimental confirmation that CH4 production is inhibited by lovastatin in A. terreus-fermented rice straw paves the way for its evaluation as a feed additive for mitigating CH4 production in ruminants.

Lovastatin in Aspergillus terreus: Fermented Rice Straw Extracts Interferes with Methane Production and Gene Expression in Methanobrevibacter smithii

Directory of Open Access Journals (Sweden)

Mohammad Faseleh Jahromi

2013-01-01

Full Text Available Lovastatin, a natural byproduct of some fungi, is able to inhibit HMG-CoA (3-hydroxy-3methyl glutaryl CoA reductase. This is a key enzyme involved in isoprenoid synthesis and essential for cell membrane formation in methanogenic Archaea. In this paper, experiments were designed to test the hypothesis that lovastatin secreted by Aspergillus terreus in fermented rice straw extracts (FRSE can inhibit growth and CH4 production in Methanobrevibacter smithii (a test methanogen. By HPLC analysis, 75% of the total lovastatin in FRSE was in the active hydroxyacid form, and in vitro studies confirmed that this had a stronger effect in reducing both growth and CH4 production in M. smithii compared to commercial lovastatin. Transmission electron micrographs revealed distorted morphological divisions of lovastatin- and FRSE-treated M. smithii cells, supporting its role in blocking normal cell membrane synthesis. Real-time PCR confirmed that both commercial lovastatin and FRSE increased (P<0.01 the expression of HMG-CoA reductase gene (hmg. In addition, expressions of other gene transcripts in M. smithii. with a key involvement in methanogenesis were also affected. Experimental confirmation that CH4 production is inhibited by lovastatin in A. terreus-fermented rice straw paves the way for its evaluation as a feed additive for mitigating CH4 production in ruminants.

Gene expression cross-profiling in genetically modified industrial Saccharomyces cerevisiae strains during high-temperature ethanol production from xylose.

Science.gov (United States)

Ismail, Ku Syahidah Ku; Sakamoto, Takatoshi; Hatanaka, Haruyo; Hasunuma, Tomohisa; Kondo, Akihiko

2013-01-10

Production of ethanol from xylose at high temperature would be an economical approach since it reduces risk of contamination and allows both the saccharification and fermentation steps in SSF to be running at elevated temperature. Eight recombinant xylose-utilizing Saccharomyces cerevisiae strains developed from industrial strains were constructed and subjected to high-temperature fermentation at 38 °C. The best performing strain was sun049T, which produced up to 15.2 g/L ethanol (63% of the theoretical production), followed by sun048T and sun588T, both with 14.1 g/L ethanol produced. Via transcriptomic analysis, expression profiling of the top three best ethanol producing strains compared to a negative control strain, sun473T, led to the discovery of genes in common that were regulated in the same direction. Identification of the 20 most highly up-regulated and the 20 most highly down-regulated genes indicated that the cells regulate their central metabolism and maintain the integrity of the cell walls in response to high temperature. We also speculate that cross-protection in the cells occurs, allowing them to maintain ethanol production at higher concentration under heat stress than the negative controls. This report provides further transcriptomics information in the interest of producing a robust microorganism for high-temperature ethanol production utilizing xylose. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of the glutaminase genes of Aspergillus sojae involved in glutamate production during soy sauce fermentation.

Science.gov (United States)

Ito, Kotaro; Koyama, Yasuji; Hanya, Yoshiki

2013-01-01

Glutaminase, an enzyme that catalyzes the conversion of L-glutamine to L-glutamate, enhances the umami taste in soy sauce. The Aspergillus sojae genome contains 10 glutaminase genes. In this study, we estimated that approximately 60% of the glutamate in soy sauce is produced through the glutaminase reaction. To determine which glutaminase is involved in soy sauce glutamate production, we prepared soy sauces using single and multiple glutaminase gene disruptants of A. sojae. The glutamate concentration in soy sauce prepared using the ΔgahA-ΔgahB-ΔggtA-Δgls disruptant was approximately 60% lower than that in the control strain, whereas it was decreased by approximately 20-30% in the ΔgahA-ΔgahB disruptant. However, the glutamate concentration was unchanged in the soy sauces prepared using the ΔgahA-ΔggtA-Δgls and ΔgahB-ΔggtA-Δgls disruptants. These results indicate that four glutaminases are involved in glutamate production in soy sauce, and that the peptidoglutaminase activities of GahA and GahB increase the glutamate concentration in soy sauce.

Transcription and translation products of the cytolysin gene psm-mec on the mobile genetic element SCCmec regulate Staphylococcus aureus virulence.

Directory of Open Access Journals (Sweden)

Chikara Kaito

Full Text Available The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA contains two bidirectionally overlapping open reading frames (ORFs, the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA strain, or into the MW2 (USA400 and FRP3757 (USA300 strains, which are community-acquired MRSA (CA-MRSA strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus.

Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing.

Science.gov (United States)

Sun, Xiaowen; Wu, Hefang; Zhao, Genhai; Li, Zhemin; Wu, Xihua; Liu, Hui; Zheng, Zhiming

2018-04-02

The mycelial morphology of Aspergillus niger, a major filamentous fungus used for citric acid production, is important for citric acid synthesis during submerged fermentation. To investigate the involvement of the chitin synthase gene, chsC, in morphogenesis and citric acid production in A. niger, an RNAi system was constructed to silence chsC and the morphological mutants were screened after transformation. The compactness of the mycelial pellets was obviously reduced in the morphological mutants, with lower proportion of dispersed mycelia. These morphological changes have caused a decrease in viscosity and subsequent improvement in oxygen and mass transfer efficiency, which may be conducive for citric acid accumulation. All the transformants exhibited improvements in citric acid production; in particular, chsC-3 showed 42.6% higher production than the original strain in the shake flask. Moreover, the high-yield strain chsC-3 exhibited excellent citric acid production potential in the scale-up process.The citric acid yield and the conversion rate of glucose of chsC-3 were both improved by 3.6%, when compared with that of the original strain in the stirred tank bioreactor.

Modulation of steroidogenic gene expression and hormone production of H295R cells by pharmaceuticals and other environmentally active compounds

International Nuclear Information System (INIS)

Gracia, Tannia; Hilscherova, Klara; Jones, Paul D.; Newsted, John L.; Higley, Eric B.; Zhang, Xiaowei; Hecker, Markus; Murphy, Margaret B.; Yu, Richard M.K.; Lam, Paul K.S.; Wu, Rudolf S.S.; Giesy, John P.

2007-01-01

The H295R cell bioassay was used to evaluate the potential endocrine disrupting effects of 18 of the most commonly used pharmaceuticals in the United States. Exposures for 48 h with single pharmaceuticals and binary mixtures were conducted; the expression of five steroidogenic genes, 3βHSD2, CYP11β1, CYP11β2, CYP17 and CYP19, was quantified by Q-RT-PCR. Production of the steroid hormones estradiol (E2), testosterone (T) and progesterone (P) was also evaluated. Antibiotics were shown to modulate gene expression and hormone production. Amoxicillin up-regulated the expression of CYP11β2 and CYP19 by more than 2-fold and induced estradiol production up to almost 3-fold. Erythromycin significantly increased CYP11β2 expression and the production of P and E2 by 3.5- and 2.4-fold, respectively, while production of T was significantly decreased. The β-blocker salbutamol caused the greatest induction of CYP17, more than 13-fold, and significantly decreased E2 production. The binary mixture of cyproterone and salbutamol significantly down-regulated expression of CYP19, while a mixture of ethynylestradiol and trenbolone, increased E2 production 3.7-fold. Estradiol production was significantly affected by changes in concentrations of trenbolone, cyproterone, and ethynylestradiol. Exposures with individual pharmaceuticals showed the possible secondary effects that drugs may exert on steroid production. Results from binary mixture exposures suggested the possible type of interactions that may occur between drugs and the joint effects product of such interactions. Dose-response results indicated that although two chemicals may share a common mechanism of action the concentration effects observed may be significantly different

Transforming growth factor-β-induced gene product-h3 inhibits odontoblastic differentiation of dental pulp cells.

Science.gov (United States)

Serita, Suguru; Tomokiyo, Atsushi; Hasegawa, Daigaku; Hamano, Sayuri; Sugii, Hideki; Yoshida, Shinichiro; Mizumachi, Hiroyuki; Mitarai, Hiromi; Monnouchi, Satoshi; Wada, Naohisa; Maeda, Hidefumi

2017-06-01

The aim of this study was to investigate transforming growth factor-β-induced gene product-h3 (βig-h3) expression in dental pulp tissue and its effects on odontoblastic differentiation of dental pulp cells (DPCs). A rat direct pulp capping model was prepared using perforated rat upper first molars capped with mineral trioxide aggregate cement. Human DPCs (HDPCs) were isolated from extracted teeth. βig-h3 expression in rat dental pulp tissue and HDPCs was assessed by immunostaining. Mineralization of HDPCs was assessed by Alizarin red-S staining. Odontoblast-related gene expression in HDPCs was analyzed by quantitative RT-PCR. Expression of βig-h3 was detected in rat dental pulp tissue, and attenuated by direct pulp capping, while expression of interleukin-1β and tumor necrosis factor-α was increased in exposed pulp tissue. βig-h3 expression was also detected in HDPCs, with reduced expression during odontoblastic differentiation. The above cytokines reduced βig-h3 expression in HDPCs, and promoted their mineralization. Recombinant βig-h3 inhibited the expression of odontoblast-related genes and mineralization of HDPCs, while knockdown of βig-h3 gene expression promoted the expression of odontoblast-related genes in HDPCs. The present findings suggest that βig-h3 in DPCs may be involved in reparative dentin formation and that its expression is likely to negatively regulate this process. Copyright © 2017 Elsevier Ltd. All rights reserved.

L-lactic acid production by Aspergillus brasiliensis overexpressing the heterologous ldha gene from Rhizopus oryzae.

Science.gov (United States)

Liaud, Nadège; Rosso, Marie-Noëlle; Fabre, Nicolas; Crapart, Sylvaine; Herpoël-Gimbert, Isabelle; Sigoillot, Jean-Claude; Raouche, Sana; Levasseur, Anthony

2015-05-03

Lactic acid is the building block of poly-lactic acid (PLA), a biopolymer that could be set to replace petroleum-based plastics. To make lactic acid production cost-effective, the production process should be carried out at low pH, in low-nutrient media, and with a low-cost carbon source. Yeasts have been engineered to produce high levels of lactic acid at low pH from glucose but not from carbohydrate polymers (e.g. cellulose, hemicellulose, starch). Aspergilli are versatile microbial cell factories able to naturally produce large amounts of organic acids at low pH and to metabolize cheap abundant carbon sources such as plant biomass. However, they have never been used for lactic acid production. To investigate the feasibility of lactic acid production with Aspergillus, the NAD-dependent lactate dehydrogenase (LDH) responsible for lactic acid production by Rhizopus oryzae was produced in Aspergillus brasiliensis BRFM103. Among transformants, the best lactic acid producer, A. brasiliensis BRFM1877, integrated 6 ldhA gene copies, and intracellular LDH activity was 9.2 × 10(-2) U/mg. At a final pH of 1.6, lactic acid titer reached 13.1 g/L (conversion yield: 26%, w/w) at 138 h in glucose-ammonium medium. This extreme pH drop was subsequently prevented by switching nitrogen source from ammonium sulfate to Na-nitrate, leading to a final pH of 3 and a lactic acid titer of 17.7 g/L (conversion yield: 47%, w/w) at 90 h of culture. Final titer was further improved to 32.2 g/L of lactic acid (conversion yield: 44%, w/w) by adding 20 g/L glucose to the culture medium at 96 h. This strain was ultimately able to produce lactic acid from xylose, arabinose, starch and xylan. We obtained the first Aspergillus strains able to produce large amounts of lactic acid by inserting recombinant ldhA genes from R. oryzae into a wild-type A. brasiliensis strain. pH regulation failed to significantly increase lactic acid production, but switching nitrogen source and changing culture feed

Nitrous oxide production and mRNA expression analysis of nitrifying and denitrifying bacterial genes under floodwater disappearance and fertilizer application.

Science.gov (United States)

Riya, Shohei; Takeuchi, Yuki; Zhou, Sheng; Terada, Akihiko; Hosomi, Masaaki

2017-06-01

A pulse of nitrous oxide (N 2 O) emission has been observed following the disappearance of floodwater by drainage. However, its mechanism is not well understood. We conducted a column study to clarify the mechanism for N 2 O production during floodwater disappearance by using a microsensor and determining the bacterial gene expression. An increase in N 2 O flux was observed following floodwater disappearance after the addition of NH 4 + , with a corresponding increase in the concentrations of NO 3 - and dissolved N 2 O in the oxic and anoxic soil layers, respectively. The transcription level of the bacterial amoA mRNA did not change, while that of nirK mRNA increased sharply after an hour of floodwater disappearance. An additional anoxic soil slurry experiment demonstrated that the addition of NO 3 - induced the expression of nirK gene and caused a concomitant increase in N 2 O production. These findings suggest that NO 3 - production in the oxic layers is important as it provides a substrate and induces the synthesis of denitrification enzymes in the anoxic layer during N 2 O production.

Uses of antimicrobial genes from microbial genome

Science.gov (United States)

Sorek, Rotem; Rubin, Edward M.

2013-08-20

We describe a method for mining microbial genomes to discover antimicrobial genes and proteins having broad spectrum of activity. Also described are antimicrobial genes and their expression products from various microbial genomes that were found using this method. The products of such genes can be used as antimicrobial agents or as tools for molecular biology.

De novo transcriptomic analysis of an oleaginous microalga: pathway description and gene discovery for production of next-generation biofuels.

Directory of Open Access Journals (Sweden)

LingLin Wan

Full Text Available Eustigmatos cf. polyphem is a yellow-green unicellular soil microalga belonging to the eustimatophyte with high biomass and considerable production of triacylglycerols (TAGs for biofuels, which is thus referred to as an oleaginous microalga. The paucity of microalgae genome sequences, however, limits development of gene-based biofuel feedstock optimization studies. Here we describe the sequencing and de novo transcriptome assembly for a non-model microalgae species, E. cf. polyphem, and identify pathways and genes of importance related to biofuel production.We performed the de novo assembly of E. cf. polyphem transcriptome using Illumina paired-end sequencing technology. In a single run, we produced 29,199,432 sequencing reads corresponding to 2.33 Gb total nucleotides. These reads were assembled into 75,632 unigenes with a mean size of 503 bp and an N50 of 663 bp, ranging from 100 bp to >3,000 bp. Assembled unigenes were subjected to BLAST similarity searches and annotated with Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG orthology identifiers. These analyses identified the majority of carbohydrate, fatty acids, TAG and carotenoids biosynthesis and catabolism pathways in E. cf. polyphem.Our data provides the construction of metabolic pathways involved in the biosynthesis and catabolism of carbohydrate, fatty acids, TAG and carotenoids in E. cf. polyphem and provides a foundation for the molecular genetics and functional genomics required to direct metabolic engineering efforts that seek to enhance the quantity and character of microalgae-based biofuel feedstock.

Super gene alternation of magnetite and pyrite and the role of their alternation products in the fixation of uranium from the circulating media. Vol. 3

Energy Technology Data Exchange (ETDEWEB)

El-Gemmizi, M A [Nuclear Materials Authority, Cairo, (Egypt)

1996-03-01

In most of the Egyptian altered radioactive granites, highly magnetic heavy particles were found to be radioactive. They are a mixture of several iron oxide minerals which are products of super gene alternation of the preexisting hypo gene iron-bearing minerals especially magnetite and pyrite. The end products of this super gene alternation are mainly hydrated iron oxide minerals limonite and/or goethite. During the alternation, deformation and defects in the mineral structure took place, thereby promoting diffusion of the substitutional and interstitial ions (uranium) towards these sites. The mechanism of the alternation of the hypo gene iron-bearing minerals, magnetite and pyrite to form the secondary mineral hematite, limonite and goethite; and the role of these secondary minerals in fixing uranium from the circulating media, and as indicators to the radioactivity of the host rocks are discussed. 2 figs.

Super gene alternation of magnetite and pyrite and the role of their alternation products in the fixation of uranium from the circulating media. Vol. 3

International Nuclear Information System (INIS)

El-Gemmizi, M.A.

1996-01-01

In most of the Egyptian altered radioactive granites, highly magnetic heavy particles were found to be radioactive. They are a mixture of several iron oxide minerals which are products of super gene alternation of the preexisting hypo gene iron-bearing minerals especially magnetite and pyrite. The end products of this super gene alternation are mainly hydrated iron oxide minerals limonite and/or goethite. During the alternation, deformation and defects in the mineral structure took place, thereby promoting diffusion of the substitutional and interstitial ions (uranium) towards these sites. The mechanism of the alternation of the hypo gene iron-bearing minerals, magnetite and pyrite to form the secondary mineral hematite, limonite and goethite; and the role of these secondary minerals in fixing uranium from the circulating media, and as indicators to the radioactivity of the host rocks are discussed. 2 figs

Ekspresi produk gen laten virus epstein-barr pada karsinoma sel skuamosa rongga mulut (The expressions of latent gene product of epstein-barr virus in oral squamous cell carcinoma

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Theresia Indah Budhy S

2005-06-01

Full Text Available Squamous cell carcinoma (SCC is a type of cancer often found in oral cavity and the area of head and neck at about 90%. Based on the geographical incidence oral SCC (OSCC has many types of different emerging. This case probably has connection with ethnic group, habit and social and economical condition. In East Java, the incidence is about 2.64% and it increases every year. The virus is known as one of the main factors that result in this disease. Epstein-Barr virus (EBV has potential capability of carcinogenesis. EBV is the family of herpesviridae that can infect cell through the linking of CD 21 receptor of the epithel with glycol protein 350/220 of the virus capsule. After primary infection, the virus will form latent-gene in human cell. Periodically, the latent-gene product can disturb proliferation and apoptotic regulator. In Indonesia, the expression of EBV latent geneproduct in the OSCC has not been reported yet. This study wanted to know the expression of EBV latent gene product found in the OSCC. This study found 25 cases of OSCC in which 17 were infected by EBV. Detection of EBV infection could be done by insitu hybridization to identify RNA EBV (EBER. To find the expression of EBV latent gene product, immunohistochemical analysis was done. The conclusion was that the emerging of expression of EBV latent gene product in OSCC were latent membrane protein-1 (LMP-1, EBV nuclear antigen-1 (EBNA-1 and RNA EBV (EBER. They were 28.28%, 25.26% and 46.47%. It was suggested to do the following research on OSCC infected by EBV and the emerging of expression of EBV latent gene product with regulator gene of proliferation and apoptotic in OSCC.

The identification of aluminium-resistance genes provides opportunities for enhancing crop production on acid soils.

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Ryan, P R; Tyerman, S D; Sasaki, T; Furuichi, T; Yamamoto, Y; Zhang, W H; Delhaize, E

2011-01-01

Acid soils restrict plant production around the world. One of the major limitations to plant growth on acid soils is the prevalence of soluble aluminium (Al(3+)) ions which can inhibit root growth at micromolar concentrations. Species that show a natural resistance to Al(3+) toxicity perform better on acid soils. Our understanding of the physiology of Al(3+) resistance in important crop plants has increased greatly over the past 20 years, largely due to the application of genetics and molecular biology. Fourteen genes from seven different species are known to contribute to Al(3+) tolerance and resistance and several additional candidates have been identified. Some of these genes account for genotypic variation within species and others do not. One mechanism of resistance which has now been identified in a range of species relies on the efflux of organic anions such as malate and citrate from roots. The genes controlling this trait are members of the ALMT and MATE families which encode membrane proteins that facilitate organic anion efflux across the plasma membrane. Identification of these and other resistance genes provides opportunities for enhancing the Al(3+) resistance of plants by marker-assisted breeding and through biotechnology. Most attempts to enhance Al(3+) resistance in plants with genetic engineering have targeted genes that are induced by Al(3+) stress or that are likely to increase organic anion efflux. In the latter case, studies have either enhanced organic anion synthesis or increased organic anion transport across the plasma membrane. Recent developments in this area are summarized and the structure-function of the TaALMT1 protein from wheat is discussed.

Integrative characterization of germ cell-specific genes from mouse spermatocyte UniGene library

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Eddy Edward M

2007-07-01

Full Text Available Abstract Background The primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes. Results We analyzed the mouse spermatocyte UniGene library containing 2155 gene-oriented transcript clusters. We predict that 11% of these genes are testis-specific and systematically identified 24 authentic genes specifically and abundantly expressed in the testis via in silico and in vitro approaches. Northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. Expression analysis revealed developmentally regulated and stage-specific expression patterns in all of the genes. We further analyzed the genes at the protein and cellular levels. Transfection assays performed using GC-2 cells provided information on the cellular characteristics of the gene products. In addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. Our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. In particular, we found for the first time that Mm.333010, predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein. Conclusion We identify 24 authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. Our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction.

RhMKK9, a rose MAP KINASE KINASE gene, is involved in rehydration-triggered ethylene production in rose gynoecia.

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Chen, Jiwei; Zhang, Qian; Wang, Qigang; Feng, Ming; Li, Yang; Meng, Yonglu; Zhang, Yi; Liu, Guoqin; Ma, Zhimin; Wu, Hongzhi; Gao, Junping; Ma, Nan

2017-02-23

Flower opening is an important process in the life cycle of flowering plants and is influenced by various endogenous and environmental factors. Our previous work demonstrated that rose (Rosa hybrida) flowers are highly sensitive to dehydration during flower opening and the water recovery process after dehydration induced ethylene production rapidly in flower gynoecia. In addition, this temporal- and spatial-specific ethylene production is attributed to a transient but robust activation of the rose MAP KINASE6-ACC SYNTHASE1 (RhMPK6-RhACS1) cascade in gynoecia. However, the upstream component of RhMPK6-RhACS1 is unknown, although RhMKK9 (MAP KINASE KINASE9), a rose homologue of Arabidopsis MKK9, could activate RhMPK6 in vitro. In this study, we monitored RhMKK2/4/5/9 expression, the potential upstream kinase to RhMPK6, in rose gynoecia during dehydration and rehydration. We found only RhMKK9 was rapidly and strongly induced by rehydration. Silencing of RhMKK9 significantly decreased rehydration-triggered ethylene production. Consistently, the expression of several ethylene-responsive genes was down regulated in the petals of RhMKK9-silenced flowers. Moreover, we detected the DNA methylation level in the promoter and gene body of RhMKK9 by Chop-PCR. The results showed that rehydration specifically elevated the DNA methylation level on the RhMKK9 gene body, whereas it resulted in hypomethylation in its promoter. Our results showed that RhMKK9 possibly acts as the upstream component of the RhMKK9-RhMPK6-RhACS1 cascade and is responsible for water recovery-triggered ethylene production in rose gynoecia, and epigenetic DNA methylation is involved in the regulation of RhMKK9 expression by rehydration.

Genetic manipulation of longevity-related genes as a tool to regulate yeast life span and metabolite production during winemaking.

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Orozco, Helena; Matallana, Emilia; Aranda, Agustín

2013-01-02

Yeast viability and vitality are essential for different industrial processes where the yeast Saccharomyces cerevisiae is used as a biotechnological tool. Therefore, the decline of yeast biological functions during aging may compromise their successful biotechnological use. Life span is controlled by a variety of molecular mechanisms, many of which are connected to stress tolerance and genomic stability, although the metabolic status of a cell has proven a main factor affecting its longevity. Acetic acid and ethanol accumulation shorten chronological life span (CLS), while glycerol extends it. Different age-related gene classes have been modified by deletion or overexpression to test their role in longevity and metabolism. Overexpression of histone deacetylase SIR2 extends CLS and reduces acetate production, while overexpression of SIR2 homolog HST3 shortens CLS, increases the ethanol level, and reduces acetic acid production. HST3 overexpression also enhances ethanol tolerance. Increasing tolerance to oxidative stress by superoxide dismutase SOD2 overexpression has only a moderate positive effect on CLS. CLS during grape juice fermentation has also been studied for mutants on several mRNA binding proteins that are regulators of gene expression at the posttranscriptional level; we found that NGR1 and UTH4 deletions decrease CLS, while PUF3 and PUB1 deletions increase it. Besides, the pub1Δ mutation increases glycerol production and blocks stress granule formation during grape juice fermentation. Surprisingly, factors relating to apoptosis, such as caspase Yca1 or apoptosis-inducing factor Aif1, play a positive role in yeast longevity during winemaking as their deletions shorten CLS. Manipulation of regulators of gene expression at both transcriptional (i.e., sirtuins) and posttranscriptional (i.e., mRNA binding protein Pub1) levels allows to modulate yeast life span during its biotechnological use. Due to links between aging and metabolism, it also influences the

Expression of a mutated SPT15 gene in Saccharomyces cerevisiae enhances both cell growth and ethanol production in microaerobic batch, fed-batch, and simultaneous saccharification and fermentations.

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Seong, Yeong-Je; Park, Haeseong; Yang, Jungwoo; Kim, Soo-Jung; Choi, Wonja; Kim, Kyoung Heon; Park, Yong-Cheol

2017-05-01

The SPT15 gene encodes a Saccharomyces cerevisiae TATA-binding protein, which is able to globally control the transcription levels of various metabolic and regulatory genes. In this study, a SPT15 gene mutant (S42N, S78R, S163P, and I212N) was expressed in S. cerevisiae BY4741 (BSPT15-M3), of which effects on fermentative yeast properties were evaluated in a series of culture types. By applying different nitrogen sources and air supply conditions in batch culture, organic nitrogen sources and microaerobic condition were decided to be more favorable for both cell growth and ethanol production of the BSPT15-M3 strain than the control S. cerevisiae BY4741 strain expressing the SPT15 gene (BSPT15wt). Microaerobic fed-batch cultures of BSPT15-M3 with glucose shock in the presence of high ethanol content resulted in a 9.5-13.4% higher glucose consumption rate and ethanol productivity than those for the BSPT15wt strain. In addition, BSPT15-M3 showed 4.5 and 3.9% increases in ethanol productivity from cassava hydrolysates and corn starch in simultaneous saccharification and fermentation processes, respectively. It was concluded that overexpression of the mutated SPT15 gene would be a potent strategy to develop robust S. cerevisiae strains with enhanced cell growth and ethanol production abilities.

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

Science.gov (United States)

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

Lack of a Functional VHL Gene Product Sensitizes Renal Cell Carcinoma Cells to the Apoptotic Effects of the Protein Synthesis Inhibitor Verrucarin A

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Girma M. Woldemichael

2012-08-01

Full Text Available Verrucarin A (VA is a small molecule derived from the fungal plant pathogen Myrothecium verrucaria and was identified as a selective inhibitor of clear cell renal cell carcinoma (CCRCC cell proliferation in a high-throughput screen of a library of naturally occurring small molecules. CCRCC arises as a result of loss-of-function mutations in the von Hippel-Lindau (VHL gene. Here we show that VA inhibits protein translation initiation culminating in apoptosis through the extrinsic signaling pathway. Reintroduction of the VHL gene in CCRCC cells afforded resistance to VA's apoptotic effects. This resistance is mediated in part by the formation of stress granules that entrap signaling molecules that initiate the apoptotic signaling cascade. The VHL gene product was found to be a component of stress granules that develop as result of VA treatment. These findings reveal an important role for the VHL gene product in cytotoxic stress response and have important implications for the rational development of VA-related compounds in chemotherapeutic targeting of CCRCC.

RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia coli strain for Cre mediated production of minicircle DNA

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Coutelle Charles

2006-03-01

Full Text Available Abstract Background Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions. Results We describe here the construction of the RecA-deficient minicircle DNA producer Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure minicircle DNA from the obtained recA- producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybrid loxP66

The Use of Cytochrome b Gene as a Specific Marker of the Rat Meat (Rattus norvegicus on Meat and Meat Products

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C. Sumantri

2012-04-01

Full Text Available Falsification of the origin of livestock meat and its processed with rat meat is a problem that must be overcome to ensure food safety. One way that is often used to detect forgeries by using cytochrome b gene as a marker. The purpose of this study was to create a specific primer derived from cytochrome b sequences in rat (Rattus norvegicus as the DNA marker to detect any contamination of rat meat on fresh livestock meat and its processed meat products. Meatballs were made from beef meat with the addition of rat 1%-25%, and the meatballs were obtained from traditional markets. DNA extraction was conducted from seven species (goat, chicken, cattle, sheep, pig, horse, and rat by using phenol-chloroform. The highest success rate in detecting the presence of rat meat in a mixture of beef meatballs at concentration of 15% was 100%. The specific fragment of cytochrome b gene in R. norvegicus has no similarity with the cytochrome b gene from six other species, so it can be used as molecular markers to detect the presence of rat meat contamination in the processed of meat products. Amplified fragment length for goats, chickens, cattle, sheep, pigs, horses, and rats 157, 227, 274, 331, 398, 439 and 603 bp respectively. The amplification of cytochrome b gene in seven species of animals with different fragment length indicated the specificity of cytochrome b gene sequences among species.

Effect of Co-overexpression of Nisin Key Genes on Nisin Production Improvement in Lactococcus lactis LS01.

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Ni, Zhi-Jian; Zhang, Xiao-Yuan; Liu, Fei; Wang, Miao; Hao, Rong-Hua; Ling, Pei-Xue; Zhu, Xi-Qiang

2017-06-01

Nisin is a small antimicrobial peptide produced by several subset strains of Lactococcus lactis. To improve nisin yield in the producer L. lactis LS01, we proposed a successive fusion of nisA with nisRK and nisFEG into a single shuttle expression vector pMG36e under the control of the native strong constitutive promoter p32. Subsequently, the recombinant vectors were transplanted into the producer cell through electroporation. Nisin productivity was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioactivity assays. Expression of nisin peptide was detected by agar diffusion bioassay, and the transcriptional levels of the target genes involved in nisin biosynthesis were investigated via semi-quantitative reverse transcription PCR expression analysis using 16S ribosomal RNA (rRNA) as an internal control. Results suggested that the introduction of empty plasmid did not affect nisin production of L. lactis LS01, whereas by our rational construction and screening, the engineered strain co-overexpressing nisA, nisRK, and nisFEG achieved a maximum increment in bioactive nisin production with a yield of 2470 IU/ml in shake flasks and 4857 IU/ml in 1.0-l fermenters, which increased by approximately 66.3 and 52.6% (P < 0.05), respectively, compared with that of the original strain under the given fermentation conditions. Meanwhile, the transcriptional analysis revealed that the expression of most of these multicopy genes except nisE at transcriptional level were upregulated in the two recombinant strains (LS01/pAR and LS01/pARF), possibly contributing to the improved nisin production. Therefore, this study would provide a potential strategy to improve the economic benefits of nisin manufacture for large-scale industrial production.

Heterologous expression of oxytetracycline biosynthetic gene cluster in Streptomyces venezuelae WVR2006 to improve production level and to alter fermentation process.

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Yin, Shouliang; Li, Zilong; Wang, Xuefeng; Wang, Huizhuan; Jia, Xiaole; Ai, Guomin; Bai, Zishang; Shi, Mingxin; Yuan, Fang; Liu, Tiejun; Wang, Weishan; Yang, Keqian

2016-12-01

Heterologous expression is an important strategy to activate biosynthetic gene clusters of secondary metabolites. Here, it is employed to activate and manipulate the oxytetracycline (OTC) gene cluster and to alter OTC fermentation process. To achieve these goals, a fast-growing heterologous host Streptomyces venezuelae WVR2006 was rationally selected among several potential hosts. It shows rapid and dispersed growth and intrinsic high resistance to OTC. By manipulating the expression of two cluster-situated regulators (CSR) OtcR and OtrR and precursor supply, the OTC production level was significantly increased in this heterologous host from 75 to 431 mg/l only in 48 h, a level comparable to the native producer Streptomyces rimosus M4018 in 8 days. This work shows that S. venezuelae WVR2006 is a promising chassis for the production of secondary metabolites, and the engineered heterologous OTC producer has the potential to completely alter the fermentation process of OTC production.

Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene

OpenAIRE

Chang, Zhenyi; Chen, Zhufeng; Wang, Na; Xie, Gang; Lu, Jiawei; Yan, Wei; Zhou, Junli; Tang, Xiaoyan; Deng, Xing Wang

2016-01-01

Nuclear male sterility is common in flowering plants, but its application in hybrid breeding and seed production is limited because of the inability to propagate a pure male sterile line for commercial hybrid seed production. Here, we characterized a rice nuclear gene essential for sporophytic male fertility and constructed a male sterility system that can propagate the pure male sterile seeds on a large scale. This system is fundamentally advantageous over the current cytoplasmic male steril...

Cloning of D-lactate dehydrogenase genes of Lactobacillus delbrueckii subsp. bulgaricus and their roles in D-lactic acid production.

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Huang, Yanna; You, Chunping; Liu, Zhenmin

2017-07-01

Lactobacillus delbrueckii subsp. bulgaricus is a heterogenous lactic acid bacterium that converts pyruvate mainly to D-lactic acid using D-lactate dehydrogenases (D-LDHs), whose functional properties remain poorly characterized. Here, the D-LDHs genes (ldb0101, ldb0813, ldb1010, ldb1147 and ldb2021) were cloned and overexpressed in Escherichia coli JM109 from an inducible pUC18 vector, respectively, and the resulting strains were compared in terms of D-lactic acid production. The strain expressing ldb0101 and ldb1010 gene individually produced more D-lactate than other three strains. Further study revealed that Ldb0101 activity was down-regulated by the oxygen and, therefore, achieved a highest titer of D-lactate (1.94 g/L) under anaerobic condition, and introduction of ldb1010 gene enhanced D-lactate formation (0.94 and 0.85 g/L, respectively) both in aerobic and anaerobic conditions due to a relatively stable q d-lactate . Our results suggested that the enzyme Ldb0101 and Ldb1010 played a role of more importance in D-lactate formation. To the best of our knowledge, we demonstrate for the first time the roles of different D-LDH homologs from L. bulgaricus in D-lactic acid production.

Survey of Expression of Aflatoxin Production Regulator Gene (aflR in Aspergillus Parasiticus by Alpinia Galanga L and Dorema Aucheri

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Zahra Arab-Mazar

2018-01-01

Full Text Available Background: Aflatoxins are one of the highly toxic secondary metabolites, which are mainly produced by Aspergillus parasiticus. This species frequently cause of food and agricultural products contamination including cereals, peanuts, and crops in the field. During recent years, researchers have considered research on elimination of aflatoxin and antifungal effects of medicinal herbals, such as Alpinia galanga L and Dorema aucheri. In this study, the effect of A.galanga L and D.aucheri a natural compound was examined on Aspergillus parasiticus growth, aflatoxins production and the aflR gene expression.Materials and Methods: Antifungal susceptibility A.galanga L and D.aucheri was performed according to CLSI document M38-A2. Quantitative changes in aflR gene level of expression were analyzed by Real-time PCR method.Results: Our result obtained that the MIC of extracts on A. parasiticus growth 250 mg/mL for D.aucheri and 800 mg/mL for A.galanga L. D.aucheri has antitoxic properties as well as its effective ability to decrease aflatoxin production. The level of aflR gene expression was decreased significantly after the exposure of fungal cell to D.aucheri extract, but A.galanga L didn’t have significant effect.Conclusion: This research indicated that D.aucheri has antifungal effects more than A.galanga L. Due to our obtained result we can suggest that D.aucheri herbal extract may have antifungal potential in medicine or agriculture.

Deletion and Gene Expression Analyses Define the Paxilline Biosynthetic Gene Cluster in Penicillium paxilli

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Emily J. Parker

2013-08-01

Full Text Available The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse. This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis.

End-to-end gene fusions and their impact on the production of multifunctional biomass degrading enzymes

International Nuclear Information System (INIS)

Rizk, Mazen; Antranikian, Garabed; Elleuche, Skander

2012-01-01

Highlights: ► Multifunctional enzymes offer an interesting approach for biomass degradation. ► Size and conformation of separate constructs play a role in the effectiveness of chimeras. ► A connecting linker allows for maximal flexibility and increased thermostability. ► Genes with functional similarities are the best choice for fusion candidates. -- Abstract: The reduction of fossil fuels, coupled with its increase in price, has made the search for alternative energy resources more plausible. One of the topics gaining fast interest is the utilization of lignocellulose, the main component of plants. Its primary constituents, cellulose and hemicellulose, can be degraded by a series of enzymes present in microorganisms, into simple sugars, later used for bioethanol production. Thermophilic bacteria have proven to be an interesting source of enzymes required for hydrolysis since they can withstand high and denaturing temperatures, which are usually required for processes involving biomass degradation. However, the cost associated with the whole enzymatic process is staggering. A solution for cost effective and highly active production is through the construction of multifunctional enzyme complexes harboring the function of more than one enzyme needed for the hydrolysis process. There are various strategies for the degradation of complex biomass ranging from the regulation of the enzymes involved, to cellulosomes, and proteins harboring more than one enzymatic activity. In this review, the construction of multifunctional biomass degrading enzymes through end-to-end gene fusions, and its impact on production and activity by choosing the enzymes and linkers is assessed.

Exploring autophagy with Gene Ontology

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2018-01-01

ABSTRACT Autophagy is a fundamental cellular process that is well conserved among eukaryotes. It is one of the strategies that cells use to catabolize substances in a controlled way. Autophagy is used for recycling cellular components, responding to cellular stresses and ridding cells of foreign material. Perturbations in autophagy have been implicated in a number of pathological conditions such as neurodegeneration, cardiac disease and cancer. The growing knowledge about autophagic mechanisms needs to be collected in a computable and shareable format to allow its use in data representation and interpretation. The Gene Ontology (GO) is a freely available resource that describes how and where gene products function in biological systems. It consists of 3 interrelated structured vocabularies that outline what gene products do at the biochemical level, where they act in a cell and the overall biological objectives to which their actions contribute. It also consists of ‘annotations’ that associate gene products with the terms. Here we describe how we represent autophagy in GO, how we create and define terms relevant to autophagy researchers and how we interrelate those terms to generate a coherent view of the process, therefore allowing an interoperable description of its biological aspects. We also describe how annotation of gene products with GO terms improves data analysis and interpretation, hence bringing a significant benefit to this field of study. PMID:29455577

Increased yield of PCR products by addition of T4 gene 32 protein to the SMART PCR cDNA synthesis system.

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Villalva, C; Touriol, C; Seurat, P; Trempat, P; Delsol, G; Brousset, P

2001-07-01

Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.

De Novo Transcriptomic Analysis of an Oleaginous Microalga: Pathway Description and Gene Discovery for Production of Next-Generation Biofuels

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Wan, LingLin; Han, Juan; Sang, Min; Li, AiFen; Wu, Hong; Yin, ShunJi; Zhang, ChengWu

2012-01-01

Background Eustigmatos cf. polyphem is a yellow-green unicellular soil microalga belonging to the eustimatophyte with high biomass and considerable production of triacylglycerols (TAGs) for biofuels, which is thus referred to as an oleaginous microalga. The paucity of microalgae genome sequences, however, limits development of gene-based biofuel feedstock optimization studies. Here we describe the sequencing and de novo transcriptome assembly for a non-model microalgae species, E. cf. polyphem, and identify pathways and genes of importance related to biofuel production. Results We performed the de novo assembly of E. cf. polyphem transcriptome using Illumina paired-end sequencing technology. In a single run, we produced 29,199,432 sequencing reads corresponding to 2.33 Gb total nucleotides. These reads were assembled into 75,632 unigenes with a mean size of 503 bp and an N50 of 663 bp, ranging from 100 bp to >3,000 bp. Assembled unigenes were subjected to BLAST similarity searches and annotated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology identifiers. These analyses identified the majority of carbohydrate, fatty acids, TAG and carotenoids biosynthesis and catabolism pathways in E. cf. polyphem. Conclusions Our data provides the construction of metabolic pathways involved in the biosynthesis and catabolism of carbohydrate, fatty acids, TAG and carotenoids in E. cf. polyphem and provides a foundation for the molecular genetics and functional genomics required to direct metabolic engineering efforts that seek to enhance the quantity and character of microalgae-based biofuel feedstock. PMID:22536352

Production of artemisinin and its derivatives in hairy roots of Artemisia dubia induced by rolA gene transformation

International Nuclear Information System (INIS)

Amanullah, M.; Mirza, B.; Zia, M.

2016-01-01

Artemisinin and its derivatives are phytochemical constituents of genus Artemisia. Demand of these plant secondary metabolitesis increasing due to their immense therapeutic significance. Besides their established antimalarial role, recent studies have also disclosed their anticancer potentials. It has made imperative to develop new and efficient sources of these compounds. Inherent synthetic challenges give biological sources preference over chemical synthesis of artemisinin and its derivatives. Therefore, genetic improvement of plants and, rather less preferentially, microbes is focus of current research to gain increase productivity of these valuable drugs. This study has analyzed A. dubiaas potential source of artemisinin and its derivatives. Transformation of Artemisia dubia was carried out using A. tumefaciens strain LBA 4404 containing rolA gene constructed on pRB 29. Healthy and acclimatizable transgenic plants were produced using optimized concentrations of BAP and NAA. Previously acclimatized rol ABC transgenic plants were also In vitro regenerated for comparative analysis of artemisinin and its derivatives. PCR amplification of rolA gene was done to confirm the integration of T-DNA in transgenic plants.TLC analysis was performed to evaluate comparative production of artemisinin and derivatives in rolA and rol ABC transgenic A. dubia. It revealed that rolA transgenic plants contain comparable amounts of these metabolites. Both type of transgenic plants manifested the enhancement of other uncharacterized compounds as well. Besides systematic optimization of In vitro regenerative protocol for Artemisia dubia, relative regeneration ability of rol transgenic and controlplants was also assessed at four regenerative stages. It was observed that unlike control, rol transgenic plants showed best root induction only on combination of auxins and cytokines. It was concluded that rol genes transformation of plants is an efficient tool to enhance their secondary

Genes Required for Free Phage Production are Essential for Pseudomonas aeruginosa Chronic Lung Infections.

Science.gov (United States)

Lemieux, Andrée-Ann; Jeukens, Julie; Kukavica-Ibrulj, Irena; Fothergill, Joanne L; Boyle, Brian; Laroche, Jérôme; Tucker, Nicholas P; Winstanley, Craig; Levesque, Roger C

2016-02-01

The opportunistic pathogen Pseudomonas aeruginosa causes chronic lung infection in patients with cystic fibrosis. The Liverpool Epidemic Strain LESB58 is highly resistant to antibiotics, transmissible, and associated with increased morbidity and mortality. Its genome contains 6 prophages and 5 genomic islands. We constructed a polymerase chain reaction (PCR)-based signature-tagged mutagenesis library of 9216 LESB58 mutants and screened the mutants in a rat model of chronic lung infection. A total of 162 mutants were identified as defective for in vivo maintenance, with 11 signature-tagged mutagenesis mutants having insertions in prophage and genomic island genes. Many of these mutants showed both diminished virulence and reduced phage production. Transcription profiling by quantitative PCR and RNA-Seq suggested that disruption of these prophages had a widespread trans-acting effect on the transcriptome. This study demonstrates that temperate phages play a pivotal role in the establishment of infection through modulation of bacterial host gene expression. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Improvement of exopolysaccharide production in Lactobacillus casei LC2W by overexpression of NADH oxidase gene.

Science.gov (United States)

Li, Nan; Wang, Yuanlong; Zhu, Ping; Liu, Zhenmin; Guo, Benheng; Ren, Jing

2015-02-01

Lactobacillus casei LC2W is an exopolysaccharide (EPS)-producing strain with probiotic effects. To investigate the regulation mechanism of EPS biosynthesis and to improve EPS production through cofactor engineering, a H₂O-forming NADH oxidase gene was cloned from Streptococcus mutans and overexpressed in L. casei LC2W under the control of constitutive promoter P₂₃. The recombinant strain LC-nox exhibited 0.854 U/mL of NADH oxidase activity, which was elevated by almost 20-fold in comparison with that of wild-type strain. As a result, overexpression of NADH oxidase resulted in a reduction in growth rate. In addition, lactate production was decreased by 22% in recombinant strain. It was proposed that more carbon source was saved and used for the biosynthesis of EPS, the production of which was reached at 219.4 mg/L, increased by 46% compared to that of wild-type strain. This work provided a novel and convenient genetic approach to manipulate metabolic flux and to increase EPS production. To the best of our knowledge, this is the first report which correlates cofactor engineering with EPS production. Copyright © 2015 Elsevier GmbH. All rights reserved.

In Silico Gene-Level Evolution Explains Microbial Population Diversity through Differential Gene Mobility

NARCIS (Netherlands)

van Dijk, Bram; Hogeweg, P.

2016-01-01

Microbial communities can show astonishing ecological and phylogenetic diversity. What is the role of pervasive horizontal gene transfer (HGT) in shaping this diversity in the presence of clonally expanding "killer strains"? Does HGT of antibiotic production and resistance genes erase phylogenetic

Postreplication repair gap filling in an Escherichia coli strain deficient in dnaB gene product

International Nuclear Information System (INIS)

Johnson, R.C.

1975-01-01

Gaps in daughter-strand DNA synthesized after exposure of Escherichia coli E279 to ultraviolet light are filled during reincubation at 30 0 C for 20 min. Escherichia coli E279 is phenotypically DnaB - when incubated at 43 0 C. Cells incubated at 43 0 C were tested for their ability to complete postreplication repair gap filling. It is concluded that the dnaB gene product is essential for postreplication repair gap filling and that the inhibition seen is not initially the result of degradation

Virulence genes in a probiotic E. coli product with a recorded long history of safe use

Science.gov (United States)

Zschüttig, Anke; Beimfohr, Claudia; Geske, Thomas; Auerbach, Christian; Cook, Helen; Zimmermann, Kurt; Gunzer, Florian

2015-01-01

The probiotic product Symbioflor2 (DSM 17252) is a bacterial concentrate of six different Escherichia coli genotypes, whose complete genome sequences are compared here, between each other as well as to other E. coli genomes. The genome sequences of Symbioflor2 E. coli components contained a number of virulence-associated genes. Their presence seems to be in conflict with a recorded history of safe use, and with the observed low frequency of adverse effects over a period of more than 6 years. The genome sequences were used to identify unique sequences for each component, for which strain-specific hybridization probes were designed. A colonization study was conducted whereby five volunteers were exposed to an exceptionally high single dose. The results showed that the probiotic E. coli could be detected for 3 months or longer in their stools, and this was in particular the case for those components containing higher numbers of virulence-associated genes. Adverse effects from this long-term colonization were absent. Thus, the presence of the identified virulence genes does not result in a pathogenic phenotype in the genetic background of these probiotic E. coli. PMID:25883796

Phylogenetic grouping and distribution of virulence genes in Escherichia coli along the production and supply chain of pork around Hubei, China.

Science.gov (United States)

Khan, Sher Bahadar; Zou, Geng; Cheng, Yu-Ting; Xiao, Ran; Li, Lu; Wu, Bin; Zhou, Rui

2017-06-01

Escherichia coli is an important foodborne zoonotic pathogen. A total of 285 strains of E. coli were isolated from the production and supply chain of pork in Hubei, China and characterized. Their phylogroups (A, B1, B2, and D) and virulence genes of public health importance become more and more diverse along the production and supply chain. Copyright © 2016. Published by Elsevier B.V.

Enhanced pest resistance and increased phenolic production in maize callus transgenically expressing a maize chalcone isomerase -3 like gene

Science.gov (United States)

Significant losses in maize production are due to damage by insects and ear rot fungi. A gene designated as chalcone-isomerase-like, located in a quantitative trait locus for resistance to Fusarium ear rot fungi, was cloned from a Fusarium ear rot resistant inbred and transgenically expressed in mai...

Caprine and ovine Greek dairy products: The official German method generates false-positive results due to κ-casein gene polymorphism.

Science.gov (United States)

Tsartsianidou, V; Triantafillidou, D; Karaiskou, N; Tarantili, P; Triantafillidis, G; Georgakis, E; Triantafyllidis, A

2017-05-01

Caseins are widely used for species identification of dairy products. Isoelectric focusing (IEF) of para-κ-casein peptide is used as the official German method for the differentiation between caprine (isoform A) and ovine (isoform B) dairy products, based on their different isoelectric points. The discrimination between Greek goat and ewe dairy products using IEF has, however, been shown to be problematic because of the existence of the ewe isoform in milk from Greek indigenous dairy goats. This could be due to nucleotide polymorphisms within the goat κ-casein gene of Greek indigenous breeds, which alter the isoelectric point of the para-κ-casein peptide and lead to false positive results. Previous DNA analysis of the goat κ-casein gene has shown high levels of polymorphism; however, no such information is available for Greek indigenous dairy goats. Therefore, 87 indigenous dairy goats were sequenced at exon IV of κ-casein gene. In total, 9 polymorphic sites were detected. Three nonsynonymous point mutations were identified, which change the isoelectric point of the goat para-κ-casein peptide so that it appears identical to that of the ewe peptide. Ten composite genotypes were reconstructed and 6 of them included the problematic point mutations. For the verification of genetic results, IEF was carried out. Both goat and ewe patterns appeared in the problematic genotypes. The frequency of these genotypes could be characterized as moderate (0.23) to high (0.60) within Greek indigenous breeds. However, this is not an issue restricted to Greece, as such genotypes have been detected in various non-Greek goat breeds. In conclusion, IEF based on the official German method is certainly inappropriate for ovine and caprine discrimination concerning Greek dairy goat products, and consequently a new method should be established. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Lack of a Functional VHL Gene Product Sensitizes Renal Cell Carcinoma Cells to the Apoptotic Effects of the Protein Synthesis Inhibitor Verrucarin A12

Science.gov (United States)

Woldemichael, Girma M; Turbyville, Thomas J; Vasselli, James R; Linehan, W Marston; McMahon, James B

2012-01-01

Verrucarin A (VA) is a small molecule derived from the fungal plant pathogen Myrothecium verrucaria and was identified as a selective inhibitor of clear cell renal cell carcinoma (CCRCC) cell proliferation in a high-throughput screen of a library of naturally occurring small molecules. CCRCC arises as a result of loss-of-function mutations in the von Hippel-Lindau (VHL) gene. Here we show that VA inhibits protein translation initiation culminating in apoptosis through the extrinsic signaling pathway. Reintroduction of the VHL gene in CCRCC cells afforded resistance to VA's apoptotic effects. This resistance is mediated in part by the formation of stress granules that entrap signaling molecules that initiate the apoptotic signaling cascade. The VHL gene product was found to be a component of stress granules that develop as result of VA treatment. These findings reveal an important role for the VHL gene product in cytotoxic stress response and have important implications for the rational development of VA-related compounds in chemotherapeutic targeting of CCRCC. PMID:22952429

Harnessing Genetic Diversity of Wild Gene Pools to Enhance Wheat Crop Production and Sustainability: Challenges and Opportunities

Directory of Open Access Journals (Sweden)

Carla Ceoloni

2017-12-01

Full Text Available Wild species are extremely rich resources of useful genes not available in the cultivated gene pool. For species providing staple food to mankind, such as the cultivated Triticum species, including hexaploid bread wheat (Triticum aestivum, 6x and tetraploid durum wheat (T. durum, 4x, widening the genetic base is a priority and primary target to cope with the many challenges that the crop has to face. These include recent climate changes, as well as actual and projected demographic growth, contrasting with reduction of arable land and water reserves. All of these environmental and societal modifications pose major constraints to the required production increase in the wheat crop. A sustainable approach to address this task implies resorting to non-conventional breeding strategies, such as “chromosome engineering”. This is based on cytogenetic methodologies, which ultimately allow for the incorporation into wheat chromosomes of targeted, and ideally small, chromosomal segments from the genome of wild relatives, containing the gene(s of interest. Chromosome engineering has been successfully applied to introduce into wheat genes/QTL for resistance to biotic and abiotic stresses, quality attributes, and even yield-related traits. In recent years, a substantial upsurge in effective alien gene exploitation for wheat improvement has come from modern technologies, including use of molecular markers, molecular cytogenetic techniques, and sequencing, which have greatly expanded our knowledge and ability to finely manipulate wheat and alien genomes. Examples will be provided of various types of stable introgressions, including pyramiding of different alien genes/QTL, into the background of bread and durum wheat genotypes, representing valuable materials for both species to respond to the needed novelty in current and future breeding programs. Challenging contexts, such as that inherent to the 4x nature of durum wheat when compared to 6x bread wheat, or

Expression of type 2 diacylglycerol acyltransferse gene DGTT1 from Chlamydomonas reinhardtii enhances lipid production in Scenedesmus obliquus.

Science.gov (United States)

Chen, Chun-Yen; Kao, Ai-Ling; Tsai, Zheng-Chia; Chow, Te-Jin; Chang, Hsin-Yueh; Zhao, Xin-Qing; Chen, Po-Ting; Su, Hsiang-Yen; Chang, Jo-Shu

2016-03-01

Microalgal strains of Scenedesmus obliquus have the great potential for the production of biofuels, CO2 fixation, and bioremediation. However, metabolic engineering of S. obliquus to improve their useful phenotypes are still not fully developed. In this study, S. obliquus strain CPC2 was genetically engineered to promote the autotrophic growth and lipid productivity. The overexpression plasmid containing the type 2 diacylglycerol acyltransferse (DGAT) gene DGTT1 from Chlamydomonas reinhardtii was constructed and transformed into S. obliquus CPC2, and the positive transformants were obtained. The expression of DGTT1 gene was confirmed by reverse transcription PCR analysis. Enhanced lipid content of the transformant S. obliquus CPC2-G1 by nearly two-fold was observed. The biomass concentration of the recombinant strains was also 29% higher than that of the wild-type strain. Furthermore, the recombinant strain CPC2-G1 was successfully grown in 40 L tubular type photobioreactor and open pond system in an outdoor environment. The lipid content, biomass concentration, and biomass productivity obtained from 40 L tubular PBR were 127.8% 20.0%, and 232.6% higher than those obtained from the wild-type strain. The major aim of this work is to develop a tool to genetically engineer an isolated S. obliquus strain for the desired purpose. This is the first report that genetic engineering of S. obliquus has been successful employed to improve both the microalgal cell growth and the lipid production. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High hydrostatic pressure activates gene expression that leads to ethanol production enhancement in a Saccharomyces cerevisiae distillery strain

Science.gov (United States)

Bravim, Fernanda; Lippman, Soyeon I.; da Silva, Lucas F.; Souza, Diego T.; Fernandes, A. Alberto R.; Masuda, Claudio A.; Broach, James R.

2016-01-01

High hydrostatic pressure (HHP) is a stress that exerts broad effects on microorganisms with characteristics similar to those of common environmental stresses. In this study, we aimed to identify genetic mechanisms that can enhance alcoholic fermentation of wild Saccharomyces cerevisiae isolated from Brazilian spirit fermentation vats. Accordingly, we performed a time course microarray analysis on a S. cerevisiae strain submitted to mild sublethal pressure treatment of 50 MPa for 30 min at room temperature, followed by incubation for 5, 10 and 15 min without pressure treatment. The obtained transcriptional profiles demonstrate the importance of post-pressurisation period on the activation of several genes related to cell recovery and stress tolerance. Based on these results, we over-expressed genes strongly induced by HHP in the same wild yeast strain and identified genes, particularly SYM1, whose over-expression results in enhanced ethanol production and stress tolerance upon fermentation. The present study validates the use of HHP as a biotechnological tool for the fermentative industries. PMID:22915193

Thermostable L-arabinose isomerase from Bacillus stearothermophilus IAM 11001 for D-tagatose production: gene cloning, purification and characterisation.

Science.gov (United States)

Cheng, Lifang; Mu, Wanmeng; Jiang, Bo

2010-06-01

D-Tagatose, as one of the rare sugars, has been found to be a natural and safe low-calorie sweetener in food products and is classified as a GRAS substance. L-Arabinose isomerase (L-AI, EC 5.3.1.4), catalysing the isomerisations of L-arabinose and D-galactose to L-ribulose and D-tagatose respectively, is considered to be the most promising enzyme for the production of D-tagatose. The araA gene encoding an L-AI from Bacillus stearothermophilus IAM 11001 was cloned, sequenced and overexpressed in Escherichia coli. The gene is composed of 1491 bp nucleotides and codes for a protein of 496 amino acid residues. The recombinant L-AI was purified to electrophoretical homogeneity by affinity chromatography. The purified enzyme was optimally active at 65 degrees C and pH 7.5 and had an absolute requirement for the divalent metal ion Mn(2+) for both catalytic activity and thermostability. The enzyme was relatively active and stable at acidic pH of 6. The bioconversion yield of D-galactose to D-tagatose by the purified L-AI after 12 h at 65 degrees C reached 36%. The purified L-AI from B. stearothermophilus IAM 11001 was characterised and shown to be a good candidate for potential application in D-tagatose production. Copyright (c) 2010 Society of Chemical Industry.

Characterization of RbmD (glycosyltransferase in ribostamycin gene cluster) through neomycin production reconstituted from the engineered Streptomyces fradiae BS1.

Science.gov (United States)

Nepal, Keshav Kumar; Oh, Tae-Jin; Subba, Bimala; Yoo, Jin Cheol; Sohng, Jae Kyung

2009-01-31

Amino acid homology analysis predicted that rbmD, a putative glycosyltransferase from Streptomyces ribosidificus ATCC 21294, has the highest homology with neoD in neomycin biosynthesis. S. fradiae BS1, in which the production of neomycin was abolished, was generated by disruption of the neoD gene in the neomycin producer S. fradiae. The restoration of neomycin by self complementation suggested that there was no polar effect in the mutant. In addition, S. fradiae BS6 was created with complementation by rbmD in S. fradiae BS1, and secondary metabolite analysis by ESI/MS, LC/MS and MS/MS showed the restoration of neomycin production in S. fradiae BS6. These gene inactivation and complementation studies suggested that, like neoD, rbmD functions as a 2-N-acetlyglucosaminyltransferase and demonstrated the potential for the generation of novel aminoglycoside antibiotics using glycosyltransferases in vivo.

Early Determination of Animals with Favorable Genes in Milk Production for Profitable Private Farms

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Daniela E. Ilie

2010-05-01

Full Text Available The primary goal of dairy industry has been to identify an efficient and economical way of increasing milk production and its constituents without increasing the size of the dairy herd. The use of milk protein polymorphisms as detectable molecular markers has been studied intensively because of their effect on the yield and processing properties of milk and its products. Thus, molecular markers are promising alternative to the current methods of trait selection once these genes are proven to be associated with traits of interest in animals. Kappa-casein (CSN3 and beta-lactoglobulin (BLG are two of the most important proteins in the milk of mammals that play a crucial role in the milk quality and coagulation, an essential process for cheese and butter. The A and B variant of k-casein and β-lactoglobulin were distinguished by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP analysis in 108 Romanian Simmental and 60 Holstein Friesian cattle.

The AINTEGUMENTA genes, MdANT1 and MdANT2, are associated with the regulation of cell production during fruit growth in apple (Malus × domestica Borkh.).

Science.gov (United States)

Dash, Madhumita; Malladi, Anish

2012-06-25

Fruit growth in apple (Malus × domestica Borkh.) is mediated by cell production and expansion. Genes involved in regulating these processes and thereby fruit growth, are not well characterized. We hypothesized that the apple homolog(s) of AINTEGUMENTA (ANT), an APETALA2-repeat containing transcription factor, regulates cell production during fruit growth in apple. Two ANT genes, MdANT1 and MdANT2, were isolated from apple and their expression was studied during multiple stages of fruit development. MdANT1 and MdANT2 expression was high during early fruit growth coincident with the period of cell production, rapidly declined during exit from cell production, and remained low during the rest of fruit development. The effects of increase in carbohydrate availability during fruit growth were characterized. Increase in carbohydrate availability enhanced fruit growth largely through an increase in cell production. Expression of MdANT1 and MdANT2 increased sharply by up to around 5-fold in response to an increase in carbohydrate availability. Expression of the ANT genes was compared across two apple genotypes, 'Gala' and 'Golden Delicious Smoothee' (GS), which differ in the extent of fruit growth, largely due to differences in cell production. In comparison to 'Gala', the larger fruit-size genotype, GS, displayed higher levels and a longer duration of MdANT1 and MdANT2 expression. Expression of the ANTs and cell cycle genes in the fruit core and cortex tissues isolated using laser capture microdissection was studied. During early fruit growth, expression of the MdANTs was higher within the cortex, the tissue that constitutes the majority of the fruit. Additionally, MdANT1 and MdANT2 expression was positively correlated with that of A- and B-type CYCLINS, B-type CYCLIN-DEPENDENT-KINASES (CDKBs) and MdDEL1. Multiple lines of evidence from this study suggest that MdANT1 and MdANT2 regulate cell production during fruit growth in apple. ANTs may coordinate the expression of

Genetic manipulation of longevity-related genes as a tool to regulate yeast life span and metabolite production during winemaking

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Orozco Helena

2013-01-01

Full Text Available Abstract Background Yeast viability and vitality are essential for different industrial processes where the yeast Saccharomyces cerevisiae is used as a biotechnological tool. Therefore, the decline of yeast biological functions during aging may compromise their successful biotechnological use. Life span is controlled by a variety of molecular mechanisms, many of which are connected to stress tolerance and genomic stability, although the metabolic status of a cell has proven a main factor affecting its longevity. Acetic acid and ethanol accumulation shorten chronological life span (CLS, while glycerol extends it. Results Different age-related gene classes have been modified by deletion or overexpression to test their role in longevity and metabolism. Overexpression of histone deacetylase SIR2 extends CLS and reduces acetate production, while overexpression of SIR2 homolog HST3 shortens CLS, increases the ethanol level, and reduces acetic acid production. HST3 overexpression also enhances ethanol tolerance. Increasing tolerance to oxidative stress by superoxide dismutase SOD2 overexpression has only a moderate positive effect on CLS. CLS during grape juice fermentation has also been studied for mutants on several mRNA binding proteins that are regulators of gene expression at the posttranscriptional level; we found that NGR1 and UTH4 deletions decrease CLS, while PUF3 and PUB1 deletions increase it. Besides, the pub1Δ mutation increases glycerol production and blocks stress granule formation during grape juice fermentation. Surprisingly, factors relating to apoptosis, such as caspase Yca1 or apoptosis-inducing factor Aif1, play a positive role in yeast longevity during winemaking as their deletions shorten CLS. Conclusions Manipulation of regulators of gene expression at both transcriptional (i.e., sirtuins and posttranscriptional (i.e., mRNA binding protein Pub1 levels allows to modulate yeast life span during its biotechnological use. Due to

FY 2000 Project of developing international standards for supporting new industries. Standardization of systems for detecting gene recombination products; 2000 nendo shinki sangyo shiengata kokusai hyojunka kaihatsu jigyo seika hokokusho. Idenshi kumikae seihin kenshutsu system no hyojunka

Energy Technology Data Exchange (ETDEWEB)

NONE

2001-03-01

Described herein are the FY 2000 results of the studies on the basic conditions for gene recombination products, and methods of determining content of the purchased gene recombination products in agricultural products, conducted to promote the standardization of the gene recombination product detection systems. Soybean is taken up as the gene recombination product to be studied, in which the samples containing the gene recombination product at 5, 0.5, 0.1 and 0% are prepared, and qualitatively analyzed by the DNA extraction and PCR method. No band is detected. For the quantitative examination by the real-time PCR method, the SYBRGeen, Taqman and hybridization probe methods are used to study the PCR conditions. The sample DNA extracted by the DNA Extraction kit for GMO Detection Ver.2 from the sample containing the RoundupReady{sup TM} soybean is quantitatively analyzed by the real-time PCR method for GMO content, which has produced the results for promotion of the standardization. (NEDO)

Expression of death-related genes and reactive oxygen species production in Skeletonema tropicum upon exposure to the polyunsaturated aldehyde octadienal

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Alessandra A. Gallina

2015-11-01

Full Text Available The effects of 4E/Z-octadienal (OCTA on ScDSP-1 and ScDSP-2 gene expression and reactive oxygen species (ROS production were investigated in the marine diatom Skeletonema tropicum (formerly costatum using qRTPCR and flow cytometry. ScDSP-1 and ScDSP-2 genes have been previously shown to be involved in cell death in ageing cells and in response to photosynthetic stress. OCTA induced a differential, concentration-dependent DSP gene expression associated to ROS production, 821.6 and 97.7 folds higher for ScDSP-1 and ScDSP-2, respectively. Among the concentrations tested, only 8 μM OCTA, which caused a reduction of 50% in cell concentrations at 24 h, was able to elicit an expression pattern consistent with a signalling role. Interestingly, only intermediate levels of reactive oxygen species (ROS (i.e., 1.5±0.1 increase were observed to be elicited by such concentration. These results suggest that ROS are key components of the molecular cascade triggered by polyunsaturated aldehydes (PUA and leading to cell death. This could have implications for bloom final stages at sea, where PUA may act as effectors of diatom population dynamics through ROS acting as modulators.

Transcription factor VdCmr1 is required for pigment production, protection from UV irradiation, and regulates expression of melanin biosynthetic genes in Verticillium dahliae.

Science.gov (United States)

Wang, Yonglin; Hu, Xiaoping; Fang, Yulin; Anchieta, Amy; Goldman, Polly H; Hernandez, Gustavo; Klosterman, Steven J

2018-04-01

Verticillium dahliae is a soilborne fungus that causes vascular wilt diseases on numerous plant species worldwide. The production of darkly melanized microsclerotia is crucial in the disease cycle of V. dahliae, as these structures allow for long-term survival in soil. Previously, transcriptomic and genomic analysis identified a cluster of genes in V. dahliae that encodes some dihydroxynaphthalene (DHN) melanin biosynthetic pathway homologues found in related fungi. In this study, we explored the roles of cluster-specific transcription factor VdCmr1, as well as two other genes within the cluster encoding a polyketide synthase (VdPKS1) and a laccase (VdLac1), enzymes at initial and endpoint steps in DHN melanin production. The results revealed that VdCmr1 and VdPKS1 are required for melanin production, but neither is required for microsclerotia production. None of the three genes were required for pathogenesis on tobacco and lettuce. Exposure of ΔVdCmr1 and wild-type strains to UV irradiation, or to high temperature (40 °C), revealed an approx. 50 % reduction of survival in the ΔVdCmr1 strain, relative to the wild-type strain, in response to either condition. Expression profiles revealed that expression of some melanin biosynthetic genes are in part dependent on VdCmr1. Combined data indicate VdCmr1 is a key regulator of melanin biosynthesis, and that via regulation of melanogenesis, VdCmr1 affects survival of V. dahliae in response to abiotic threats. We conclude with a model showing regulation of VdCmr1 by a high osmolarity glycerol response (Hog)-type MAP kinase pathway.

Dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis suppressed the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor.

Science.gov (United States)

Lee, Hyun Jae; Park, Jin Sung; Yoon, Yong Pill; Shin, Ye Jin; Lee, Sang Kook; Kim, Yeong Shik; Hong, Jang-Hee; Son, Kun Ho; Lee, Choong Jae

2015-05-15

The root of Asparagus cochinchinensis (Lour.) Merr. has been utilized as mucoregulators and expectorants for controlling the airway inflammatory diseases in folk medicine. We investigated whether dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis (Lour.) Merr. suppress the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor. Confluent NCI-H292 cells were pretreated with dioscin or methylprotodioscin for 30 min and then stimulated with EGF or PMA for 24 h. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA. (1) Dioscin and methylprotodioscin suppressed the expression of MUC5AC mucin gene induced by EGF or PMA; (2) dioscin suppressed the production of MUC5AC mucin induced by either EGF at 10(-5) M (p Asparagus cochinchinensis suppress the gene expression and production of MUC5AC mucin, by directly acting on airway epithelial cells, and the results are consistent with the traditional use of Asparagus cochinchinensis as remedy for diverse inflammatory pulmonary diseases. Copyright © 2015 Elsevier GmbH. All rights reserved.

Quantitative trait loci linked to PRNP gene controlling health and production traits in INRA 401 sheep

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Brunel Jean-Claude

2007-07-01

Full Text Available Abstract In this study, the potential association of PrP genotypes with health and productive traits was investigated. Data were recorded on animals of the INRA 401 breed from the Bourges-La Sapinière INRA experimental farm. The population consisted of 30 rams and 852 ewes, which produced 1310 lambs. The animals were categorized into three PrP genotype classes: ARR homozygous, ARR heterozygous, and animals without any ARR allele. Two analyses differing in the approach considered were carried out. Firstly, the potential association of the PrP genotype with disease (Salmonella resistance and production (wool and carcass traits was studied. The data used included 1042, 1043 and 1013 genotyped animals for the Salmonella resistance, wool and carcass traits, respectively. The different traits were analyzed using an animal model, where the PrP genotype effect was included as a fixed effect. Association analyses do not indicate any evidence of an effect of PrP genotypes on traits studied in this breed. Secondly, a quantitative trait loci (QTL detection approach using the PRNP gene as a marker was applied on ovine chromosome 13. Interval mapping was used. Evidence for one QTL affecting mean fiber diameter was found at 25 cM from the PRNP gene. However, a linkage between PRNP and this QTL does not imply unfavorable linkage disequilibrium for PRNP selection purposes.

Discovering gene annotations in biomedical text databases

Directory of Open Access Journals (Sweden)

Ozsoyoglu Gultekin

2008-03-01

Full Text Available Abstract Background Genes and gene products are frequently annotated with Gene Ontology concepts based on the evidence provided in genomics articles. Manually locating and curating information about a genomic entity from the biomedical literature requires vast amounts of human effort. Hence, there is clearly a need forautomated computational tools to annotate the genes and gene products with Gene Ontology concepts by computationally capturing the related knowledge embedded in textual data. Results In this article, we present an automated genomic entity annotation system, GEANN, which extracts information about the characteristics of genes and gene products in article abstracts from PubMed, and translates the discoveredknowledge into Gene Ontology (GO concepts, a widely-used standardized vocabulary of genomic traits. GEANN utilizes textual "extraction patterns", and a semantic matching framework to locate phrases matching to a pattern and produce Gene Ontology annotations for genes and gene products. In our experiments, GEANN has reached to the precision level of 78% at therecall level of 61%. On a select set of Gene Ontology concepts, GEANN either outperforms or is comparable to two other automated annotation studies. Use of WordNet for semantic pattern matching improves the precision and recall by 24% and 15%, respectively, and the improvement due to semantic pattern matching becomes more apparent as the Gene Ontology terms become more general. Conclusion GEANN is useful for two distinct purposes: (i automating the annotation of genomic entities with Gene Ontology concepts, and (ii providing existing annotations with additional "evidence articles" from the literature. The use of textual extraction patterns that are constructed based on the existing annotations achieve high precision. The semantic pattern matching framework provides a more flexible pattern matching scheme with respect to "exactmatching" with the advantage of locating approximate

Early and Late Trisporoids Differentially Regulate β-Carotene Production and Gene Transcript Levels in the Mucoralean Fungi Blakeslea trispora and Mucor mucedo

Science.gov (United States)

Sahadevan, Yamuna; Richter-Fecken, Mareike; Kaerger, Kerstin; Voigt, Kerstin

2013-01-01

The multistep cleavage of carotenoids in Mucorales during the sexual phase results in a cocktail of trisporic acid (C18) sex pheromones. We hypothesized that the C18 trisporoid intermediates have a specific regulatory function for sex pheromone production and carotenogenesis that varies with genus/species and vegetative and sexual phases of their life cycles. Real-time quantitative PCR kinetics determined for Blakeslea trispora displayed a very high transcript turnover in the gene for carotenoid cleavage dioxygenase, tsp3, during the sexual phase. An in vivo enzyme assay and chromatographic analysis led to the identification of β-apo-12′-carotenal as the first apocarotenoid involved in trisporic acid biosynthesis in B. trispora. Supplementation of C18 trisporoids, namely D'orenone, methyl trisporate C, and trisporin C, increased tsp3 transcripts in the plus compared to minus partners. Interestingly, the tsp1 gene, which is involved in trisporic acid biosynthesis, was downregulated compared to tsp3 irrespective of asexual or sexual phase. Only the minus partners of both B. trispora and Mucor mucedo had enhanced β-carotene production after treatment with C20 apocarotenoids, 15 different trisporoids, and their analogues. We conclude that the apocarotenoids and trisporoids influence gene transcription and metabolite production, depending upon the fungal strain, corresponding genus, and developmental phase, representing a “chemical dialect” during sexual communication. PMID:24056470

Cell surface expression system for the display of heterologous gene products using chimeric flagellin fusions of bacillus halodurans isolate

CSIR Research Space (South Africa)

Du Plessis, A

2006-10-01

Full Text Available system for the display of heterologous gene products using chimeric flagellin fusions of a Bacillus halodurans isolate Slide 2 © CSIR 2006 www.csir.co.za Bacillus halodurans Alk 36 xrhombus Ability to over-produce cell... for functionality of the His-tag for metal binding. Slide 13 © CSIR 2006 www.csir.co.za PAGE gel showing over-production of chimeric poly-His flagellin proteins 66.2 kDa 45.0 kDa 31.0 kDa 1. LMW ladder 2. NC3 3. NHisC3 4. NC6 5...

Classifying genes to the correct Gene Ontology Slim term in Saccharomyces cerevisiae using neighbouring genes with classification learning

Directory of Open Access Journals (Sweden)

Tsatsoulis Costas

2010-05-01

Full Text Available Abstract Background There is increasing evidence that gene location and surrounding genes influence the functionality of genes in the eukaryotic genome. Knowing the Gene Ontology Slim terms associated with a gene gives us insight into a gene's functionality by informing us how its gene product behaves in a cellular context using three different ontologies: molecular function, biological process, and cellular component. In this study, we analyzed if we could classify a gene in Saccharomyces cerevisiae to its correct Gene Ontology Slim term using information about its location in the genome and information from its nearest-neighbouring genes using classification learning. Results We performed experiments to establish that the MultiBoostAB algorithm using the J48 classifier could correctly classify Gene Ontology Slim terms of a gene given information regarding the gene's location and information from its nearest-neighbouring genes for training. Different neighbourhood sizes were examined to determine how many nearest neighbours should be included around each gene to provide better classification rules. Our results show that by just incorporating neighbour information from each gene's two-nearest neighbours, the percentage of correctly classified genes to their correct Gene Ontology Slim term for each ontology reaches over 80% with high accuracy (reflected in F-measures over 0.80 of the classification rules produced. Conclusions We confirmed that in classifying genes to their correct Gene Ontology Slim term, the inclusion of neighbour information from those genes is beneficial. Knowing the location of a gene and the Gene Ontology Slim information from neighbouring genes gives us insight into that gene's functionality. This benefit is seen by just including information from a gene's two-nearest neighbouring genes.

The effect of frizzle gene and dwarf gene on reproductive performance of broiler breeder dams under high and normal ambient temperatures.

Science.gov (United States)

Sharifi, A R; Horst, P; Simianer, H

2010-11-01

In 3 experimental runs, the influence of genotype × temperature interactions on the reproductive traits (sexual maturity, egg production, fertility, hatchability, and chick production) of hens of a broiler breeder dam line carrying major genes for dwarfism (dw-) and frizzle (F) was investigated. In experiments 1 and 2, the hens were caged individually under hot (30°C) and temperate (19°C) temperatures, from wk 18 to 72 of age, whereas in experiment 3, hens were kept under moderate temperature (24°C). Hens in experiment 1 were heterozygous for the frizzle gene, and those in experiments 2 and 3 were homozygous, both with and without the dwarf gene. Hens without the above-mentioned major genes (ffDw-) served as control lines. In experiment 1, the frizzle gene (Ff) had no significant effect on sexual maturity, egg production, fertility, hatchability, and chick number under the 2 environmental conditions. In experiment 2, there was a significant interaction between feathering genotype (FF) and environmental temperature for all traits except sexual maturity. Under heat stress, there was a distinct reduction in all reproductive traits except sexual maturity for normally feathered hens compared with frizzle-feathered hens, whereas under temperate conditions, egg production and number of chicks of the FF genotype were reduced and sexual maturity was delayed. In experiments 1 and 2, the dw- gene showed a depressive effect on the growth of hens. In experiment 1, the interaction between dwarf genotype and environmental temperature for egg production was significant. Under temperate conditions, the egg production of dwarf hens was inferior to that of normally sized birds, whereas under hot temperatures, the egg production of the 2 body sizes did not differ. In experiment 2, for sexual maturity, egg production and fertility locus × locus interactions could be determined. The genotype combining the 2 major genes (FFdw-) proved to be inferior to the normally feathered dwarf

Deletion of meso-2,3-butanediol dehydrogenase gene budC for enhanced D-2,3-butanediol production in Bacillus licheniformis

Science.gov (United States)

2014-01-01

Background D-2,3-butanediol has many industrial applications such as chiral reagents, solvents, anti-freeze agents, and low freezing point fuels. Traditional D-2,3-butanediol producing microorganisms, such as Klebsiella pneumonia and K. xoytoca, are pathogenic and not capable of producing D-2,3-butanediol at high optical purity. Bacillus licheniformis is a potential 2,3-butanediol producer but the wild type strain (WX-02) produces a mix of D- and meso-type isomers. BudC in B. licheniformis is annotated as 2,3-butanediol dehydrogenase or acetoin reductase, but no pervious experiment was performed to verify this hypothesis. Results We developed a genetically modified strain of B. licheniformis (WX-02 ΔbudC) as a D-2,3-butanediol producer with high optimal purity. A marker-less gene deletion protocol based on a temperature sensitive knock-out plasmid T2-Ori was used to knock out the budC gene in B. licheniformis WX-02. The budC knock-out strain successfully abolished meso-2,3-butanediol production with enhanced D-2,3-butanediol production. No meso-BDH activity was detectable in cells of this strain. On the other hand, the complementary strain restored the characteristics of wild strain, and produced meso-2,3-butanediol and possessed meso-BDH activity. All of these data suggested that budC encoded the major meso-BDH catalyzing the reversible reaction from acetoin to meso-2,3-butanediol in B. licheniformis. The budC knock-out strain produced D-2,3-butanediol isomer only with a high yield of 30.76 g/L and a productivity of 1.28 g/L-h. Conclusions We confirmed the hypothesis that budC gene is responsible to reversibly transfer acetoin to meso-2,3-butanediol in B. licheniformis. A mutant strain of B. licheniformis with depleted budC gene was successfully developed and produced high level of the D-2,3-butanediol with high optimal purity. PMID:24475980

Astrocyte production of the chemokine macrophage inflammatory protein-2 is inhibited by the spice principle curcumin at the level of gene transcription

Directory of Open Access Journals (Sweden)

Santoro Thomas J

2005-02-01

Full Text Available Abstract Background In neuropathological processes associated with neutrophilic infiltrates, such as experimental allergic encephalitis and traumatic injury of the brain, the CXC chemokine, macrophage inflammatory protein-2 (MIP-2 is thought to play a pivotal role in the induction and perpetuation of inflammation in the central nervous system (CNS. The origin of MIP-2 in inflammatory disorders of the brain has not been fully defined but astrocytes appear to be a dominant source of this chemokine. Curcumin is a spice principle in, and constitutes approximately 4 percent of, turmeric. Curcumin's immunomodulating and antioxidant activities suggest that it might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation. Relatively unexplored, but relevant to its potential therapeutic efficacy in neuroinflammatory syndromes is the effect of curcumin on chemokine production. To examine the possibility that curcumin may influence CNS inflammation by mechanisms distinct from its known anti-oxidant activities, we studied the effect of this spice principle on the synthesis of MIP-2 by astrocytes. Methods Primary astrocytes were prepared from neonatal brains of CBA/CaJ mice. The cells were stimulated with lipopolysaccharide in the presence or absence of various amount of curcumin or epigallocatechin gallate. MIP-2 mRNA was analyzed using semi-quantitative PCR and MIP-2 protein production in the culture supernatants was quantified by ELISA. Astrocytes were transfected with a MIP-2 promoter construct, pGL3-MIP-2, and stimulated with lipopolysaccharide in the presence or absence of curcumin. Results The induction of MIP-2 gene expression and the production of MIP-2 protein were inhibited by curcumin. Curcumin also inhibited lipopolysaccharide-induced transcription of the MIP-2 promoter reporter gene construct in primary astrocytes. However MIP-2 gene induction by lipopolysaccharide was not inhibited by another anti

Astrocyte production of the chemokine macrophage inflammatory protein-2 is inhibited by the spice principle curcumin at the level of gene transcription.

Science.gov (United States)

Tomita, Michiyo; Holman, Brita J; Santoro, Christopher P; Santoro, Thomas J

2005-02-25

BACKGROUND: In neuropathological processes associated with neutrophilic infiltrates, such as experimental allergic encephalitis and traumatic injury of the brain, the CXC chemokine, macrophage inflammatory protein-2 (MIP-2) is thought to play a pivotal role in the induction and perpetuation of inflammation in the central nervous system (CNS). The origin of MIP-2 in inflammatory disorders of the brain has not been fully defined but astrocytes appear to be a dominant source of this chemokine.Curcumin is a spice principle in, and constitutes approximately 4 percent of, turmeric. Curcumin's immunomodulating and antioxidant activities suggest that it might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation. Relatively unexplored, but relevant to its potential therapeutic efficacy in neuroinflammatory syndromes is the effect of curcumin on chemokine production. To examine the possibility that curcumin may influence CNS inflammation by mechanisms distinct from its known anti-oxidant activities, we studied the effect of this spice principle on the synthesis of MIP-2 by astrocytes. METHODS: Primary astrocytes were prepared from neonatal brains of CBA/CaJ mice. The cells were stimulated with lipopolysaccharide in the presence or absence of various amount of curcumin or epigallocatechin gallate. MIP-2 mRNA was analyzed using semi-quantitative PCR and MIP-2 protein production in the culture supernatants was quantified by ELISA. Astrocytes were transfected with a MIP-2 promoter construct, pGL3-MIP-2, and stimulated with lipopolysaccharide in the presence or absence of curcumin. RESULTS: The induction of MIP-2 gene expression and the production of MIP-2 protein were inhibited by curcumin. Curcumin also inhibited lipopolysaccharide-induced transcription of the MIP-2 promoter reporter gene construct in primary astrocytes. However MIP-2 gene induction by lipopolysaccharide was not inhibited by another anti-oxidant, epigallocatechin

Bioengineering of the Plant Culture of Capsicum frutescens with Vanillin Synthase Gene for the Production of Vanillin.

Science.gov (United States)

Chee, Marcus Jenn Yang; Lycett, Grantley W; Khoo, Teng-Jin; Chin, Chiew Foan

2017-01-01

Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively. As the ferulic acid precursor and vanillin were found to be the intermediates in the phenylpropanoid biosynthetic pathway of Capsicum species, this work serves as a proof-of-concept for vanillin production using Capsicum frutescens (C. frutescens or hot chili pepper). The cells of C. frutescens were genetically transformed with a codon optimized VpVAN gene via biolistics. Transformed explants were selected and regenerated into callus. Successful integration of the gene cassette into the plant genome was confirmed by polymerase chain reaction. High-performance liquid chromatography was used to quantify the phenolic compounds detected in the callus tissues. The vanillin content of transformed calli was 0.057% compared to 0.0003% in untransformed calli.

Phytase-producing capacity of yeasts isolated from traditional African fermented food products and PHYPk gene expression of Pichia kudriavzevii strains.

Science.gov (United States)

Greppi, Anna; Krych, Łukasz; Costantini, Antonella; Rantsiou, Kalliopi; Hounhouigan, D Joseph; Arneborg, Nils; Cocolin, Luca; Jespersen, Lene

2015-07-16

Phytate is known as a strong chelate of minerals causing their reduced uptake by the human intestine. Ninety-three yeast isolates from traditional African fermented food products, belonging to nine species (Pichia kudriavzevii, Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces marxianus, Millerozyma farinosa, Candida glabrata, Wickerhamomyces anomalus, Hanseniaspora guilliermondii and Debaryomyces nepalensis) were screened for phytase production on solid and liquid media. 95% were able to grow in the presence of phytate as sole phosphate source, P. kudriavzevii being the best growing species. A phytase coding gene of P. kudriavzevii (PHYPk) was identified and its expression was studied during growth by RT-qPCR. The expression level of PHYPk was significantly higher in phytate-medium, compared to phosphate-medium. In phytate-medium expression was seen in the lag phase. Significant differences in gene expression were detected among the strains as well as between the media. A correlation was found between the PHYPk expression and phytase extracellular activity. Copyright © 2015 Elsevier B.V. All rights reserved.

Tissue-specific production of limonene in Camelina sativa with the Arabidopsis promoters of genes BANYULS and FRUITFULL.

Science.gov (United States)

Borghi, Monica; Xie, De-Yu

2016-02-01

Arabidopsis promoters of genes BANYULS and FRUITFULL are transcribed in Camelina. They triggered the transcription of limonene synthase and induced higher limonene production in seeds and fruits than CaMV 35S promoter. Camelina sativa (Camelina) is an oilseed crop of relevance for the production of biofuels and the plant has been target of a recent and intense program of genetic manipulation aimed to increase performance, seed yield and to modify the fatty acid composition of the oil. Here, we have explored the performance of two Arabidopsis thaliana (Arabidopsis) promoters in triggering transgene expression in Camelina. The promoters of two genes BANYULS (AtBAN pro ) and FRUITFULL (AtFUL pro ), which are expressed in seed coat and valves of Arabidopsis, respectively, have been chosen to induce the expression of limonene synthase (LS) from Citrus limon. In addition, the constitutive CaMV 35S promoter was utilized to overexpress LS in Camelina . The results of experiments revealed that AtBAN pro and AtFUL pro are actively transcribed in Camelina where they also retain specificity of expression in seeds and valves as previously observed in Arabidopsis. LS induced by AtBAN pro and AtFUL pro leads to higher limonene production in seeds and fruits than when the CaMV 35S was used to trigger the expression. In conclusion, the results of experiments indicate that AtBAN pro and AtFUL pro can be successfully utilized to induce the expression of the transgenes of interest in seeds and fruits of Camelina.

Transgenic expression of an unedited mitochondrial orfB gene product from wild abortive (WA) cytoplasm of rice (Oryza sativa L.) generates male sterility in fertile rice lines.

Science.gov (United States)

Chakraborty, Anirban; Mitra, Joy; Bhattacharyya, Jagannath; Pradhan, Subrata; Sikdar, Narattam; Das, Srirupa; Chakraborty, Saikat; Kumar, Sachin; Lakhanpaul, Suman; Sen, Soumitra K

2015-06-01

Over-expression of the unedited mitochondrial orfB gene product generates male sterility in fertile indica rice lines in a dose-dependent manner. Cytoplasmic male sterility (CMS) and nuclear-controlled fertility restoration are widespread developmental features in plant reproductive systems. In self-pollinated crop plants, these processes often provide useful tools to exploit hybrid vigour. The wild abortive CMS has been employed in the majority of the "three-line" hybrid rice production since 1970s. In the present study, we provide experimental evidence for a positive functional relationship between the 1.1-kb unedited orfB gene transcript, and its translated product in the mitochondria with male sterility. The generation of the 1.1-kb unedited orfB gene transcripts increased during flowering, resulting in low ATP synthase activity in sterile plants. Following insertion of the unedited orfB gene into the genome of male-fertile plants, the plants became male sterile in a dose-dependent manner with concomitant reduction of ATPase activity of F1F0-ATP synthase (complex V). Fertility of the transgenic lines and normal activity of ATP synthase were restored by down-regulation of the unedited orfB gene expression through RNAi-mediated silencing. The genetic elements deciphered in this study could further be tested for their use in hybrid rice development.

Overexpression of Genes Encoding Glycolytic Enzymes in Corynebacterium glutamicum Enhances Glucose Metabolism and Alanine Production under Oxygen Deprivation Conditions

Science.gov (United States)

Yamamoto, Shogo; Gunji, Wataru; Suzuki, Hiroaki; Toda, Hiroshi; Suda, Masako; Jojima, Toru; Inui, Masayuki

2012-01-01

We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159–165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD+ ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses. PMID

Ameliorative Effects of Allium sativum Extract on iNOS Gene Expression and NO Production in Liver of Streptozotocin + Nicotinamide-Induced Diabetic Rats.

Science.gov (United States)

Ziamajidi, Nasrin; Behrouj, Hamid; Abbasalipourkabir, Roghayeh; Lotfi, Fatemeh

2018-04-01

Diabetes mellitus (DM) is one of the most prevalent diseases in the world, which is strongly associated with liver dysfunction. Hyperglycemia, through an oxidative stress pathway, damages various tissues. Herbal medicine is a good candidate to ameliorate hyperglycemia and oxidative stress. In this study, the effects of aqueous Allium sativum (garlic) extract (AGE) on gene expression of inducible nitric oxide synthases (iNOS) and production of nitric oxide (NO) were evaluated in the liver tissue of diabetic rats. Four groups of rats contained normal control rats, garlic control rats (AGE), Streptozotocin (STZ) + nicotinamide-induced diabetic rats (DM), and diabetic rats treated with garlic (DM + AGE). Glucose levels and liver enzymes activities were determined by colorimetric assay in the serum. Gene expression of iNOS by real-time PCR, NO levels by Griess method, oxidative stress parameters by spectrophotometric method and histopathological examination by hematoxylin and eosin staining method were evaluated in the liver tissues. Glucose levels, activities of liver enzymes, oxidative stress markers, iNOS gene expression, and NO production increased significantly in diabetic rats in comparison with control rats, whereas after oral administration of garlic, these parameters decreased significantly, close to the normal levels. Hence, the beneficial effects of garlic on the liver injury of diabetes could be included in the hypoglycaemic and antioxidant properties of garlic via a decrease in gene expression of iNOS and subsequent NO production.

An intronic microRNA silences genes that are functionally antagonistic to its host gene.

Science.gov (United States)

Barik, Sailen

2008-09-01

MicroRNAs (miRNAs) are short noncoding RNAs that down-regulate gene expression by silencing specific target mRNAs. While many miRNAs are transcribed from their own genes, nearly half map within introns of 'host' genes, the significance of which remains unclear. We report that transcriptional activation of apoptosis-associated tyrosine kinase (AATK), essential for neuronal differentiation, also generates miR-338 from an AATK gene intron that silences a family of mRNAs whose protein products are negative regulators of neuronal differentiation. We conclude that an intronic miRNA, transcribed together with the host gene mRNA, may serve the interest of its host gene by silencing a cohort of genes that are functionally antagonistic to the host gene itself.

Human gene therapy: novel approaches to improve the current gene delivery systems.

Science.gov (United States)

Cucchiarini, Magali

2016-06-01

Even though gene therapy made its way through the clinics to treat a number of human pathologies since the early years of experimental research and despite the recent approval of the first gene-based product (Glybera) in Europe, the safe and effective use of gene transfer vectors remains a challenge in human gene therapy due to the existence of barriers in the host organism. While work is under active investigation to improve the gene transfer systems themselves, the use of controlled release approaches may offer alternative, convenient tools of vector delivery to achieve a performant gene transfer in vivo while overcoming the various physiological barriers that preclude its wide use in patients. This article provides an overview of the most significant contributions showing how the principles of controlled release strategies may be adapted for human gene therapy.

Phytoalexin detoxification genes and gene products: Implication for the evolution of host specific traits for pathogenicity. Final report

International Nuclear Information System (INIS)

VanEtten, H.

1997-01-01

The overall objectives of this research were to determine which differences among PDA genes were associated with different levels of virulence on pea and to clone and characterize a MAK gene. The authors also proposed to characterize the pisatin detoxifying system in pea pathogens in addition to N. haematococca to assess whether pathogens of a common host had evolved similar pathogenicity genes

Phytoalexin detoxification genes and gene products: Implication for the evolution of host specific traits for pathogenicity. Final report

Energy Technology Data Exchange (ETDEWEB)

VanEtten, H.

1997-06-01

The overall objectives of this research were to determine which differences among PDA genes were associated with different levels of virulence on pea and to clone and characterize a MAK gene. The authors also proposed to characterize the pisatin detoxifying system in pea pathogens in addition to N. haematococca to assess whether pathogens of a common host had evolved similar pathogenicity genes.

Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species

DEFF Research Database (Denmark)

Nielsen, Jens Christian; Grijseels, Sietske; Prigent, Sylvain

2017-01-01

Filamentous fungi produce a wide range of bioactive compounds with important pharmaceutical applications, such as antibiotic penicillins and cholesterol-lowering statins. However, less attention has been paid to fungal secondary metabolites compared to those from bacteria. In this study, we...... sequenced the genomes of 9 Penicillium species and, together with 15 published genomes, we investigated the secondary metabolism of Penicillium and identified an immense, unexploited potential for producing secondary metabolites by this genus. A total of 1,317 putative biosynthetic gene clusters (BGCs) were......-referenced the predicted pathways with published data on the production of secondary metabolites and experimentally validated the production of antibiotic yanuthones in Penicillia and identified a previously undescribed compound from the yanuthone pathway. This study is the first genus-wide analysis of the genomic...

Cloning and knockout of formate hydrogen lyase and H{sub 2}-uptake hydrogenase genes in Enterobacter aerogenes for enhanced hydrogen production

Energy Technology Data Exchange (ETDEWEB)

Zhao, Hongxin; Ma, Kun; Lu, Yuan; Zhang, Chong; Wang, Liyan; Xing, Xin-Hui [Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Tsinghua Yuan, Beijing 100084 (China)

2009-01-15

A 5431-bp DNA fragment partially encoding the formate hydrogen lyase (FHL) gene cluster hycABCDE was isolated and identified from Enterobacter aerogenes IAM1183 chromosomal DNA. All the five putative gene products showed a high degree of homology to the reported bacterial FHL proteins. The gene hycA, encoding the FHL repressor protein, and hybO, encoding the small subunit of the uptake hydrogenase, were targeted for genetic knockout for improving the hydrogen production. The pYM-Red recombination system was adopted to form insertional mutations in the E. aerogenes genome, thereby creating mutant strains of IAM1183-A ({delta} hycA), IAM1183-O ({delta} hybO), and IAM1183-AO ({delta} hycA/ {delta} hybO double knockout). The hydrogen production experiments with these mutants showed that the maximum specific hydrogen productivities of IAM1183-A, IAM1183-O, and IAM1183-AO were 2879.466 {+-} 38.59, 2747.203 {+-} 13.25 and 3372.019 {+-} 4.39 (ml h{sup -1} g{sup -1}dry cell weight), respectively, higher than that of the wild strain (2321.861 {+-} 15.34 ml h{sup -1} g{sup -1}dry cell weight). The total H{sub 2} yields by the three mutants IAM1183-A, IAM1183-O and IAM1183-AO were 0.73, 0.78, and 0.83 mol-H{sub 2}/mol glucose, respectively, while the wild-type IAM1183 was only 0.65 mol-H{sub 2}/mol glucose. The metabolites of the mutants including acetate, ethanol, 2,3-butanediol and succinate were all increased compared with that of the wild type, implying the changed metabolic flux by the mutation. In the fermentor cultivation with IAM1183 {delta} hycA/ {delta} hybO, the total hydrogen volume after 16 h cultivation reached 4.4 L, while that for the wild type was only 2.9 L. (author)

Non-functional genes repaired at the RNA level.

Science.gov (United States)

Burger, Gertraud

2016-01-01

Genomes and genes continuously evolve. Gene sequences undergo substitutions, deletions or nucleotide insertions; mobile genetic elements invade genomes and interleave in genes; chromosomes break, even within genes, and pieces reseal in reshuffled order. To maintain functional gene products and assure an organism's survival, two principal strategies are used - either repair of the gene itself or of its product. I will introduce common types of gene aberrations and how gene function is restored secondarily, and then focus on systematically fragmented genes found in a poorly studied protist group, the diplonemids. Expression of their broken genes involves restitching of pieces at the RNA-level, and substantial RNA editing, to compensate for point mutations. I will conclude with thoughts on how such a grotesquely unorthodox system may have evolved, and why this group of organisms persists and thrives since tens of millions of years. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells

Directory of Open Access Journals (Sweden)

Roni M. Shtein

2012-01-01

Full Text Available Purpose. To determine time course of effect of lipopolysaccharide (LPS on production of interleukin-8 (IL-8 and monocyte chemotactic protein (MCP by cultured human corneal stromal cells. Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student's t-test. Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases (P<0.05 after corneal stromal cell stimulation with LPS. Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS.

Interaction of maternal atopy, CTLA-4 gene polymorphism and gender on antenatal immunoglobulin E production.

Science.gov (United States)

Yang, K D; Ou, C-Y; Hsu, T-Y; Chang, J-C; Chuang, H; Liu, C-A; Liang, H-M; Kuo, H-C; Chen, R-F; Huang, E-Y

2007-05-01

Genetic heritability and maternal atopy have been correlated to antenatal IgE production, but very few studies have studied gene-maternal atopy interaction on antenatal IgE production. This study investigated the interaction of CTLA-4 polymorphism with prenatal factors on the elevation of cord blood IgE (CBIgE). Pregnant women were antenatally recruited for collection of prenatal environmental factors by a questionnaire. Umbilical cord blood samples were collected for CBIgE detection by fluorescence-linked enzyme assay and CTLA-4 polymorphism measurement by restriction fragment length polymorphism. A total of 1104 pregnant women initially participated in this cohort study, and 898 of them completed cord blood collection. 21.4% of the newborns had elevation of CBIgE (>or=0.5 kU/L). The CTLA-4+49A allele (P=0.021), maternal atopy (Ppaternal atopy, were significantly correlated with the CBIgE elevation in multivariate analysis. A dichotomous analysis of gene-maternal atopy interactions identified maternal atopy and CTLA-4+49A allele had an additive effect on the CBIgE elevation, especially prominent in male newborns; and in the absence of maternal atopy, CTLA-4+49GG genotype had a protective effect on CBIgE elevation in female newborns. Maternal but not paternal atopy has significant impacts on CBIgE elevation depending on gender and CTLA-4+49A/G polymorphism of newborns. Control of maternal atopy and modulation of CTLA-4 expression in the prenatal stage may be a target for the early prevention of perinatal allergy sensitization.

Complex Formation by the mrpABCDEFG Gene Products, Which Constitute a Principal Na+/H+ Antiporter in Bacillus subtilis▿

OpenAIRE

Kajiyama, Yusuke; Otagiri, Masato; Sekiguchi, Junichi; Kosono, Saori; Kudo, Toshiaki

2007-01-01

The Bacillus subtilis Mrp (also referred to as Sha) is a particularly unusual Na+/H+ antiporter encoded by mrpABCDEFG. Using His tagging of Mrp proteins, we showed complex formation by the mrpABCDEFG gene products by pull-down and blue native polyacrylamide gel electrophoresis analyses. This is the first molecular evidence that the Mrp is a multicomponent antiporter in the cation-proton antiporter 3 family.

The L locus, one of complementary genes required for anthocyanin production in onions (Allium cepa), encodes anthocyanidin synthase.

Science.gov (United States)

Kim, Sunggil; Jones, Rick; Yoo, Kil-Sun; Pike, Leonard M

2005-06-01

Bulb color in onions (Allium cepa) is an important trait, but its complex, unclear mechanism of inheritance has been a limiting factor in onion cultivar improvement. The identity of the L locus, which is involved in the color difference between Brazilian yellow and red onions, is revealed in this study. A cross was made between a US-type yellow breeding line and a Brazilian yellow cultivar. The segregation ratio of nine red to seven yellow onions in the F(2) population supports the involvement of two complementary genes in anthocyanin production in the F(1) hybrids. The high-performance liquid chromatography (HPLC) and reverse-transcriptase (RT)-PCR analysis of the Brazilian yellow onions indicated that the genes are involved late in the anthocyanin synthesis pathway. The genomic sequence of the anthocyanidin synthase (ANS) gene in Brazilian yellow onions showed a point mutation, which results in an amino acid change of a glycine to an arginine at residue 229. Because this residue is located adjacent to a highly conserved iron-binding active site, this mutation is likely responsible for the inactivation of the ANS gene in Brazilian yellow onions. Following the isolation of the promoter sequence of the mutant allele, a PCR-based marker for allelic selection of the ANS gene was designed. This assay is based on an insertion (larger than 3 kb) mutation. The marker perfectly co-segregated with the color phenotypes in the F(2) populations, thereby indicating that the L locus encodes ANS.

The Aspergillus flavus Spermidine Synthase (spds Gene, Is Required for Normal Development, Aflatoxin Production, and Pathogenesis During Infection of Maize Kernels

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Rajtilak Majumdar

2018-03-01

Full Text Available Aspergillus flavus is a soil-borne saprophyte and an opportunistic pathogen of both humans and plants. This fungus not only causes disease in important food and feed crops such as maize, peanut, cottonseed, and tree nuts but also produces the toxic and carcinogenic secondary metabolites (SMs known as aflatoxins. Polyamines (PAs are ubiquitous polycations that influence normal growth, development, and stress responses in living organisms and have been shown to play a significant role in fungal pathogenesis. Biosynthesis of spermidine (Spd is critical for cell growth as it is required for hypusination-mediated activation of eukaryotic translation initiation factor 5A (eIF5A, and other biochemical functions. The tri-amine Spd is synthesized from the diamine putrescine (Put by the enzyme spermidine synthase (Spds. Inactivation of spds resulted in a total loss of growth and sporulation in vitro which could be partially restored by addition of exogenous Spd. Complementation of the Δspds mutant with a wild type (WT A. flavus spds gene restored the WT phenotype. In WT A. flavus, exogenous supply of Spd (in vitro significantly increased the production of sclerotia and SMs. Infection of maize kernels with the Δspds mutant resulted in a significant reduction in fungal growth, sporulation, and aflatoxin production compared to controls. Quantitative PCR of Δspds mutant infected seeds showed down-regulation of aflatoxin biosynthetic genes in the mutant compared to WT A. flavus infected seeds. Expression analyses of PA metabolism/transport genes during A. flavus-maize interaction showed significant increase in the expression of arginine decarboxylase (Adc and S-adenosylmethionine decarboxylase (Samdc genes in the maize host and PA uptake transporters in the fungus. The results presented here demonstrate that Spd biosynthesis is critical for normal development and pathogenesis of A. flavus and pre-treatment of a Δspds mutant with Spd or Spd uptake from the

From gene engineering to gene modulation and manipulation: can we prevent or detect gene doping in sports?

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Fischetto, Giuseppe; Bermon, Stéphane

2013-10-01

During the last 2 decades, progress in deciphering the human gene map as well as the discovery of specific defective genes encoding particular proteins in some serious human diseases have resulted in attempts to treat sick patients with gene therapy. There has been considerable focus on human recombinant proteins which were gene-engineered and produced in vitro (insulin, growth hormone, insulin-like growth factor-1, erythropoietin). Unfortunately, these substances and methods also became improper tools for unscrupulous athletes. Biomedical research has focused on the possible direct insertion of gene material into the body, in order to replace some defective genes in vivo and/or to promote long-lasting endogenous synthesis of deficient proteins. Theoretically, diabetes, anaemia, muscular dystrophies, immune deficiency, cardiovascular diseases and numerous other illnesses could benefit from such innovative biomedical research, though much work remains to be done. Considering recent findings linking specific genotypes and physical performance, it is tempting to submit the young athletic population to genetic screening or, alternatively, to artificial gene expression modulation. Much research is already being conducted in order to achieve a safe transfer of genetic material to humans. This is of critical importance since uncontrolled production of the specifically coded protein, with serious secondary adverse effects (polycythaemia, acute cardiovascular problems, cancer, etc.), could occur. Other unpredictable reactions (immunogenicity of vectors or DNA-vector complex, autoimmune anaemia, production of wild genetic material) also remain possible at the individual level. Some new substances (myostatin blockers or anti-myostatin antibodies), although not gene material, might represent a useful and well-tolerated treatment to prevent progression of muscular dystrophies. Similarly, other molecules, in the roles of gene or metabolic activators [5-aminoimidazole-4

Gene-Editing: Interpretation of Current Law and Legal Policy.

Science.gov (United States)

Kim, Na-Kyoung

2017-09-01

With the development of the third-generation gene scissors, CRISPR-Cas9, concerns are being raised about ethical and social repercussions of the new gene-editing technology. In this situation, this article explores the legislation and interpretation of the positive laws in South Korea. The BioAct does not specify and regulate 'gene editing' itself. However, assuming that genetic editing is used in the process of research and treatment, we can look to the specific details of the regulations for research on humans as well as gene therapy research in order to see how genetic editing is regulated under the BioAct. BioAct differentiates the regulation between (born) humans and embryos etc. and the regulation differ entirely in the manner and scope. Moreover, due to the fact that gene therapy products are regarded as drugs, they fall under different regulations. The Korean Pharmacopoeia Act put stringent sanctions on clinical trials for gene therapy products and the official Notification "Approval and Examination Regulations for Biological Products, etc." by Food and Drug Safety Administration may be applied to gene editing for gene therapy purposes.

Effects of Thyme Extract Oils (from Thymus vulgaris, Thymus zygis, and Thymus hyemalis on Cytokine Production and Gene Expression of oxLDL-Stimulated THP-1-Macrophages

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A. Ocaña

2012-01-01

Full Text Available Properties of thyme extracts from three different species (Thymus vulgaris, Thymus zygis, and Thymus hyemalis were examined. Two oil fractions from each species were obtained by CO2 supercritical fluid extraction. Main compounds presented in the supercritical extracts of the three thyme varieties were 1,8 cineole, thymol, camphor, borneol, and carvacrol. As a cellular model of inflammation/atherogenesis, we use human macrophages derived from THP-1 monocytes and activated by oxidized LDLs. These cells were incubated with the thyme fraction oils, and the productions and gene expressions of the inflammatory mediators TNF-α, IL-1B, IL-6, and IL-10 were determined. Thyme extracts significantly reduced production and gene expression of the proinflammatory mediators TNF-α, IL-1B, and IL-6 and highly increased these parameters on the anti-inflammatory IL-10 cytokine. Changes on production and gene expressions were dose dependent and according to the thyme content of each species. Taken together, these results may suggest that thyme extracts could have anti-inflammatory effects.

Flagellar-phase variation: isolation of the rh1 gene

International Nuclear Information System (INIS)

Silverman, M.; Zieg, J.; Simon, M.

1979-01-01

In Salmonella, expression of flagellar antigen alternates between two serotypes (phases) encoded by two genes, H1 and H2. The mechanism which controls the alternative expression of the H1 and H2 genes was examined by cloning these genes and the genetic elements which control their activity on hybrid vehicles in Escherichia coli. H2 gene activity was shown to be controlled by a recombinational switch located adjacent to the H2 gene. Activity of the H1 gene is thought to be repressed, when the H2 gene is expressed, by the product of another gene, rhl (repressor of H1), which is controlled coordinately with the H2 gene. In this report, we describe the construction of hybrid lambda vehicles which contain, in addition to the H2 gene, a genetic activity corresponding to rhl. Variation of flagellar antigens analogous to that observed in Salmonella was observed when E. coli strains were transduced with the hybrid lambda. By using the lambda H2rhl hybrid to program protein syntheis in uv-irradiated cells, the synthesis of a polypeptide was correlated with rhl gene product activity. We conclude that the H2 region consists of two cotranscribed genes, H2 and rhl. The expression of both gene products is regulated by the same recombinational event

Intracellular delivery of potential therapeutic genes: prospects in cancer gene therapy.

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Bakhtiar, Athirah; Sayyad, Mustak; Rosli, Rozita; Maruyama, Atsushi; Chowdhury, Ezharul H

2014-01-01

Conventional therapies for malignant cancer such as chemotherapy and radiotherapy are associated with poor survival rates owing to the development of cellular resistance to cancer drugs and the lack of targetability, resulting in unwanted adverse effects on healthy cells and necessitating the lowering of therapeutic dose with consequential lower efficacy of the treatment. Gene therapy employing different types of viral and non-viral carriers to transport gene(s) of interest and facilitating production of the desirable therapeutic protein(s) has tremendous prospects in cancer treatments due to the high-level of specificity in therapeutic action of the expressed protein(s) with diminished off-target effects, although cancer cell-specific delivery of transgene(s) still poses some challenges to be addressed. Depending on the potential therapeutic target genes, cancer gene therapy could be categorized into tumor suppressor gene replacement therapy, immune gene therapy and enzyme- or prodrug-based therapy. This review would shed light on the current progress of delivery of potentially therapeutic genes into various cancer cells in vitro and animal models utilizing a variety of viral and non-viral vectors.

De novo assembly, gene annotation, and marker discovery in stored-product pest Liposcelis entomophila (Enderlein using transcriptome sequences.

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Dan-Dan Wei

Full Text Available BACKGROUND: As a major stored-product pest insect, Liposcelis entomophila has developed high levels of resistance to various insecticides in grain storage systems. However, the molecular mechanisms underlying resistance and environmental stress have not been characterized. To date, there is a lack of genomic information for this species. Therefore, studies aimed at profiling the L. entomophila transcriptome would provide a better understanding of the biological functions at the molecular levels. METHODOLOGY/PRINCIPAL FINDINGS: We applied Illumina sequencing technology to sequence the transcriptome of L. entomophila. A total of 54,406,328 clean reads were obtained and that de novo assembled into 54,220 unigenes, with an average length of 571 bp. Through a similarity search, 33,404 (61.61% unigenes were matched to known proteins in the NCBI non-redundant (Nr protein database. These unigenes were further functionally annotated with gene ontology (GO, cluster of orthologous groups of proteins (COG, and Kyoto Encyclopedia of Genes and Genomes (KEGG databases. A large number of genes potentially involved in insecticide resistance were manually curated, including 68 putative cytochrome P450 genes, 37 putative glutathione S-transferase (GST genes, 19 putative carboxyl/cholinesterase (CCE genes, and other 126 transcripts to contain target site sequences or encoding detoxification genes representing eight types of resistance enzymes. Furthermore, to gain insight into the molecular basis of the L. entomophila toward thermal stresses, 25 heat shock protein (Hsp genes were identified. In addition, 1,100 SSRs and 57,757 SNPs were detected and 231 pairs of SSR primes were designed for investigating the genetic diversity in future. CONCLUSIONS/SIGNIFICANCE: We developed a comprehensive transcriptomic database for L. entomophila. These sequences and putative molecular markers would further promote our understanding of the molecular mechanisms underlying

Enhancing the laccase production and laccase gene expression in the white-rot fungus Trametes velutina 5930 with great potential for biotechnological applications by different metal ions and aromatic compounds.

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Yang Yang

Full Text Available Laccase is useful for various biotechnological and industrial applications. The white-rot fungus Trametes velutina 5930 and its laccase, isolated from the Shennongjia Nature Reserve in China by our laboratory, has great potential for practical application in environmental biotechnology. However, the original level of laccase produced by Trametes velutina 5930 was relatively low in the absence of any inducer. Therefore, in order to enhance the laccase production by Trametes velutina 5930 and make better use of this fungus in the field of environmental biotechnology, the regulation of laccase production and laccase gene expression in Trametes velutina 5930 were investigated in this study. Different metal ions such as Cu(2+ and Fe(2+ could stimulate the laccase synthesis and laccase gene transcription in Trametes velutina 5930. Some aromatic compounds structurally related to lignin, such as tannic acid, syringic acid, cinnamic acid, gallic acid and guaiacol, could also enhance the level of laccase activity and laccase gene transcription. We also found that there existed a positive synergistic effect of aromatic compound and metal ion on the laccase production and laccase gene transcription in Trametes velutina 5930. Taken together, our study may contribute to the improvement of laccase productivity by Trametes velutina 5930.

Enhancing the laccase production and laccase gene expression in the white-rot fungus Trametes velutina 5930 with great potential for biotechnological applications by different metal ions and aromatic compounds.

Science.gov (United States)

Yang, Yang; Wei, Fuxiang; Zhuo, Rui; Fan, Fangfang; Liu, Huahua; Zhang, Chen; Ma, Li; Jiang, Mulan; Zhang, Xiaoyu

2013-01-01

Laccase is useful for various biotechnological and industrial applications. The white-rot fungus Trametes velutina 5930 and its laccase, isolated from the Shennongjia Nature Reserve in China by our laboratory, has great potential for practical application in environmental biotechnology. However, the original level of laccase produced by Trametes velutina 5930 was relatively low in the absence of any inducer. Therefore, in order to enhance the laccase production by Trametes velutina 5930 and make better use of this fungus in the field of environmental biotechnology, the regulation of laccase production and laccase gene expression in Trametes velutina 5930 were investigated in this study. Different metal ions such as Cu(2+) and Fe(2+) could stimulate the laccase synthesis and laccase gene transcription in Trametes velutina 5930. Some aromatic compounds structurally related to lignin, such as tannic acid, syringic acid, cinnamic acid, gallic acid and guaiacol, could also enhance the level of laccase activity and laccase gene transcription. We also found that there existed a positive synergistic effect of aromatic compound and metal ion on the laccase production and laccase gene transcription in Trametes velutina 5930. Taken together, our study may contribute to the improvement of laccase productivity by Trametes velutina 5930.

Cloning, reassembling and integration of the entire nikkomycin biosynthetic gene cluster into Streptomyces ansochromogenes lead to an improved nikkomycin production

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Yang Haihua

2010-01-01

Full Text Available Abstract Background Nikkomycins are a group of peptidyl nucleoside antibiotics produced by Streptomyces ansochromogenes. They are competitive inhibitors of chitin synthase and show potent fungicidal, insecticidal, and acaricidal activities. Nikkomycin X and Z are the main components produced by S. ansochromogenes. Generation of a high-producing strain is crucial to scale up nikkomycins production for further clinical trials. Results To increase the yields of nikkomycins, an additional copy of nikkomycin biosynthetic gene cluster (35 kb was introduced into nikkomycin producing strain, S. ansochromogenes 7100. The gene cluster was first reassembled into an integrative plasmid by Red/ET technology combining with classic cloning methods and then the resulting plasmid(pNIKwas introduced into S. ansochromogenes by conjugal transfer. Introduction of pNIK led to enhanced production of nikkomycins (880 mg L-1, 4 -fold nikkomycin X and 210 mg L-1, 1.8-fold nikkomycin Z in the resulting exconjugants comparing with the parent strain (220 mg L-1 nikkomycin X and 120 mg L-1 nikkomycin Z. The exconjugants are genetically stable in the absence of antibiotic resistance selection pressure. Conclusion A high nikkomycins producing strain (1100 mg L-1 nikkomycins was obtained by introduction of an extra nikkomycin biosynthetic gene cluster into the genome of S. ansochromogenes. The strategies presented here could be applicable to other bacteria to improve the yields of secondary metabolites.

Branched-chain alpha-keto acid catabolism via the gene products of the bkd operon in Enterococcus faecalis: a ne, secreted metabolite serving as a temporary redox sink.

NARCIS (Netherlands)

Ward, D.E.; van der Weijden, C.C.; van der Merwe, M.J.; Westerhoff, H.V.; Claiborne, A.; Snoep, J.L.

2000-01-01

Recently the bkd gene cluster from Enterococcus faecalis was sequenced, and it was shown that the gene products constitute a pathway for the catabolism of branched-chain α-keto acids. We have now investigated the regulation and physiological role of this pathway. Primer extension analysis identified

Id-1 and Id-2 genes and products as therapeutic targets for treatment of breast cancer and other types of carcinoma

Science.gov (United States)

Desprez, Pierre-Yves; Campisi, Judith

2014-09-30

A method for treatment and amelioration of breast, cervical, ovarian, endometrial, squamous cells, prostate cancer and melanoma in a patient comprising targeting Id-1 or Id-2 gene expression with a delivery vehicle comprising a product which modulates Id-1 or Id-2 expression.

Changes in transcript levels of starch hydrolysis genes and raising citric acid production via carbon ion irradiation mutagenesis of Aspergillus niger.

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Wei Hu

Full Text Available The filamentous ascomycete Aspergillus niger is well known for its ability to accumulate citric acid for the hydrolysis of starchy materials. To improve citric acid productivity, heavy ion beam mutagenesis was utilized to produce mutant A.niger strains with enhanced production of citric acid in this work. It was demonstrated that a mutant HW2 with high concentration of citric acid was isolated after carbon ion irradiation with the energy of 80Mev/μ, which was obvious increase higher than the original strain from liquefied corn starch as a feedstock. More importantly, with the evidence from the expression profiles of key genes and enzyme activity involved in the starch hydrolysis process between original strain and various phenotype mutants, our results confirmed that different transcript levels of key genes involving in starch hydrolysis process between original strain and mutants could be a significant contributor to different citric acid concentration in A.niger, such as, amyR and glaA, which therefore opened a new avenue for constructing genetically engineered A.niger mutants for high-yield citric acid accumulation in the future. As such, this work demonstrated that heavy ion beam mutagenesis presented an efficient alternative strategy to be developed to generate various phenotype microbe species mutants for functional genes research.

Changes in transcript levels of starch hydrolysis genes and raising citric acid production via carbon ion irradiation mutagenesis of Aspergillus niger.

Science.gov (United States)

Hu, Wei; Li, Wenjian; Chen, Hao; Liu, Jing; Wang, Shuyang; Chen, Jihong

2017-01-01

The filamentous ascomycete Aspergillus niger is well known for its ability to accumulate citric acid for the hydrolysis of starchy materials. To improve citric acid productivity, heavy ion beam mutagenesis was utilized to produce mutant A.niger strains with enhanced production of citric acid in this work. It was demonstrated that a mutant HW2 with high concentration of citric acid was isolated after carbon ion irradiation with the energy of 80Mev/μ, which was obvious increase higher than the original strain from liquefied corn starch as a feedstock. More importantly, with the evidence from the expression profiles of key genes and enzyme activity involved in the starch hydrolysis process between original strain and various phenotype mutants, our results confirmed that different transcript levels of key genes involving in starch hydrolysis process between original strain and mutants could be a significant contributor to different citric acid concentration in A.niger, such as, amyR and glaA, which therefore opened a new avenue for constructing genetically engineered A.niger mutants for high-yield citric acid accumulation in the future. As such, this work demonstrated that heavy ion beam mutagenesis presented an efficient alternative strategy to be developed to generate various phenotype microbe species mutants for functional genes research.

Changes in transcript levels of starch hydrolysis genes and raising citric acid production via carbon ion irradiation mutagenesis of Aspergillus niger

Science.gov (United States)

Li, Wenjian; Chen, Hao; Liu, Jing; Wang, Shuyang; Chen, Jihong

2017-01-01

The filamentous ascomycete Aspergillus niger is well known for its ability to accumulate citric acid for the hydrolysis of starchy materials. To improve citric acid productivity, heavy ion beam mutagenesis was utilized to produce mutant A.niger strains with enhanced production of citric acid in this work. It was demonstrated that a mutant HW2 with high concentration of citric acid was isolated after carbon ion irradiation with the energy of 80Mev/μ, which was obvious increase higher than the original strain from liquefied corn starch as a feedstock. More importantly, with the evidence from the expression profiles of key genes and enzyme activity involved in the starch hydrolysis process between original strain and various phenotype mutants, our results confirmed that different transcript levels of key genes involving in starch hydrolysis process between original strain and mutants could be a significant contributor to different citric acid concentration in A.niger, such as, amyR and glaA, which therefore opened a new avenue for constructing genetically engineered A.niger mutants for high-yield citric acid accumulation in the future. As such, this work demonstrated that heavy ion beam mutagenesis presented an efficient alternative strategy to be developed to generate various phenotype microbe species mutants for functional genes research. PMID:28650980

Prokaryotic Expression of Rice Ospgip1 Gene and Bioinformatic Analysis of Encoded Product

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Xi-jun CHEN

2011-12-01

Full Text Available Using the reference sequences of pgip genes in GenBank, a fragment of 930 bp covering the open reading frame (ORF of rice Ospgip1 (Oryza sativa polygalacturonase-inhibiting protein 1 was amplified. The prokaryotic expression product of the gene inhibited the growth of Rhizoctonia solani, the causal agent of rice sheath blight, and reduced its polygalacturonase activity. Bioinformatic analysis showed that OsPGIP1 is a hydrophobic protein with a molecular weight of 32.8 kDa and an isoelectric point (pI of 7.26. The protein is mainly located in the cell wall of rice, and its signal peptide cleavage site is located between the 17th and 18th amino acids. There are four cysteines in both the N- and C-termini of the deduced protein, which can form three disulfide bonds (between the 56th and 63rd, the 278th and 298th, and the 300th and 308th amino acids. The protein has a typical leucine-rich repeat (LRR domain, and its secondary structure comprises α-helices, β-sheets and irregular coils. Compared with polygalacturonase-inhibiting proteins (PGIPs from other plants, the 7th LRR is absent in OsPGIP1. The nine LRRs could form a cleft that might associate with proteins from pathogenic fungi, such as polygalacturonase.

Yeast genes involved in regulating cysteine uptake affect production of hydrogen sulfide from cysteine during fermentation.

Science.gov (United States)

Huang, Chien-Wei; Walker, Michelle E; Fedrizzi, Bruno; Gardner, Richard C; Jiranek, Vladimir

2017-08-01

An early burst of hydrogen sulfide (H2S) produced by Saccharomyces cerevisiae during fermentation could increase varietal thiols and therefore enhance desirable tropical aromas in varieties such as Sauvignon Blanc. Here we attempted to identify genes affecting H2S formation from cysteine by screening yeast deletion libraries via a colony colour assay on media resembling grape juice. Both Δlst4 and Δlst7 formed lighter coloured colonies and produced significantly less H2S than the wild type on high concentrations of cysteine, likely because they are unable to take up cysteine efficiently. We then examined the nine known cysteine permeases and found that deletion of AGP1, GNP1 and MUP1 led to reduced production of H2S from cysteine. We further showed that deleting genes involved in the SPS-sensing pathway such as STP1 and DAL81 also reduced H2S from cysteine. Together, this study indirectly confirms that Agp1p, Gnp1p and Mup1p are the major cysteine permeases and that they are regulated by the SPS-sensing and target of rapamycin pathways under the grape juice-like, cysteine-supplemented, fermentation conditions. The findings highlight that cysteine transportation could be a limiting factor for yeast to generate H2S from cysteine, and therefore selecting wine yeasts without defects in cysteine uptake could maximise thiol production potential. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Metagenomic Analysis of the Sponge Discodermia Reveals the Production of the Cyanobacterial Natural Product Kasumigamide by 'Entotheonella'.

Science.gov (United States)

Nakashima, Yu; Egami, Yoko; Kimura, Miki; Wakimoto, Toshiyuki; Abe, Ikuro

2016-01-01

Sponge metagenomes are a useful platform to mine cryptic biosynthetic gene clusters responsible for production of natural products involved in the sponge-microbe association. Since numerous sponge-derived bioactive metabolites are biosynthesized by the symbiotic bacteria, this strategy may concurrently reveal sponge-symbiont produced compounds. Accordingly, a metagenomic analysis of the Japanese marine sponge Discodermia calyx has resulted in the identification of a hybrid type I polyketide synthase-nonribosomal peptide synthetase gene (kas). Bioinformatic analysis of the gene product suggested its involvement in the biosynthesis of kasumigamide, a tetrapeptide originally isolated from freshwater free-living cyanobacterium Microcystis aeruginosa NIES-87. Subsequent investigation of the sponge metabolic profile revealed the presence of kasumigamide in the sponge extract. The kasumigamide producing bacterium was identified as an 'Entotheonella' sp. Moreover, an in silico analysis of kas gene homologs uncovered the presence of kas family genes in two additional bacteria from different phyla. The production of kasumigamide by distantly related multiple bacterial strains implicates horizontal gene transfer and raises the potential for a wider distribution across other bacterial groups.

78 FR 70307 - Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy...

Science.gov (United States)

2013-11-25

...] Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products... Assessment of Investigational Cellular and Gene Therapy Products'' dated November 2013. The guidance document... products reviewed by the Office of Cellular, Tissue and Gene Therapies (OCTGT). The product areas covered...

Dynamics of antigen presentation to transgene product-specific CD4+ T cells and of Treg induction upon hepatic AAV gene transfer

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George Q Perrin

2016-01-01

Full Text Available The tolerogenic hepatic microenvironment impedes clearance of viral infections but is an advantage in viral vector gene transfer, which often results in immune tolerance induction to transgene products. Although the underlying tolerance mechanism has been extensively studied, our understanding of antigen presentation to transgene product-specific CD4+ T cells remains limited. To address this, we administered hepatotropic adeno-associated virus (AAV8 vector expressing cytoplasmic ovalbumin (OVA into wt mice followed by adoptive transfer of transgenic OVA-specific T cells. We find that that the liver-draining lymph nodes (celiac and portal are the major sites of MHC II presentation of the virally encoded antigen, as judged by in vivo proliferation of DO11.10 CD4+ T cells (requiring professional antigen-presenting cells, e.g., macrophages and CD4+CD25+FoxP3+ Treg induction. Antigen presentation in the liver itself contributes to activation of CD4+ T cells egressing from the liver. Hepatic-induced Treg rapidly disseminate through the systemic circulation. By contrast, a secreted OVA transgene product is presented in multiple organs, and OVA-specific Treg emerge in both the thymus and periphery. In summary, liver draining lymph nodes play an integral role in hepatic antigen presentation and peripheral Treg induction, which results in systemic regulation of the response to viral gene products.

Key gene regulating cell wall biosynthesis and recalcitrance in Populus, gene Y

Science.gov (United States)

Chen, Jay; Engle, Nancy; Gunter, Lee E.; Jawdy, Sara; Tschaplinski, Timothy J.; Tuskan, Gerald A.

2015-12-08

This disclosure provides methods and transgenic plants for improved production of renewable biofuels and other plant-derived biomaterials by altering the expression and/or activity of Gene Y, an O-acetyltransferase. This disclosure also provides expression vectors containing a nucleic acid (Gene Y) which encodes the polypeptide of SEQ ID NO: 1 and is operably linked to a heterologous promoter.

Development of Peptidomimetic Inhibitors of the ERG Gene Fusion Product in Prostate Cancer.

Science.gov (United States)

Wang, Xiaoju; Qiao, Yuanyuan; Asangani, Irfan A; Ateeq, Bushra; Poliakov, Anton; Cieślik, Marcin; Pitchiaya, Sethuramasundaram; Chakravarthi, Balabhadrapatruni V S K; Cao, Xuhong; Jing, Xiaojun; Wang, Cynthia X; Apel, Ingrid J; Wang, Rui; Tien, Jean Ching-Yi; Juckette, Kristin M; Yan, Wei; Jiang, Hui; Wang, Shaomeng; Varambally, Sooryanarayana; Chinnaiyan, Arul M

2017-04-10

Transcription factors play a key role in the development of diverse cancers, and therapeutically targeting them has remained a challenge. In prostate cancer, the gene encoding the transcription factor ERG is recurrently rearranged and plays a critical role in prostate oncogenesis. Here, we identified a series of peptides that interact specifically with the DNA binding domain of ERG. ERG inhibitory peptides (EIPs) and derived peptidomimetics bound ERG with high affinity and specificity, leading to proteolytic degradation of the ERG protein. The EIPs attenuated ERG-mediated transcription, chromatin recruitment, protein-protein interactions, cell invasion and proliferation, and tumor growth. Thus, peptidomimetic targeting of transcription factor fusion products may provide a promising therapeutic strategy for prostate cancer as well as other malignancies. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of Genes Involved in Bacteriocin Production and Self-Resistance in Lactobacillus brevis 174A Is Mediated by Two Regulatory Proteins.

Science.gov (United States)

Noda, Masafumi; Miyauchi, Rumi; Danshiitsoodol, Narandalai; Matoba, Yasuyuki; Kumagai, Takanori; Sugiyama, Masanori

2018-04-01

We have previously shown that the lactic acid bacterium Lactobacillus brevis 174A, isolated from Citrus iyo fruit, produces a bacteriocin designated brevicin 174A, which is comprised of two antibacterial polypeptides (designated brevicins 174A-β and 174A-γ). We have also found a gene cluster, composed of eight open reading frames (ORFs), that contains genes for the biosynthesis of brevicin 174A, self-resistance to its own bacteriocin, and two transcriptional regulatory proteins. Some lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth. Generally, the system consists of a membrane-bound histidine protein kinase (HPK) that senses a specific environmental stimulus and a corresponding response regulator (RR) that mediates the cellular response. We have previously shown that although the HPK- and RR-encoding genes are not found on the brevicin 174A biosynthetic gene cluster in the 174A strain, two putative regulatory genes, designated breD and breG , are in the gene cluster. In the present study, we demonstrate that the expression of brevicin 174A production and self-resistance is positively controlled by two transcriptional regulatory proteins, designated BreD and BreG. BreD is expressed together with BreE as the self-resistance determinant of L. brevis 174A. DNase I footprinting analysis and a promoter assay demonstrated that BreD binds to the breED promoter as a positive autoregulator. The present study also demonstrates that BreG, carrying a transmembrane domain, binds to the common promoter of breB and breC , encoding brevicins 174A-β and 174A-γ, respectively, for positive regulation. IMPORTANCE The problem of the appearance of bacteria that are resistant to practical antibiotics and the increasing demand for safe foods have increased interest in replacing conventional antibiotics with bacteriocin produced by the lactic acid bacteria. This antibacterial substance can inhibit the growth of pathogenic

Tumor targeted gene therapy

International Nuclear Information System (INIS)

Kang, Joo Hyun

2006-01-01

Knowledge of molecular mechanisms governing malignant transformation brings new opportunities for therapeutic intervention against cancer using novel approaches. One of them is gene therapy based on the transfer of genetic material to an organism with the aim of correcting a disease. The application of gene therapy to the cancer treatment had led to the development of new experimental approaches such as suicidal gene therapy, inhibition of oncogenes and restoration of tumor-suppressor genes. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a prodrug into a toxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1-tk) and cytosine deaminase (CD). Especially, physicians and scientists of nuclear medicine field take an interest in suicidal gene therapy because they can monitor the location and magnitude, and duration of expression of HSV1-tk and CD by PET scanner

Genes2FANs: connecting genes through functional association networks

Science.gov (United States)

2012-01-01

Background Protein-protein, cell signaling, metabolic, and transcriptional interaction networks are useful for identifying connections between lists of experimentally identified genes/proteins. However, besides physical or co-expression interactions there are many ways in which pairs of genes, or their protein products, can be associated. By systematically incorporating knowledge on shared properties of genes from diverse sources to build functional association networks (FANs), researchers may be able to identify additional functional interactions between groups of genes that are not readily apparent. Results Genes2FANs is a web based tool and a database that utilizes 14 carefully constructed FANs and a large-scale protein-protein interaction (PPI) network to build subnetworks that connect lists of human and mouse genes. The FANs are created from mammalian gene set libraries where mouse genes are converted to their human orthologs. The tool takes as input a list of human or mouse Entrez gene symbols to produce a subnetwork and a ranked list of intermediate genes that are used to connect the query input list. In addition, users can enter any PubMed search term and then the system automatically converts the returned results to gene lists using GeneRIF. This gene list is then used as input to generate a subnetwork from the user’s PubMed query. As a case study, we applied Genes2FANs to connect disease genes from 90 well-studied disorders. We find an inverse correlation between the counts of links connecting disease genes through PPI and links connecting diseases genes through FANs, separating diseases into two categories. Conclusions Genes2FANs is a useful tool for interpreting the relationships between gene/protein lists in the context of their various functions and networks. Combining functional association interactions with physical PPIs can be useful for revealing new biology and help form hypotheses for further experimentation. Our finding that disease genes in

GO(vis), a gene ontology visualization tool based on multi-dimensional values.

Science.gov (United States)

Ning, Zi; Jiang, Zhenran

2010-05-01

Most of gene product similarity measurements concentrate on the information content of Gene Ontology (GO) terms or use a path-based similarity between GO terms, which may ignore other important information contained in the structure of the ontology. In our study, we integrate different GO similarity measure approaches to analyze the functional relationship of genes and gene products with a new triangle-based visualization tool called GO(Vis). The purpose of this tool is to demonstrate the effect of three important information factors when measuring the similarity between gene products. One advantage of this tool is that its important ratio can be adjusted to meet different measuring requirements according to the biological knowledge of each factor. The experimental results demonstrate that GO(Vis) can display diagrams of the functional relationship for gene products effectively.

Photobiological hydrogen production with switchable photosystem-II designer algae

Science.gov (United States)

Lee, James Weifu

2014-02-18

A process for enhanced photobiological H.sub.2 production using transgenic alga. The process includes inducing exogenous genes in a transgenic alga by manipulating selected environmental factors. In one embodiment inducing production of an exogenous gene uncouples H.sub.2 production from existing mechanisms that would downregulate H.sub.2 production in the absence of the exogenous gene. In other embodiments inducing an exogenous gene triggers a cascade of metabolic changes that increase H.sub.2 production. In some embodiments the transgenic alga are rendered non-regenerative by inducing exogenous transgenes for proton channel polypeptides that are targeted to specific algal membranes.

Clinical significance of productive immunoglobulin heavy chain gene rearrangements in childhood acute lymphoblastic leukemia.

Science.gov (United States)

Katsibardi, Katerina; Braoudaki, Maria; Papathanasiou, Chrissa; Karamolegou, Kalliopi; Tzortzatou-Stathopoulou, Fotini

2011-09-01

We analyzed the CDR3 region of 80 children with B-cell acute lymphoblastic leukemia (B-ALL) using the ImMunoGeneTics Information System and JOINSOLVER. In total, 108 IGH@ rearrangements were analyzed. Most of them (75.3%) were non-productive. IGHV@ segments proximal to IGHD-IGHJ@ were preferentially rearranged (45.3%). Increased utilization of IGHV3 segments IGHV3-13 (11.3%) and IGHV3-15 (9.3%), IGHD3 (30.5%), and IGHJ4 (34%) was noted. In pro-B ALL more frequent were IGHV3-11 (33.3%) and IGHV6-1 (33.3%), IGHD2-21 (50%), IGHJ4 (50%), and IGHJ6 (50%) segments. Shorter CDR3 length was observed in IGHV@6, IGHD7, and IGHJ1 segments, whereas increased CDR3 length was related to IGHV3, IGHD2, and IGHJ4 segments. Increased risk of relapse was found in patients with productive sequences. Specifically, the relapse-free survival rate at 5 years in patients with productive sequences at diagnosis was 75% (standard error [SE] ±9%), whereas in patients with non-productive sequences it was 97% (SE ±1.92%) (p-value =0.0264). Monoclonality and oligoclonality were identified in 81.2% and 18.75% cases at diagnosis, respectively. Sequence analysis revealed IGHV@ to IGHDJ joining only in 6.6% cases with oligoclonality. The majority (75%) of relapsed patients had monoclonal IGH@ rearrangements. The preferential utilization of IGHV@ segments proximal to IGHDJ depended on their location on the IGHV@ locus. Molecular mechanisms occurring during IGH@ rearrangement might play an essential role in childhood ALL prognosis. In our study, the productivity of the rearranged sequences at diagnosis proved to be a significant prognostic factor.

Evaluation of gene expression and alginate production in response to oxygen transfer in continuous culture of Azotobacter vinelandii.

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Alvaro Díaz-Barrera

Full Text Available Alginates are polysaccharides used as food additives and encapsulation agents in biotechnology, and their functional properties depend on its molecular weight. In this study, different steady-states in continuous cultures of A. vinelandii were established to determine the effect of the dilution rate (D and the agitation rate on alginate production and expression of genes involved in alginate polymerization and depolymerization. Both, the agitation and dilution rates, determined the partitioning of the carbon utilization from sucrose into alginate and CO2 under oxygen-limiting conditions. A low D (0.07 h(-1 and 500 rpm resulted in the highest carbon utilization into alginate (25%. Quantitative real-time polymerase chain reaction was used to determine the transcription level of six genes involved in alginate polymerization and depolymerization. In chemostat cultures at 0.07 h(-1, the gene expression was affected by changes in the agitation rate. By increasing the agitation rate from 400 to 600 rpm, the algE7 gene expression decreased tenfold, whereas alyA1, algL and alyA2 gene expression increased between 1.5 and 2.8 times under similar conditions evaluated. Chemostat at 0.07 h(-1 showed a highest alginate molecular weight (580 kDa at 500 rpm whereas similar molecular weights (480 kDa were obtained at 400 and 600 rpm. The highest molecular weight was not explained by changes in the expression of alg8 and alg44 (genes involved in alginate polymerization. Nonetheless, a different expression pattern observed for lyases could explain the highest alginate molecular weight obtained. Overall, the results suggest that the control of alginate molecular weight in A. vinelandii cells growing in continuous mode is determined by a balance between the gene expression of intracellular and extracellular lyases in response to oxygen availability. These findings better our understanding of the biosynthesis of bacterial alginate and help us progress toward obtain

[Prokaryotic expression of trigeminy artificial fusion gene of Leptospira interrogans and the immunogenicity of its products].

Science.gov (United States)

Luo, Dong-jiao; Qiu, Xiao-feng; Wang, Jiang; Yan, Jin; Wang, Hai-bin; Zhou, Jin-cheng; Yan, Jie

2008-11-01

To construct lipL32/1-lipL21-OmpL1/2 fusion gene of Leptospira interrogans and its prokaryotic expression system, and to identify the immunogenicity of its products. PCR using linking primers was applied to construct lipL32/1-lipL21-OmpL1/2 fusion gene and a prokaryotic expression system of the fusion gene was then established using routine genetic engineering technique. SDS-PAGE was used to examine output of the target recombinant protein rLipL32/1-LipL21-OmpL1/2. Double immunodiffusion and Western Blot assay were applied to identify immunogenicity of rLipL32/1-LipL21-OmpL1/2. lipL32/1-lipL21-OmpL1/2 fusion gene with correct sequence and its prokaryotic expression system E.coli BL21DE3pET42a-lipL32/1-lipL21-ompL1/2 was obtained in this study. The output of rLipL32/1-LipL21- OmpL1/2 after optimisation was 37.78 mg/L. The immunodiffusion titer of rabbit antiserum against rLipL32/1-LipL21-OmpL1/2 was 1:4. The rLipL32/1-LipL21-OmpL1/2 antiserum was able to recognize rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 and rOmpL1/2. Positive Western hybridization signals were found among rLipL32/1-LipL21-OmpL1/2 and rabbit antiserum against whole cell of strain 56601 and serum from patients infected with L.interrogans serogroups Icterohaemorrhagiae, Grippotyphosa, Autumnalis and Pomona. The fusion gene lipL32/1-lipL21-OmpL1/2 and its prokaryotic expression system were successfully constructed in this study. The expressed fusion protein can be used as the antigen for developing universal genetic engineering vaccine and universal serological tests of leptospirosis.

Vector Production in an Academic Environment: A Tool to Assess Production Costs

Science.gov (United States)

Boeke, Aaron; Doumas, Patrick; Reeves, Lilith; McClurg, Kyle; Bischof, Daniela; Sego, Lina; Auberry, Alisha; Tatikonda, Mohan

2013-01-01

Abstract Generating gene and cell therapy products under good manufacturing practices is a complex process. When determining the cost of these products, researchers must consider the large number of supplies used for manufacturing and the personnel and facility costs to generate vector and maintain a cleanroom facility. To facilitate cost estimates, the Indiana University Vector Production Facility teamed with the Indiana University Kelley School of Business to develop a costing tool that, in turn, provides pricing. The tool is designed in Microsoft Excel and is customizable to meet the needs of other core facilities. It is available from the National Gene Vector Biorepository. The tool allows cost determinations using three different costing methods and was developed in an effort to meet the A21 circular requirements for U.S. core facilities performing work for federally funded projects. The costing tool analysis reveals that the cost of vector production does not have a linear relationship with batch size. For example, increasing the production from 9 to18 liters of a retroviral vector product increases total costs a modest 1.2-fold rather than doubling in total cost. The analysis discussed in this article will help core facilities and investigators plan a cost-effective strategy for gene and cell therapy production. PMID:23360377

Contribution of V(H replacement products in mouse antibody repertoire.

Directory of Open Access Journals (Sweden)

Lin Huang

Full Text Available VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS near the 3' end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified VH replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

A Study on Enhanced Expression of 3-Hydroxypropionic Acid Pathway Genes and Impact on Its Production in Lactobacillus reuteri

Directory of Open Access Journals (Sweden)

Gopal Ramakrishnan Gopi

2015-01-01

Full Text Available 3-Hydroxypropionic acid (3-HP is a novel antimicrobial agent against foodborne pathogens like Salmonella and Staphylococcus species. Lactobacillus reuteri converts glycerol into 3-HP using a coenzyme A-dependent pathway, which is encoded by propanediol utilization operon (pdu subjected to catabolite repression. In a catabolite repression-deregulated L. reuteri RPRB3007, quantitative PCR revealed a 2.5-fold increase in the transcripts of the genes pduP, pduW and pduL during the mid-log phase of growth. The production of 3-HP was tested in resting cells in phosphate buff er and growing batch cultures in MRS broth of various glucose/glycerol ratios. Due to the upregulation of pathway genes, specific formation rate of 3-HP in the mutant strain was found to be enhanced from 0.167 to 0.257 g per g of cell dry mass per h. Furthermore, formation of 3-HP in resting cells was limited due to the substrate inhibition by reuterin at a concentration of (30±5 mM. In batch cultures, the formation of 3-HP was not observed during the logarithmic and stationary phases of growth of wild-type and mutant strains, which was confi rmed by NMR spectroscopy. However, the cells collected in these phases were found to produce 3-HP aft er washing and converting them to resting cells. Lactate and acetate, the primary end products of glucose catabolism, might be the inhibiting elements for 3-HP formation in batch cultures. This was confirmed when lactate (25±5 mM or acetate (20±5 mM were added to biotransformation medium, which prevented the 3-HP formation. Moreover, the removal of sodium acetate and glucose (carbon source for lactic acid production was found to restore 3-HP formation in the MRS broth in a similar manner to that of the phosphate buff er. Even though the genetic repression was circumvented by the up-regulation of pathway genes using a mutant strain, 3-HP formation was further limited by the substrate and catabolite inhibition.

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

Science.gov (United States)

Pyeon, Hye-Rim; Nah, Hee-Ju; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

2017-05-31

Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem

Putrescine production by Lactococcus lactis subsp. cremoris CECT 8666 is reduced by NaCl via a decrease in bacterial growth and the repression of the genes involved in putrescine production.

Science.gov (United States)

Del Rio, Beatriz; Redruello, Begoña; Ladero, Victor; Fernandez, Maria; Martin, Maria Cruz; Alvarez, Miguel A

2016-09-02

The reduction of NaCl in food is a public health priority; high NaCl intakes have been associated with serious health problems. However, it is reported that reducing the NaCl content of cheeses may lead to an increase in the content of biogenic amines (BAs). The present work examines the effect of NaCl on the accumulation of putrescine (one of the BAs often detected at high concentration in cheese) in experimental Cabrales-like cheeses containing Lactococcus lactis subsp. cremoris CECT 8666, a dairy strain that catabolises agmatine to putrescine via the agmatine deiminase (AGDI) pathway. The genes responsible for this pathway are grouped in the AGDI cluster. This comprises a regulatory gene (aguR) (transcribed independently), followed by the catabolic genes that together form an operon (aguBDAC). Reducing the NaCl concentration of the cheese led to increased putrescine accumulation. In contrast, increasing the NaCl concentration of both pH-uncontrolled and pH-controlled (pH 6) cultures of L. lactis subsp. cremoris CECT 8666 significantly inhibited its growth and the production of putrescine. Such production appeared to be inhibited via a reduction in the transcription of the aguBDAC operon; no effect on the transcription of aguR was recorded. The present results suggest that low-sodium cheeses are at risk of accumulating higher concentrations of putrescine. Copyright © 2016 Elsevier B.V. All rights reserved.

GeneViTo: Visualizing gene-product functional and structural features in genomic datasets

Directory of Open Access Journals (Sweden)

Promponas Vasilis J

2003-10-01

Full Text Available Abstract Background The availability of increasing amounts of sequence data from completely sequenced genomes boosts the development of new computational methods for automated genome annotation and comparative genomics. Therefore, there is a need for tools that facilitate the visualization of raw data and results produced by bioinformatics analysis, providing new means for interactive genome exploration. Visual inspection can be used as a basis to assess the quality of various analysis algorithms and to aid in-depth genomic studies. Results GeneViTo is a JAVA-based computer application that serves as a workbench for genome-wide analysis through visual interaction. The application deals with various experimental information concerning both DNA and protein sequences (derived from public sequence databases or proprietary data sources and meta-data obtained by various prediction algorithms, classification schemes or user-defined features. Interaction with a Graphical User Interface (GUI allows easy extraction of genomic and proteomic data referring to the sequence itself, sequence features, or general structural and functional features. Emphasis is laid on the potential comparison between annotation and prediction data in order to offer a supplement to the provided information, especially in cases of "poor" annotation, or an evaluation of available predictions. Moreover, desired information can be output in high quality JPEG image files for further elaboration and scientific use. A compilation of properly formatted GeneViTo input data for demonstration is available to interested readers for two completely sequenced prokaryotes, Chlamydia trachomatis and Methanococcus jannaschii. Conclusions GeneViTo offers an inspectional view of genomic functional elements, concerning data stemming both from database annotation and analysis tools for an overall analysis of existing genomes. The application is compatible with Linux or Windows ME-2000-XP operating

Polymorphism screening of four genes encoding advanced glycation end-product putative receptors. Association study with nephropathy in type 1 diabetic patients

DEFF Research Database (Denmark)

Poirier, Odette; Nicaud, Viviane; Vionnet, N

2001-01-01

Advanced glycation end-products (AGEs) may play an important role in the pathogenesis and progression of cardiovascular and renal complications of diabetes. Four putative AGE receptors (RAGEs), AGE-R1, AGE-R2, and AGE-R3 have been described. In this study, we scanned the sequence of the genes enc...

Characterization of Antibiotic Resistance Genes from Lactobacillus Isolated from Traditional Dairy Products.

Science.gov (United States)

Guo, Huiling; Pan, Lin; Li, Lina; Lu, Jie; Kwok, Laiyu; Menghe, Bilige; Zhang, Heping; Zhang, Wenyi

2017-03-01

Lactobacilli are widely used as starter cultures or probiotics in yoghurt, cheese, beer, wine, pickles, preserved food, and silage. They are generally recognized as safe (GRAS). However, recent studies have shown that some lactic acid bacteria (LAB) strains carry antibiotic resistance genes and are resistant to antibiotics. Some of them may even transfer their intrinsic antibiotic resistance genes to other LAB or pathogens via horizontal gene transfer, thus threatening human health. A total of 33 Lactobacillus strains was isolated from fermented milk collected from different areas of China. We analyzed (1) their levels of antibiotic resistance using a standardized dilution method, (2) their antibiotic resistance gene profiles by polymerase chain reaction (PCR) using gene-specific primers, and (3) the transferability of some of the detected resistance markers by a filter mating assay. All Lactobacillus strains were found to be resistant to vancomycin, but susceptible to gentamicin, linezolid, neomycin, erythromycin, and clindamycin. Their susceptibilities to tetracycline, kanamycin, ciprofloxacin, streptomycin, quinupristin/dalfopristin, trimethoprim, ampicillin, rifampicin, and chloramphenicol was different. Results from our PCR analysis revealed 19 vancomycin, 10 ciprofloxacin, and 1 tetracycline-resistant bacteria that carried the van(X), van(E), gyr(A), and tet(M) genes, respectively. Finally, no transferal of the monitored antibiotic resistance genes was observed in the filter mating assay. Taken together, our study generated the antibiotic resistance profiles of some milk-originated lactobacilli isolates and preliminarily assessed their risk of transferring antibiotic gene to other bacteria. The study may provide important data concerning the safe use of LAB. © 2017 Institute of Food Technologists®.

Co-expression of Exo-inulinase and Endo-inulinase Genes in the Oleaginous Yeast Yarrowia lipolytica for Efficient Single Cell Oil Production from Inulin.

Science.gov (United States)

Shi, Nianci; Mao, Weian; He, Xiaoxia; Chi, Zhe; Chi, Zhenming; Liu, Guanglei

2018-05-01

Yarrowia lipolytica is a promising platform for the single cell oil (SCO) production. In this study, a transformant X+N8 in which exo- and endo-inulinase genes were co-expressed could produce an inulinase activity of 124.33 U/mL within 72 h. However, the inulinase activity of a transformant X2 carrying a single exo-inulinase gene was only 47.33 U/mL within 72 h. Moreover, the transformant X+N8 could accumulate 48.13% (w/w) SCO from inulin and the cell dry weight reached 13.63 g/L within 78 h, which were significantly higher than those of the transformant X2 (41.87% (w/w) and 11.23 g/L) under the same conditions. In addition, inulin hydrolysis and utilization of the transformant X+N8 were also more efficient than those of the transformant X2 during the fermentation process. These results demonstrated that the co-expression of the exo- and endo-inulinase genes significantly enhanced the SCO production from inulin due to the improvement of the inulinase activity and the synergistic action of exo- and endo-inulinase. Besides, over 95.01% of the fatty acids from the transformant X+N8 were C16-C18, especially C18:1 (53.10%), suggesting that the fatty acids could be used as feedstock for biodiesel production.

The Crc protein participates in down-regulation of the Lon gene to promote rhamnolipid production and rhl quorum sensing in Pseudomonas aeruginosa.

Science.gov (United States)

Yang, Nana; Ding, Shuting; Chen, Feifei; Zhang, Xue; Xia, Yongjie; Di, Hongxia; Cao, Qiao; Deng, Xin; Wu, Min; Wong, Catherine C L; Tian, Xiao-Xu; Yang, Cai-Guang; Zhao, Jing; Lan, Lefu

2015-05-01

Rhamnolipid acts as a virulence factor during Pseudomonas aeruginosa infection. Here, we show that deletion of the catabolite repression control (crc) gene in P. aeruginosa leads to a rhamnolipid-negative phenotype. This effect is mediated by the down-regulation of rhl quorum sensing (QS). We discover that a disruption of the gene encoding the Lon protease entirely offsets the effect of crc deletion on the production of both rhamnolipid and rhl QS signal C4-HSL. Crc is unable to bind lon mRNA in vitro in the absence of the RNA chaperon Hfq, while Crc contributes to Hfq-mediated repression of the lon gene expression at a posttranscriptional level. Deletion of crc, which results in up-regulation of lon, significantly reduces the in vivo stability and abundance of the RhlI protein that synthesizes C4-HSL, causing the attenuation of rhl QS. Lon is also capable of degrading the RhlI protein in vitro. In addition, constitutive expression of rhlI suppresses the defects of the crc deletion mutant in rhamnolipid, C4-HSL and virulence on lettuce leaves. This study therefore uncovers a novel posttranscriptional regulatory cascade, Crc-Hfq/Lon/RhlI, for the regulation of rhamnolipid production and rhl QS in P. aeruginosa. © 2015 John Wiley & Sons Ltd.

Influence of zinc on biogas production and antibiotic resistance gene profiles during anaerobic digestion of swine manure.

Science.gov (United States)

Zhang, Ranran; Wang, Xiaojuan; Gu, Jie; Zhang, Yajun

2017-11-01

This study determined the accumulated biogas, methane content, and absolute abundances (AAs) of 14 common antibiotic resistance genes (ARGs) and two integrons during the anaerobic digestion of swine manure for 52days with different amounts of added zinc. The accumulated biogas increased by 51.2% and 56.0% with 125mgL -1 (L) and 1250mgL -1 (H) zinc, respectively, compared with the control with no added zinc (CK), but there was no significant difference between L and H. Compared with CK, excluding tetW and tetC, all the other ARGs detected in this study increased in the L and H reactors. However, the low concentration of zinc (L reactor) caused greater increases in the AAs of ARGs in the AD products. Redundancy analysis showed that NO 3 -N and bio-zinc significantly explained the changes in genes, where they accounted for 60.9% and 20.3% of the total variation in the environmental factors, respectively. Copyright © 2017. Published by Elsevier Ltd.

Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae

Science.gov (United States)

2013-01-01

Background Secondary metabolite production, a hallmark of filamentous fungi, is an expanding area of research for the Aspergilli. These compounds are potent chemicals, ranging from deadly toxins to therapeutic antibiotics to potential anti-cancer drugs. The genome sequences for multiple Aspergilli have been determined, and provide a wealth of predictive information about secondary metabolite production. Sequence analysis and gene overexpression strategies have enabled the discovery of novel secondary metabolites and the genes involved in their biosynthesis. The Aspergillus Genome Database (AspGD) provides a central repository for gene annotation and protein information for Aspergillus species. These annotations include Gene Ontology (GO) terms, phenotype data, gene names and descriptions and they are crucial for interpreting both small- and large-scale data and for aiding in the design of new experiments that further Aspergillus research. Results We have manually curated Biological Process GO annotations for all genes in AspGD with recorded functions in secondary metabolite production, adding new GO terms that specifically describe each secondary metabolite. We then leveraged these new annotations to predict roles in secondary metabolism for genes lacking experimental characterization. As a starting point for manually annotating Aspergillus secondary metabolite gene clusters, we used antiSMASH (antibiotics and Secondary Metabolite Analysis SHell) and SMURF (Secondary Metabolite Unknown Regions Finder) algorithms to identify potential clusters in A. nidulans, A. fumigatus, A. niger and A. oryzae, which we subsequently refined through manual curation. Conclusions This set of 266 manually curated secondary metabolite gene clusters will facilitate the investigation of novel Aspergillus secondary metabolites. PMID:23617571

Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

Science.gov (United States)

Pyne, C; Bognar, A L

1992-03-01

The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.

Association study of molecular polymorphisms in candidate genes related to stress responses with production and meat quality traits in pigs.

Science.gov (United States)

Terenina, E; Babigumira, B M; Le Mignon, G; Bazovkina, D; Rousseau, S; Salin, F; Bendixen, C; Mormede, P

2013-02-01

The hypothalamic-pituitary-adrenal (HPA) axis exerts a large range of effects on metabolism, the immune system, inflammatory processes, and brain functions. Together with the sympathetic nervous system, it is also the most important stress-responsive neuroendocrine system. Both systems influence production traits, carcass composition, and meat quality. The HPA axis may be a critical target for genetic selection of more robust animals. Indeed, numerous studies in various species have demonstrated the importance of genetic factors in shaping the individual HPA axis phenotype, and genetic polymorphism can be found at each level of the axis, including hormone production by the adrenal cortices under stimulation by adrenocorticotropic hormone (ACTH), hormone bioavailability, or receptor and postreceptor mechanisms. The aim of the present experiment was to extend these findings to the brain neurochemical systems involved in stress responses. To this end, a number of candidate genes were sequenced for molecular polymorphisms and their association was studied with stress neuroendocrine and production traits in a genetically diverse population consisting of 100 female pigs from an advanced intercross (F10-F12) between 2 highly divergent breeds, Large White (LW) and Meishan (MS). The LW breed has a high production potential for lean meat and a low HPA axis activity, and the MS breed has low growth rate, fat carcasses-but large litters of highly viable piglets-and a high HPA axis activity. Candidate genes were chosen in the catecholaminergic and serotonergic pathways, in the pituitary control of cortisol production, among genes previously demonstrated to be differentially expressed in ACTH-stimulated adrenal glands from LW and MS pigs, and in cortisol receptors. Sixty new polymorphisms were found. The association study with carcass and meat quality traits and with endocrine traits showed a number of significant results, such as monoamine oxidase (MAOA) polymorphisms with

Gene-based vaccine development for improving animal production in developing countries. Possibilities and constraints

International Nuclear Information System (INIS)

Egerton, J.R.

2005-01-01

For vaccine production, recombinant antigens must be protective. Identifying protective antigens or candidate antigens is an essential precursor to vaccine development. Even when a protective antigen has been identified, cloning of its gene does not lead directly to vaccine development. The fimbrial protein of Dichelobacter nodosus, the agent of foot-rot in ruminants, was known to be protective. Recombinant vaccines against this infection are ineffective if expressed protein subunits are not assembled as mature fimbriae. Antigenic competition between different, but closely related, recombinant antigens limited the use of multivalent vaccines based on this technology. Recombinant antigens may need adjuvants to enhance response. DNA vaccines, potentiated with genes for different cytokines, may replace the need for aggressive adjuvants, and especially where cellular immunity is essential for protection. The expression of antigens from animal pathogens in plants and the demonstration of some immunity to a disease like rinderpest after ingestion of these, suggests an alternative approach to vaccination by injection. Research on disease pathogenesis and the identification of candidate antigens is specific to the disease agent. The definition of expression systems and the formulation of a vaccine for each disease must be followed by research to establish safety and efficacy. Where vaccines are based on unique gene sequences, the intellectual property is likely to be protected by patent. Organizations, licensed to produce recombinant vaccines, expect to recover their costs and to make a profit. The consequence is that genetically-derived vaccines are expensive. The capacity of vaccines to help animal owners of poorer countries depends not only on quality and cost but also on the veterinary infrastructure where they are used. Ensuring the existence of an effective animal health infrastructure in developing countries is as great a challenge for the developed world as

Think big--giant genes in bacteria.

Science.gov (United States)

Reva, Oleg; Tümmler, Burkhard

2008-03-01

Long genes should be rare in archaea and eubacteria because of the demanding costs of time and resources for protein production. The search in 580 sequenced prokaryotic genomes, however, revealed 0.2% of all genes to be longer than 5 kb (absolute number: 3732 genes). Eighty giant bacterial genes of more than 20 kb in length were identified in 47 taxa that belong to the phyla Thermotogae (1), Chlorobi (3), Planctomycetes (1), Cyanobacteria (2), Firmicutes (7), Actinobacteria (9), Proteobacteria (23) or Euryarchaeota (1) (number of taxa in brackets). Giant genes are strain-specific, differ in their tetranucleotide usage from the bulk genome and occur preferentially in non-pathogenic environmental bacteria. The two longest bacterial genes known to date were detected in the green sulfur bacterium Chlorobium chlorochromatii CaD3 encoding proteins of 36 806 and 20 647 amino acids, being surpassed in length only by the human titin coding sequence. More than 90% of bacterial giant genes either encode a surface protein or a polyketide/non-ribosomal peptide synthetase. Most surface proteins are acidic, threonine-rich, lack cystein and harbour multiple amino acid repeats. Giant proteins increase bacterial fitness by the production of either weapons towards or shields against animate competitors or hostile environments.

Overexpression and rapid purification of the orfE/rph gene product, RNase PH of Escherichia coli

DEFF Research Database (Denmark)

Jensen, Kaj Frank; Andersen, J T; Poulsen, Peter

1992-01-01

acid residue protein which was recently identified as the phosphorolytic ribonuclease, RNase PH, that removes nucleotides from the 3' ends of tRNA precursors. In this paper we report the construction of a plasmid, which overexpresses the orfE and pyrE gene products substantially, as well....../min/mg, as characteristic for RNase PH. OrfE/RNase PH contains helix-turn-helix motifs resembling those in DNA-binding proteins, and it binds nonspecifically to DNA. On SDS gels, OrfE/RNase PH migrates as two distinct protein bands. This heterogeneity might be caused by post-translational modification other than...

Mitochondrial DNA Variants Mediate Energy Production and Expression Levels for CFH, C3 and EFEMP1 Genes: Implications for Age-Related Macular Degeneration

Science.gov (United States)

Kenney, M. Cristina; Chwa, Marilyn; Atilano, Shari R.; Pavlis, Janelle M.; Falatoonzadeh, Payam; Ramirez, Claudio; Malik, Deepika; Hsu, Tiffany; Woo, Grace; Soe, Kyaw; Nesburn, Anthony B.; Boyer, David S.; Kuppermann, Baruch D.; Jazwinski, S. Michal; Miceli, Michael V.; Wallace, Douglas C.; Udar, Nitin

2013-01-01

Background Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD). Methodology/Principal Findings Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism. Conclusion/Significance Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD. PMID:23365660

Mitochondrial DNA variants mediate energy production and expression levels for CFH, C3 and EFEMP1 genes: implications for age-related macular degeneration.

Directory of Open Access Journals (Sweden)

M Cristina Kenney

Full Text Available Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD. Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt DNA haplogroups (as defined by combinations of mtDNA polymorphisms that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD versus J haplogroup (high risk for AMD.Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19 that was devoid of mitochondrial DNA (Rho0. In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism.Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.

Analysis of the structural genes encoding M-factor in the fission yeast Schizosaccharomyces pombe: identification of a third gene, mfm3

DEFF Research Database (Denmark)

Kjaerulff, S; Davey, William John; Nielsen, O

1994-01-01

We previously identified two genes, mfm1 and mfm2, with the potential to encode the M-factor mating pheromone of the fission yeast Schizosaccharomyces pombe (J. Davey, EMBO J. 11:951-960, 1992), but further analysis revealed that a mutant strain lacking both genes still produced active M-factor. ......We previously identified two genes, mfm1 and mfm2, with the potential to encode the M-factor mating pheromone of the fission yeast Schizosaccharomyces pombe (J. Davey, EMBO J. 11:951-960, 1992), but further analysis revealed that a mutant strain lacking both genes still produced active M...... that is not rescued by addition of exogenous M-factor. A mutational analysis reveals that all three mfm genes contribute to the production of M-factor. Their transcription is limited to M cells and requires the mat1-Mc and ste11 gene products. Each gene is induced when the cells are starved of nitrogen and further...

Evaluating PHA productivity of bioengineered Rhodosprillum rubrum.

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Huanan Jin

Full Text Available This study explored the potential of using Rhodosprillum rubrum as the biological vehicle to convert chemically simple carbon precursors to a value-added bio-based product, the biopolymer PHA. R. rubrum strains were bioengineered to overexpress individually or in various combinations, six PHA biosynthetic genes (phaC1, phaA, phaB, phaC2, phaC3, and phaJ, and the resulting nine over-expressing strains were evaluated to assess the effect on PHA content, and the effect on growth. These experiments were designed to genetically evaluate: 1 the role of each apparently redundant PHA polymerase in determining PHA productivity; 2 identify the key gene(s within the pha biosynthetic operon that determines PHA productivity; and 3 the role of phaJ to support PHA productivity. The result of overexpressing each PHA polymerase-encoding gene indicates that phaC1 and phaC2 are significant contributors to PHA productivity, whereas phaC3 has little effect. Similarly, over-expressing individually or in combination the three PHA biosynthesis genes located in the pha operon indicates that phaB is the key determinant of PHA productivity. Finally, analogous experiments indicate that phaJ does not contribute significantly to PHA productivity. These bioengineering strains achieved PHA productivity of up to 30% of dry biomass, which is approximately 2.5-fold higher than the non-engineered control strain, indicating the feasibility of using this approach to produce value added bio-based products.

In Vivo Production of Monoclonal Antibodies by Gene Transfer via Electroporation Protects against Lethal Influenza and Ebola Infections

Directory of Open Access Journals (Sweden)

Chasity D. Andrews

2017-12-01

Full Text Available Monoclonal antibodies (mAbs have wide clinical utility, but global access is limited by high costs and impracticalities associated with repeated passive administration. Here, we describe an optimized electroporation-based DNA gene transfer platform technology that can be utilized for production of functional mAbs in vivo, with the potential to reduce costs and administration burdens. We demonstrate that multiple mAbs can be simultaneously expressed at protective concentrations for a protracted period of time using DNA doses and electroporation conditions that are feasible clinically. The expressed mAbs could also protect mice against lethal influenza or Ebola virus challenges. Our findings suggest that this DNA gene transfer platform technology could be a game-changing advance that expands access to effective mAb therapeutics globally.

Deleting multiple lytic genes enhances biomass yield and production of recombinant proteins by Bacillus subtilis.

Science.gov (United States)

Wang, Yi; Chen, Zhenmin; Zhao, Ruili; Jin, Tingting; Zhang, Xiaoming; Chen, Xiangdong

2014-08-31

Bacillus subtilis is widely used in agriculture and industrial biotechnology; however, cell autolysis significantly decreases its yield in liquid cultures. Numerous factors mediate the lysis of B. subtilis, such as cannibalism factors, prophages, and peptidoglycan (PG) hydrolases. The aim of this work was to use molecular genetic techniques to develop a new strategy to prevent cell lysis and enhance biomass as well as the production of recombinant proteins. Five genes or genetic elements representing three different functional categories were studied as follows: lytC encoding PG hydrolases, the prophage genes xpf and yqxG-yqxH-cwlA (yGlA), and skfA and sdpC that encode cannibalism factors. Cell lysis was reduced and biomass was enhanced by deleting individually skfA, sdpC, xpf, and lytC. We constructed the multiple deletion mutant LM2531 (skfA sdpC lytC xpf) and found that after 4 h of culture, its biomass yield was significantly increased compared with that of prototypical B. subtilis 168 (wild-type) strain and that 15% and 92% of the cells were lysed in cultures of LM2531 and wild-type, respectively. Moreover, two expression vectors were constructed for producing recombinant proteins (β-galactosidase and nattokinase) under the control of the P43 promoter. Cultures of LM2531 and wild-type transformants produced 13741 U/ml and 7991 U/ml of intracellular β-galactosidase, respectively (1.72-fold increase). Further, the level of secreted nattokinase produced by strain LM2531 increased by 2.6-fold compared with wild-type (5226 IU/ml vs. 2028 IU/ml, respectively). Our novel, systematic multigene deletion approach designed to inhibit cell lysis significantly increased the biomass yield and the production of recombinant proteins by B. subtilis. These findings show promise for guiding efforts to manipulate the genomes of other B. subtilis strains that are used for industrial purposes.

The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking.

Science.gov (United States)

Warner, T S; Sinclair, D A; Fitzpatrick, K A; Singh, M; Devlin, R H; Honda, B M

1998-04-01

Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.

Optimization to the Culture Conditions for Phellinus Production with Regression Analysis and Gene-Set Based Genetic Algorithm

Science.gov (United States)

Li, Zhongwei; Xin, Yuezhen; Wang, Xun; Sun, Beibei; Xia, Shengyu; Li, Hui

2016-01-01

Phellinus is a kind of fungus and is known as one of the elemental components in drugs to avoid cancers. With the purpose of finding optimized culture conditions for Phellinus production in the laboratory, plenty of experiments focusing on single factor were operated and large scale of experimental data were generated. In this work, we use the data collected from experiments for regression analysis, and then a mathematical model of predicting Phellinus production is achieved. Subsequently, a gene-set based genetic algorithm is developed to optimize the values of parameters involved in culture conditions, including inoculum size, PH value, initial liquid volume, temperature, seed age, fermentation time, and rotation speed. These optimized values of the parameters have accordance with biological experimental results, which indicate that our method has a good predictability for culture conditions optimization. PMID:27610365

Identification of gene products suppressed by human immunodeficiency virus type 1 infection or gp120 exposure of primary human astrocytes by rapid subtraction hybridization.

Science.gov (United States)

Su, Zao-Zhong; Kang, Dong-Chul; Chen, Yinming; Pekarskaya, Olga; Chao, Wei; Volsky, David J; Fisher, Paul B

2003-06-01

Neurodegeneration and human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) are the major disease manifestations of HIV-1 colonization of the central nervous system (CNS). In the brain, HIV-1 replicates in microglial cells and infiltrating macrophages and it persists in a low-productive, noncytolytic state in astrocytes. Astrocytes play critical roles in the maintenance of the brain microenvironment, responses to injury, and in neuronal signal transmission, and disruption of these functions by HIV-1 could contribute to HAD. To better understand the potential effects of HIV-1 on astrocyte biology, the authors investigated changes in gene expression using an efficient and sensitive rapid subtraction hybridization approach, RaSH. Primary human astrocytes were isolated from abortus brain tissue, low-passage cells were infected with HIV-1 or mock infected, and total cellular RNAs were isolated at multiple time points over a period of 1 week. This approach is designed to identify gene products modulated early and late after HIV-1 infection and limits the cloning of genes displaying normal cell-cycle fluctuations in astrocytes. By subtracting temporal cDNAs derived from HIV-1-infected astrocytes from temporal cDNAs made from uninfected cells, 10 genes displaying reduced expression in infected cells, termed astrocyte suppressed genes (ASGs), were identified and their suppression was confirmed by Northern blot hybridization. Both known and novel ASGs, not reported in current DNA databases, that are down-regulated by HIV-1 infection are described. Northern blotting confirms suppression of the same panel of ASGs by treatment of astrocytes with recombinant HIV-1 envelope glycoprotein, gp120. These results extend our previous analysis of astrocyte genes induced or enhanced by HIV-1 infection and together they suggest that HIV-1 and viral proteins have profound effects on astrocyte physiology, which may influence their function in the CNS.

Primary structure of the tms and prs genes of Bacillus subtilis

DEFF Research Database (Denmark)

Nilsson, Dan; Hove-Jensen, Bjarne; Arnvig, Kirsten

1989-01-01

The nucleotide sequence was determined of a 3211 nucleotide pair EcoRI-PvuII DNA fragment containing the tms and prs genes as well as a part of the ctc gene of Bacillus subtilis. The prs gene encodes phosphoribosylpyrophosphate (PRPP) synthetase, whereas the functioning of the tms and ctc gene...... products remains to be established. The prs gene contains an open reading frame of 317 codons resulting in a subunit Mr of 34828. An open reading frame comprising the tms gene contained 456 codons resulting in a putative translation product with an Mr of 49,554. Comparison of the deduced B. subtilis PRPP...

Functional mitochondrial ATP synthase proteolipid gene produced by recombination of parental genes in a petunia somatic hybrid

International Nuclear Information System (INIS)

Rothenberg, M.; Hanson, M.R.

1988-01-01

A novel ATP synthase subunit 9 gene (atp9) was identified in the mitochondrial genome of a Petunia somatic hybrid line (13-133) which was produced from a fusion between Petunia lines 3688 and 3704. The novel gene was generated by intergenomic recombination between atp9 genes from the two parental plant lines. The entire atp9 coding region is represented on the recombinant gene. Comparison of gene sequences using electrophoresis and autoradiography, indicate that the 5' transcribed region is contributed by an atp9 gene from 3704 and the 3' transcribed region is contributed by an atp9 gene from 3688. The recombinant atp9 gene is transcriptionally active. The location of the 5' and 3' transcript termini are conserved with respect to the parental genes, resulting in the production of hybrid transcripts

Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene.

Science.gov (United States)

Chang, Zhenyi; Chen, Zhufeng; Wang, Na; Xie, Gang; Lu, Jiawei; Yan, Wei; Zhou, Junli; Tang, Xiaoyan; Deng, Xing Wang

2016-12-06

The breeding and large-scale adoption of hybrid seeds is an important achievement in agriculture. Rice hybrid seed production uses cytoplasmic male sterile lines or photoperiod/thermo-sensitive genic male sterile lines (PTGMS) as female parent. Cytoplasmic male sterile lines are propagated via cross-pollination by corresponding maintainer lines, whereas PTGMS lines are propagated via self-pollination under environmental conditions restoring male fertility. Despite huge successes, both systems have their intrinsic drawbacks. Here, we constructed a rice male sterility system using a nuclear gene named Oryza sativa No Pollen 1 (OsNP1). OsNP1 encodes a putative glucose-methanol-choline oxidoreductase regulating tapetum degeneration and pollen exine formation; it is specifically expressed in the tapetum and miscrospores. The osnp1 mutant plant displays normal vegetative growth but complete male sterility insensitive to environmental conditions. OsNP1 was coupled with an α-amylase gene to devitalize transgenic pollen and the red fluorescence protein (DsRed) gene to mark transgenic seed and transformed into the osnp1 mutant. Self-pollination of the transgenic plant carrying a single hemizygous transgene produced nontransgenic male sterile and transgenic fertile seeds in 1:1 ratio that can be sorted out based on the red fluorescence coded by DsRed Cross-pollination of the fertile transgenic plants to the nontransgenic male sterile plants propagated the male sterile seeds of high purity. The male sterile line was crossed with ∼1,200 individual rice germplasms available. Approximately 85% of the F1s outperformed their parents in per plant yield, and 10% out-yielded the best local cultivars, indicating that the technology is promising in hybrid rice breeding and production.

Re-engineering bacteria for ethanol production

Science.gov (United States)

Yomano, Lorraine P; York, Sean W; Zhou, Shengde; Shanmugam, Keelnatham; Ingram, Lonnie O

2014-05-06

The invention provides recombinant bacteria, which comprise a full complement of heterologous ethanol production genes. Expression of the full complement of heterologous ethanol production genes causes the recombinant bacteria to produce ethanol as the primary fermentation product when grown in mineral salts medium, without the addition of complex nutrients. Methods for producing the recombinant bacteria and methods for producing ethanol using the recombinant bacteria are also disclosed.

Putative carotenoid genes expressed under the regulation of Shine-Dalgarno regions in Escherichia coli for efficient lycopene production.

Science.gov (United States)

Jin, Weiyue; Xu, Xian; Jiang, Ling; Zhang, Zhidong; Li, Shuang; Huang, He

2015-11-01

Putative genes crtE, crtB, and crtI from Deinococcus wulumiqiensis R12, a novel species, were identified by genome mining and were co-expressed using the optimized Shine-Dalgarno (SD) regions to improve lycopene yield. A lycopene biosynthesis pathway was constructed by co-expressing these three genes in Escherichia coli. After optimizing the upstream SD regions and the culture medium, the recombinant strain EDW11 produced 88 mg lycopene g(-1) dry cell wt (780 mg lycopene l(-1)) after 40 h fermentation without IPTG induction, while the strain EDW without optimized SD regions only produced 49 mg lycopene g(-1) dry cell wt (417 mg lycopene l(-1)). Based on the optimization of the upstream SD regions and culture medium, the yield of the strain EDW11 reached a high level during microbial lycopene production until now.

Relationships between functional genes in Lactobacillus delbrueckii ssp. bulgaricus isolates and phenotypic characteristics associated with fermentation time and flavor production in yogurt elucidated using multilocus sequence typing.

Science.gov (United States)

Liu, Wenjun; Yu, Jie; Sun, Zhihong; Song, Yuqin; Wang, Xueni; Wang, Hongmei; Wuren, Tuoya; Zha, Musu; Menghe, Bilige; Heping, Zhang

2016-01-01

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is well known for its worldwide application in yogurt production. Flavor production and acid producing are considered as the most important characteristics for starter culture screening. To our knowledge this is the first study applying functional gene sequence multilocus sequence typing technology to predict the fermentation and flavor-producing characteristics of yogurt-producing bacteria. In the present study, phenotypic characteristics of 35 L. bulgaricus strains were quantified during the fermentation of milk to yogurt and during its subsequent storage; these included fermentation time, acidification rate, pH, titratable acidity, and flavor characteristics (acetaldehyde concentration). Furthermore, multilocus sequence typing analysis of 7 functional genes associated with fermentation time, acid production, and flavor formation was done to elucidate the phylogeny and genetic evolution of the same L. bulgaricus isolates. The results showed that strains significantly differed in fermentation time, acidification rate, and acetaldehyde production. Combining functional gene sequence analysis with phenotypic characteristics demonstrated that groups of strains established using genotype data were consistent with groups identified based on their phenotypic traits. This study has established an efficient and rapid molecular genotyping method to identify strains with good fermentation traits; this has the potential to replace time-consuming conventional methods based on direct measurement of phenotypic traits. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Evolutionary history of chordate PAX genes: dynamics of change in a complex gene family.

Directory of Open Access Journals (Sweden)

Vanessa Rodrigues Paixão-Côrtes

Full Text Available Paired box (PAX genes are transcription factors that play important roles in embryonic development. Although the PAX gene family occurs in animals only, it is widely distributed. Among the vertebrates, its 9 genes appear to be the product of complete duplication of an original set of 4 genes, followed by an additional partial duplication. Although some studies of PAX genes have been conducted, no comprehensive survey of these genes across the entire taxonomic unit has yet been attempted. In this study, we conducted a detailed comparison of PAX sequences from 188 chordates, which revealed restricted variation. The absence of PAX4 and PAX8 among some species of reptiles and birds was notable; however, all 9 genes were present in all 74 mammalian genomes investigated. A search for signatures of selection indicated that all genes are subject to purifying selection, with a possible constraint relaxation in PAX4, PAX7, and PAX8. This result indicates asymmetric evolution of PAX family genes, which can be associated with the emergence of adaptive novelties in the chordate evolutionary trajectory.

Gram-scale production of plasmid pUDK-HGF with current good manufacturing practices for gene therapy of critical limb ischemia.

Science.gov (United States)

Hu, ChunSheng; Cheng, XiaoChen; Lu, YuXin; Wu, ZuZe; Zhang, QingLin

2016-11-16

The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24 ± 0.41 g of pharmaceutical pUDK-HGF from 1.0 kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of > 95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications.

An efficient and reproducible protocol for the production of salt tolerant transgenic wheat plants expressing the Arabidopsis AtNHX1 gene.

Science.gov (United States)

Moghaieb, Reda E A; Sharaf, Ahmed N; Soliman, Mohamed H; El-Arabi, Nagwa I; Momtaz, Osama A

2014-01-01

We present an efficient method for the production of transgenic salt tolerant hexaploid wheat plants expressing the Arabidopsis AtNHX1 gene. Wheat mature zygotic embryos were isolated from two hexaploid bread wheat (Triticum aestivum) cultivars (namely: Gemmeiza 9 and Gemmeiza 10) and were transformed with the A. tumefaciens LBA4404 harboring the pBI-121 vector containing the AtNHX1 gene. Transgenic wheat lines that express the gus intron was obtained and used as control. The results confirmed that npt-II gene could be transmitted and expressed in the T2 following 3:1 Mendelian segregation while the control plant couldn't. The data indicate that, the AtNHX1 gene was integrated in a stable manner into the wheat genome and the corresponding transcripts were expressed. The transformation efficiency was 5.7 and 7.5% for cultivars Gemmeiza 10 and Gemmeiza 9, respectively. A greenhouse experiment was conducted to investigate the effect of AtNHX1 gene in wheat salt tolerance. The transgenic wheat lines could maintain high growth rate under salt stress condition (350 mM NaCl) while the control plant couldn't. The results confirmed that Na(+)/H(+) antiporter gene AtNHX1 increased salt tolerance by increasing Na(+) accumulation and keeping K+/Na(+) balance. Thus, transgenic plants showed high tolerance to salt stress and can be considered as a new genetic resource in breeding programs.

Liver upregulation of genes involved in cortisol production and action is associated with metabolic syndrome in morbidly obese patients.

Science.gov (United States)

Torrecilla, Esther; Fernández-Vázquez, Gumersindo; Vicent, David; Sánchez-Franco, Franco; Barabash, Ana; Cabrerizo, Lucio; Sánchez-Pernaute, Andrés; Torres, Antonio J; Rubio, Miguel Angel

2012-03-01

Hepatic 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activity, which converts cortisone (inactive) to cortisol, is downregulated in obesity. However, this compensation fails in obese with metabolic abnormalities, such as diabetes. To further characterize the tissue-specific cortisol regeneration in obesity, we have investigated the mRNA expression of genes related to local cortisol production, i.e., 11β-HSD1, hexose-6-phosphate dehydrogenase (H6PDH) and cortisol action, glucocorticoid receptor (GR) and a cortisol target gene, phosphoenolpyruvate carboxykinase (PEPCK) in the liver, and visceral (VAT) and subcutaneous (SAT) adipose tissues from morbidly obese patients with and without metabolic syndrome (MS). Fifty morbidly obese patients undergoing bariatric surgery, 14 men (mean age, 41.3 ± 3.5 years; BMI, 48.0 ± 3.6 kg/m(2)) and 36 women (mean age, 44.6 ± 1.9 years; BMI, 44.9 ± 1.2 kg/m(2)), were classified as having MS (MS+, n = 20) or not (MS-, n = 30). Tissue mRNA levels were measured by real-time polymerase chain reaction. Hepatic mRNA levels of these genes were higher in obese patients with MS (11β-HSD1, P = 0.002; H6PDH, P = 0.043; GR, P = 0.033; PEPCK, P = 0.032) and positively correlated with the number of clinical characteristics that define the MS. The expression of the four genes positively correlated among them. In contrast to the liver, these genes were not differently expressed in VAT or SAT, when MS+ and MS- obese patients were compared. Coordinated liver-specific upregulation of genes involved in local cortisol regeneration and action support the concept that local hepatic hypercortisolism contributes to development of MS in morbidly obese patients.

Analysis of β-galactosidase production and their genes of two strains of Lactobacillus bulgaricus.

Science.gov (United States)

Zhang, Wen; Wang, Chuan; Huang, Cheng-Yu; Yu, Qian; Liu, Heng-Chuan; Zhang, Chao-Wu; Pei, Xiao-Fang; Xu, Xin; Wang, Guo-Qing

2012-06-01

A bacterial β-galactosidase delivery system is a potential therapy for lactose intolerance. Currently, two Lactobacillus bulgaricus strains with different biological characteristics are under consideration as potential sources. However, differences in these β-galactosidase genes and their resulting production levels are poorly characterized. The β-galactosidase ORF of L. bulgaricus yogurt isolate had high variability and was terminated at site 1924 due to a stop codon. However, the full 114 kDa β-galactosidase band was still resolved by SDS-PAGE, which may indicate that the interrupted ORF was translated into more than one peptide, and they together were folded into the complete enzyme protein that showed much higher β-galactosidase activity (6.2 U/mg protein) than the enzyme generated from L. bulgaricus reference strain (2.5 U/mg protein).

Loss of retrovirus production in JB/RH melanoma cells transfected with H-2Kb and TAP-1 genes.

Science.gov (United States)

Li, M; Xu, F; Muller, J; Huang, X; Hearing, V J; Gorelik, E

1999-01-20

JB/RH1 melanoma cells, as well as other melanomas of C57BL/6 mice (B16 and JB/MS), express a common melanoma-associated antigen (MAA) encoded by an ecotropic melanoma-associated retrovirus (MelARV). JB/RH1 cells do not express the H-2Kb molecules due to down-regulation of the H-2Kb and TAP-1 genes. When JB/RH1 cells were transfected with the H-2Kb and cotransfected with the TAP-1 gene, it resulted in the appearance of H-2Kb molecules and an increase in their immunogenicity, albeit they lost expression of retrovirus-encoded MAA recognized by MM2-9B6 mAb. Loss of MAA was found to result from a complete and stable elimination of ecotropic MelARV production in the H-2Kb/TAP-1-transfected JB/RH1 cells. Northern blot analysis showed no differences in ecotropic retroviral messages in MelARV-producing and -nonproducing melanoma cells, suggesting that loss of MelARV production was not due to down-regulation of MelARV transcription. Southern blot analysis revealed several rearrangements in the proviral DNA of H-2Kb-positive JB/RH1 melanoma cells. Sequence analysis of the ecotropic proviral DNA from these cells showed numerous nucleotide substitutions, some of which resulted in the appearance of a novel intraviral PstI restriction site and the loss of a HindIII restriction site in the pol region. PCR amplification of the proviral DNAs indicates that an ecotropic provirus found in the H-2Kb-positive cells is novel and does not preexist in the parental H-2Kb-negative melanoma cells. Conversely, the ecotropic provirus of the parental JB/RH1 cells was not amplifable from the H-2Kb-positive cells. Our data indicate that stable loss of retroviral production in the H-2Kb/TAP-1-transfected melanoma cells is probably due to the induction of recombination between a productive ecotropic MelARV and a defective nonecotropic provirus leading to the generation of a defective ecotropic provirus and the loss of MelARV production and expression of the retrovirus-encoded MAA. Copyright 1999

Construction of a fusion gene containing hepatitis B virus L gene ...

African Journals Online (AJOL)

Jane

2011-10-05

Oct 5, 2011 ... the successful construction of a recombinant yeast expression vector containing gene coding L protein and Ag85B ..... the production of memory T cells, promote cytokine secretion and ... Dual DNA vaccination of rainbow trout.

Gene Ontology Consortium: going forward.

Science.gov (United States)

2015-01-01

The Gene Ontology (GO; http://www.geneontology.org) is a community-based bioinformatics resource that supplies information about gene product function using ontologies to represent biological knowledge. Here we describe improvements and expansions to several branches of the ontology, as well as updates that have allowed us to more efficiently disseminate the GO and capture feedback from the research community. The Gene Ontology Consortium (GOC) has expanded areas of the ontology such as cilia-related terms, cell-cycle terms and multicellular organism processes. We have also implemented new tools for generating ontology terms based on a set of logical rules making use of templates, and we have made efforts to increase our use of logical definitions. The GOC has a new and improved web site summarizing new developments and documentation, serving as a portal to GO data. Users can perform GO enrichment analysis, and search the GO for terms, annotations to gene products, and associated metadata across multiple species using the all-new AmiGO 2 browser. We encourage and welcome the input of the research community in all biological areas in our continued effort to improve the Gene Ontology. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification of the G13 (cAMP-response-element-binding protein-related protein) gene product related to activating transcription factor 6 as a transcriptional activator of the mammalian unfolded protein response.

Science.gov (United States)

Haze, K; Okada, T; Yoshida, H; Yanagi, H; Yura, T; Negishi, M; Mori, K

2001-04-01

Eukaryotic cells control the levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element consisting of 19 nt (CCAATN(9)CCACG), the CCACG part of which is considered to provide specificity. We recently identified the basic leucine zipper (bZIP) protein ATF6 as a mammalian UPR-specific transcription factor; ATF6 is activated by ER stress-induced proteolysis and binds directly to CCACG. Here we report that eukaryotic cells express another bZIP protein closely related to ATF6 in both structure and function. This protein encoded by the G13 (cAMP response element binding protein-related protein) gene is constitutively synthesized as a type II transmembrane glycoprotein anchored in the ER membrane and processed into a soluble form upon ER stress as occurs with ATF6. The proteolytic processing of ATF6 and the G13 gene product is accompanied by their relocation from the ER to the nucleus; their basic regions seem to function as a nuclear localization signal. Overexpression of the soluble form of the G13 product constitutively activates the UPR, whereas overexpression of a mutant lacking the activation domain exhibits a strong dominant-negative effect. Furthermore, the soluble forms of ATF6 and the G13 gene product are unable to bind to several point mutants of the cis-acting ER stress response element in vitro that hardly respond to ER stress in vivo. We thus concluded that the two related bZIP proteins are crucial transcriptional regulators of the mammalian UPR, and propose calling the ATF6 gene product ATF6alpha and the G13 gene product ATF6beta.

Molecular cloning of cellulase genes from indigenous bacterial isolates

International Nuclear Information System (INIS)

Jong Bor Chyan; Pauline Liew Woan Ying; Mat Rasol Awang

2006-01-01

Indigenous cellulolytic bacterial isolates having high activities in degrading carboxymethyl cellulose (CMC) were isolated from local environments. Identification of these isolates were performed by molecular techniques. By using polymerase chain reaction (PCR) techniques, PCR products encoding cellulase gene were amplified from the total genomic DNAs. Purified PCR product was successfully cloned and expressed in Escherichia coli host system. The complete nucleotide sequences of the cellulase genes determined. The analysis of amino acid sequences deduced from the genes indicated that the cloned DNA fragments show high homology to those of endoglucanase genes of family GH5. All cloned genes consist of an N-terminal signal peptide, a catalytic domain of family 5 glycosyl hydrolase and a cellulose-binding domain of family III. (Author)

In silico analysis of miRNA-mediated gene regulation in OCA and OA genes.

Science.gov (United States)

Kamaraj, Balu; Gopalakrishnan, Chandrasekhar; Purohit, Rituraj

2014-12-01

Albinism is an autosomal recessive genetic disorder due to low secretion of melanin. The oculocutaneous albinism (OCA) and ocular albinism (OA) genes are responsible for melanin production and also act as a potential targets for miRNAs. The role of miRNA is to inhibit the protein synthesis partially or completely by binding with the 3'UTR of the mRNA thus regulating gene expression. In this analysis, we predicted the genetic variation that occurred in 3'UTR of the transcript which can be a reason for low melanin production thus causing albinism. The single nucleotide polymorphisms (SNPs) in 3'UTR cause more new binding sites for miRNA which binds with mRNA which leads to inhibit the translation process either partially or completely. The SNPs in the mRNA of OCA and OA genes can create new binding sites for miRNA which may control the gene expression and lead to hypopigmentation. We have developed a computational procedure to determine the SNPs in the 3'UTR region of mRNA of OCA (TYR, OCA2, TYRP1 and SLC45A2) and OA (GPR143) genes which will be a potential cause for albinism. We identified 37 SNPs in five genes that are predicted to create 87 new binding sites on mRNA, which may lead to abrogation of the translation process. Expression analysis confirms that these genes are highly expressed in skin and eye regions. It is well supported by enrichment analysis that these genes are mainly involved in eye pigmentation and melanin biosynthesis process. The network analysis also shows how the genes are interacting and expressing in a complex network. This insight provides clue to wet-lab researches to understand the expression pattern of OCA and OA genes and binding phenomenon of mRNA and miRNA upon mutation, which is responsible for inhibition of translation process at genomic levels.

Differential Gene Expression of Fibroblasts: Keloid versus Normal

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Michael F. Angel

2002-11-01

Full Text Available Abstract: This study investigated gene regulation and unique gene products in both keloid (KDF and normal (NDF dermal fibroblasts in established cell lines. For gene regulation, NDF versus KDF were compared using Clontech's Atlas™ Human cDNA Expression Array while unique gene products were studied using RNA Fingerprinting Kit. RNA from each sample was converted to cDNA using oligo-dT primers. Down-regulated genes using Atlas Array in KDF were 1 60 S ribosomal protein, 2 Thioredoxin dependent peroxidase, 3 Nuclease sensitive element DNA binding protein, 4 c-myc purine-binding transcription factor, 5 c-AMP dependent protein kinase, and, 6 Heat Shock Protein 90 kDa. Genes that are up regulated in KDF were 1 Tubulin and 2 Heat Shock Protein 27 kDa. With the differential display, we found 17 bands unique to both KDF and NDF. The specific gene and the manner in which they were differentially regulated have direct implications to understanding keloid fibroblast proliferation.

Normal Collagen and Bone Production by Gene-targeted Human Osteogenesis Imperfecta iPSCs

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Deyle, David R; Khan, Iram F; Ren, Gaoying; Wang, Pei-Rong; Kho, Jordan; Schwarze, Ulrike; Russell, David W

2012-01-01

Osteogenesis imperfecta (OI) is caused by dominant mutations in the type I collagen genes. In principle, the skeletal abnormalities of OI could be treated by transplantation of patient-specific, bone-forming cells that no longer express the mutant gene. Here, we develop this approach by isolating mesenchymal cells from OI patients, inactivating their mutant collagen genes by adeno-associated virus (AAV)-mediated gene targeting, and deriving induced pluripotent stem cells (iPSCs) that were expanded and differentiated into mesenchymal stem cells (iMSCs). Gene-targeted iMSCs produced normal collagen and formed bone in vivo, but were less senescent and proliferated more than bone-derived MSCs. To generate iPSCs that would be more appropriate for clinical use, the reprogramming and selectable marker transgenes were removed by Cre recombinase. These results demonstrate that the combination of gene targeting and iPSC derivation can be used to produce potentially therapeutic cells from patients with genetic disease. PMID:22031238

Bovine Host Genetic Variation Influences Rumen Microbial Methane Production with Best Selection Criterion for Low Methane Emitting and Efficiently Feed Converting Hosts Based on Metagenomic Gene Abundance.

Directory of Open Access Journals (Sweden)

Rainer Roehe

2016-02-01

Full Text Available Methane produced by methanogenic archaea in ruminants contributes significantly to anthropogenic greenhouse gas emissions. The host genetic link controlling microbial methane production is unknown and appropriate genetic selection strategies are not developed. We used sire progeny group differences to estimate the host genetic influence on rumen microbial methane production in a factorial experiment consisting of crossbred breed types and diets. Rumen metagenomic profiling was undertaken to investigate links between microbial genes and methane emissions or feed conversion efficiency. Sire progeny groups differed significantly in their methane emissions measured in respiration chambers. Ranking of the sire progeny groups based on methane emissions or relative archaeal abundance was consistent overall and within diet, suggesting that archaeal abundance in ruminal digesta is under host genetic control and can be used to genetically select animals without measuring methane directly. In the metagenomic analysis of rumen contents, we identified 3970 microbial genes of which 20 and 49 genes were significantly associated with methane emissions and feed conversion efficiency respectively. These explained 81% and 86% of the respective variation and were clustered in distinct functional gene networks. Methanogenesis genes (e.g. mcrA and fmdB were associated with methane emissions, whilst host-microbiome cross talk genes (e.g. TSTA3 and FucI were associated with feed conversion efficiency. These results strengthen the idea that the host animal controls its own microbiota to a significant extent and open up the implementation of effective breeding strategies using rumen microbial gene abundance as a predictor for difficult-to-measure traits on a large number of hosts. Generally, the results provide a proof of principle to use the relative abundance of microbial genes in the gastrointestinal tract of different species to predict their influence on traits e

Evolution of Chemical Diversity in a Group of Non-Reduced Polyketide Gene Clusters: Using Phylogenetics to Inform the Search for Novel Fungal Natural Products

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Kurt Throckmorton

2015-09-01

Full Text Available Fungal polyketides are a diverse class of natural products, or secondary metabolites (SMs, with a wide range of bioactivities often associated with toxicity. Here, we focus on a group of non-reducing polyketide synthases (NR-PKSs in the fungal phylum Ascomycota that lack a thioesterase domain for product release, group V. Although widespread in ascomycete taxa, this group of NR-PKSs is notably absent in the mycotoxigenic genus Fusarium and, surprisingly, found in genera not known for their secondary metabolite production (e.g., the mycorrhizal genus Oidiodendron, the powdery mildew genus Blumeria, and the causative agent of white-nose syndrome in bats, Pseudogymnoascus destructans. This group of NR-PKSs, in association with the other enzymes encoded by their gene clusters, produces a variety of different chemical classes including naphthacenediones, anthraquinones, benzophenones, grisandienes, and diphenyl ethers. We discuss the modification of and transitions between these chemical classes, the requisite enzymes, and the evolution of the SM gene clusters that encode them. Integrating this information, we predict the likely products of related but uncharacterized SM clusters, and we speculate upon the utility of these classes of SMs as virulence factors or chemical defenses to various plant, animal, and insect pathogens, as well as mutualistic fungi.

Smokeless tobacco products harbor diverse bacterial microbiota that differ across products and brands.

Science.gov (United States)

Smyth, Eoghan M; Kulkarni, Prachi; Claye, Emma; Stanfill, Stephen; Tyx, Robert; Maddox, Cynthia; Mongodin, Emmanuel F; Sapkota, Amy R

2017-07-01

Smokeless tobacco products contain numerous chemical compounds, including known human carcinogens. Other smokeless tobacco constituents, including bacteria, may also contribute to adverse health effects among smokeless tobacco users. However, there is a lack of data regarding the microbial constituents of smokeless tobacco. Our goal was to characterize the bacterial microbiota of different smokeless tobacco products and evaluate differences across product types and brands. DNA was extracted from 15 brands of smokeless tobacco products (including dry snuff, moist snuff, snus, and Swedish snus) and 6 handmade products (e.g., toombak) using an enzymatic and mechanical lysis approach. Bacterial community profiling was performed using PCR amplification of the V1-V2 hypervariable region of the 16S rRNA gene, followed by 454 pyrosequencing of the resulting amplicons and sequence analysis using the QIIME package. Total viable counts were also determined to estimate the number of viable bacteria present in each product. Average total viable counts ranged from 0 to 9.35 × 10 7  CFU g -1 . Analysis of the 16S rRNA gene sequences revealed high bacterial diversity across the majority of products tested: dry snuff products where characterized by the highest diversity indices compared to other products. The most dominant bacterial phyla across all products were Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes. Significant differences in both bacterial community composition and in silico predicted gene content were observed between smokeless tobacco product types and between brands of specific smokeless tobacco products. These data are useful in order to comprehensively address potential health risks associated with the use of smokeless tobacco products.

Oil production at different stages of leaf development in Lippia alba

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Diego Pandeló

2012-06-01

Full Text Available The aim of this work was to analyze terpene oil production and terpene synthases (TPS gene expression from leaves at different developmental stages of different chemotypes of Lippia alba (Mill. N.E. Br. ex Britton & P. Wilson, Verbenaceae. Hydro-distilled essential oil were used for chemical analysis and gene expression of three monoterpene synthase genes called LaTPS12, LaTPS23 and LaTPS25 were used for analyses of gene expression associated to oil production. The putative genes were associated to TPS-b gene class. Semi-quantitative PCR and quantitative PCR (qPCR analysis were used to investigate the expression profile of those three putative genes in different leaf stages and different chemotypes. Additionally, total oil production and gene expression of putative TPS genes cloned from L. alba chemotype linalool were evaluated at different stages of leaf development. The expression level of those three genes was higher when the highest oil production was observed, mainly in young leaves at the fourth nodal segment for all evaluated chemotypes. Total oil production was higher at leaves that had unopened trichomes. We also observed that the 1mM of MeJA treatment increased the gene expression in all chemotypes after 24 h elicitation.

Oil production at different stages of leaf development in Lippia alba

Directory of Open Access Journals (Sweden)

Diego Pandeló

2012-01-01

Full Text Available The aim of this work was to analyze terpene oil production and terpene synthases (TPS gene expression from leaves at different developmental stages of different chemotypes of Lippia alba (Mill. N.E. Br. ex Britton & P. Wilson, Verbenaceae. Hydro-distilled essential oil were used for chemical analysis and gene expression of three monoterpene synthase genes called LaTPS12, LaTPS23 and LaTPS25 were used for analyses of gene expression associated to oil production. The putative genes were associated to TPS-b gene class. Semi-quantitative PCR and quantitative PCR (qPCR analysis were used to investigate the expression profile of those three putative genes in different leaf stages and different chemotypes. Additionally, total oil production and gene expression of putative TPS genes cloned from L. alba chemotype linalool were evaluated at different stages of leaf development. The expression level of those three genes was higher when the highest oil production was observed, mainly in young leaves at the fourth nodal segment for all evaluated chemotypes. Total oil production was higher at leaves that had unopened trichomes. We also observed that the 1mM of MeJA treatment increased the gene expression in all chemotypes after 24 h elicitation.

Learning gene regulatory networks from gene expression data using weighted consensus

KAUST Repository

Fujii, Chisato; Kuwahara, Hiroyuki; Yu, Ge; Guo, Lili; Gao, Xin

2016-01-01

An accurate determination of the network structure of gene regulatory systems from high-throughput gene expression data is an essential yet challenging step in studying how the expression of endogenous genes is controlled through a complex interaction of gene products and DNA. While numerous methods have been proposed to infer the structure of gene regulatory networks, none of them seem to work consistently over different data sets with high accuracy. A recent study to compare gene network inference methods showed that an average-ranking-based consensus method consistently performs well under various settings. Here, we propose a linear programming-based consensus method for the inference of gene regulatory networks. Unlike the average-ranking-based one, which treats the contribution of each individual method equally, our new consensus method assigns a weight to each method based on its credibility. As a case study, we applied the proposed consensus method on synthetic and real microarray data sets, and compared its performance to that of the average-ranking-based consensus and individual inference methods. Our results show that our weighted consensus method achieves superior performance over the unweighted one, suggesting that assigning weights to different individual methods rather than giving them equal weights improves the accuracy. © 2016 Elsevier B.V.

Learning gene regulatory networks from gene expression data using weighted consensus

KAUST Repository

Fujii, Chisato

2016-08-25

An accurate determination of the network structure of gene regulatory systems from high-throughput gene expression data is an essential yet challenging step in studying how the expression of endogenous genes is controlled through a complex interaction of gene products and DNA. While numerous methods have been proposed to infer the structure of gene regulatory networks, none of them seem to work consistently over different data sets with high accuracy. A recent study to compare gene network inference methods showed that an average-ranking-based consensus method consistently performs well under various settings. Here, we propose a linear programming-based consensus method for the inference of gene regulatory networks. Unlike the average-ranking-based one, which treats the contribution of each individual method equally, our new consensus method assigns a weight to each method based on its credibility. As a case study, we applied the proposed consensus method on synthetic and real microarray data sets, and compared its performance to that of the average-ranking-based consensus and individual inference methods. Our results show that our weighted consensus method achieves superior performance over the unweighted one, suggesting that assigning weights to different individual methods rather than giving them equal weights improves the accuracy. © 2016 Elsevier B.V.

Evolutionary dynamics of human autoimmune disease genes and malfunctioned immunological genes

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Podder Soumita

2012-01-01

Full Text Available Abstract Background One of the main issues of molecular evolution is to divulge the principles in dictating the evolutionary rate differences among various gene classes. Immunological genes have received considerable attention in evolutionary biology as candidates for local adaptation and for studying functionally important polymorphisms. The normal structure and function of immunological genes will be distorted when they experience mutations leading to immunological dysfunctions. Results Here, we examined the fundamental differences between the genes which on mutation give rise to autoimmune or other immune system related diseases and the immunological genes that do not cause any disease phenotypes. Although the disease genes examined are analogous to non-disease genes in product, expression, function, and pathway affiliation, a statistically significant decrease in evolutionary rate has been found in autoimmune disease genes relative to all other immune related diseases and non-disease genes. Possible ways of accumulation of mutation in the three steps of the central dogma (DNA-mRNA-Protein have been studied to trace the mutational effects predisposed to disease consequence and acquiring higher selection pressure. Principal Component Analysis and Multivariate Regression Analysis have established the predominant role of single nucleotide polymorphisms in guiding the evolutionary rate of immunological disease and non-disease genes followed by m-RNA abundance, paralogs number, fraction of phosphorylation residue, alternatively spliced exon, protein residue burial and protein disorder. Conclusions Our study provides an empirical insight into the etiology of autoimmune disease genes and other immunological diseases. The immediate utility of our study is to help in disease gene identification and may also help in medicinal improvement of immune related disease.